Upinder Singh

All Publications

• 2024 Update of the RECOVER-Adult Long COVID Research Index. JAMA Geng, L. N., Erlandson, K. M., Hornig, M., Letts, R., Selvaggi, C., Ashktorab, H., Atieh, O., Bartram, L., Brim, H., Brosnahan, S. B., Brown, J., Castro, M., Charney, A., Chen, P., Deeks, S. G., Erdmann, N., Flaherman, V. J., Ghamloush, M. A., Goepfert, P., Goldman, J. D., Han, J. E., Hess, R., Hirshberg, E., Hoover, S. E., Katz, S. D., Kelly, J. D., Klein, J. D., Krishnan, J. A., Lee-Iannotti, J., Levitan, E. B., Marconi, V. C., Metz, T. D., Modes, M. E., Nikolich, J. Ž., Novak, R. M., Ofotokun, I., Okumura, M. J., Parthasarathy, S., Patterson, T. F., Peluso, M. J., Poppas, A., Quintero Cardona, O., Scott, J., Shellito, J., Sherif, Z. A., Singer, N. G., Taylor, B. S., Thaweethai, T., Verduzco-Gutierrez, M., Wisnivesky, J., McComsey, G. A., Horwitz, L. I., Foulkes, A. S. 2024

  Abstract

  Classification of persons with long COVID (LC) or post-COVID-19 condition must encompass the complexity and heterogeneity of the condition. Iterative refinement of the classification index for research is needed to incorporate newly available data as the field rapidly evolves.To update the 2023 research index for adults with LC using additional participant data from the Researching COVID to Enhance Recovery (RECOVER-Adult) study and an expanded symptom list based on input from patient communities.Prospective, observational cohort study including adults 18 years or older with or without known prior SARS-CoV-2 infection who were enrolled at 83 sites in the US and Puerto Rico. Included participants had at least 1 study visit taking place 4.5 months after first SARS-CoV-2 infection or later, and not within 30 days of a reinfection. The study visits took place between October 2021 and March 2024.SARS-CoV-2 infection.Presence of LC and participant-reported symptoms.A total of 13 647 participants (11 743 with known SARS-CoV-2 infection and 1904 without known prior SARS-CoV-2 infection; median age, 45 years [IQR, 34-69 years]; and 73% were female) were included. Using the least absolute shrinkage and selection operator analysis regression approach from the 2023 model, symptoms contributing to the updated 2024 index included postexertional malaise, fatigue, brain fog, dizziness, palpitations, change in smell or taste, thirst, chronic cough, chest pain, shortness of breath, and sleep apnea. For the 2024 LC research index, the optimal threshold to identify participants with highly symptomatic LC was a score of 11 or greater. The 2024 index classified 20% of participants with known prior SARS-CoV-2 infection and 4% of those without known prior SARS-CoV-2 infection as having likely LC (vs 21% and 5%, respectively, using the 2023 index) and 39% of participants with known prior SARS-CoV-2 infection as having possible LC, which is a new category for the 2024 model. Cluster analysis identified 5 LC subtypes that tracked quality-of-life measures.The 2024 LC research index for adults builds on the 2023 index with additional data and symptoms to help researchers classify symptomatic LC and its symptom subtypes. Continued future refinement of the index will be needed as the understanding of LC evolves.

  View details for DOI 10.1001/jama.2024.24184

  View details for PubMedID 39693079

• Identification of two transcription factors that work coordinately to regulate early development inEntamoeba. mBio Lozano-Amado, D., Singh, U. 2024: e0225024

  Abstract

  The protozoan parasite Entamoeba has a life cycle that switches between infective cysts and invasive trophozoites. Encystation, a crucial process in parasite biology, is controlled by different mechanisms including transcriptional control. We identified two nuclear proteins in Entamoeba invadens, EIN_066100 and EIN_085620, that regulate parasite development by binding to a DNA motif (TCACTTTC) in the promoter regions of genes upregulated in the first 8 h of stage conversion. Overexpression of EIN_066100, a homolog of MAK16 protein, resulted in reduced amoebic proliferation without affecting encystation efficiency. Overexpression of EIN_085620, a protein with an RNA-recognition motif (RRM), led to increased encystation efficiency. Glutathione S-transferase (GST) pull down assays revealed that EIN_066100 interacts with EIN_085620 both in vivo and in vitro, and this interaction is mediated by the EIN_085620 RRM domain. By evaluating truncated proteins with deletions at either the N-terminal or C-terminal regions of EIN_066100, we elucidated the importance of its N-terminal region in proper protein localization, proliferation, encystation, and interaction with EIN_085620. Taken together, these results indicate a coordinated role of EIN_066100 and EIN_085620 in regulating Entamoeba development. This work sheds light on the molecular mechanisms in the earliest stages of Entamoeba encystation.IMPORTANCEAn important biological process in the biology of Entamoeba is stage conversion, which plays a crucial role in disease propagation, facilitating parasite survival outside the host and spreading to new hosts. Multiple mechanisms contribute to controlling the expression of amebic stage-specific genes such as epigenetic and transcriptional control. Identification of early transcriptional control regulators is crucial to understanding the initiation of the encystation cascade. We identified two nuclear proteins, EIN_066100 and EIN_085620, involved in the proliferation and developmental regulation of E. invadens. These proteins work by direct binding to each other and mediating encystation efficiency. Study of new regulators involved in Entamoeba development represents an important advance in a critical aspect of parasite biology.

  View details for DOI 10.1128/mbio.02250-24

  View details for PubMedID 39540742

• Sex differences and immune correlates of Long Covid development, symptom persistence, and resolution. Science translational medicine Hamlin, R. E., Pienkos, S. M., Chan, L., Stabile, M. A., Pinedo, K., Rao, M., Grant, P., Bonilla, H., Holubar, M., Singh, U., Jacobson, K. B., Jagannathan, P., Maldonado, Y., Holmes, S. P., Subramanian, A., Blish, C. A. 2024; 16 (773): eadr1032

  Abstract

  Sex differences have been observed in acute coronavirus disease 2019 (COVID-19) and Long Covid (LC) outcomes, with greater disease severity and mortality during acute infection in males and greater proportions of females developing LC. We hypothesized that sex-specific immune dysregulation contributes to LC pathogenesis. To investigate the immunologic underpinnings of LC development and symptom persistence, we performed multiomic analyses on blood samples obtained during acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and 3 and 12 months after infection in a cohort of 45 participants who either developed LC or recovered. Several sex-specific immune pathways were associated with LC. Males who would later develop LC exhibited increases in transforming growth factor-β (TGF-β) signaling during acute infection, whereas females who would go on to develop LC had reduced TGFB1 expression. Females who developed LC demonstrated increased expression of XIST, an RNA gene implicated in autoimmunity, during acute infection compared with females who recovered. Many immune features of LC were also conserved across sexes, such as alterations in monocyte phenotype and activation state. Nuclear factor κB (NF-κB) transcription factors were up-regulated in many cell types at acute and convalescent time points. Those with ongoing LC demonstrated reduced ETS1 expression across lymphocyte subsets and elevated intracellular IL-4 in T cell subsets, suggesting that ETS1 alterations may drive aberrantly elevated T helper cell 2-like responses in LC. Altogether, this study describes multiple innate and adaptive immune correlates of LC, some of which differ by sex, and offers insights toward the pursuit of tailored therapeutics.

  View details for DOI 10.1126/scitranslmed.adr1032

  View details for PubMedID 39536117

• Engaging communities in therapeutics clinical research during pandemics: Experiences and lessons from the ACTIV COVID-19 therapeutics research initiative JOURNAL OF CLINICAL AND TRANSLATIONAL SCIENCE Wohl, D. A., Adam, S. J., Gibbs, K. W., Moskowitz, A. L., Ortel, T. L., Singh, U., Jilg, N., Evering, T. H., Fischer, W. A., Taiwo, B. O., Daar, E. S., Lindsell, C. J., Naggie, S., Rothman, R. L., Dunsmore, S. E., McAdams, M., Vail, J., Jayaweera, D. 2024; 8 (1)

  View details for DOI 10.1017/cts.2024.561

  View details for Web of Science ID 001331196100001

• Engaging communities in therapeutics clinical research during pandemics: Experiences and lessons from the ACTIV COVID-19 therapeutics research initiative. Journal of clinical and translational science Wohl, D. A., Adam, S. J., Gibbs, K. W., Moskowitz, A. L., Ortel, T. L., Singh, U., Jilg, N., Evering, T. H., Fischer, W. A., Taiwo, B. O., Daar, E. S., Lindsell, C. J., Naggie, S., Rothman, R. L., Dunsmore, S. E., McAdams, M. P., Vail, J., Jayaweera, D. 2024; 8 (1): e156

  Abstract

  This manuscript addresses a critical topic: navigating complexities of conducting clinical trials during a pandemic. Central to this discussion is engaging communities to ensure diverse participation. The manuscript elucidates deliberate strategies employed to recruit minority communities with poor social drivers of health for participation in COVID-19 trials. The paper adopts a descriptive approach, eschewing analysis of data-driven efficacy of these efforts, and instead provides a comprehensive account of strategies utilized. The Accelerate COVID-19 Treatment Interventions and Vaccines (ACTIV) public-private partnership launched early in the COVID-19 pandemic to develop clinical trials to advance SARS-CoV-2 treatments. In this paper, ACTIV investigators share challenges in conducting research during an evolving pandemic and approaches selected to engage communities when traditional strategies were infeasible. Lessons from this experience include importance of community representatives' involvement early in study design and implementation and integration of well-developed public outreach and communication strategies with trial launch. Centralization and coordination of outreach will allow for efficient use of resources and the sharing of best practices. Insights gleaned from the ACTIV program, as outlined in this paper, shed light on effective strategies for involving communities in treatment trials amidst rapidly evolving public health emergencies. This underscores critical importance of community engagement initiatives well in advance of the pandemic.

  View details for DOI 10.1017/cts.2024.561

  View details for PubMedID 39540112

  View details for PubMedCentralID PMC11557280

• Time to Sustained Recovery Among Outpatients With COVID-19 Receiving Montelukast vs Placebo: The ACTIV-6 Randomized Clinical Trial. JAMA network open Rothman, R. L., Stewart, T. G., Mourad, A., Boulware, D. R., McCarthy, M. W., Thicklin, F., Garcia Del Sol, I. T., Garcia, J. L., Bramante, C. T., Shah, N. S., Singh, U., Williamson, J. C., Rebolledo, P. A., Jagannathan, P., Schwasinger-Schmidt, T., Ginde, A. A., Castro, M., Jayaweera, D., Sulkowski, M., Gentile, N., McTigue, K., Felker, G. M., DeLong, A., Wilder, R., Collins, S., Dunsmore, S. E., Adam, S. J., Hanna, G. J., Shenkman, E., Hernandez, A. F., Naggie, S., Lindsell, C. J., Accelerating COVID-19 Therapeutic Interventions and Vaccines-6 Study Group and Investigators, Hanna, G., Fraser, R., Ward, M., Gamboa Jackson, J., McAdams, M. P., Vail, J., Korzekwinski, K., Oyelakin, M., Chopp, J., Randle, D., Dockery, S., Adkins, R., Crow, M., Nowell, E., Wells, K., Herbert, A., Stone, A., Heavlin, H., Brown, L., Harding, T., Harrington, A., Beauchaine, M., Lindblom, K., Burns, A., Aamodt, D., Collins, J., Dixon, S., Gao, Y., Graves, J., Grindstaff, J., Harrell, F., Lai, J., Liao, V., Lopez, I., Manis, E., Mankowski, K., Marlin, J., Merkel, A., Nwosu, S., Obregon, S., Orozco, D., Prato, N., Rhode, M., Shirey-Rice, J., Vermillion, K., Smith, J., Tan, H., Vance, M., Weir, M., Bianchi, R., Premas, J., Gupta, M., Karawan, G., Lima, S., Ziomek, C., Arena, J., DeAlmeida, S., Malik, A., Bryce, J., Swint, S., Ramin, S., Nataraj, J., Deider, J., Cruz, R., Ramirez, A. M., Henault, L., Marcus, J., Southwell, A., Jacques, G., Sexton, C., Tiffany, B., Tanner, C., Sahelian, A., George-Adebayo, C., Adebayo, A., Zapatero, J., Clement, J., Ronan, T., Woods, A., Gallegos, C., Flys, T., Sloan, O., Olofintuyi, A., Samraj, J., Vasbinder, A., Averett, A., Slandzicki, A., Wallen, J., Vogel, C., Munoz, S., Kavtaradze, D., Watson, C., Singleton, D., Sevier, M., Rivon, M., Del Pilar, A., Spangler, A., Rao, S., Cantu, L., Krishna, A., Daugherty, H., Kerr, B., Evans, K., Spees, R., Marta, M., Dolor, R., Vergara, L., Jordan, J., Burruss, V., Hurst, T., Ofotokun, I., Zhang, C., Traenkner, J., Atha, M. M., Prabhu, R., Klicka, K., Lightfeather, A., James, V., Rogers, M., Oragwu, C., Oguego, N., Pillai, R., Gabriel, A., Ghaly, E., Michal, M., Vasquez, M., Mamon, A., Sheets, M., Hassanien, G., Ismail, S., Samir, Y., Meltzer, A., Shahamatdar, S., Heidish, R. S., Loganathan, A., Brehaut, S., Roche, A., Mehta, M., Koppinger, N., Baez, J., Pagan, I., Abdelsayed, D., Aziz, M., Robinson, P., Lozinski, G., Nguyen, J., Griffin, A., Morris, M., Love, N., Mattox, B., Martin, R., Pardue, V., Rowland, T., Ruiz-Unger, J., Reyes, L., Zamora, Y., Bacallao, N., Cienki, J., Cohen, J., Yuan, Y., Li, J., Szeto, J., Stelmash, L., Mekhael, S., Morales Castillo, L., Gutierrez, A., Prieto, S., Amon, A., Barbera, A., Bugajski, A., Willis, W., Jacklin, K., Lamb, D., Harper, A., Stout, E., Griffin, M., Pyram-Bernard, N., Quintero, A., Clark, N., Barsanti-Sekhar, M., Carbrera-Mendez, C., Evans, M. R., Adhami, E., Carillo, G., Maria, J., Paudel, D., Raymond, O., Summers, J., Turner, T., Lenert, L., Panaccione, E., Szwast, E., Reynolds, A., Abdulghani, A., Vasoya, P., Miller, C., Wiley, H., Chan, A., Khizer, S., Adeyemi, O., Chi, W. N., Chen, J., Morton-Jost, M., Castex, J., Quirch, A., Belani, H., Machicado, R., Bjornsson, B., Olivo, J., Maldonado, M., Vecchiarelli, A., Gaytan-Alvarez, D., Cherukuri, V., Alicic, R., Lambert, A. A., Urbat, C., Baxter, J., Cooper, A., Linn, D., Fisher, L., Patel, V., Talati, R., Patel, P., Ellison, L., Roman, A., Harrison, J., Moy, J., Naquiallah, D., Shah, B., Quintero, O., Scott, J., Jazayeri, Y., O'Donnell, A., Pathak, D., Gupta, A., Chandrasekar, N., Curtis, C., White, B., Dockery, M., Fortt, T., Fortt, A., Jones-Ince, I., McKee, A., Wilson, J., Marcelin, J., Farlow, B., Grady, C., Richwine, R., Pazier, P., Michelson, E., Watts, S., Kariyawasam, D., Rodriguez, L., Manresa, I., Achong, A. A., Garcia, M. C., Khetpal, S., Posey, F., Mahadevan, A., Gnoni, M., Van de Weerd, C., Lowenkron, J., Sappington, E., Roberts, M., Wang, J., Adams, M., Ding, X., Co, M., D'Andrea, M., Lim, S., Swink, W., Bozant, E., Young, M., Wilson, M., Eastin, C., Cheathem, A., Nadeem, A., Walters, C., Powers-Fletcher, M., Brown, D., Miller, D., Mukunzi, S., Manning, B., Terry-White, M., Crizaldo, M. C., Isache, C., Bowman, J., Callaghan-Brown, A., Martin, D., Ast, A., Duran, B., Cornejo, A., Archer, A., Almanzar, M., Motel, V., Pullen, M., Anderson, B., Bhat, N., Parra, D., Campora, P., Robinson, M., Seithel, M., Kendrick, L., Helming, D., Pollock, K., Sekikawa, A., Klawson, E., Arnold, J., Weiland, N., Ostrosky-Zeichner, L., Patel, B., Umana, V., Nielsen, L., Grimes, C. Z., Patterson, T. F., Tragus, R., Soileau, B. T., Heath, T., Hinjosa, E., Gutierrez, C., Jackson, P. E., Hallowell, C., Haughey, H. M., Vaidya-Tank, B., Gould, C., Goyal, P., Sommers, S., Pangburn, H., Jones, C., Michalowski, L., Wortham, B., Abbott, R., Umana, U., Alleyne, C., Witting, B., Armas, E., Perez Landaburo, R. O., De La Cruz, M., Ballmajo, M., Alvarez, J. 2024; 7 (10): e2439332

  Abstract

  Importance: The effect of montelukast in reducing symptom duration among outpatients with mild to moderate COVID-19 is uncertain.Objective: To assess the effectiveness of montelukast compared with placebo in treating outpatients with mild to moderate COVID-19.Design, Setting, and Participants: This randomized clinical trial (Accelerating COVID-19 Therapeutic Interventions and Vaccines [ACTIV]-6) was conducted from January 27 through June 23, 2023, during the circulation of Omicron subvariants. Participants aged 30 years or older with confirmed SARS-CoV-2 infection and 2 or more acute COVID-19 symptoms for less than 7 days were included across 104 US sites.Interventions: Participants were randomized 1:1 to receive montelukast, 10 mg once daily, or matched placebo for 14 days.Main Outcomes and Measures: The primary outcome was time to sustained recovery (defined as ≥3 consecutive days without symptoms). Secondary outcomes included time to death; time to hospitalization or death; a composite of health care utilization events (hospitalization, urgent care clinic visit, emergency department visit, or death); COVID-19 clinical progression scale score; and difference in mean time unwell. A modified intention-to-treat approach was used for the analysis.Results: Among 1250 participants who were randomized and received the study drug or placebo, the median age was 53 years (IQR, 42-62 years), 753 (60.2%) were female, and 704 (56.3%) reported receiving 2 or more doses of a SARS-CoV-2 vaccine. Among 628 participants who received montelukast and 622 who received placebo, differences in time to sustained recovery were not observed (adjusted hazard ratio [AHR], 1.02; 95% credible interval [CrI], 0.92-1.12; P=.63 for efficacy). Unadjusted median time to sustained recovery was 10 days (95% CI, 10-11 days) in both groups. No deaths occurred, and hospitalizations were reported for 2 participants (0.3%) in each group; the composite of health care utilization events was reported for 18 participants (2.9%) in the montelukast group and 18 (2.9%) in the placebo group (AHR, 1.01; 95% CrI, 0.45-1.84; P=.48 for efficacy). Five participants (0.4%) experienced serious adverse events (3 [0.5%] in the montelukast group and 2 [0.3%] in the placebo group).Conclusions and Relevance: In this randomized clinical trial of outpatients with mild to moderate COVID-19, treatment with montelukast did not reduce duration of COVID-19 symptoms. These findings do not support the use of montelukast for the treatment of mild to moderate COVID-19.Trial Registration: ClinicalTrials.gov Identifier: NCT04885530.

  View details for DOI 10.1001/jamanetworkopen.2024.39332

  View details for PubMedID 39422912

• Differentiation of Prior SARS-CoV-2 Infection and Postacute Sequelae by Standard Clinical Laboratory Measurements in the RECOVER Cohort. Annals of internal medicine Erlandson, K. M., Geng, L. N., Selvaggi, C. A., Thaweethai, T., Chen, P., Erdmann, N. B., Goldman, J. D., Henrich, T. J., Hornig, M., Karlson, E. W., Katz, S. D., Kim, C., Cribbs, S. K., Laiyemo, A. O., Letts, R., Lin, J. Y., Marathe, J., Parthasarathy, S., Patterson, T. F., Taylor, B. D., Duffy, E. R., Haack, M., Julg, B., Maranga, G., Hernandez, C., Singer, N. G., Han, J., Pemu, P., Brim, H., Ashktorab, H., Charney, A. W., Wisnivesky, J., Lin, J. J., Chu, H. Y., Go, M., Singh, U., Levitan, E. B., Goepfert, P. A., Nikolich, J. Ž., Hsu, H., Peluso, M. J., Kelly, J. D., Okumura, M. J., Flaherman, V. J., Quigley, J. G., Krishnan, J. A., Scholand, M. B., Hess, R., Metz, T. D., Costantine, M. M., Rouse, D. J., Taylor, B. S., Goldberg, M. P., Marshall, G. D., Wood, J., Warren, D., Horwitz, L., Foulkes, A. S., McComsey, G. A. 2024

  Abstract

  There are currently no validated clinical biomarkers of postacute sequelae of SARS-CoV-2 infection (PASC).To investigate clinical laboratory markers of SARS-CoV-2 and PASC.Propensity score-weighted linear regression models were fitted to evaluate differences in mean laboratory measures by prior infection and PASC index (≥12 vs. 0). (ClinicalTrials.gov: NCT05172024).83 enrolling sites.RECOVER-Adult cohort participants with or without SARS-CoV-2 infection with a study visit and laboratory measures 6 months after the index date (or at enrollment if >6 months after the index date). Participants were excluded if the 6-month visit occurred within 30 days of reinfection.Participants completed questionnaires and standard clinical laboratory tests.Among 10 094 participants, 8746 had prior SARS-CoV-2 infection, 1348 were uninfected, 1880 had a PASC index of 12 or higher, and 3351 had a PASC index of zero. After propensity score adjustment, participants with prior infection had a lower mean platelet count (265.9 × 109 cells/L [95% CI, 264.5 to 267.4 × 109 cells/L]) than participants without known prior infection (275.2 × 109 cells/L [CI, 268.5 to 282.0 × 109 cells/L]), as well as higher mean hemoglobin A1c (HbA1c) level (5.58% [CI, 5.56% to 5.60%] vs. 5.46% [CI, 5.40% to 5.51%]) and urinary albumin-creatinine ratio (81.9 mg/g [CI, 67.5 to 96.2 mg/g] vs. 43.0 mg/g [CI, 25.4 to 60.6 mg/g]), although differences were of modest clinical significance. The difference in HbA1c levels was attenuated after participants with preexisting diabetes were excluded. Among participants with prior infection, no meaningful differences in mean laboratory values were found between those with a PASC index of 12 or higher and those with a PASC index of zero.Whether differences in laboratory markers represent consequences of or risk factors for SARS-CoV-2 infection could not be determined.Overall, no evidence was found that any of the 25 routine clinical laboratory values assessed in this study could serve as a clinically useful biomarker of PASC.National Institutes of Health.

  View details for DOI 10.7326/M24-0737

  View details for PubMedID 39133923

• Sex differences and immune correlates of Long COVID development, persistence, and resolution. bioRxiv : the preprint server for biology Hamlin, R. E., Pienkos, S. M., Chan, L., Stabile, M. A., Pinedo, K., Rao, M., Grant, P., Bonilla, H., Holubar, M., Singh, U., Jacobson, K. B., Jagannathan, P., Maldonado, Y., Holmes, S. P., Subramanian, A., Blish, C. A. 2024

  Abstract

  Sex differences have been observed in acute COVID-19 and Long COVID (LC) outcomes, with greater disease severity and mortality during acute infection in males and a greater proportion of females developing LC. We hypothesized that sex-specific immune dysregulation contributes to the pathogenesis of LC. To investigate the immunologic underpinnings of LC development and persistence, we used single-cell transcriptomics, single-cell proteomics, and plasma proteomics on blood samples obtained during acute SARS-CoV-2 infection and at 3 and 12 months post-infection in a cohort of 45 patients who either developed LC or recovered. Several sex-specific immune pathways were associated with LC. Specifically, males who would develop LC at 3 months had widespread increases in TGF-β signaling during acute infection in proliferating NK cells. Females who would develop LC demonstrated increased expression of XIST, an RNA gene implicated in autoimmunity, and increased IL1 signaling in monocytes at 12 months post infection. Several immune features of LC were also conserved across sexes. Both males and females with LC had reduced co-stimulatory signaling from monocytes and broad upregulation of NF-κB transcription factors. In both sexes, those with persistent LC demonstrated increased LAG3, a marker of T cell exhaustion, reduced ETS1 transcription factor expression across lymphocyte subsets, and elevated intracellular IL-4 levels in T cell subsets, suggesting that ETS1 alterations may drive an aberrantly elevated Th2-like response in LC. Altogether, this study describes multiple innate and adaptive immune correlates of LC, some of which differ by sex, and offers insights toward the pursuit of tailored therapeutics.

  View details for DOI 10.1101/2024.06.18.599612

  View details for PubMedID 38948732

  View details for PubMedCentralID PMC11212991

• Nirmatrelvir-Ritonavir and Symptoms in Adults With Postacute Sequelae of SARS-CoV-2 Infection: The STOP-PASC Randomized Clinical Trial. JAMA internal medicine Geng, L. N., Bonilla, H., Hedlin, H., Jacobson, K. B., Tian, L., Jagannathan, P., Yang, P. C., Subramanian, A. K., Liang, J. W., Shen, S., Deng, Y., Shaw, B. J., Botzheim, B., Desai, M., Pathak, D., Jazayeri, Y., Thai, D., O'Donnell, A., Mohaptra, S., Leang, Z., Reynolds, G. Z., Brooks, E. F., Bhatt, A. S., Shafer, R. W., Miglis, M. G., Quach, T., Tiwari, A., Banerjee, A., Lopez, R. N., De Jesus, M., Charnas, L. R., Utz, P. J., Singh, U. 2024

  Abstract

  There is an urgent need to identify treatments for postacute sequelae of SARS-CoV-2 infection (PASC).To assess the efficacy of a 15-day course of nirmatrelvir-ritonavir in reducing the severity of select PASC symptoms.This was a 15-week blinded, placebo-controlled, randomized clinical trial conducted from November 2022 to September 2023 at Stanford University (California). The participants were adults with moderate to severe PASC symptoms of 3 months or longer duration.Participants were randomized 2:1 to treatment with oral nirmatrelvir-ritonavir (NMV/r, 300 mg and 100 mg) or with placebo-ritonavir (PBO/r) twice daily for 15 days.Primary outcome was a pooled severity of 6 PASC symptoms (fatigue, brain fog, shortness of breath, body aches, gastrointestinal symptoms, and cardiovascular symptoms) based on a Likert scale score at 10 weeks. Secondary outcomes included symptom severity at different time points, symptom burden and relief, patient global measures, Patient-Reported Outcomes Measurement Information System (PROMIS) measures, orthostatic vital signs, and sit-to-stand test change from baseline.Of the 155 participants (median [IQR] age, 43 [34-54] years; 92 [59%] females), 102 were randomized to the NMV/r group and 53 to the PBO/r group. Nearly all participants (n = 153) had received the primary series for COVID-19 vaccination. Mean (SD) time between index SARS-CoV-2 infection and randomization was 17.5 (9.1) months. There was no statistically significant difference in the model-derived severity outcome pooled across the 6 core symptoms at 10 weeks between the NMV/r and PBO/r groups. No statistically significant between-group differences were found at 10 weeks in the Patient Global Impression of Severity or Patient Global Impression of Change scores, summative symptom scores, and change from baseline to 10 weeks in PROMIS fatigue, dyspnea, cognitive function, and physical function measures. Adverse event rates were similar in NMV/r and PBO/r groups and mostly of low grade.The results of this randomized clinical trial showed that a 15-day course of NMV/r in a population of patients with PASC was generally safe but did not demonstrate a significant benefit for improving select PASC symptoms in a mostly vaccinated cohort with protracted symptom duration. Further studies are needed to determine the role of antivirals in the treatment of PASC.ClinicalTrials.gov Identifier: NCT05576662.

  View details for DOI 10.1001/jamainternmed.2024.2007

  View details for PubMedID 38848477

• Development of a Definition of Postacute Sequelae of SARS-CoV-2 Infection (vol 329, pg 1934, 2023) JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Thaweethai, T., Jolley, S. E., Karlson, E. W., Levitan, E. B., Levy, B., McComsey, G. A., McCorkell, L., Nadkarni, G. N., Parthasarathy, S., Singh, U., Walker, T. A., Selvaggi, C. A., Shinnick, D. J., Schulte, C. M., Atchley-Challenner, R., Horwitz, L. I., Foulkes, A. S., RECOVER Consortium 2024

  View details for DOI 10.1001/jama.2024.2984

  View details for Web of Science ID 001201448800001

• 2023: Looking Back and Looking Ahead JOURNAL OF INFECTIOUS DISEASES Li, J. Z., Clancy, C. J., Singh, U., Sears, C. L. 2024: 619-620

  View details for DOI 10.1093/infdis/jiae076

  View details for Web of Science ID 001179515200001

  View details for PubMedID 38386686

• Extracellular Vesicles Derived FromEntamoeba histolyticaHave an Immunomodulatory Effect on THP-1 Macrophages. Journal of parasitology research Chowdhury, D., Sharma, M., Jahng, J. W., Singh, U. 2024; 2024: 7325606

  Abstract

  Recent studies have shown that extracellular vesicles (EVs) secreted by various parasites are capable of modulating the host's innate immune responses, such as by altering macrophage (Mϕ) phenotypes and functions. Studies have shown that Mϕ promote early host responses to amoebic infection by releasing proinflammatory cytokines that are crucial to combating amoebiasis. Here, we are reporting for the first time the effect of EVs released by Entamoeba histolytica (EhEVs) on human THP-1 differentiated Mϕ (THP-1 Mϕ). We show that the EhEVs are internalized by THP-1 Mϕ which leads to differential regulation of various cytokines associated with both M1 and M2 Mϕ. We also saw that EhEV treatment thwarted Type 2 immune-response-related transcriptome pSTAT6 in the THP-1 Mϕ. Furthermore, EhEVs stimulated Mϕ to reduce their energy demand by suppressing oxidative phosphorylation (OXPHOS) and adenosine triphosphate (ATP) production. Hence, the human parasite E. histolytica-derived EVs are capable of eliciting an immune response from Mϕ that may contribute to overall infection status.

  View details for DOI 10.1155/2024/7325606

  View details for PubMedID 39502090

• Higher-Dose Fluvoxamine and Time to Sustained Recovery in Outpatients With COVID-19: The ACTIV-6 Randomized Clinical Trial. JAMA Stewart, T. G., Rebolledo, P. A., Mourad, A., Lindsell, C. J., Boulware, D. R., McCarthy, M. W., Thicklin, F., Garcia Del Sol, I. T., Bramante, C. T., Lenert, L. A., Lim, S., Williamson, J. C., Cardona, O. Q., Scott, J., Schwasinger-Schmidt, T., Ginde, A. A., Castro, M., Jayaweera, D., Sulkowski, M., Gentile, N., McTigue, K., Felker, G. M., DeLong, A., Wilder, R., Rothman, R. L., Collins, S., Dunsmore, S. E., Adam, S. J., Hanna, G. J., Shenkman, E., Hernandez, A. F., Naggie, S. 2023

  Abstract

  The effect of higher-dose fluvoxamine in reducing symptom duration among outpatients with mild to moderate COVID-19 remains uncertain.To assess the effectiveness of fluvoxamine, 100 mg twice daily, compared with placebo, for treating mild to moderate COVID-19.The ACTIV-6 platform randomized clinical trial aims to evaluate repurposed medications for mild to moderate COVID-19. Between August 25, 2022, and January 20, 2023, a total of 1175 participants were enrolled at 103 US sites for evaluating fluvoxamine; participants were 30 years or older with confirmed SARS-CoV-2 infection and at least 2 acute COVID-19 symptoms for 7 days or less.Participants were randomized to receive fluvoxamine, 50 mg twice daily on day 1 followed by 100 mg twice daily for 12 additional days (n = 601), or placebo (n = 607).The primary outcome was time to sustained recovery (defined as at least 3 consecutive days without symptoms). Secondary outcomes included time to death; time to hospitalization or death; a composite of hospitalization, urgent care visit, emergency department visit, or death; COVID-19 clinical progression scale score; and difference in mean time unwell. Follow-up occurred through day 28.Among 1208 participants who were randomized and received the study drug, the median (IQR) age was 50 (40-60) years, 65.8% were women, 45.5% identified as Hispanic/Latino, and 76.8% reported receiving at least 2 doses of a SARS-CoV-2 vaccine. Among 589 participants who received fluvoxamine and 586 who received placebo included in the primary analysis, differences in time to sustained recovery were not observed (adjusted hazard ratio [HR], 0.99 [95% credible interval, 0.89-1.09]; P for efficacy = .40]). Additionally, unadjusted median time to sustained recovery was 10 (95% CI, 10-11) days in both the intervention and placebo groups. No deaths were reported. Thirty-five participants reported health care use events (a priori defined as death, hospitalization, or emergency department/urgent care visit): 14 in the fluvoxamine group compared with 21 in the placebo group (HR, 0.69 [95% credible interval, 0.27-1.21]; P for efficacy = .86) There were 7 serious adverse events in 6 participants (2 with fluvoxamine and 4 with placebo) but no deaths.Among outpatients with mild to moderate COVID-19, treatment with fluvoxamine does not reduce duration of COVID-19 symptoms.ClinicalTrials.gov Identifier: NCT04885530.

  View details for DOI 10.1001/jama.2023.23363

  View details for PubMedID 37976072

• Safety and efficacy of inhaled interferon-β1a (SNG001) in adults with mild-to-moderate COVID-19: a randomized, controlled, phase II trial. EClinicalMedicine Jagannathan, P., Chew, K. W., Giganti, M. J., Hughes, M. D., Moser, C., Main, M. J., Monk, P. D., Javan, A. C., Li, J. Z., Fletcher, C. V., McCarthy, C., Wohl, D. A., Daar, E. S., Eron, J. J., Currier, J. S., Singh, U., Smith, D. M., Fischer, W. 2023; 65: 102250

  Abstract

  With the emergence of SARS-CoV-2 variants resistant to monoclonal antibody therapies and limited global access to therapeutics, the evaluation of novel therapeutics to prevent progression to severe COVID-19 remains a critical need.Safety, clinical and antiviral efficacy of inhaled interferon-β1a (SNG001) were evaluated in a phase II randomized controlled trial on the ACTIV-2/A5401 platform (ClinicalTrials.govNCT04518410). Adult outpatients with confirmed SARS-CoV-2 infection within 10 days of symptom onset were randomized and initiated either orally inhaled nebulized SNG001 given once daily for 14 days (n = 110) or blinded pooled placebo (n = 110) between February 10 and August 18, 2021.The proportion of participants reporting premature treatment discontinuation was 9% among SNG001 and 13% among placebo participants. There were no differences between participants who received SNG001 or placebo in the primary outcomes of treatment emergent Grade 3 or higher adverse events (3.6% and 8.2%, respectively), time to symptom improvement (median 13 and 9 days, respectively), or proportion with unquantifiable nasopharyngeal SARS-CoV-2 RNA at days 3 (28% [26/93] vs. 39% [37/94], respectively), 7 (65% [60/93] vs. 66% [62/94]) and 14 (91% [86/95] vs. 91% [83/81]). There were fewer hospitalizations with SNG001 (n = 1; 1%) compared with placebo (n = 7; 6%), representing an 86% relative risk reduction (p = 0.07). There were no deaths in either arm.In this trial, SNG001 was safe and associated with a non-statistically significant decrease in hospitalization for COVID-19 pneumonia.The ACTIV-2 platform study is funded by the NIH. Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number UM1 AI068634, UM1 AI068636 and UM1 AI106701. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  View details for DOI 10.1016/j.eclinm.2023.102250

  View details for PubMedID 37855026

  View details for PubMedCentralID PMC10579289

• Extracellular Vesicles and Their Impact on the Biology of Protozoan Parasites. Tropical medicine and infectious disease Sharma, M., Lozano-Amado, D., Chowdhury, D., Singh, U. 2023; 8 (9)

  Abstract

  Extracellular vesicles (EVs) are lipid-membrane-bound structures produced naturally by all cells and have a variety of functions. EVs act as vehicles for transporting important molecular signals from one cell to another. Several parasites have been shown to secrete EVs, and their biological functions have been extensively studied. EVs have been shown to facilitate communication with the host cells (such as modulation of the host's immune system or promoting attachment and invasion into the host cells) or for communication between parasitic cells (e.g., transferring drug-resistance genes or factors modulating stage conversion). It is clear that EVs play an important role in host-parasite interactions. In this review, we summarized the latest research on the EVs secreted by protozoan parasites and their role in host-parasite and parasite-parasite communications.

  View details for DOI 10.3390/tropicalmed8090448

  View details for PubMedID 37755909

• The Tomato Brown Rugose Fruit Virus Movement Protein Gene Is a Novel Microbial Source Tracking Marker. Applied and environmental microbiology Natarajan, A., Fremin, B. J., Schmidtke, D. T., Wolfe, M. K., Zlitni, S., Graham, K. E., Brooks, E. F., Severyn, C. J., Sakamoto, K. M., Lacayo, N. J., Kuersten, S., Koble, J., Caves, G., Kaplan, I., Singh, U., Jagannathan, P., Rezvani, A. R., Bhatt, A. S., Boehm, A. B. 2023: e0058323

  Abstract

  Microbial source tracking (MST) identifies sources of fecal contamination in the environment using host-associated fecal markers. While there are numerous bacterial MST markers that can be used herein, there are few such viral markers. Here, we designed and tested novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States. Next, we developed two novel probe-based reverse transcription-PCR (RT-PCR) assays based on conserved regions of the ToBRFV genome and tested the markers' sensitivities and specificities using human and non-human animal stool as well as wastewater. The ToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a commonly used viral marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We used the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, these results indicate that ToBRFV is a promising viral human-associated MST marker. IMPORTANCE Human exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of host-associated MST markers. Here, we designed and tested novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool and highly abundant in human stool and wastewater samples.

  View details for DOI 10.1128/aem.00583-23

  View details for PubMedID 37404180

• Researching COVID to Enhance Recovery (RECOVER) adult study protocol: Rationale, objectives, and design. PloS one Horwitz, L. I., Thaweethai, T., Brosnahan, S. B., Cicek, M. S., Fitzgerald, M. L., Goldman, J. D., Hess, R., Hodder, S. L., Jacoby, V. L., Jordan, M. R., Krishnan, J. A., Laiyemo, A. O., Metz, T. D., Nichols, L., Patzer, R. E., Sekar, A., Singer, N. G., Stiles, L. E., Taylor, B. S., Ahmed, S., Algren, H. A., Anglin, K., Aponte-Soto, L., Ashktorab, H., Bassett, I. V., Bedi, B., Bhadelia, N., Bime, C., Bind, M. C., Black, L. J., Blomkalns, A. L., Brim, H., Castro, M., Chan, J., Charney, A. W., Chen, B. K., Chen, L. Q., Chen, P., Chestek, D., Chibnik, L. B., Chow, D. C., Chu, H. Y., Clifton, R. G., Collins, S., Costantine, M. M., Cribbs, S. K., Deeks, S. G., Dickinson, J. D., Donohue, S. E., Durstenfeld, M. S., Emery, I. F., Erlandson, K. M., Facelli, J. C., Farah-Abraham, R., Finn, A. V., Fischer, M. S., Flaherman, V. J., Fleurimont, J., Fonseca, V., Gallagher, E. J., Gander, J. C., Gennaro, M. L., Gibson, K. S., Go, M., Goodman, S. N., Granger, J. P., Greenway, F. L., Hafner, J. W., Han, J. E., Harkins, M. S., Hauser, K. S., Heath, J. R., Hernandez, C. R., Ho, O., Hoffman, M. K., Hoover, S. E., Horowitz, C. R., Hsu, H., Hsue, P. Y., Hughes, B. L., Jagannathan, P., James, J. A., John, J., Jolley, S., Judd, S. E., Juskowich, J. J., Kanjilal, D. G., Karlson, E. W., Katz, S. D., Kelly, J. D., Kelly, S. W., Kim, A. Y., Kirwan, J. P., Knox, K. S., Kumar, A., Lamendola-Essel, M. F., Lanca, M., Lee-Lannotti, J. K., Lefebvre, R. C., Levy, B. D., Lin, J. Y., Logarbo, B. P., Logue, J. K., Longo, M. T., Luciano, C. A., Lutrick, K., Malakooti, S. K., Mallett, G., Maranga, G., Marathe, J. G., Marconi, V. C., Marshall, G. D., Martin, C. F., Martin, J. N., May, H. T., McComsey, G. A., McDonald, D., Mendez-Figueroa, H., Miele, L., Mittleman, M. A., Mohandas, S., Mouchati, C., Mullington, J. M., Nadkarni, G. N., Nahin, E. R., Neuman, R. B., Newman, L. T., Nguyen, A., Nikolich, J. Z., Ofotokun, I., Ogbogu, P. U., Palatnik, A., Palomares, K. T., Parimon, T., Parry, S., Parthasarathy, S., Patterson, T. F., Pearman, A., Peluso, M. J., Pemu, P., Pettker, C. M., Plunkett, B. A., Pogreba-Brown, K., Poppas, A., Porterfield, J. Z., Quigley, J. G., Quinn, D. K., Raissy, H., Rebello, C. J., Reddy, U. M., Reece, R., Reeder, H. T., Rischard, F. P., Rosas, J. M., Rosen, C. J., Rouphael, N. G., Rouse, D. J., Ruff, A. M., Saint Jean, C., Sandoval, G. J., Santana, J. L., Schlater, S. M., Sciurba, F. C., Selvaggi, C., Seshadri, S., Sesso, H. D., Shah, D. P., Shemesh, E., Sherif, Z. A., Shinnick, D. J., Simhan, H. N., Singh, U., Sowles, A., Subbian, V., Sun, J., Suthar, M. S., Teunis, L. J., Thorp, J. M., Ticotsky, A., Tita, A. T., Tragus, R., Tuttle, K. R., Urdaneta, A. E., Utz, P. J., VanWagoner, T. M., Vasey, A., Vernon, S. D., Vidal, C., Walker, T., Ward, H. D., Warren, D. E., Weeks, R. M., Weiner, S. J., Weyer, J. C., Wheeler, J. L., Whiteheart, S. W., Wiley, Z., Williams, N. J., Wisnivesky, J. P., Wood, J. C., Yee, L. M., Young, N. M., Zisis, S. N., Foulkes, A. S. 2023; 18 (6): e0286297

  Abstract

  SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or other health effects after the acute phase of infection; termed post-acute sequelae of SARS-CoV-2 infection (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are ill-defined. The objectives of the Researching COVID to Enhance Recovery (RECOVER) Multi-site Observational Study of PASC in Adults (RECOVER-Adult) are to: (1) characterize PASC prevalence; (2) characterize the symptoms, organ dysfunction, natural history, and distinct phenotypes of PASC; (3) identify demographic, social and clinical risk factors for PASC onset and recovery; and (4) define the biological mechanisms underlying PASC pathogenesis.RECOVER-Adult is a combined prospective/retrospective cohort currently planned to enroll 14,880 adults aged ≥18 years. Eligible participants either must meet WHO criteria for suspected, probable, or confirmed infection; or must have evidence of no prior infection. Recruitment occurs at 86 sites in 33 U.S. states, Washington, DC and Puerto Rico, via facility- and community-based outreach. Participants complete quarterly questionnaires about symptoms, social determinants, vaccination status, and interim SARS-CoV-2 infections. In addition, participants contribute biospecimens and undergo physical and laboratory examinations at approximately 0, 90 and 180 days from infection or negative test date, and yearly thereafter. Some participants undergo additional testing based on specific criteria or random sampling. Patient representatives provide input on all study processes. The primary study outcome is onset of PASC, measured by signs and symptoms. A paradigm for identifying PASC cases will be defined and updated using supervised and unsupervised learning approaches with cross-validation. Logistic regression and proportional hazards regression will be conducted to investigate associations between risk factors, onset, and resolution of PASC symptoms.RECOVER-Adult is the first national, prospective, longitudinal cohort of PASC among US adults. Results of this study are intended to inform public health, spur clinical trials, and expand treatment options.NCT05172024.

  View details for DOI 10.1371/journal.pone.0286297

  View details for PubMedID 37352211

  View details for PubMedCentralID PMC10289397

• Transparent Reporting at the Journal of Infectious Diseases. The Journal of infectious diseases Clancy, C. J., Li, J. Z., Singh, U., Sears, C. L. 2023

  View details for DOI 10.1093/infdis/jiad129

  View details for PubMedID 37134129

• Diversity and Plasticity of Virulent Characteristics of Entamoeba histolytica. Tropical medicine and infectious disease Yanagawa, Y., Singh, U. 2023; 8 (5)

  Abstract

  The complexity of clinical syndromes of amebiasis, caused by the parasite Entamoeba histolytica, stems from the intricate interplay between the host immune system, the virulence of the invading parasite, and the surrounding environment. Although there is still a relative paucity of information about the precise relationship between virulence factors and the pathogenesis of Entamoeba histolytica, by accumulating data from clinical and basic research, researchers have identified essential pathogenic factors that play a critical role in the pathogenesis of amebiasis, providing important insights into disease development through animal models. Moreover, the parasite's genetic variability has been associated with differences in virulence and disease outcomes, making it important to fully understand the epidemiology and pathogenesis of amebiasis. Deciphering the true mechanism of disease progression in humans caused by this parasite is made more difficult through its ability to demonstrate both genomic and pathological plasticity. The objective of this article is to underscore the heterogeneous nature of disease states and the malleable virulence characteristics in experimental models, while also identifying persistent scientific issues that need to be addressed.

  View details for DOI 10.3390/tropicalmed8050255

  View details for PubMedID 37235303

• Stress Response in Entamoeba histolytica Is Associated with Robust Processing of tRNA to tRNA Halves. mBio Sharma, M., Zhang, H., Ehrenkaufer, G., Singh, U. 2023: e0345022

  Abstract

  tRNA-derived fragments have been reported in many different organisms and have diverse cellular roles, such as regulating gene expression, inhibiting protein translation, silencing transposable elements, and modulating cell proliferation. In particular, tRNA halves, a class of tRNA fragments produced by the cleavage of tRNAs in the anti-codon loop, have been widely reported to accumulate under stress and regulate translation in cells. Here, we report the presence of tRNA-derived fragments in Entamoeba, with tRNA halves being the most abundant. We further established that tRNA halves accumulate in the parasites upon different stress stimuli such as oxidative stress, heat shock, and serum starvation. We also observed differential expression of tRNA halves during developmental changes of trophozoite-to-cyst conversion, with various tRNA halves accumulating during early encystation. In contrast to other systems, the stress response does not appear to be mediated by a few specific tRNA halves, as multiple tRNAs appear to be processed during the various stresses. Furthermore, we identified some tRNA-derived fragments associated with Entamoeba Argonaute proteins, EhAgo2-2 and EhAgo2-3, which have a preference for different tRNA-derived fragment species. Finally, we show that tRNA halves are packaged inside extracellular vesicles secreted by amoebas. The ubiquitous presence of tRNA-derived fragments, their association with the Argonaute proteins, and the accumulation of tRNA halves during multiple different stresses, including encystation, suggest a nuanced level of gene expression regulation mediated by different tRNA-derived fragments in Entamoeba. IMPORTANCE In the present study, we report for the first time the presence of tRNA-derived fragments in Entamoeba. tRNA-derived fragments were identified by bioinformatics analyses of small-RNA sequencing data sets from the parasites and also confirmed experimentally. We found that tRNA halves accumulated in parasites exposed to environmental stress or during the developmental process of encystation. We also found that shorter tRNA-derived fragments are bound to Entamoeba Argonaute proteins, indicating that they may have a potential role in the Argonaute-mediated RNA-interference pathway, which mediates robust gene silencing in Entamoeba. We noticed that in response to heat shock, the protein translation levels were elevated in the parasites. This effect was reversed in the presence of an analog of leucine, which also reduced the levels of the tRNA halves in the stressed cells. Our results suggest that tRNA-derived fragments in Entamoeba have a possible role in regulating gene expression during environmental stress.

  View details for DOI 10.1128/mbio.03450-22

  View details for PubMedID 36809068

• Autoantibodies are highly prevalent in non-SARS-CoV-2 respiratory infections and critical illness. JCI insight Feng, A., Yang, E. Y., Moore, A. R., Dhingra, S., Chang, S. E., Yin, X., Pi, R., Mack, E. K., Völkel, S., Geßner, R., Gündisch, M., Neubauer, A., Renz, H., Tsiodras, S., Fragkou, P. C., Asuni, A. A., Levitt, J. E., Wilson, J. G., Leong, M., Lumb, J. H., Mao, R., Pinedo, K., Roque, J., Richards, C. M., Stabile, M., Swaminathan, G., Salagianni, M. L., Triantafyllia, V., Bertrams, W., Blish, C. A., Carette, J. E., Frankovich, J., Meffre, E., Nadeau, K. C., Singh, U., Wang, T. T., Luning Prak, E. T., Herold, S., Andreakos, E., Schmeck, B., Skevaki, C., Rogers, A. J., Utz, P. J. 2023; 8 (3)

  Abstract

  The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

  View details for DOI 10.1172/jci.insight.163150

  View details for PubMedID 36752204

• Challenges in Harnessing Shared Within-Host Severe Acute Respiratory Syndrome Coronavirus 2 Variation for Transmission Inference. Open forum infectious diseases Walter, K. S., Kim, E., Verma, R., Altamirano, J., Leary, S., Carrington, Y. J., Jagannathan, P., Singh, U., Holubar, M., Subramanian, A., Khosla, C., Maldonado, Y., Andrews, J. R. 2023; 10 (2): ofad001

  Abstract

  The limited variation observed among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference.We performed tiled amplicon sequencing on 307 SARS-CoV-2 samples, including 130 samples from 32 individuals in 14 households and 47 longitudinally sampled individuals, from 4 prospective studies with household membership data, a proxy for transmission linkage.Consensus sequences from households had limited diversity (mean pairwise distance, 3.06 single-nucleotide polymorphisms [SNPs]; range, 0-40). Most (83.1%, 255 of 307) samples harbored at least 1 intrahost single-nucleotide variant ([iSNV] median, 117; interquartile range [IQR], 17-208), above a minor allele frequency threshold of 0.2%. Pairs in the same household shared significantly more iSNVs (mean, 1.20 iSNVs; 95% confidence interval [CI], 1.02-1.39) than did pairs in different households infected with the same viral clade (mean, 0.31 iSNVs; 95% CI, .28-.34), a signal that decreases with increasingly stringent minor allele frequency thresholds. The number of shared iSNVs was significantly associated with an increased odds of household membership (adjusted odds ratio, 1.35; 95% CI, 1.23-1.49). However, the poor concordance of iSNVs detected across sequencing replicates (24.8% and 35.0% above a 0.2% and 1% threshold) confirms technical concerns that current sequencing and bioinformatic workflows do not consistently recover low-frequency within-host variants.Shared within-host variation may augment the information in consensus sequences for predicting transmission linkages. Improving sensitivity and specificity of within-host variant identification will improve the informativeness of within-host variation.

  View details for DOI 10.1093/ofid/ofad001

  View details for PubMedID 36751652

  View details for PubMedCentralID PMC9898879

• Effect of Fluvoxamine vs Placebo on Time to Sustained Recovery in Outpatients With Mild to Moderate COVID-19: A Randomized Clinical Trial. JAMA McCarthy, M. W., Naggie, S., Boulware, D. R., Lindsell, C. J., Stewart, T. G., Felker, G. M., Jayaweera, D., Sulkowski, M., Gentile, N., Bramante, C., Singh, U., Dolor, R. J., Ruiz-Unger, J., Wilson, S., DeLong, A., Remaly, A., Wilder, R., Collins, S., Dunsmore, S. E., Adam, S. J., Thicklin, F., Hanna, G., Ginde, A. A., Castro, M., McTigue, K., Shenkman, E., Hernandez, A. F. 2023

  Abstract

  The effectiveness of fluvoxamine to shorten symptom duration or prevent hospitalization among outpatients with mild to moderate symptomatic COVID-19 is unclear.To evaluate the efficacy of low-dose fluvoxamine (50 mg twice daily) for 10 days compared with placebo for the treatment of mild to moderate COVID-19 in the US.The ongoing Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV-6) platform randomized clinical trial was designed to test repurposed medications in outpatients with mild to moderate COVID-19. A total of 1288 participants aged 30 years or older with test-confirmed SARS-CoV-2 infection and experiencing 2 or more symptoms of acute COVID-19 for 7 days or less were enrolled between August 6, 2021, and May 27, 2022, at 91 sites in the US.Participants were randomized to receive 50 mg of fluvoxamine twice daily for 10 days or placebo.The primary outcome was time to sustained recovery (defined as the third day of 3 consecutive days without symptoms). There were 7 secondary outcomes, including a composite outcome of hospitalization, urgent care visit, emergency department visit, or death through day 28.Among 1331 participants who were randomized (median age, 47 years [IQR, 38-57 years]; 57% were women; and 67% reported receiving ≥2 doses of a SARS-CoV-2 vaccine), 1288 completed the trial (674 in the fluvoxamine group and 614 in the placebo group). The median time to sustained recovery was 12 days (IQR, 11-14 days) in the fluvoxamine group and 13 days (IQR, 12-13 days) in the placebo group (hazard ratio [HR], 0.96 [95% credible interval, 0.86-1.06], posterior P = .21 for the probability of benefit [determined by an HR >1]). For the composite outcome, 26 participants (3.9%) in the fluvoxamine group were hospitalized, had an urgent care visit, had an emergency department visit, or died compared with 23 participants (3.8%) in the placebo group (HR, 1.1 [95% credible interval, 0.5-1.8], posterior P = .35 for the probability of benefit [determined by an HR <1]). One participant in the fluvoxamine group and 2 participants in the placebo group were hospitalized; no deaths occurred in either group. Adverse events were uncommon in both groups.Among outpatients with mild to moderate COVID-19, treatment with 50 mg of fluvoxamine twice daily for 10 days, compared with placebo, did not improve time to sustained recovery. These findings do not support the use of fluvoxamine at this dose and duration in patients with mild to moderate COVID-19.ClinicalTrials.gov Identifier: NCT04885530.

  View details for DOI 10.1001/jama.2022.24100

  View details for PubMedID 36633838

• Development of a Definition of Postacute Sequelae of SARS-CoV-2 Infection. JAMA Thaweethai, T., Jolley, S. E., Karlson, E. W., Levitan, E. B., Levy, B., McComsey, G. A., McCorkell, L., Nadkarni, G. N., Parthasarathy, S., Singh, U., Walker, T. A., Selvaggi, C. A., Shinnick, D. J., Schulte, C. C., Atchley-Challenner, R., Horwitz, L. I., Foulkes, A. S., RECOVER Consortium, Alba, G. A., Alicic, R., Altman, N., Anglin, K., Argueta, U., Ashktorab, H., Baslet, G., Bassett, I. V., Bateman, L., Bedi, B., Bhattacharyya, S., Bind, M., Blomkalns, A. L., Bonilla, H., Bush, P. A., Castro, M., Chan, J., Charney, A. W., Chen, P., Chibnik, L. B., Chu, H. Y., Clifton, R. G., Costantine, M. M., Cribbs, S. K., Davila Nieves, S. I., Deeks, S. G., Duven, A., Emery, I. F., Erdmann, N., Erlandson, K. M., Ernst, K. C., Farah-Abraham, R., Farner, C. E., Feuerriegel, E. M., Fleurimont, J., Fonseca, V., Franko, N., Gainer, V., Gander, J. C., Gardner, E. M., Geng, L. N., Gibson, K. S., Go, M., Goldman, J. D., Grebe, H., Greenway, F. L., Habli, M., Hafner, J., Han, J. E., Hanson, K. A., Heath, J., Hernandez, C., Hess, R., Hodder, S. L., Hoffman, M. K., Hoover, S. E., Huang, B., Hughes, B. L., Jagannathan, P., John, J., Jordan, M. R., Katz, S. D., Kaufman, E. S., Kelly, J. D., Kelly, S. W., Kemp, M. M., Kirwan, J. P., Klein, J. D., Knox, K. S., Krishnan, J. A., Kumar, A., Laiyemo, A. O., Lambert, A. A., Lanca, M., Lee-Iannotti, J. K., Logarbo, B. P., Longo, M. T., Luciano, C. A., Lutrick, K., Maley, J. H., Marathe, J. G., Marconi, V., Marshall, G. D., Martin, C. F., Matusov, Y., Mehari, A., Mendez-Figueroa, H., Mermelstein, R., Metz, T. D., Morse, R., Mosier, J., Mouchati, C., Mullington, J., Murphy, S. N., Neuman, R. B., Nikolich, J. Z., Ofotokun, I., Ojemakinde, E., Palatnik, A., Palomares, K., Parimon, T., Parry, S., Patterson, J. E., Patterson, T. F., Patzer, R. E., Peluso, M. J., Pemu, P., Pettker, C. M., Plunkett, B. A., Pogreba-Brown, K., Poppas, A., Quigley, J. G., Reddy, U., Reece, R., Reeder, H., Reeves, W. B., Reiman, E. M., Rischard, F., Rosand, J., Rouse, D. J., Ruff, A., Saade, G., Sandoval, G. J., Schlater, S. M., Shepherd, F., Sherif, Z. A., Simhan, H., Singer, N. G., Skupski, D. W., Sowles, A., Sparks, J. A., Sukhera, F. I., Taylor, B. S., Teunis, L., Thomas, R. J., Thorp, J. M., Thuluvath, P., Ticotsky, A., Tita, A. T., Tuttle, K. R., Urdaneta, A. E., Valdivieso, D., VanWagoner, T. M., Vasey, A., Verduzco-Gutierrez, M., Wallace, Z. S., Ward, H. D., Warren, D. E., Weiner, S. J., Welch, S., Whiteheart, S. W., Wiley, Z., Wisnivesky, J. P., Yee, L. M., Zisis, S. 2023

  Abstract

  Importance: SARS-CoV-2 infection is associated with persistent, relapsing, or new symptoms or other health effects occurring after acute infection, termed postacute sequelae of SARS-CoV-2 infection (PASC), also known as long COVID. Characterizing PASC requires analysis of prospectively and uniformly collected data from diverse uninfected and infected individuals.Objective: To develop a definition of PASC using self-reported symptoms and describe PASC frequencies across cohorts, vaccination status, and number of infections.Design, Setting, and Participants: Prospective observational cohort study of adults with and without SARS-CoV-2 infection at 85 enrolling sites (hospitals, health centers, community organizations) located in 33 states plus Washington, DC, and Puerto Rico. Participants who were enrolled in the RECOVER adult cohort before April 10, 2023, completed a symptom survey 6 months or more after acute symptom onset or test date. Selection included population-based, volunteer, and convenience sampling.Exposure: SARS-CoV-2 infection.Main Outcomes and Measures: PASC and 44 participant-reported symptoms (with severity thresholds).Results: A total of 9764 participants (89% SARS-CoV-2 infected; 71% female; 16% Hispanic/Latino; 15% non-Hispanic Black; median age, 47 years [IQR, 35-60]) met selection criteria. Adjusted odds ratios were 1.5 or greater (infected vs uninfected participants) for 37 symptoms. Symptoms contributing to PASC score included postexertional malaise, fatigue, brain fog, dizziness, gastrointestinal symptoms, palpitations, changes in sexual desire or capacity, loss of or change in smell or taste, thirst, chronic cough, chest pain, and abnormal movements. Among 2231 participants first infected on or after December 1, 2021, and enrolled within 30 days of infection, 224 (10% [95% CI, 8.8%-11%]) were PASC positive at 6 months.Conclusions and Relevance: A definition of PASC was developed based on symptoms in a prospective cohort study. As a first step to providing a framework for other investigations, iterative refinement that further incorporates other clinical features is needed to support actionable definitions of PASC.

  View details for DOI 10.1001/jama.2023.8823

  View details for PubMedID 37278994

• Turning the page: a welcome from the new leadership of the Journal of Infectious Diseases. The Journal of infectious diseases Sears, C. L., Li, J. Z., Singh, U. 2022; 227 (1): 1-3

  View details for DOI 10.1093/infdis/jiac400

  View details for PubMedID 36576495

• Early immune markers of clinical, virological, and immunological outcomes in patients with COVID-19: a multi-omics study. eLife Hu, Z., van der Ploeg, K., Chakraborty, S., Arunachalam, P. S., Mori, D. A., Jacobson, K. B., Bonilla, H., Parsonnet, J., Andrews, J. R., Holubar, M., Subramanian, A., Khosla, C., Maldonado, Y., Hedlin, H., de la Parte, L., Press, K., Ty, M., Tan, G. S., Blish, C., Takahashi, S., Rodriguez-Barraquer, I., Greenhouse, B., Butte, A. J., Singh, U., Pulendran, B., Wang, T. T., Jagannathan, P. 2022; 11

  Abstract

  The great majority of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, there is substantial heterogeneity in SARS-CoV-2-specific memory immune responses following infection. There remains a critical need to identify host immune biomarkers predictive of clinical and immunological outcomes in SARS-CoV-2-infected patients.Leveraging longitudinal samples and data from a clinical trial (N=108) in SARS-CoV-2-infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients. We characterized the association between early immune markers and subsequent disease progression, control of viral shedding, and SARS-CoV-2-specific T cell and antibody responses measured up to 7 months after enrollment. We further compared associations between early immune markers and subsequent T cell and antibody responses following natural infection with those following mRNA vaccination. We developed machine-learning models to predict patient outcomes and validated the predictive model using data from 54 individuals enrolled in an independent clinical trial.We identify early immune signatures, including plasma RIG-I levels, early IFN signaling, and related cytokines (CXCL10, MCP1, MCP-2, and MCP-3) associated with subsequent disease progression, control of viral shedding, and the SARS-CoV-2-specific T cell and antibody response measured up to 7 months after enrollment. We found that several biomarkers for immunological outcomes are shared between individuals receiving BNT162b2 (Pfizer-BioNTech) vaccine and COVID-19 patients. Finally, we demonstrate that machine-learning models using 2-7 plasma protein markers measured early within the course of infection are able to accurately predict disease progression, T cell memory, and the antibody response post-infection in a second, independent dataset.Early immune signatures following infection can accurately predict clinical and immunological outcomes in outpatients with COVID-19 using validated machine-learning models.Support for the study was provided from National Institute of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) (U01 AI150741-01S1 and T32-AI052073), the Stanford's Innovative Medicines Accelerator, National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA) DP1DA046089, and anonymous donors to Stanford University. Peginterferon lambda provided by Eiger BioPharmaceuticals.

  View details for DOI 10.7554/eLife.77943

  View details for PubMedID 36239699

• Successful recruitment of monolingual Spanish speaking Latinos to university phase II and III outpatient COVID-19 clinical treatment trials in Northern California. Contemporary clinical trials Levy, V., Bengoa, R. Y., Romero, P. P., Bollyky, J., Singh, U. 2022: 106891

  Abstract

  Through a public County/University partnership, we employed a Spanish/English bilingual research coordinator to increase awareness of newly available treatments with FDA Emergency Use Authorization and clinical trial opportunities for Latino outpatients with mild to moderate COVID-19. Out of the 550 San Mateo County outpatients with COVID-19 referred to Stanford University between July 2020 and April 2022, 9.5% elected to receive monoclonal antibody EUA treatment. COVID-19 treatment trial enrollment of County patients, 5% of those recruited, was commensurate with non-County populations enrollment. Recruitment models such as ours have the potential to increase US Latino populations' recruitment in outpatient COVID-19 treatment trials and contribute to decreasing COVID-19 health disparities.

  View details for DOI 10.1016/j.cct.2022.106891

  View details for PubMedID 36002110

• TNF-alpha+ CD4+ Tcells dominate the SARS-CoV-2 specific T cell response in COVID-19 outpatients and are associated with durable antibodies. Cell reports. Medicine van der Ploeg, K., Kirosingh, A. S., Mori, D. A., Chakraborty, S., Hu, Z., Sievers, B. L., Jacobson, K. B., Bonilla, H., Parsonnet, J., Andrews, J. R., Press, K. D., Ty, M. C., Ruiz-Betancourt, D. R., de la Parte, L., Tan, G. S., Blish, C. A., Takahashi, S., Rodriguez-Barraquer, I., Greenhouse, B., Singh, U., Wang, T. T., Jagannathan, P. 2022: 100640

  Abstract

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific CD4+ Tcells are likely important in immunity against coronavirus 2019 (COVID-19), but our understanding of CD4+ longitudinal dynamics following infection and of specific features that correlate with the maintenance of neutralizing antibodies remains limited. Here, we characterize SARS-CoV-2-specific CD4+ Tcells in a longitudinal cohort of 109 COVID-19 outpatients enrolled during acute infection. The quality of the SARS-CoV-2-specific CD4+ response shifts from cells producing interferon gamma (IFNgamma) to tumor necrosis factor alpha (TNF-alpha) from 5days to 4months post-enrollment, with IFNgamma-IL-21-TNF-alpha+ CD4+ Tcells the predominant population detected at later time points. Greater percentages of IFNgamma-IL-21-TNF-alpha+ CD4+ Tcells on day 28 correlate with SARS-CoV-2-neutralizing antibodies measured 7months post-infection (⍴= 0.4, p= 0.01). mRNA vaccination following SARS-CoV-2 infection boosts both IFNgamma- and TNF-alpha-producing, spike-protein-specific CD4+ Tcells. These data suggest that SARS-CoV-2-specific, TNF-alpha-producing CD4+ Tcells may play an important role in antibody maintenance following COVID-19.

  View details for DOI 10.1016/j.xcrm.2022.100640

  View details for PubMedID 35588734

• Favipiravir for treatment of outpatients with asymptomatic or uncomplicated COVID-19: a double-blind randomized, placebo-controlled, phase 2 trial. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Holubar, M., Subramanian, A., Purington, N., Hedlin, H., Bunning, B., Walter, K. S., Bonilla, H., Boumis, A., Chen, M., Clinton, K., Dewhurst, L., Epstein, C., Jagannathan, P., Kaszynski, R. H., Panu, L., Parsonnet, J., Ponder, E. L., Quintero, O., Sefton, E., Singh, U., Soberanis, L., Truong, H., Andrews, J. R., Desai, M., Khosla, C., Maldonado, Y. 2022

  Abstract

  Favipiravir is an oral, RNA-dependent RNA polymerase inhibitor with in vitro activity against SARS-CoV2. Despite limited data, favipiravir is administered to patients with COVID-19 in several countries.We conducted a phase 2 double-blind randomized controlled outpatient trial of favipiravir in asymptomatic or mildly symptomatic adults with a positive SARS-CoV2 RT-PCR within 72 hours of enrollment. Participants were randomized 1: 1 to receive placebo or favipiravir (1800mg BID Day 1, 800 mg BID Days 2-10). The primary outcome was SARS-CoV-2 shedding cessation in a modified intention-to-treat (mITT) cohort of participants with positive enrollment RT-PCRs. Using SARS-CoV-2 amplicon-based sequencing, we assessed favipiravir's impact on mutagenesis.From July 8, 2020 - March 23, 2021, we randomized 149 participants with 116 included in the mITT cohort. The participants' mean age was 43 years (SD 12.5) and 57 (49%) were women. We found no difference in time to shedding cessation by treatment arm overall (HR 0.76 favoring placebo, 95% confidence interval [CI] 0.48-1.20) or in sub-group analyses (age, sex, high-risk comorbidities, seropositivity or symptom duration at enrollment). We observed no difference in time to symptom resolution (initial: HR 0.84, 95% CI 0.54-1.29; sustained: HR 0.87, 95% CI 0.52-1.45). We detected no difference in accumulation of transition mutations in the viral genome during treatment.Our data do not support favipiravir use at commonly used doses in outpatients with uncomplicated COVID-19. Further research is needed to ascertain if higher doses of favipiravir are effective and safe for patients with COVID-19.

  View details for DOI 10.1093/cid/ciac312

  View details for PubMedID 35446944

• Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA suggest prolonged gastrointestinal infection. Med (New York, N.Y.) Natarajan, A., Zlitni, S., Brooks, E. F., Vance, S. E., Dahlen, A., Hedlin, H., Park, R. M., Han, A., Schmidtke, D. T., Verma, R., Jacobson, K. B., Parsonnet, J., Bonilla, H. F., Singh, U., Pinsky, B. A., Andrews, J. R., Jagannathan, P., Bhatt, A. S. 2022

  Abstract

  COVID-19 manifests with respiratory, systemic, and gastrointestinal (GI) symptoms.1,2 SARS-CoV-2 RNA is detected in respiratory and fecal samples, and recent reports demonstrate viral replication in both the lung and intestinal tissue.3-5 Although much is known about early fecal RNA shedding, little is known about the long term shedding, especially in those with mild COVID-19. Furthermore, most reports of fecal RNA shedding do not correlate these findings with GI symptoms.6.We analyze the dynamics of fecal RNA shedding up to 10 months after COVID-19 diagnosis in 113 individuals with mild to moderate disease. We also correlate shedding with disease symptoms.Fecal SARS-CoV-2 RNA is detected in 49.2% [95% Confidence interval = 38.2%-60.3%] of participants within the first week after diagnosis. Whereas there was no ongoing oropharyngeal SARS-CoV-2 RNA shedding in subjects at and after 4 months, 12.7% [8.5%-18.4%] of participants continued to shed SARS-CoV-2 RNA in the feces at 4 months after diagnosis and 3.8% [2.0%-7.3%] shed at 7 months. Finally, we find that GI symptoms (abdominal pain, nausea, vomiting) are associated with fecal shedding of SARS-CoV-2 RNA.The extended presence of viral RNA in feces, but not respiratory samples, along with the association of fecal viral RNA shedding with GI symptoms suggest that SARS-CoV-2 infects the GI tract, and that this infection can be prolonged in a subset of individuals with COVID-19.

  View details for DOI 10.1016/j.medj.2022.04.001

  View details for PubMedID 35434682

  View details for PubMedCentralID PMC9005383

• Early immune responses have long-term associations with clinical, virologic, and immunologic outcomes in patients with COVID-19. Research square Hu, Z., van der Ploeg, K., Chakraborty, S., Arunachalam, P., Mori, D., Jacobson, K., Bonilla, H., Parsonnet, J., Andrews, J., Hedlin, H., de la Parte, L., Dantzler, K., Ty, M., Tan, G., Blish, C., Takahashi, S., Rodriguez-Barraquer, I., Greenhouse, B., Butte, A., Singh, U., Pulendran, B., Wang, T., Jagannathan, P. 2022

  Abstract

  The great majority of SARS-CoV-2 infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, there is substantial heterogeneity in SARS-CoV-2-specific memory immune responses following infection. There remains a critical need to identify host immune biomarkers predictive of clinical and immunologic outcomes in SARS-CoV-2-infected patients. Leveraging longitudinal samples and data from a clinical trial in SARS-CoV-2 infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients within the first 2 weeks of symptom onset. We identify early immune signatures, including plasma RIG-I levels, early interferon signaling, and related cytokines (CXCL10, MCP1, MCP-2 and MCP-3) associated with subsequent disease progression, control of viral shedding, and the SARS-CoV-2 specific T cell and antibody response measured up to 7 months after enrollment. We found that several biomarkers for immunological outcomes are shared between individuals receiving BNT162b2 (Pfizera"BioNTech) vaccine and COVID-19 patients. Finally, we demonstrate that machine learning models using 7-10 plasma protein markers measured early within the course of infection are able to accurately predict disease progression, T cell memory, and the antibody response post-infection in a second, independent dataset.

  View details for DOI 10.21203/rs.3.rs-847082/v1

  View details for PubMedID 35132407

• SARS-CoV-2 Neutralizing Monoclonal Antibodies for the Treatment of COVID-19 in Kidney Transplant Recipients. Kidney360 Wang, A. X., Busque, S., Kuo, J., Singh, U., Roeltgen, K., Pinsky, B. A., Chertow, G. M., Scandling, J. D., Lenihan, C. R. 2022; 3 (1): 133-143

  Abstract

  Background: Morbidity and mortality associated with coronavirus disease 2019 (COVID-19) infection in kidney transplant recipients are high and early outpatient interventions to prevent progression to severe disease are needed. SARS-CoV-2 neutralizing mAbs, including bamlanivimab and casirivimab-imdevimab, received emergency use authorization in the United States in November 2020 for treatment of mild to moderate COVID-19 disease.Methods: We performed a retrospective analysis of 27 kidney transplant recipients diagnosed with COVID-19 between July 2020 and February 2021 who were treated with bamlanivimab or casirivimab-imdevimab and immunosuppression reduction. We additionally identified 13 kidney transplant recipients with COVID-19 who had mild to moderate disease at presentation, who did not receive mAbs, and had SARS-CoV-2 serology testing available.Results: There were no deaths or graft failures in either group. Both infusions were well tolerated. Four of the 27 patients treated with mAbs required hospitalization due to COVID-19. Four of 13 patients who did not receive mAbs required hospitalization due to COVID-19. Patients who received mAbs demonstrated measurable anti-SARS-CoV-2 IgG with angiotensin-converting enzyme 2 (ACE2) receptor blocking activity at the highest level detectable at 90 days postinfusion, whereas ACE2 blocking activity acquired from natural immunity in the mAb-untreated group was weak.Conclusions: Bamlanivimab and casirivimab-imdevimab combined with immunosuppression reduction were well tolerated and associated with favorable clinical outcomes in kidney transplant recipients diagnosed with mild to moderate COVID-19.

  View details for DOI 10.34067/KID.0005732021

  View details for PubMedID 35368573

• Autoantibodies targeting cytokines and connective tissue disease autoantigens are common in acute non-SARS-CoV-2 infections. Research square Feng, A., Yang, E., Moore, A., Dhingra, S., Chang, S., Yin, X., Pi, R., Mack, E., Völkel, S., Geßner, R., Gundisch, M., Neubauer, A., Renz, H., Tsiodras, S., Fragkou, P., Asuni, A., Levitt, J., Wilson, J., Leong, M., Lumb, J., Mao, R., Pinedo, K., Roque, J., Richards, C., Stabile, M., Swaminathan, G., Salagianni, M., Triantafyllia, V., Bertrams, W., Blish, C., Carette, J., Frankovich, J., Meffre, E., Nadeau, K. C., Singh, U., Wang, T., Prak, E. L., Herold, S., Andreakos, E., Schmeck, B., Skevaki, C., Rogers, A., Utz, P. 2022

  Abstract

  The widespread presence of autoantibodies in acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is increasingly recognized, but the prevalence of autoantibodies in infections with organisms other than SARS-CoV-2 has not yet been reported. We used protein arrays to profile IgG autoantibodies from 317 samples from 268 patients across a spectrum of non-SARS-CoV-2 infections, many of whom were critically ill with pneumonia. Anti-cytokine antibodies (ACA) were identified in > 50% of patients infected with non-SARS-CoV-2 viruses and other pathogens, including patients with pneumonia attributed to bacterial causes. In cell-based functional assays, some ACA blocked binding to surface receptors for type I interferons (Type I IFN), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6). Autoantibodies against traditional autoantigens associated with connective tissue diseases (CTDs) were also commonly observed in these cohorts, including newly-detected antibodies that emerged in longitudinal samples from patients infected with influenza. We conclude that autoantibodies, some of which are functionally active, may be much more prevalent than previously appreciated in patients who are symptomatically infected with diverse pathogens.

  View details for DOI 10.21203/rs.3.rs-1233038/v1

  View details for PubMedID 35075455

  View details for PubMedCentralID PMC8786233

• Early non-neutralizing, afucosylated antibody responses are associated with COVID-19 severity. Science translational medicine Chakraborty, S., Gonzalez, J. C., Sievers, B. L., Mallajosyula, V., Chakraborty, S., Dubey, M., Ashraf, U., Cheng, B. Y., Kathale, N., Tran, K. Q., Scallan, C., Sinnott, A., Cassidy, A., Chen, S. T., Gelbart, T., Gao, F., Golan, Y., Ji, X., Kim-Schulze, S., Prahl, M., Gaw, S. L., Gnjatic, S., Marron, T. U., Merad, M., Arunachalam, P. S., Boyd, S. D., Davis, M. M., Holubar, M., Khosla, C., Maecker, H. T., Maldonado, Y., Mellins, E. D., Nadeau, K. C., Pulendran, B., Singh, U., Subramanian, A., Utz, P. J., Sherwood, R., Zhang, S., Jagannathan, P., Tan, G. S., Wang, T. T. 1800: eabm7853

  Abstract

  A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated IgG antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were instead highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc gamma receptor (FcgammaR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from COVID-19 patients induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine-elicited IgG did not promote an inflammatory lung response. Together, these results show that IgG-FcgammaR interactions are able to regulate inflammation in the lung and may define distinct lung activities associated with the IgG that are associated with severe COVID-19 and protection against infection with SARS-CoV-2.

  View details for DOI 10.1126/scitranslmed.abm7853

  View details for PubMedID 35040666

• Antibodies elicited by SARS-CoV-2 infection or mRNA vaccines have reduced neutralizing activity against Beta and Omicron pseudoviruses. Science translational medicine Sievers, B. L., Chakraborty, S., Xue, Y., Gelbart, T., Gonzalez, J. C., Cassidy, A. G., Golan, Y., Prahl, M., Gaw, S. L., Arunachalam, P. S., Blish, C. A., Boyd, S. D., Davis, M. M., Jagannathan, P., Nadeau, K. C., Pulendran, B., Singh, U., Scheuermann, R. H., Frieman, M. B., Vashee, S., Wang, T. T., Tan, G. S. 1800: eabn7842

  Abstract

  Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that possess mutations associated with increased transmission and antibody escape have arisen over the course of the current pandemic. Although the current vaccines have largely been effective against past variants, the number of mutations found on the Omicron (B.1.1.529) spike protein appear to diminish the protection conferred by pre-existing immunity. Using vesicular stomatitis virus (VSV) pseudoparticles expressing the spike protein of several SARS-CoV-2 variants, we evaluated the magnitude and breadth of the neutralizing antibody response over time in individuals after infection and in mRNA-vaccinated individuals. We observed that boosting increases the magnitude of the antibody response to wildtype (D614), Beta, Delta, and Omicron variants; however, the Omicron variant was the most resistant to neutralization. We further observed that vaccinated healthy adults had robust and broad antibody responses whereas responses may have been reduced in vaccinated pregnant women, underscoring the importance of learning how to maximize mRNA vaccine responses in pregnant populations. Findings from this study show substantial heterogeneity in the magnitude and breadth of responses after infection and mRNA vaccination and may support the addition of more conserved viral antigens to existing SARS-CoV-2 vaccines.

  View details for DOI 10.1126/scitranslmed.abn7842

  View details for PubMedID 35025672

• Long Term Accuracy of SARS-CoV-2 Interferon-γ Release Assay and its Application in Household Investigation. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Murugesan, K., Jagannathan, P., Altamirano, J., Maldonado, Y. A., Bonilla, H. F., Jacobson, K. B., Parsonnet, J., Andrews, J. R., Shi, R. Z., Boyd, S., Pinsky, B. A., Singh, U., Banaei, N. 2022

  Abstract

  An immunodiagnostic assay that sensitively detects a cell-mediated immune response to SARS-CoV-2 is needed for epidemiological investigation and for clinical assessment of T cell-mediated immune response to vaccines, particularly in the context of emerging variants that might escape antibody responses.The performance of a whole blood interferon-gamma (IFN-γ) release assay (IGRA) for the detection of SARS-CoV-2 antigen-specific T cells was evaluated in COVID-19 convalescents tested serially up to 10 months post-infection and in healthy blood donors. SARS-CoV-2 IGRA was applied in contacts of households with index cases. Freshly collected blood in the lithium heparin tube was left unstimulated, stimulated with a SARS-CoV-2 peptide pool, and stimulated with mitogen.The overall sensitivity and specificity of IGRA were 84.5% (153/181; 95% confidence interval [CI] 79.0-89.0) and 86.6% (123/142; 95% CI;80.0-91.2), respectively. The sensitivity declined from 100% (16/16; 95% CI 80.6-100) at 0.5-month post-infection to 79.5% (31/39; 95% CI 64.4-89.2) at 10 months post-infection (P<0.01). The IFN-γ response remained relatively robust at 10 months post-infection (3.8 vs. 1.3 IU/mL, respectively). In 14 households, IGRA showed a positivity rate of 100% (12/12) and 65.2% (15/23), and IgG of 50.0% (6/12) and 43.5% (10/23) in index cases and contacts, respectively, exhibiting a difference of +50% (95% CI +25.4-+74.6) and +21.7% (95% CI, +9.23-+42.3), respectively. Either IGRA or IgG was positive in 100% (12/12) of index cases and 73.9% (17/23) of contacts.The SARS-CoV-2 IGRA is a useful clinical diagnostic tool for assessing cell-mediated immune response to SARS-CoV-2.

  View details for DOI 10.1093/cid/ciac045

  View details for PubMedID 35079772

• Variation in Severe Acute Respiratory Syndrome Coronavirus 2 Bioaerosol Production in Exhaled Breath. Open forum infectious diseases Verma, R., Kim, E., Degner, N., Walter, K. S., Singh, U., Andrews, J. R. 2022; 9 (1): ofab600

  Abstract

  We developed a simple, noninvasive mask sampling method to quantify and sequence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from exhaled breath. We found substantial variation between individuals in SARS-CoV-2 copies exhaled over a 15-minute period, which moderately correlated with nasal swab viral load. Talking was associated with a median of 2 log10 greater exhaled viral copies. Exposure varies substantially between individuals but may be risk stratified by nasal swab viral load and whether the exposure involved conversation.

  View details for DOI 10.1093/ofid/ofab600

  View details for PubMedID 35028332

  View details for PubMedCentralID PMC8753034

• Ponatinib, Lestaurtinib and mTOR/PI3K inhibitors are promising repurposing candidates against Entamoeba histolytica. Antimicrobial agents and chemotherapy Kangussu-Marcolino, M. M., Singh, U. 2021: AAC0120721

  Abstract

  Dysentery caused by Entamoeba histolytica affects millions of people annually. Current treatment regimens are based on metronidazole to treat invasive parasites combined with paromomycin for luminal parasites. Issues with treatment include significant side effects, inability to easily treat breastfeeding and pregnant women, the use of two sequential agents, and concern that all therapy is based on nitroimidazole agents with no alternatives if clinical resistance emerges. Thus, the need for new drugs against amebiasis is urgent. To identify new therapeutic candidates, we screened the ReFRAME library (11,948 compounds assembled for Repurposing, Focused Rescue, and Accelerated Medchem) against E. histolytica trophozoites. We identified 159 hits in the primary screen at 10 muM and 46 compounds were confirmed in secondary assays. Overall, 26 were selected as priority molecules for further investigation including 6 FDA approved, 5 orphan designation, and 15 which are currently in clinical trials (3 phase III, 7 phase II and 5 phase I). We found that all 26 compounds are active against metronidazole resistant E. histolytica and 24 are able to block parasite recrudescence after drug removal. Additionally, 14 are able to inhibit encystation and 2 (lestaurtinib and LY-2874455) are active against mature cysts. Two classes of compounds are most interesting for further investigations: the Bcr-Abl TK inhibitors, with the ponatinib (EC50 0.39) as most potent and mTOR or PI3K inhibitors with 8 compounds in clinical development, of which 4 have nanomolar potency. Overall, these are promising candidates and represent a significant advance for drug development against E. histolytica.

  View details for DOI 10.1128/AAC.01207-21

  View details for PubMedID 34871094

• New-onset IgG autoantibodies in hospitalized patients with COVID-19. Nature communications Chang, S. E., Feng, A., Meng, W., Apostolidis, S. A., Mack, E., Artandi, M., Barman, L., Bennett, K., Chakraborty, S., Chang, I., Cheung, P., Chinthrajah, S., Dhingra, S., Do, E., Finck, A., Gaano, A., GeSSner, R., Giannini, H. M., Gonzalez, J., Greib, S., Gundisch, M., Hsu, A. R., Kuo, A., Manohar, M., Mao, R., Neeli, I., Neubauer, A., Oniyide, O., Powell, A. E., Puri, R., Renz, H., Schapiro, J., Weidenbacher, P. A., Wittman, R., Ahuja, N., Chung, H., Jagannathan, P., James, J. A., Kim, P. S., Meyer, N. J., Nadeau, K. C., Radic, M., Robinson, W. H., Singh, U., Wang, T. T., Wherry, E. J., Skevaki, C., Luning Prak, E. T., Utz, P. J. 2021; 12 (1): 5417

  Abstract

  COVID-19 is associated with a wide range of clinical manifestations, including autoimmune features and autoantibody production. Here we develop three protein arrays to measure IgG autoantibodies associated with connective tissue diseases, anti-cytokine antibodies, and anti-viral antibody responses in serum from 147 hospitalized COVID-19 patients. Autoantibodies are identified in approximately 50% of patients but in less than 15% of healthy controls. When present, autoantibodies largely target autoantigens associated with rare disorders such as myositis, systemic sclerosis and overlap syndromes. A subset of autoantibodies targeting traditional autoantigens or cytokines develop de novo following SARS-CoV-2 infection. Autoantibodies track with longitudinal development of IgG antibodies recognizing SARS-CoV-2 structural proteins and a subset of non-structural proteins, but not proteins from influenza, seasonal coronaviruses or other pathogenic viruses. We conclude that SARS-CoV-2 causes development of new-onset IgG autoantibodies in a significant proportion of hospitalized COVID-19 patients and are positively correlated with immune responses to SARS-CoV-2 proteins.

  View details for DOI 10.1038/s41467-021-25509-3

  View details for PubMedID 34521836

• RISC in Entamoeba histolytica: Identification of a Protein-Protein Interaction Network for the RNA Interference Pathway in a Deep-Branching Eukaryote. mBio Zhang, H., Veira, J., Bauer, S. T., Yip, C., Singh, U. 2021: e0154021

  Abstract

  Entamoeba histolytica is a protozoan parasite that causes amebiasis in humans and is a major health concern in developing countries. Our previous work revealed a functional RNA interference (RNAi) pathway in Entamoeba. Several unusual features encompass the RNAi pathway in the parasite, including small RNAs (sRNAs) with a 5'-polyphosphate structure (identified to date only in Entamoeba and nematodes) and the conspicuous absence of a canonical Dicer enzyme. Currently, little is known about the Entamoeba RNA-induced silencing complex (RISC), which is critical in understanding how RNAi is achieved in the parasite. In this study, we examined the RISC of EhAgo2-2, the most highly expressed Argonaute protein in Entamoeba. We identified 43 protein components of EhAgo2-2 RISC with a broad range of functional activities. Two proteins with nucleosome assembly protein (NAP) domains, not previously observed in other RNAi systems, were identified as novel core members of amebic RISC. We further demonstrated the interaction of these NAPs with Ago using an in vitro recombinant system. Finally, we characterized the interaction network of five RISC components identified in this study to further elucidate the interactions of these RNAi pathway proteins. Our data suggest the presence of closely interacting protein groups within RISC and allowed us to build a map of protein-protein interactions in relation to Ago. Our work is the first to elucidate RISC components in Entamoeba and expands the current knowledge of RISC to a deep-branching single-celled eukaryote. IMPORTANCE Entamoeba histolytica is a leading parasitic cause of death in developing countries, and our efforts are focused on defining the molecular basis of RNA interference (RNAi) gene regulation in this parasite. The Entamoeba RNAi pathway effectively silences a subset of endogenous genes and has also been harnessed as a gene silencing tool to study gene function in this organism. However, little is known about the components of the Entamoeba RNA-induced silencing complex (RISC), which is critical in understanding how gene silencing is achieved in the parasite. This study characterizes, for the first time, the RISC components in Entamoeba and provides new insights in understanding the molecular regulatory mechanisms of RNAi in this parasite, including the demonstration of novel Ago protein-interacting partners. From an evolutionary point of view, our findings expand the current knowledge of RISC to a deep-branching single-celled eukaryote.

  View details for DOI 10.1128/mBio.01540-21

  View details for PubMedID 34488447

• New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19 Chang, S., Feng, A., Meng, W., Apostolidis, S., Mack, E., Artandi, M., Barman, L., Bennett, K., Chakraborty, S., Chang, I., Cheung, P., Chinthrajah, S., Dhingra, S., Do, E., Finck, A., Gaano, A., Gessner, R., Giannini, H., Gonzalez, J., Greib, S., Gundisch, M., Hsu, A., Kuo, A., Manohar, M., Mao, R., Neeli, I., Neubauer, A., Oniyide, O., Powell, A., Puri, R., Renz, H., Schapiro, J., Weidenbacher, P., Wittman, R., Ahuja, N., Chung, H., Jagannathan, P., James, J., Kim, P., Meyer, N., Nadeau, K., Radic, M., Robinson, W., Singh, U., Wang, T., Wherry, J., Skevaki, C., Prak, E., Utz, P. WILEY. 2021: 3202-3205

  View details for Web of Science ID 000744545206097

• The COVID-19 Outpatient Pragmatic Platform Study (COPPS): Study design of a multi-center pragmatic platform trial. Contemporary clinical trials Bunning, B., Hedlin, H., Purington, N., Sundaram, V., Kapphahn, K., Weng, Y., Cunanan, K., Maldonado, Y., Singh, U., Khosla, C., O'Hara, R., Nicolls, M., Springman, E., Parsonnet, J., Rogers, A., Levitt, J., Desai, M. 2021: 106509

  Abstract

  More than 3000 clinical trials related to COVID-19 have been registered through clinicaltrials.gov. With so many trials, there is a risk that many will be inconclusive due to being underpowered or due to an inability to recruit patients. At academic medical centers, multiple trials are competing for the same resources; the success of one may come at the expense of another. The COVID-19 Outpatient Pragmatic Protocol Study (COPPS) is a flexible phase 2, multi-site, randomized, blinded trial based at Stanford University designed to overcome these issues by simultaneously evaluating multiple COVID-19 treatments in the outpatient setting in one common platform with shared controls. This approach reduces the overall number of patients required for statistical power, while improving the likelihood that any enrolled patient receives active treatment. The platform study has two main domains designed to evaluate COVID-19 treatments by assessing their ability to reduce viral shedding (Viral Domain), measured with self-collected nasal swabs, or improve clinical outcomes (Clinical Domain), measured through self-reported symptomology data. Data are collected on both domains for all participants enrolled. Participants are followed over a 28-day period. COPPS has the advantage of pragmatism created around its workflow that is also appealing to potential participants because of a lower probability of inactive treatment. At the conclusion of this clinical trial we expect to have identified potentially effective therapeutic strategy/ies for treating COVID-19 in the outpatient setting, which will have a transformative impact on medicine and public health.

  View details for DOI 10.1016/j.cct.2021.106509

  View details for PubMedID 34274494

• Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA. medRxiv : the preprint server for health sciences Natarajan, A., Han, A., Zlitni, S., Brooks, E. F., Vance, S. E., Wolfe, M., Singh, U., Jagannathan, P., Pinsky, B. A., Boehm, A., Bhatt, A. S. 2021

  Abstract

  COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

  View details for DOI 10.1101/2021.04.10.21255250

  View details for PubMedID 33880485

• Patients with uncomplicated COVID-19 have long-term persistent symptoms and functional impairment similar to patients with severe COVID-19: a cautionary tale during a global pandemic. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Jacobson, K. B., Rao, M., Bonilla, H., Subramanian, A., Hack, I., Madrigal, M., Singh, U., Jagannathan, P., Grant, P. 2021

  Abstract

  To assess the prevalence of persistent functional impairment after COVID-19, we assessed 118 individuals 3-4 months after their initial COVID-19 diagnosis with a symptom survey, work productivity and activity index questionnaire, and 6-minute walk test. We found significant persistent symptoms and functional impairment, even in non-hospitalized patients with COVID-19.

  View details for DOI 10.1093/cid/ciab103

  View details for PubMedID 33624010

• Peginterferon Lambda-1a for treatment of outpatients with uncomplicated COVID-19: a randomized placebo-controlled trial. Nature communications Jagannathan, P. n., Andrews, J. R., Bonilla, H. n., Hedlin, H. n., Jacobson, K. B., Balasubramanian, V. n., Purington, N. n., Kamble, S. n., de Vries, C. R., Quintero, O. n., Feng, K. n., Ley, C. n., Winslow, D. n., Newberry, J. n., Edwards, K. n., Hislop, C. n., Choong, I. n., Maldonado, Y. n., Glenn, J. n., Bhatt, A. n., Blish, C. n., Wang, T. n., Khosla, C. n., Pinsky, B. A., Desai, M. n., Parsonnet, J. n., Singh, U. n. 2021; 12 (1): 1967

  Abstract

  Type III interferons have been touted as promising therapeutics in outpatients with coronavirus disease 2019 (COVID-19). We conducted a randomized, single-blind, placebo-controlled trial (NCT04331899) in 120 outpatients with mild to moderate COVID-19 to determine whether a single, 180 mcg subcutaneous dose of Peginterferon Lambda-1a (Lambda) within 72 hours of diagnosis could shorten the duration of viral shedding (primary endpoint) or symptoms (secondary endpoint). In both the 60 patients receiving Lambda and 60 receiving placebo, the median time to cessation of viral shedding was 7 days (hazard ratio [HR] = 0.81; 95% confidence interval [CI] 0.56 to 1.19). Symptoms resolved in 8 and 9 days in Lambda and placebo, respectively, and symptom duration did not differ significantly between groups (HR 0.94; 95% CI 0.64 to 1.39). Both Lambda and placebo were well-tolerated, though liver transaminase elevations were more common in the Lambda vs. placebo arm (15/60 vs 5/60; p = 0.027). In this study, a single dose of subcutaneous Peginterferon Lambda-1a neither shortened the duration of SARS-CoV-2 viral shedding nor improved symptoms in outpatients with uncomplicated COVID-19.

  View details for DOI 10.1038/s41467-021-22177-1

  View details for PubMedID 33785743

• Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA. Nature communications Natarajan, A., Han, A., Zlitni, S., Brooks, E. F., Vance, S. E., Wolfe, M., Singh, U., Jagannathan, P., Pinsky, B. A., Boehm, A., Bhatt, A. S. 2021; 12 (1): 5753

  Abstract

  Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

  View details for DOI 10.1038/s41467-021-25576-6

  View details for PubMedID 34599164

• Publisher Correction: Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA. Nature communications Natarajan, A., Han, A., Zlitni, S., Brooks, E. F., Vance, S. E., Wolfe, M., Singh, U., Jagannathan, P., Pinsky, B. A., Boehm, A., Bhatt, A. S. 2021; 12 (1): 7100

  View details for DOI 10.1038/s41467-021-27392-4

  View details for PubMedID 34853336

• SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples Open Forum Infectious Diseases Verma, R., Kim, E., Martinez, G., Jagannathan, ., Rustagi, A., Parsonnet, J., Bonilla, H., Khosla, C., Holubar, M., Subramanian, A., Singh, ., Maldonado, Y., Blish, C., Andrews, J. 2021

  View details for DOI 10.1093/ofid/ofab310

• Inflammatory but not respiratory symptoms are associated with ongoing upper airway viral shedding in outpatients with uncomplicated COVID-19. Diagnostic microbiology and infectious disease Jacobson, K. B., Purington, N., Parsonnet, J., Andrews, J., Balasubramanian, V., Bonilla, H., Edwards, K., Desai, M., Singh, U., Hedlin, H., Jagannathan, P. 2021; 102 (3): 115612

  Abstract

  Although the vast majority of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are uncomplicated, our understanding of predictors of symptom resolution and viral shedding cessation remains limited. We characterized symptom trajectories and oropharyngeal viral shedding among 120 outpatients with uncomplicated Coronavirus Disease of 2019 (COVID-19) enrolled in a clinical trial of Peginterferon Lambda, which demonstrated no clinical or virologic benefit compared with placebo. In the combined trial cohort, objective fever was uncommon, inflammatory symptoms (myalgias, fatigue) peaked at 4 to 5 days postsymptom onset, and cough peaked at 9 days. The median time to symptom resolution from earliest symptom onset was 17 days (95% confidence interval 14-18). SARS-CoV-2 IgG seropositivity at enrollment was associated with hastened resolution of viral shedding (hazard ratio 1.80, 95% confidence interval 1.05-3.1, P = 0.03), but not with symptom resolution. Inflammatory symptoms were associated with a significantly greater odds of oropharyngeal SARS-CoV-2 RNA detection; respiratory symptoms were not. These findings have important implications for COVID-19 screening approaches and trial design.

  View details for DOI 10.1016/j.diagmicrobio.2021.115612

  View details for PubMedID 34974350

• Identification of oligo-adenylated small RNAs in the parasite Entamoeba and a potential role for small RNA control. BMC genomics Zhang, H., Ehrenkaufer, G. M., Hall, N., Singh, U. 2020; 21 (1): 879

  Abstract

  BACKGROUND: The RNA interference (RNAi) pathway is a gene regulation mechanism that utilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27nt antisense sRNA populations which associate with EhAgo2-2 protein. However, there is lack of understanding about the sRNAs that are bound to two other EhAgos (EhAgo2-1 and 2-3), and the mechanism of sRNA regulation itself is unclear in this parasite. Therefore, identification of the entire pool of sRNA species and their sub-populations that associate with each individual EhAgo protein would be a major step forward.RESULTS: In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31nt sRNAs that results from the addition of a non-templated 3-4 adenosine nucleotides at the 3'-end of the 27nt sRNAs, indicating a non-templated RNA-tailing event in the parasite. The relative abundance of these two sRNA populations is linked to the efficacy of gene silencing for the target gene when parasites are transfected with an RNAi-trigger construct, indicating that non-templated sRNA-tailing likely play a role in sRNA regulation in this parasite. We found that both sRNA populations (27nt and 31nt) are present in the related parasite Entamoeba invadens, and are unchanged during the development. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27nt sRNAs with 5'-polyphosphate (5'-polyP) structure and share a largely overlapping sRNA repertoire. In addition, our data showed that a fraction of 31nt sRNAs associate with EhAgo2-2 but not with its mutant protein (C-terminal deletion), nor other two EhAgos, indicating a specific EhAgo site may be required for sRNA modification process in the parasite.CONCLUSION: We identified a new population of sRNA with non-templated oligo-adenylation modification, which is the first such observation amongst single celled protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.

  View details for DOI 10.1186/s12864-020-07275-6

  View details for PubMedID 33297948

• Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept. International journal for parasitology Kangussu-Marcolino, M. M., Morgado, P., Manna, D., Yee, H., Singh, U. 2020

  Abstract

  The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimized CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilizing domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.

  View details for DOI 10.1016/j.ijpara.2020.09.005

  View details for PubMedID 33264648

• Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity PLOS NEGLECTED TROPICAL DISEASES Ehrenkaufer, G., Li, P., Stebbins, E. E., Kangussu-Marcolino, M. M., Debnath, A., White, C., Moser, M. S., DeRisi, J., Gisselberg, J., Yeh, E., Wang, S. C., Company, A., Monti, L., Caffrey, C. R., Huston, C. D., Wang, B., Singh, U. 2020; 14 (3)

  View details for DOI 10.1371/journal.pntd.0008150.r004

  View details for Web of Science ID 000528655400050

• Entamoeba stage conversion: progress and new insights. Current opinion in microbiology Manna, D. n., Ehrenkaufer, G. M., Lozano-Amado, D. n., Singh, U. n. 2020; 58: 62–68

  Abstract

  Entamoeba histolytica, an anaerobic protozoan, is an important global health problem. This parasite has a biphasic life cycle consisting of a dormant cyst stage which is environmentally resistant and transmits the infection, and the proliferative trophozoite stage which is motile and causes invasive disease. The stage conversion process remains poorly understood despite being central to amoebic biology. In this review, we will highlight recent progress in our understanding of Entamoeba stage conversion including dissecting transcriptome analysis in development, characterization of transcriptional networks, demonstration of epigenetic regulation, and role of small molecules that regulate Entamoeba development.

  View details for DOI 10.1016/j.mib.2020.09.005

  View details for PubMedID 33032142

• The NAD+ Responsive Transcription Factor ERM-BP Functions Downstream of Cellular Aggregation and Is an Early Regulator of Development and Heat Shock Response in Entamoeba. Frontiers in cellular and infection microbiology Manna, D. n., Lozano-Amado, D. n., Ehrenkaufer, G. n., Singh, U. n. 2020; 10: 363

  Abstract

  Entamoeba histolytica is a protozoan parasite and a major cause of dysentery and diarrheal disease in developing countries. Disease transmission from one host to another occurs via cysts which can survive in environmental extremes and are transmitted through contaminated food and water. Recent studies in our lab identified a novel transcription factor, Encystation Regulatory Motif- Binding Protein (ERM-BP), which is responsive to NAD+ and has an important role in encystation. The key residues important for ERM-BP function were demonstrated in vitro using recombinant protein. In this study we demonstrate the in vivo functional consequences of mutations in key domains and their impact on Entamoeba encystation. Our results show that mutations in the DNA binding domain (ERM-BP-DBM) and in the nicotinamidase domain (ERM-BP-C198A) lead to protein mis-localization in both trophozoites and cysts and significantly reduce encystation efficiency. Additionally, we showed that silencing of ERM-BP significantly decreased the size and number of multi-nucleated giant cells (MGC) that form during encystation, indicating that ERM-BP functions upstream of the cellular aggregation that precedes stage conversion. Dissection of epistatic interactions between ERM-BP and a second encystation-related transcription factor, NF-Y revealed that ERM-BP is upstream of NF-Y in controlling the developmental cascade and appears to be one of the earliest regulators of development identified to date in Entamoeba. We also demonstrated that ERM-BP is upregulated during heat stress in Entamoeba, another condition which increases intracellular NAD+ levels and that overexpression of ERM-BP makes E. histolytica and E. invadens parasites more resistant to heat stress. Overexpression of ERM-BP in E. histolytica also induced the formation of cyst-like quadrinucleated cells and formation of MGCs. Overall, our work has identified an important role of ERM-BP in Entamoeba stress response and links an NAD+-responsive transcription factor to both development and heat shock response. Characterization of stress and developmental cascades are important avenues to investigate for Entamoeba, an important human parasitic pathogen.

  View details for DOI 10.3389/fcimb.2020.00363

  View details for PubMedID 32766170

  View details for PubMedCentralID PMC7379229

• Interferon-gamma release assay for accurate detection of SARS-CoV-2 T cell response. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Murugesan, K. n., Jagannathan, P. n., Pham, T. D., Pandey, S. n., Bonilla, H. F., Jacobson, K. n., Parsonnet, J. n., Andrews, J. R., Weiskopf, D. n., Sette, A. n., Pinsky, B. A., Singh, U. n., Banaei, N. n. 2020

  Abstract

  We investigated feasibility and accuracy of an interferon-gamma release assay (IGRA) for detection of T cell responses to SARS-CoV-2. Whole blood IGRA accurately distinguished between convalescents and uninfected healthy blood donors with a predominantly CD4+ T cell response. SARS-CoV-2 IGRA may serve as a useful diagnostic tool in managing the COVID-19 pandemic.

  View details for DOI 10.1093/cid/ciaa1537

  View details for PubMedID 33035306

• Proinflammatory IgG Fc structures in patients with severe COVID-19 Nature Immunology Chakraborty, S., Gonzales, J., Edwards, K., Mallajosyulla, V., Buzzanco, A. S., Sherwood, R., Buffone, C., Kathale, N., Providenza, S., Xie, M. M., Andrews, J. R., Blish, C. A., Singh, U., Dugan, H., Wilson, P. C., Pham, T. D., Boyd, S. D., Nadeau, K. C., Pinsky, B. A., Zhang, S., Memoli, M. J., Taubenberger, J. K., Morales, T., Schapiro, J. M., Tan, G. S., et al 2020

  View details for DOI 10.1038/s41590-020-00828-7

• Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity. PLoS neglected tropical diseases Ehrenkaufer, G. n., Li, P. n., Stebbins, E. E., Kangussu-Marcolino, M. M., Debnath, A. n., White, C. V., Moser, M. S., DeRisi, J. n., Gisselberg, J. n., Yeh, E. n., Wang, S. C., Company, A. H., Monti, L. n., Caffrey, C. R., Huston, C. D., Wang, B. n., Singh, U. n. 2020; 14 (3): e0008150

  Abstract

  Parasitic infections are a major source of human suffering, mortality, and economic loss, but drug development for these diseases has been stymied by the significant expense involved in bringing a drug though clinical trials and to market. Identification of single compounds active against multiple parasitic pathogens could improve the economic incentives for drug development as well as simplifying treatment regimens. We recently performed a screen of repurposed compounds against the protozoan parasite Entamoeba histolytica, causative agent of amebic dysentery, and identified four compounds (anisomycin, prodigiosin, obatoclax and nithiamide) with low micromolar potency and drug-like properties. Here, we extend our investigation of these drugs. We assayed the speed of killing of E. histolytica trophozoites and found that all four have more rapid action than the current drug of choice, metronidazole. We further established a multi-institute collaboration to determine whether these compounds may have efficacy against other parasites and opportunistic pathogens. We found that anisomycin, prodigiosin and obatoclax all have broad-spectrum antiparasitic activity in vitro, including activity against schistosomes, T. brucei, and apicomplexan parasites. In several cases, the drugs were found to have significant improvements over existing drugs. For instance, both obatoclax and prodigiosin were more efficacious at inhibiting the juvenile form of Schistosoma than the current standard of care, praziquantel. Additionally, low micromolar potencies were observed against pathogenic free-living amebae (Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba castellanii), which cause CNS infection and for which there are currently no reliable treatments. These results, combined with the previous human use of three of these drugs (obatoclax, anisomycin and nithiamide), support the idea that these compounds could serve as the basis for the development of broad-spectrum anti-parasitic drugs.

  View details for DOI 10.1371/journal.pntd.0008150

  View details for PubMedID 32196500

• Characterization of extracellular vesicles from Entamoeba histolytica identifies roles in intercellular communication that regulates parasite growth and development. Infection and immunity Sharma, M. n., Morgado, P. n., Zhang, H. n., Ehrenkaufer, G. n., Manna, D. n., Singh, U. n. 2020

  Abstract

  Extracellular vesicles (EVs) secreted by eukaryotic and prokaryotic cells to transport lipids, proteins and nucleic acids to the external environment have important roles in cell-cell communication through cargo transfer. We identified and characterized EVs from Entamoeba histolytica, a protozoan parasite and a human pathogen. Conditioned medium from amebic parasites contained particles consistent with the expected size and morphology of EVs. Mass spectrometry was used to characterize the EV proteome and showed that it was enriched in common exosome marker proteins including proteins associated with vesicle formation, cell-signaling, metabolism, and cytoskeletal proteins. Additionally, the EVs were found to selectively package small RNAs (sRNA), which were protected within the vesicles against RNase treatment. Sequencing analysis of the sRNA contained in EVs revealed that the majority were 27nt in size and represented a subset of the cellular antisense small RNA population that has previously been characterized in Entamoeba RNAi pathway proteins, including Argonaute were also present in amebic EVs. Interestingly, we found that the amebic EVs impact inter-cellular communication between parasites and altered encystation efficiency. EVs isolated from encysting parasites promoted encystation in other parasites whereas EVs from metabolically active trophozoites impeded encystation. Overall, the data reveals that Entamoeba secrete EVs that are similar in size and shape to previously characterized exosomes from other organisms and that these EVs contain a defined protein and small RNA cargo and have roles in intercellular communication amongst parasites and influence growth kinetics.

  View details for DOI 10.1128/IAI.00349-20

  View details for PubMedID 32719158

• Identification of plicamycin, TG02, panobinostat, lestaurtinib, and GDC-0084 as promising compounds for the treatment of central nervous system infections caused by the free-living amebae Naegleria, Acanthamoeba and Balamuthia. International journal for parasitology. Drugs and drug resistance Kangussu-Marcolino, M. M., Ehrenkaufer, G. M., Chen, E., Debnath, A., Singh, U. 2019; 11: 80–94

  Abstract

  The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis.

  View details for DOI 10.1016/j.ijpddr.2019.10.003

  View details for PubMedID 31707263

• Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization. mSphere Zhang, H., Tran, V., Manna, D., Ehrenkaufer, G., Singh, U. 2019; 4 (5)

  Abstract

  The RNA interference (RNAi) pathway regulates gene expression in many eukaryotic organisms. Argonaute (Ago) proteins, together with bound small RNAs (sRNAs), are key effectors that mediate gene silencing function. However, there is limited knowledge of Ago proteins and their functions in nonmodel systems. In the protozoan parasite Entamoeba histolytica, RNAi is a robust means for stable gene silencing mediated via large populations of antisense sRNAs. Here, we report functional characterization of three Ago proteins in E. histolytica (EhAgo2-1, EhAgo2-2, and EhAgo2-3). Our data show that each EhAgo protein has a distinct subcellular localization and binds 27-nucleotide (nt) sRNAs and that the localization of EhAgo proteins is altered in response to stress conditions. Via mutagenesis analyses, we demonstrated that the Ago PAZ (Piwi/Argonaute/Zwille) domain in all three EhAgos is essential for sRNA binding. With mutation of the PAZ domain in EhAgo2-2, there was no effect on the nuclear localization of the protein but a strong phenotype and a growth defect. We further show that EhAgo2-2 contains an unusual repetitive DR-rich (aspartic acid, arginine-rich) motif region which functions as a nuclear localization signal (NLS) and is both necessary and sufficient to mediate nuclear localization. Overall, our data delineate the localization and sRNA binding features of the three E. histolytica Ago proteins and demonstrate that the PAZ domain is necessary for sRNA binding. The repetitive DR-rich motif region in EhAgo2-2 has not previously been defined in other systems, which adds to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.IMPORTANCE The protozoan parasite Entamoeba histolytica, which causes amebiasis and affects over 50 million people worldwide, contains an important RNAi pathway for gene silencing. Gene silencing via the RNAi pathway is mediated by the Argonaute (Ago) proteins. However, we lack knowledge on Ago function(s) in this nonmodel system. In this paper, we discovered that three E. histolytica Ago proteins (EhAgo2-1, EhAgo2-2, and EhAgo2-3) all bind 27-nt small RNAs and have distinct subcellular localizations, which change in response to stress conditions. The EhAgos bind small RNA populations via their PAZ domains. An unusual repetitive DR-rich motif region is identified in EhAgo2-2 that functions as a nuclear localization signal. Our results show for the first time an active nuclear transport process of the EhAgo2-2 RNA-induced silencing complex (RISC) in this parasite. These data add to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.

  View details for DOI 10.1128/mSphere.00580-19

  View details for PubMedID 31619501

• Drug treatment algorithms for water-borne parasitic pathogens Singh, U. AMER CHEMICAL SOC. 2019

  View details for Web of Science ID 000525055501648

• Nuclear Factor Y (NF-Y) Modulates Encystation in Entamoeba via Stage-Specific Expression of the NF-YB and NF-YC Subunits. mBio Manna, D., Singh, U. 2019; 10 (3)

  Abstract

  Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of three subunits, namely, NF-YA, NF-YB, and NF-YC, which are conserved throughout evolution. In higher eukaryotes, NF-Y plays important roles in several cellular processes (development, cell cycle regulation, apoptosis, and response to growth, stress, and DNA damage) by controlling gene expression through binding to a CCAAT promoter motif. We demonstrated that NF-Y subunits in the protist Entamoeba, while significantly divergent from those of higher eukaryotes, have well-conserved domains important for subunit interactions and DNA binding and that NF-YB and NF-YC are developmentally expressed during encystation. Electrophoretic mobility shift assays confirmed that the NF-Y protein(s) from Entamoeba cysts binds to a CCAAT motif. Consistent with a role as a transcription factor, the NF-Y proteins show nuclear localization during development. Additionally, we demonstrated that NF-YC localizes to the chromatoid body (an RNA processing center) during development, indicating that it may have a role in RNA processing. Finally, silencing of the NF-YC subunit resulted in reduced stability of the NF-Y complex and decreased encystation efficiency. We demonstrated that the NF-Y complex functions at a time point subsequent to the NAD+ flux and expression of the transcription factor encystation regulatory motif-binding protein, both of which are early regulators of Entamoeba development. Taken together, our results demonstrate that the NF-Y complex plays an important role in regulating encystation in Entamoeba and add to our understanding of the transcriptional networks and signals that control this essential developmental pathway in an important human pathogen.IMPORTANCE The human parasite Entamoeba histolytica is an important pathogen with significant global impact and is a leading cause of parasitic death in humans. Since only the cyst form can be transmitted, blocking encystation would prevent new infections, making the encystation pathway an attractive target for the development of new drugs. Identification of the genetic signals and transcriptional regulatory networks that control encystation would be an important advance in understanding the developmental cascade. We show that the Entamoeba NF-Y complex plays a crucial role in regulating the encystation process in Entamoeba.

  View details for DOI 10.1128/mBio.00737-19

  View details for PubMedID 31213550

• An NAD+-dependent novel transcription factor controls stage conversion in Entamoeba. eLife Manna, D., Lentz, C. S., Ehrenkaufer, G. M., Suresh, S., Bhat, A., Singh, U. 2018; 7

  Abstract

  Developmental switching between life-cycle stages is a common feature among parasitic pathogens to facilitate disease transmission and pathogenesis. The protozoan parasite Entamoeba switches between invasive trophozoites and dormant cysts, but the encystation process remains poorly understood despite being central to amoebic biology. We identify a transcription factor, Encystation Regulatory Motif-Binding Protein (ERM-BP), that regulates encystation. Down-regulation of ERM-BP decreases encystation efficiency resulting in abnormal cysts with defective cyst walls. We demonstrate that direct binding of NAD+ to ERM-BP affects ERM-BP conformation and facilitates its binding to promoter DNA. Additionally, cellular NAD+ levels increase during encystation and exogenous NAD+ enhances encystation consistent with the role of carbon source depletion in triggering Entamoeba encystation. Furthermore, ERM-BP catalyzes conversion of nicotinamide to nicotinic acid, which might have second messenger effects on stage conversion. Our findings link the metabolic cofactors nicotinamide and NAD+ to transcriptional regulation via ERM-BP and provide the first mechanistic insights into Entamoeba encystation.

  View details for PubMedID 30375973

• Supporting Research Career Development of Physician-Scientists JOURNAL OF INFECTIOUS DISEASES Singh, U. 2018; 218: S36–S39

  View details for PubMedID 30124974

• High-Throughput Screening of Entamoeba Identifies Compounds Which Target Both Life Cycle Stages and Which Are Effective Against Metronidazole Resistant Parasites FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY Ehrenkaufer, G. M., Suresh, S., Solow-Cordero, D., Singh, U. 2018; 8

  View details for DOI 10.3389/fcimb.2018.00276

  View details for Web of Science ID 000441933600001

• High-Throughput Screening of Entamoeba Identifies Compounds Which Target Both Life Cycle Stages and Which Are Effective Against Metronidazole Resistant Parasites. Frontiers in cellular and infection microbiology Ehrenkaufer, G. M., Suresh, S., Solow-Cordero, D., Singh, U. 2018; 8: 276

  Abstract

  Neglected tropical diseases, especially those caused by parasites, are significantly underserved by current drug development efforts, mostly due to the high costs and low economic returns. One method for lowering the costs of drug discovery and development for these diseases is to repurpose drugs developed for other indications. Here, we present the results of a screen of five repurposed drug libraries to identify potential new lead compounds to treat amebiasis, a disease that affects tens of millions of people and causes ~100,000 deaths annually. E. histolytica, the causative agent of amebiasis, has two major life cycle stages, the trophozoite and the cyst. The current primary treatment for amebiasis, nitroimidazole compounds, do not eliminate parasites from the colonic lumen, necessitating a multi-drug treatment regimen. We aimed to address this problem by screening against both life stages, with the aim of identifying a single drug that targets both. We successfully identified eleven compounds with activity against both cysts and trophozoites, as well as multiple compounds that killed trophozoites with improved efficacy over existing drugs. Two lead compounds (anisomycin and prodigiosin) were further characterized for activity against metronidazole (MNZ) resistant parasites and mature cysts. Anisomycin and prodigiosin were both able to kill MNZ resistant parasites while prodigiosin and its analog obatoclax were active against mature cysts. This work confirms the feasibility of identifying drugs that target both Entamoeba trophozoites and cysts, and is an important step toward developing improved treatment regimens for Entamoeba infection.

  View details for DOI 10.3389/fcimb.2018.00276

  View details for PubMedID 30175074

  View details for PubMedCentralID PMC6107840

• Policy Recommendations for Optimizing the Infectious Diseases Physician-Scientist Workforce. The Journal of infectious diseases Singh, U., Levy, J., Armstrong, W., Bedimo, R., Creech, C. B., Lautenbach, E., Popovich, K. J., Snowden, J., Vyas, J. M., Infectious Diseases Society of America, H. M. 2018; 218 (suppl_1): S49–S54

  Abstract

  The Infectious Diseases Society of America, HIV Medicine Association, and Pediatric Infectious Diseases Society are concerned by the continued decline in the number of infectious diseases trainees pursuing careers as physician-scientists and the attrition of junior and midcareer physician-scientists. The inability to replace the aging physician-scientist workforce will have a negative, long-lasting impact our biomedical research enterprise and its ability to drive the discovery of new treatments for important infectious diseases. We discuss policy recommendations for securing and optimizing the infectious diseases physician-scientist workforce in the areas of education, training, compensation, and mentorship, as well as ways to improve federal research funding, cross-sector collaboration, and workforce diversity.

  View details for PubMedID 30124981

• Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens. Infection and immunity Suresh, S., Ehrenkaufer, G., Zhang, H., Singh, U. 2016; 84 (4): 964-975

  Abstract

  Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission.Entamoeba invadensis the model system to studyEntamoebadevelopmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools inE. invadenshas limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway inEntamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence twoE. invadensgenes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis ofEntamoebastage conversion is now possible, representing an important technical advance for the system.

  View details for DOI 10.1128/IAI.01161-15

  View details for PubMedID 26787723

  View details for PubMedCentralID PMC4807475

• Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica INTERNATIONAL JOURNAL FOR PARASITOLOGY Khalil, M. I., Foda, B. M., Suresh, S., Singh, U. 2016; 46 (3): 205-212

  Abstract

  Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community.

  View details for DOI 10.1016/j.ijpara.2015.11.004

  View details for Web of Science ID 000371951900006

  View details for PubMedID 26747561

  View details for PubMedCentralID PMC4767557

• biology: RNA interference, drug discovery, and gut microbiome. F1000Research Morgado, P., Manna, D., Singh, U. 2016; 5: 2578-?

  Abstract

  In recent years, substantial progress has been made in understanding the molecular and cell biology of the human parasite Entamoeba histolytica, an important pathogen with significant global impact. This review outlines some recent advances in the Entamoeba field in the last five years, focusing on areas that have not recently been discussed in detail: (i) molecular mechanisms regulating parasite gene expression, (ii) new efforts at drug discovery using high-throughput drug screens, and (iii) the effect of gut microbiota on amoebiasis.

  View details for PubMedID 27853522

• Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway JOURNAL OF BIOLOGICAL CHEMISTRY Foda, B. M., Singh, U. 2015; 290 (34): 21114-21130

  Abstract

  RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica.

  View details for DOI 10.1074/jbc.M115.647263

  View details for Web of Science ID 000360011800043

  View details for PubMedCentralID PMC4543668

• Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway. The Journal of biological chemistry Foda, B. M., Singh, U. 2015; 290 (34): 21114-30

  Abstract

  RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica.

  View details for DOI 10.1074/jbc.M115.647263

  View details for PubMedID 26149683

  View details for PubMedCentralID PMC4543668

• High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress PLOS ONE Zhang, H., Ehrenkaufer, G. M., Manna, D., Hall, N., Singh, U. 2015; 10 (8)

  Abstract

  The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

  View details for DOI 10.1371/journal.pone.0134481

  View details for Web of Science ID 000359062300039

  View details for PubMedID 26248204

• A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System PLOS ONE Pompey, J. M., Foda, B., Singh, U. 2015; 10 (7)

  Abstract

  Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

  View details for DOI 10.1371/journal.pone.0133740

  View details for Web of Science ID 000358838400054

  View details for PubMedID 26230096

  View details for PubMedCentralID PMC4521922

• Entamoeba histolytica rhomboid protease 1 has a role in migration and motility as validated by two independent genetic approaches EXPERIMENTAL PARASITOLOGY Rastew, E., Morf, L., Singh, U. 2015; 154: 33-42

  Abstract

  Rhomboid proteins represent a recently discovered family of intramembrane proteases present in a broad range of organisms and with increasing links to human diseases. The enteric parasite Entamoeba histolytica has evolved multiple mechanisms to adapt to the human host environment and establish infection. Our recent studies identified EhROM1 as a functional E. histolytica rhomboid protease with roles in adhesion to and phagocytosis of host cells. Since those studies were performed in a non-virulent strain, roles in parasite virulence could not be assessed. We focused this study on the comparison and validation of two genetic manipulation techniques: overexpression of a dominant-negative catalytic mutant of EhROM1 and knock down of EhROM1 using a RNAi-based silencing approach followed by functional studies of phenotypic analyses in virulent parasites. Both the EhROM1 catalytic mutant and parasites with EhROM1 downregulation were reduced in cytotoxicity, hemolytic activity, and directional and non-directional transwell migration. Importantly, the role for EhROM1 in cell migration mimics similar roles for rhomboid proteases from mammalian and apicomplexan systems. However, the EhROM1 catalytic mutant and EhROM1 downregulation parasites had different phenotypes for erythrophagocytosis, while complement resistance was not affected in either strain. In summary, in this study we genetically manipulated E. histolytica rhomboid protease EhROM1 by two different approaches and identified similarly attenuated phenotypes by both approaches, suggesting a novel role for EhROM1 in amebic motility.

  View details for DOI 10.1016/j.exppara.2015.04.004

  View details for Web of Science ID 000355640500006

  View details for PubMedID 25889553

  View details for PubMedCentralID PMC4444385

• High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress. PloS one Zhang, H., Ehrenkaufer, G. M., Manna, D., Hall, N., Singh, U. 2015; 10 (8)

  Abstract

  The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

  View details for DOI 10.1371/journal.pone.0134481

  View details for PubMedID 26248204

• Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns INTERNATIONAL JOURNAL FOR PARASITOLOGY Manna, D., Ehrenkaufer, G. M., Singh, U. 2014; 44 (11): 837-845

  Abstract

  Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points. In this study RNA-Seq data was utilised to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5' and 3' untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3'-U-Rich Motif (Ei3'-URM) (TTTGTT) in the 5' and 3' flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analysed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E. invadens and allowing further dissection of factors controlling Entamoeba developmental biology.

  View details for DOI 10.1016/j.ijpara.2014.06.008

  View details for Web of Science ID 000343361100009

• Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns. International journal for parasitology Manna, D., Ehrenkaufer, G. M., Singh, U. 2014; 44 (11): 837-845

  Abstract

  Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points. In this study RNA-Seq data was utilised to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5' and 3' untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3'-U-Rich Motif (Ei3'-URM) (TTTGTT) in the 5' and 3' flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analysed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E. invadens and allowing further dissection of factors controlling Entamoeba developmental biology.

  View details for DOI 10.1016/j.ijpara.2014.06.008

  View details for PubMedID 25075445

• RNAi Pathway Genes Are Resistant to Small RNA Mediated Gene Silencing in the Protozoan Parasite Entamoeba histolytica PLOS ONE Pompey, J. M., Morf, L., Singh, U. 2014; 9 (9)

  Abstract

  The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.

  View details for DOI 10.1371/journal.pone.0106477

  View details for Web of Science ID 000341304700029

  View details for PubMedCentralID PMC4157801

• Destabilization domain approach adapted for regulated protein expression in the protozoan parasite Entamoeba histolytica. International journal for parasitology Liu, Y., Singh, U. 2014; 44 (10): 729-735

  Abstract

  A plethora of information has been gained by sequencing the genome of the human parasite Entamoeba histolytica, however a lack of robust genetic tools hampers experimental elucidation of gene functions. We adapted the destabilization domain approach for modulation of protein levels in E. histolytica using the destabilization domains of FK506 binding protein (ddFKBP) and dihydrofolate reductase (ddDHFR), respectively. In our studies, the ddFKBP appears to be more tightly regulated than ddDHFR, with minimal detectable protein in trophozoites in the absence of the stabilizing compound. The on- and off-rate kinetics for ddFKBP were rapid, with stabilization and degradation within 3h of addition or removal of stabilizing compound, respectively. The kinetics for ddDHFR was different, with rapid stabilization (within 3h of stabilizing compound being added) but much slower degradation (protein not destabilized until 24h after compound removal). Furthermore, we demonstrated that for the ddFKBP, the standard stabilizing compound Shield-1 could be effectively replaced by two cheaper alternatives (rapamycin and FK506), indicating that the more cost-effective alternatives are viable options for use with E. histolytica. Thus, the destabilization domain approach represents a powerful method to study protein functions in E. histolytica and adds to the catalog of genetic tools that could be used to study this important human pathogen.

  View details for DOI 10.1016/j.ijpara.2014.05.002

  View details for PubMedID 24929134

  View details for PubMedCentralID PMC4138259

• RNAi pathway genes are resistant to small RNA mediated gene silencing in the protozoan parasite Entamoeba histolytica. PloS one Pompey, J. M., Morf, L., Singh, U. 2014; 9 (9)

  Abstract

  The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.

  View details for DOI 10.1371/journal.pone.0106477

  View details for PubMedID 25198343

• Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica. Nucleic acids research Morf, L., Pearson, R. J., Wang, A. S., Singh, U. 2013; 41 (20): 9424-9437

  Abstract

  RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5'-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a 'trigger' and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5'-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects.

  View details for DOI 10.1093/nar/gkt717

  View details for PubMedID 23935116

  View details for PubMedCentralID PMC3814356

• The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation. Genome biology Ehrenkaufer, G. M., Weedall, G. D., Williams, D., Lorenzi, H. A., Caler, E., Hall, N., Singh, U. 2013; 14 (7): R77

  Abstract

  Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion.We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains.Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens.

  View details for DOI 10.1186/gb-2013-14-7-r77

  View details for PubMedID 23889909

• Regulation of H2O2 Stress-responsive Genes through a Novel Transcription Factor in the Protozoan Pathogen Entamoeba histolytica JOURNAL OF BIOLOGICAL CHEMISTRY Pearson, R. J., Morf, L., Singh, U. 2013; 288 (6): 4462-4474

  Abstract

  Outcome of infection depends upon complex interactions between the invading pathogen and the host. As part of the host's innate immune response, the release of reactive oxygen and nitrogen species by phagocytes represents a major obstacle to the establishment of infection. The ability of the human parasite Entamoeba histolytica to survive reactive oxygen and nitrogen species is central to its pathogenic potential and contributes to disease outcome. In order to define the transcriptional network associated with oxidative stress, we utilized the MEME and MAST programs to analyze the promoter regions of 57 amoebic genes that had increased expression specifically in response to H(2)O(2) exposure. We functionally characterized an H(2)O(2)-regulatory motif (HRM) ((1)AAACCTCAATGAAGA(15)), which was enriched in these promoters and specifically bound amoebic nuclear protein(s). Assays with promoter-luciferase fusions established the importance of key residues and that the HRM motif directly impacted the ability of H(2)O(2)-responsive promoters to drive gene expression. DNA affinity chromatography and mass spectrometry identified EHI_108720 as an HRM DNA-binding protein. Overexpression and down-regulation of EHI_108720 demonstrated the specificity of EHI_108720 protein binding to the HRM, and overexpression increased basal expression from an H(2)O(2)-responsive wild-type promoter but not from its mutant counterpart. Thus, EHI_108720, or HRM-binding protein, represents a new stress-responsive transcription factor in E. histolytica that controls a transcriptional regulatory network associated with oxidative stress. Overexpression of EHI_108720 increased parasite virulence. Insight into how E. histolytica responds to oxidative stress increases our understanding of how this important human pathogen establishes invasive disease.

  View details for DOI 10.1074/jbc.M112.423467

  View details for Web of Science ID 000314845000072

  View details for PubMedID 23250742

  View details for PubMedCentralID PMC3567695

• Small RNA pyrosequencing in the protozoan parasite Entamoeba histolytica reveals strain-specific small RNAs that target virulence genes BMC GENOMICS Zhang, H., Ehrenkaufer, G. M., Hall, N., Singh, U. 2013; 14

  Abstract

  Small RNA mediated gene silencing is a well-conserved regulatory pathway. In the parasite Entamoeba histolytica an endogenous RNAi pathway exists, however, the depth and diversity of the small RNA population remains unknown.To characterize the small RNA population that associates with E. histolytica Argonaute-2 (EhAGO2-2), we immunoprecipitated small RNAs that associate with it and performed one full pyrosequencing run. Data analysis revealed new features of the 27nt small RNAs including the 5'-G predominance, distinct small RNA distribution patterns on protein coding genes, small RNAs mapping to both introns and exon-exon junctions, and small RNA targeted genes that are clustered particularly in sections of genome duplication. Characterization of genomic loci to which both sense and antisense small RNAs mapped showed that both sets of small RNAs have 5'-polyphosphate termini; strand-specific RT-PCR detected transcripts in both directions at these loci suggesting that both transcripts may serve as template for small RNA generation. In order to determine whether small RNA abundance patterns account for strain-specific gene expression profiles of E. histolytica virulent and non-virulent strains, we sequenced small RNAs from a non-virulent strain and found that small RNAs mapped to genes in a manner consistent with their regulation of strain-specific virulence genes.We provided a full spectrum analysis for E. histolytica AGO2-2 associated 27nt small RNAs. Additionally, comparative analysis of small RNA populations from virulent and non-virulent amebic strains indicates that small RNA populations may regulate virulence genes.

  View details for DOI 10.1186/1471-2164-14-53

  View details for Web of Science ID 000316679600001

  View details for PubMedID 23347563

  View details for PubMedCentralID PMC3610107

• Distinct Distal Gut Microbiome Diversity and Composition in Healthy Children from Bangladesh and the United States PLOS ONE Lin, A., Bik, E. M., Costello, E. K., Dethlefsen, L., Haque, R., Relman, D. A., Singh, U. 2013; 8 (1)

  Abstract

  Our current understanding of the composition and stability of the human distal gut microbiota is based largely on studies of infants and adults living in developed countries. In contrast, little is known about the gut microbiota and its variation over time in older children and adolescents, especially in developing countries.We compared the diversity, composition, and temporal stability of the fecal microbiota of healthy children, ages 9 to 14 years, living in an urban slum in Bangladesh with that of children of the same age range in an upper-middle class suburban community in the United States. We analyzed >8,000 near full-length 16S rRNA gene sequences and over 845,000 pyrosequencing reads of the 16S rRNA V1-V3 region. The distal gut of Bangladeshi children harbored significantly greater bacterial diversity than that of U.S. children, including novel lineages from several bacterial phyla. Bangladeshi and U.S. children had distinct fecal bacterial community membership and structure; the microbiota of Bangladeshi children was enriched in Prevotella, Butyrivibrio, and Oscillospira and depleted in Bacteroides relative to U.S. children (although similar to Bangladeshi adults). Furthermore, community membership and structure in Bangladeshi children was significantly less stable month-to-month than U.S. children.Together, these results suggest that differing environmental or genetic factors may shape the microbiota of healthy children in the two countries. Further investigation is necessary to understand the mechanisms and factors that underlie these differences, and to incorporate these findings into new strategies for the prevention and treatment of childhood and adolescent diseases.

  View details for DOI 10.1371/journal.pone.0053838

  View details for Web of Science ID 000314019100034

  View details for PubMedID 23349750

  View details for PubMedCentralID PMC3551965

• Distinct distal gut microbiome diversity and composition in healthy children from Bangladesh and the United States. PloS one Lin, A., Bik, E. M., Costello, E. K., Dethlefsen, L., Haque, R., Relman, D. A., Singh, U. 2013; 8 (1)

  Abstract

  Our current understanding of the composition and stability of the human distal gut microbiota is based largely on studies of infants and adults living in developed countries. In contrast, little is known about the gut microbiota and its variation over time in older children and adolescents, especially in developing countries.We compared the diversity, composition, and temporal stability of the fecal microbiota of healthy children, ages 9 to 14 years, living in an urban slum in Bangladesh with that of children of the same age range in an upper-middle class suburban community in the United States. We analyzed >8,000 near full-length 16S rRNA gene sequences and over 845,000 pyrosequencing reads of the 16S rRNA V1-V3 region. The distal gut of Bangladeshi children harbored significantly greater bacterial diversity than that of U.S. children, including novel lineages from several bacterial phyla. Bangladeshi and U.S. children had distinct fecal bacterial community membership and structure; the microbiota of Bangladeshi children was enriched in Prevotella, Butyrivibrio, and Oscillospira and depleted in Bacteroides relative to U.S. children (although similar to Bangladeshi adults). Furthermore, community membership and structure in Bangladeshi children was significantly less stable month-to-month than U.S. children.Together, these results suggest that differing environmental or genetic factors may shape the microbiota of healthy children in the two countries. Further investigation is necessary to understand the mechanisms and factors that underlie these differences, and to incorporate these findings into new strategies for the prevention and treatment of childhood and adolescent diseases.

  View details for DOI 10.1371/journal.pone.0053838

  View details for PubMedID 23349750

  View details for PubMedCentralID PMC3551965

• The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation GENOME BIOLOGY Ehrenkaufer, G. M., Weedall, G. D., Williams, D., Lorenzi, H. A., Caler, E., Hall, N., Singh, U. 2013; 14 (7)

  Abstract

  Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion.We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains.Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens.

  View details for DOI 10.1186/gb-2013-14-7-r77

  View details for Web of Science ID 000328194900009

• Oxidative stress resistance genes contribute to the pathogenic potential of the anaerobic protozoan parasite, Entamoeba histolytica INTERNATIONAL JOURNAL FOR PARASITOLOGY Rastew, E., Vicente, J. B., Singh, U. 2012; 42 (11): 1007-1015

  Abstract

  The protozoan parasite, Entamoeba histolytica, invades the host colon causing significant tissue destruction and inflammation. Upon host infection, the parasite is confronted with reactive oxygen and nitrogen species (ROS/RNS) that cause large-scale changes in gene expression profiles, which likely support the parasite's adaptation to the host environment. We have previously identified oxidative and nitrosative stress responsive genes using whole-genome expression profiling. Functional studies on two such genes are now reported and demonstrate that they have roles in parasite virulence. EHI_056680 encodes a small hypothetical protein named E. histolytica stress-induced adhesion factor (EhSIAF); EHI_188210 encodes a putative phospholipid transporting P-type ATPase/flippase (EhPTPA). Over-expression of each protein in E. histolytica trophozoites enhanced parasite survival in response to oxidative stress. Exposure to oxidative and nitrosative stress did not affect the localization of EhSIAF or EhPTPA but markedly increased EhPTPA protein levels. Interestingly, over-expression of each gene resulted in parasites with increased adherence to healthy mammalian cells, but increased adherence to apoptotic cells was noted only in EhSIAF over-expressing parasites. However, despite having increased adherence to both healthy and apoptotic host cells, EhSIAF-over-expressing parasites were reduced in their ability to destroy mammalian cell monolayers, raising the intriguing possibility that EhSIAF over-expression caused signaling defects or resulted in a dominant negative phenotype. Over-expression of EhSIAF and EhPTPA also resulted in decreased motility in a transwell motility assay. Thus, we have confirmed that two genes that are upregulated by ROS confer increased resistance to oxidative stress and have identified an unexpected role of EhSIAF and EhPTPA in host cell adherence and a role of EhSIAF in parasite virulence. Our data imply that stress response genes may play multi-factorial roles in amoebic pathogenesis.

  View details for DOI 10.1016/j.ijpara.2012.08.006

  View details for Web of Science ID 000311331200006

  View details for PubMedID 23009748

  View details for PubMedCentralID PMC3483436

• A Detoxifying Oxygen Reductase in the Anaerobic Protozoan Entamoeba histolytica EUKARYOTIC CELL Vicente, J. B., Vy Tran, V., Pinto, L., Teixeira, M., Singh, U. 2012; 11 (9): 1112-1118

  Abstract

  We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ~5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.

  View details for DOI 10.1128/EC.00149-12

  View details for Web of Science ID 000308446200004

  View details for PubMedID 22798391

  View details for PubMedCentralID PMC3445978

• Entamoeba histolytica: a snapshot of current research and methods for genetic analysis CURRENT OPINION IN MICROBIOLOGY Morf, L., Singh, U. 2012; 15 (4): 469-475

  Abstract

  Entamoeba histolytica represents one of the leading causes of parasitic death worldwide. Although identified as the causative agent of amebiasis since 1875, the molecular mechanisms by which the parasite causes disease are still not fully understood. Studying Entamoeba reveals insights into a eukaryotic cell that differs in many ways from better-studied model organisms. Thus, much can be learned from this protozoan parasite on evolution, cell biology, and RNA biology. In this review we discuss selected research highlights in Entamoeba research and focus on the development of molecular biological techniques to study this pathogen. We end by highlighting some of the many questions that remain to be answered in order to fully understand this important human pathogen.

  View details for DOI 10.1016/j.mib.2012.04.011

  View details for Web of Science ID 000308622300012

  View details for PubMedID 22664276

  View details for PubMedCentralID PMC3424301

• Transient and stable transfection in the protozoan parasite Entamoeba invadens MOLECULAR AND BIOCHEMICAL PARASITOLOGY Ehrenkaufer, G. M., Singh, U. 2012; 184 (1): 59-62

  Abstract

  Entamoeba histolytica is an important human pathogen and a major health problem worldwide. Many aspects of parasite biology can be studied with the exception of stage conversion, which cannot be reproduced adequately in E. histolytica. The reptile parasite Entamoeba invadens is a vital model system for studying stage conversion since it can be induced to undergo both encystation and excystation with high efficiency in vitro. However, functional studies using E. invadens have been limited by the lack of genetic tools in this species. Here, we report a new method for both transient and stable transfection of E. invadens. These new tools will greatly enhance research into Entamoeba development.

  View details for DOI 10.1016/j.molbiopara.2012.04.007

  View details for Web of Science ID 000305112200011

  View details for PubMedID 22561071

  View details for PubMedCentralID PMC3358517

• Nucleus-localized Antisense Small RNAs with 5 '-Polyphosphate Termini Regulate Long Term Transcriptional Gene Silencing in Entamoeba histolytica G3 Strain JOURNAL OF BIOLOGICAL CHEMISTRY Zhang, H., Alramini, H., Vy Tran, V., Singh, U. 2011; 286 (52): 44467-44479

  Abstract

  In the deep-branching eukaryotic parasite Entamoeba histolytica, transcriptional gene silencing (TGS) of the Amoebapore A gene (ap-a) in the G3 strain has been reported with subsequent development of this parasite strain for gene silencing. However, the mechanisms underlying this gene silencing approach are poorly understood. Here we report that antisense small RNAs (sRNAs) specific to the silenced ap-a gene can be identified in G3 parasites. Furthermore, when additional genes are silenced in the G3 strain, antisense sRNAs to the newly silenced genes can also be detected. Characterization of these sRNAs demonstrates that they are ~27 nucleotides in size, have 5'-polyphosphate termini, and persist even after removal of the silencing plasmid. Immunofluorescence analysis (IFA) and fluorescence in situ hybridization (FISH) show that both the Argonaute protein EhAGO2-2 and antisense sRNAs to the silenced genes are localized to the parasite nucleus. Furthermore, α-EhAGO2-2 immunoprecipitation confirmed the direct association of the antisense sRNAs with EhAGO2-2. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that the loci of the silenced genes are enriched for histone H3 and EhAGO2-2, indicating that both chromatin modification and the RNA-induced transcriptional silencing complex are involved in permanent gene silencing in G3 parasites. In conclusion, our data demonstrate that G3-based gene silencing in E. histolytica is mediated by an siRNA pathway, which utilizes antisense 5'-polyphosphate sRNAs. To our knowledge, this is the first study to show that 5'- polyphosphate antisense sRNAs can mediate TGS, and it is the first example of RNAi-mediated TGS in protozoan parasites.

  View details for DOI 10.1074/jbc.M111.278184

  View details for Web of Science ID 000298645500019

  View details for PubMedID 22049083

  View details for PubMedCentralID PMC3247957

• Antiparasitic Therapy MAYO CLINIC PROCEEDINGS Kappagoda, S., Singh, U., Blackburn, B. G. 2011; 86 (6): 561-583

  Abstract

  Parasitic diseases affect more than 2 billion people globally and cause substantial morbidity and mortality, particularly among the world's poorest people. This overview focuses on the treatment of the major protozoan and helminth infections in humans. Recent developments in antiparasitic therapy include the expansion of artemisinin-based therapies for malaria, new drugs for soil-transmitted helminths and intestinal protozoa, expansion of the indications for antiparasitic drug treatment in patients with Chagas disease, and the use of combination therapy for leishmaniasis and human African trypanosomiasis.

  View details for DOI 10.4065/mcp.2011.0203

  View details for Web of Science ID 000291288400012

  View details for PubMedID 21628620

  View details for PubMedCentralID PMC3104918

• RNA interference in Entamoeba histolytica: implications for parasite biology and gene silencing FUTURE MICROBIOLOGY Zhang, H., Pompey, J. M., Singh, U. 2011; 6 (1): 103-117

  Abstract

  Entamoeba histolytica is a major health threat to people in developing countries, where it causes invasive diarrhea and liver abscesses. The study of this important human pathogen has been hindered by a lack of tools for genetic manipulation. Recently, a number of genetic approaches based on variations of the RNAi method have been successfully developed and cloning of endogenous small-interfering RNAs from E. histolytica revealed an abundant population of small RNAs with an unusual 5´-polyphosphate structure. However, little is known about the implications of these findings to amebic biology or the mechanisms of gene silencing in this organism. In this article we review the literature relevant to RNAi in E. histolytica, discuss its implications for advances in gene silencing in this organism and outline potential future directions towards understanding the repertoire of RNAi and its impact on the biology of this deep-branching eukaryotic parasite.

  View details for DOI 10.2217/FMB.10.154

  View details for Web of Science ID 000286686500013

  View details for PubMedID 21162639

  View details for PubMedCentralID PMC3038252

• Approaches to characterizing Entamoeba histolytica transcriptional regulation CELLULAR MICROBIOLOGY Pearson, R. J., Singh, U. 2010; 12 (12): 1681-1690

  Abstract

  Entamoeba histolytica causes an estimated 100,000 deaths per year and is one of the leading causes of death among parasitic infections. Studies using E. histolytica-specific polymerase chain reaction identified that 13.8% of adults in a rural Mexican community and 11.2% of adults in central Vietnam are asymptomatically colonized. Such high incidents of asymptomatic infection suggest that only a minority of infections proceed to invasive disease. Understanding the mechanisms that underpin variable disease outcome will be critical in developing therapeutic strategies. In recent years there have been a plethora of gene expression profiling data documenting the transcriptome differences between virulent and non-virulent strains of E. histolytica as well as changes induced by external environmental changes or stimuli. While these studies have successfully identified co-regulated genes and potential virulence factors, there is still little known about the transcriptional mechanisms that induce the changes observed in this non-model organism. In this review, we have looked at how molecular technological advances have shaped our understanding of transcriptional regulation in amoeba and what we may expect from the application of powerful new techniques.

  View details for DOI 10.1111/j.1462-5822.2010.01524.x

  View details for Web of Science ID 000284318600001

  View details for PubMedID 20812994

• Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis EUKARYOTIC CELL Baxt, L. A., Rastew, E., Bracha, R., Mirelman, D., Singh, U. 2010; 9 (8): 1283-1293

  Abstract

  Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

  View details for DOI 10.1128/EC.00015-10

  View details for Web of Science ID 000280577300014

  View details for PubMedID 20581296

  View details for PubMedCentralID PMC2918930

• Entamoeba histolytica Genomic Analyses ANAEROBIC PARASITIC PROTOZOA: GENOMICS AND MOLECULAR BIOLOGY Singh, U., Ehrenkaufer, G. M., Clark, C. G., Johnson, P. J., Adam, R. D. 2010: 157–73

  View details for Web of Science ID 000277629500006

• A developmentally regulated Myb domain protein regulates expression of a subset of stage-specific genes in Entamoeba histolytica CELLULAR MICROBIOLOGY Ehrenkaufer, G. M., Hackney, J. A., Singh, U. 2009; 11 (6): 898-910

  Abstract

  Conversion between a cyst and trophozoite stage is essential to disease transmission and pathogenesis in the parasitic protist Entamoeba histolytica. A transcriptomic analysis of E. histolytica cysts and trophozoites has recently been accomplished, but the molecular basis of the regulation of encystation is not known. We have now identified a developmentally regulated Myb protein (belonging to the SHAQKY family of Myb proteins), which controls expression of a subset of amoebic stage-specific genes. Overexpression of the nuclear localized Myb protein resulted in a transcriptome that overlapped significantly with the expression profile of amoebic cysts. Analysis of promoters from genes regulated by the Myb protein identified a CCCCCC promoter motif to which amoebic nuclear protein(s) bind in a sequence-specific manner. Chromatin immunoprecipitation demonstrated that the E. histolytica Myb protein binds to promoters of genes which contain the CCCCCC motif and which are regulated by the Myb protein. This work is the first identification of a transcription factor, which regulates expression of a subset of stage-specific genes in E. histolytica. Identification of transcriptional regulatory networks that control developmental pathways will provide novel insights into the biology of this important human pathogen.

  View details for DOI 10.1111/j.1462-5822.2009.01300.x

  View details for Web of Science ID 000265883500005

  View details for PubMedID 19239479

  View details for PubMedCentralID PMC3424735

• A Pseudouridine Synthase Homologue Is Critical to Cellular Differentiation in Toxoplasma gondii EUKARYOTIC CELL Anderson, M. Z., Brewer, J., Singh, U., Boothroyd, J. C. 2009; 8 (3): 398-409

  Abstract

  Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system, and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite-to-bradyzoite differentiation (Tbd(-)) was created through insertional mutagenesis. This library contains mutants that, compared to the wild type, are between 20% and 74% as efficient at stage conversion. Two mutants, TBD5 and TBD8, with disruptions in a gene encoding a putative pseudouridine synthase, PUS1, were identified. The disruption in TBD8 is in the 5' end of the PUS1 gene and appears to produce a null allele with a 50% defect in differentiation. This is about the same switch efficiency as obtained with an engineered pus1 deletion mutant (Deltapus1). The insertion in TBD5 is within the PUS1 coding region, and this appears to result in a more extreme phenotype of only approximately 10% switch efficiency. Complementation of TBD8 with the genomic PUS1 allele restored wild-type differentiation efficiency. Infection of mice with pus1 mutant strains results in increased mortality during the acute phase and higher cyst burdens during the chronic infection, demonstrating an aberrant differentiation phenotype in vivo due to PUS1 disruption. Our results suggest a surprising and important role for RNA modification in this biological process.

  View details for DOI 10.1128/EC.00329-08

  View details for Web of Science ID 000263935200014

  View details for PubMedID 19124578

  View details for PubMedCentralID PMC2653242

• Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica BMC MICROBIOLOGY Linford, A. S., Moreno, H., Good, K. R., Zhang, H., Singh, U., Petri, W. A. 2009; 9

  Abstract

  Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica.An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%.Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.

  View details for DOI 10.1186/1471-2180-9-38

  View details for Web of Science ID 000264160800001

  View details for PubMedID 19222852

  View details for PubMedCentralID PMC2652455

• Recent insights into Entamoeba development: Identification of transcriptional networks associated with stage conversion INTERNATIONAL JOURNAL FOR PARASITOLOGY Singh, U., Ehrenkaufer, G. M. 2009; 39 (1): 41-47

  Abstract

  Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death globally. The parasite life cycle alternates between the trophozoite form, which is motile and causes invasive disease and the cyst stage, which is environmentally resistant and transmits infection. Understanding the triggers that initiate stage conversion is an important yet understudied area of investigation. Recent progress in dissecting the transcriptional networks that regulate E. histolytica development is outlined in this paper.

  View details for DOI 10.1016/j.ijpara.2008.09.004

  View details for Web of Science ID 000262735000004

  View details for PubMedID 18938171

  View details for PubMedCentralID PMC2648836

• Entamoeba histolytica modulates a complex repertoire of novel genes in response to oxidative and nitrosative stresses: implications for amebic pathogenesis CELLULAR MICROBIOLOGY Vicente, J. B., Ehrenkaufer, G. M., Saraiva, L. M., Teixeira, M., Singh, U. 2009; 11 (1): 51-69

  Abstract

  Upon host infection, the protozoan parasite Entamoeba histolytica is confronted with reactive oxygen and nitrogen species and must survive these stresses in order to cause invasive disease. We analysed the parasite's response to oxidative and nitrosative stresses, probing the transcriptional changes of trophozoites of a pathogenic strain after a 60 min exposure to H2O2 (1 mM) or a NO donor (dipropylenetriamine-NONOate, 200 microM), using whole-genome DNA microarrays. Genes encoding reactive oxygen and nitrogen species detoxification enzymes had high transcriptional levels under basal conditions and upon exposure to both stresses. On a whole-genome level, there was significant modulation of gene expression by H2O2 (286 genes regulated) and dipropylenetriamine-NONOate (1036 genes regulated) with a significant overlap of genes modulated under both conditions (164 genes). A number of transcriptionally regulated genes were in signalling/regulatory and repair/metabolic pathways. However, the majority of genes with altered transcription encode unknown proteins, suggesting as yet unraveled response pathways in E. histolytica. Trophozoites of a non-pathogenic E. histolytica strain had a significantly muted transcriptional response to H2O2 compared with the pathogenic strain, hinting that differential response to oxidative stress may be one factor that contributes to the pathogenic potential of E. histolytica.

  View details for DOI 10.1111/j.1462-5822.2008.01236.x

  View details for Web of Science ID 000262150100005

  View details for PubMedID 18778413

  View details for PubMedCentralID PMC3418052

• Small RNAs with 5 '- Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica PLOS PATHOGENS Zhang, H., Ehrenkaufer, G. M., Pompey, J. M., Hackney, J. A., Singh, U. 2008; 4 (11)

  Abstract

  Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5'-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5'-polyphosphate siRNAs extends to single-celled eukaryotic organisms.

  View details for DOI 10.1371/journal.ppat.1000219

  View details for Web of Science ID 000261481200023

  View details for PubMedID 19043551

  View details for PubMedCentralID PMC2582682

• Transcriptional Regulatory Networks in Entamoeba histolytica CURRENT DRUG TARGETS Ehrenkaufer, G. M., Singh, U. 2008; 9 (11): 931-937

  Abstract

  Expression profiling with microarray technology has revolutionized exploration of transcriptional regulatory networks on a genome-wide scale. This approach has been successfully applied to the study of Entamoeba histolytica, which causes dysentery and liver abscesses and is a leading parasitic cause of death globally. A variety of microarray platforms have been developed for this system including those generated from genomic DNA, long oligonucleotides, and short oligonucleotides. Using these tools researchers have identified parasite genes whose transcript abundance is differentially regulated during stress, host invasion, and stage conversion. Additionally, novel virulence factors have been identified by identifying genes that are highly expressed in virulent but with low expression in non-virulent Entamoeba strains. All combined, these studies have provided new data on molecular aspects of amebic biology, pathogenic potential and stage conversion and provide investigators with the first insights into potential novel drug targets against amebic disease.

  View details for Web of Science ID 000261495900003

  View details for PubMedID 18991605

• New insights into Entamoeba histolytica pathogenesis CURRENT OPINION IN INFECTIOUS DISEASES Baxt, L. A., Singh, U. 2008; 21 (5): 489-494

  Abstract

  Entamoeba histolytica is an important global pathogen and a leading cause of parasitic death worldwide. This article summarizes significant research findings over the last year.Efforts have focused primarily on identification of novel virulence determinants in E. histolytica, transcriptional profiling during tissue invasion and stage conversion, and characterization of basic cell biological processes. Additionally, new techniques for gene silencing have been identified.A comprehensive examination of the parasite lifestyle on a whole genome level has been undertaken, allowing identification of new virulence genes and signaling pathways and processes relevant to amebic biology.

  View details for DOI 10.1097/QCO.0b013e32830ce75f

  View details for Web of Science ID 000259078900007

  View details for PubMedID 18725798

  View details for PubMedCentralID PMC2688559

• An Entamoeba histolytica rhomboid protease with atypical specificity cleaves a surface lectin involved in phagocytosis and immune evasion GENES & DEVELOPMENT Baxt, L. A., Baker, R. P., Singh, U., Urban, S. 2008; 22 (12): 1636-1646

  Abstract

  Rhomboid proteases are membrane-embedded enzymes conserved in all kingdoms of life, but their cellular functions across evolution are largely unknown. Prior work has uncovered a role for rhomboid enzymes in host cell invasion by malaria and related intracellular parasites, but this is unlikely to be a widespread function, even in pathogens, since rhomboid proteases are also conserved in unrelated protozoa that maintain an extracellular existence. We examined rhomboid function in Entamoeba histolytica, an extracellular, parasitic ameba that is second only to malaria in medical burden globally. Despite its large genome, E. histolytica encodes only one rhomboid (EhROM1) with residues necessary for protease activity. EhROM1 displayed atypical substrate specificity, being able to cleave Plasmodium adhesins but not the canonical substrate Drosophila Spitz. We searched for substrates encoded in the ameba genome and found EhROM1 was able to cleave a cell surface lectin specifically. In E. histolytica trophozoites, EhROM1 changed localization to vesicles during phagocytosis and to the posterior cap structure during surface receptor shedding for immune evasion, in both cases colocalizing with lectins. Collectively these results implicate rhomboid proteases for the first time in immune evasion and suggest that a common function of rhomboid enzymes in widely divergent protozoan pathogens is to break down adhesion proteins.

  View details for DOI 10.1101/gad.1667708

  View details for Web of Science ID 000256797300009

  View details for PubMedID 18559479

  View details for PubMedCentralID PMC2428061

• Loss of dsRNA-based gene silencing in Entamoeba histolytica: Implications for approaches to genetic analysis EXPERIMENTAL PARASITOLOGY MacFarlane, R. C., Singh, U. 2008; 119 (2): 296-300

  Abstract

  The ability to regulate gene expression in the protozoan parasite Entamoeba histolytica is critical in determining gene function. We previously published that expression of dsRNA specific to E. histolytica serine threonine isoleucine rich protein (EhSTIRP) resulted in reduction of gene expression [MacFarlane, R.C., Singh, U., 2007. Identification of an Entamoeba histolytica serine, threonine, isoleucine, rich protein with roles in adhesion and cytotoxicity. Eukaryotic Cell 6, 2139-2146]. However, after approximately one year of continuous drug selection, the expression of EhSTIRP reverted to wild-type levels. We confirmed that the parasites (i) contained the appropriate dsRNA plasmid, (ii) were not contaminated with other plasmids, (iii) the drug selectable marker was functional, and (iv) sequenced the dsRNA portion of the construct. This work suggests that in E. histolytica long term cultivation of parasites expressing dsRNA can lead to the loss of dsRNA based silencing through the selection of "RNAi" negative parasites. Thus, users of the dsRNA silencing approach should proceed with caution and regularly confirm gene down regulation. The development and use of constructs for inducible expression of dsRNA may help alleviate this potential problem.

  View details for DOI 10.1016/j.exppara.2008.02.001

  View details for Web of Science ID 000256607700016

  View details for PubMedID 18346737

  View details for PubMedCentralID PMC2426738

• Identification of an Entamoeba histolytica serine-, threonine-, and isoleucine-rich protein with roles in adhesion and cytotoxicity EUKARYOTIC CELL MacFarlane, R. C., Singh, U. 2007; 6 (11): 2139-2146

  Abstract

  Entamoeba histolytica is a leading cause of parasitic death globally. However, the molecular framework regulating pathogenesis is poorly understood. We have previously used expression profiling to identify Entamoeba genes whose expressions were strictly associated with virulent strains (R. C. MacFarlane and U. Singh, Infect. Immun. 74:340-351, 2006). One gene, which we have named EhSTIRP (Entamoeba histolytica serine-, threonine-, and isoleucine-rich protein), was exclusively expressed in virulent but not in nonvirulent Entamoeba strains. EhSTIRP is predicted to be a transmembrane protein and is encoded by a multigene family. In order to characterize its function in amebic biology, we used a double-stranded RNA-based approach and were able to selectively down-regulate expression of this gene family. Upon EhSTIRP down-regulation, we were able to ascribe cytotoxic and adhesive properties to the protein family using lactate dehydrogenase release and Chinese hamster ovary cell adhesion assays. EhSTIRP thus likely represents a novel determinant of virulence in Entamoeba histolytica. This work validates the fact that genes expressed exclusively in virulent strains may represent virulence determinants and highlights the need for further functional analyses of other genes with similar expression profiles.

  View details for DOI 10.1128/EC.00174-07

  View details for Web of Science ID 000251410200021

  View details for PubMedID 17827347

  View details for PubMedCentralID PMC2168410

• Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica BMC GENOMICS Ehrenkaufer, G. M., Eichinger, D. J., Singh, U. 2007; 8

  Abstract

  Histone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome. However, the overall extent of gene regulation tied to histone acetylation is not known.In order to identify the genome-wide effects of histone acetylation in regulating E. histolytica gene expression, we used whole-genome expression profiling of parasites treated with SCFA and Trichostatin A (TSA). Despite significant changes in histone acetylation patterns, exposure of parasites to SCFA resulted in minimal transcriptional changes (11 out of 9,435 genes transcriptionally regulated). In contrast, exposure to TSA, a more specific inhibitor of histone deacetylases, significantly affected transcription of 163 genes (122 genes upregulated and 41 genes downregulated). Genes modulated by TSA were not regulated by treatment with 5-Azacytidine, an inhibitor of DNA-methyltransferase, indicating that in E. histolytica the crosstalk between DNA methylation and histone modification is not substantial. However, the set of genes regulated by TSA overlapped substantially with genes regulated during parasite development: 73/122 genes upregulated by TSA exposure were upregulated in E. histolytica cysts (p-value = 6 x 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 x 10(-7)).This work represents the first genome-wide analysis of histone acetylation and its effects on gene expression in E. histolytica. The data indicate that SCFAs, despite their ability to influence histone acetylation, have minimal effects on gene transcription in cultured parasites. In contrast, the effect of TSA on E. histolytica gene expression is more substantial and includes genes involved in the encystation pathway. These observations will allow further dissection of the effects of histone acetylation and the genetic pathways regulating stage conversion in this pathogenic parasite.

  View details for DOI 10.1186/1471-2164-8-216

  View details for Web of Science ID 000248701500001

  View details for PubMedID 17612405

  View details for PubMedCentralID PMC1940012

• Identification of developmentally regulated genes in Entamoeba histolytica: insights into mechanisms of stage conversion in a protozoan parasite CELLULAR MICROBIOLOGY Ehrenkaufer, G. M., Haque, R., Hackney, J. A., Eichinger, D. J., Singh, U. 2007; 9 (6): 1426-1444

  Abstract

  Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. The protozoan parasite Entamoeba histolytica converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Identification of genes involved in the developmental pathway has been severely hindered by the inability to generate E. histolytica cysts in vitro. Using parasite strains derived from recent human infections and whole-genome transcriptional profiling, we determined that 1439 genes (approximately 15% of annotated genes) were potentially developmentally regulated. Genes enriched in cysts (672 in total) included cysteine proteinases and transmembrane protein kinases, which may be involved in signal transduction. Genes enriched in trophozoites (767 in total) included genes typically thought of as important in tissue invasion by trophozoites, including the Gal/GalNAc lectin light subunit and cysteine protease 1. Putative regulators of differentiation including possible G-protein coupled receptors, signal transduction proteins and transcription factors were identified. A number of E. histolytica stage-specific genes were also developmentally regulated in the reptilian parasite E. invadens, indicating that they likely have conserved functions in Entamoeba development. These advances lay the groundwork for dissection of the molecular signals that initiate stage conversion and development of novel diagnostic and therapeutic measures targeting E. histolytica cysts.

  View details for DOI 10.1111/j.1462-5822.2006.00882.x

  View details for Web of Science ID 000246577400006

  View details for PubMedID 17250591

• Functional characterization of spliceosomal introns and identification of U2, U4, and U5 snRNAs in the deep-branching eukaryote Entamoeba histolytica EUKARYOTIC CELL Davis, C. A., Brown, M. P., Singh, U. 2007; 6 (6): 940-948

  Abstract

  Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5' splice site and a UAG 3' splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression.

  View details for DOI 10.1128/EC.00059-07

  View details for Web of Science ID 000247439700006

  View details for PubMedID 17468393

  View details for PubMedCentralID PMC1951529

• Identification of putative transcriptional regulatory networks in Entamoeba histolytica using Bayesian inference NUCLEIC ACIDS RESEARCH Hackney, J. A., Ehrenkaufer, G. M., Singh, U. 2007; 35 (7): 2141-2152

  Abstract

  Few transcriptional regulatory networks have been described in non-model organisms. In Entamoeba histolytica seminal aspects of pathogenesis are transcriptionally controlled, however, little is known about transcriptional regulatory networks that effect gene expression in this parasite. We used expression data from two microarray experiments, cis-regulatory motif elucidation, and a naïve Bayesian classifier to identify genome-wide transcriptional regulatory patterns in E. histolytica. Our algorithm identified promoter motifs that accurately predicted the gene expression level of 68% of genes under trophozoite conditions. We identified a promoter motif ((A)/(T)AAACCCT) associated with high gene expression, which is highly enriched in promoters of ribosomal protein genes and tRNA synthetases. Additionally, we identified three promoter motifs (GAATGATG, AACTATTTAAACAT(C)/(T)C and TGAACTTATAAACATC) associated with low gene expression. The promoters of a large gene family were highly enriched for these motifs, and in these genes the presence of >/=2 motifs predicted low baseline gene expression and transcriptional activation by heat shock. We demonstrate that amebic nuclear protein(s) bind specifically to four of the motifs identified herein. Our analysis suggests that transcriptional regulatory networks can be identified using limited expression data. Thus, this approach is applicable to the multitude of systems for which microarray and genome sequence data are emerging.

  View details for DOI 10.1093/nar/gkm028

  View details for Web of Science ID 000246294700013

  View details for PubMedID 17355990

  View details for PubMedCentralID PMC1874630

• Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression BMC GENOMICS Ali, I. K., Ehrenkaufer, G. M., Hackney, J. A., Singh, U. 2007; 8

  Abstract

  In higher eukaryotes DNA methylation regulates important biological functions including silencing of gene expression and protection from adverse effects of retrotransposons. In the protozoan parasite Entamoeba histolytica, a DNA methyltransferase has been identified and treatment with 5-azacytidine (5-AzaC), a potent inhibitor of DNA methyltransferase, has been reported to attenuate parasite virulence. However, the overall extent of DNA methylation and its subsequent effects on global gene expression in this parasite are currently unknown.In order to identify the genome-wide effects of DNA methylation in E. histolytica, we used a short oligonucleotide microarray representing 9,435 genes (approximately 95% of all annotated amebic genes) and compared the expression profile of E. histolytica HM-1:IMSS parasites with those treated with 23 microM 5-AzaC for up to one week. Overall, 2.1% of genes tested were transcriptionally modulated under these conditions. 68 genes were upregulated and 131 genes down regulated (2-fold change; p-value < 0.05). Sodium-bisulfite treatment and sequencing of genes indicated that there were at least two subsets of genes with genomic DNA methylation in E. histolytica: (i) genes that were endogenously silenced by genomic DNA methylation and for which 5-AzaC treatment induced transcriptional de-repression, and (ii) genes that have genomic DNA methylation, but which were not endogenously silenced by the methylation. We identified among the genes down regulated by 5-AzaC treatment a cysteine proteinase (2.m00545) and lysozyme (52.m00148) both of which have known roles in amebic pathogenesis. Decreased expression of these genes in the 5-AzaC treated E. histolytica may account in part for the parasites reduced cytolytic abilities.This work represents the first genome-wide analysis of DNA-methylation in Entamoeba histolytica and indicates that DNA methylation has relatively limited effects on gene expression in this parasite.

  View details for DOI 10.1186/1471-2164-8-7

  View details for PubMedID 17207281

• Structure and content of the Entamoeba histolytica genome ADVANCES IN PARASITOLOGY, VOL 65 Clark, C. G., Alsmark, U. C., Tazreiter, M., Saito-Nakano, Y., Ali, V., Marion, S., Weber, C., Mukherjee, C., Bruchhaus, I., Tannich, E., Leippe, M., Sicheritz-Ponten, T., Foster, P. G., Samuelson, J., NOEL, C. J., Hirt, R. P., Embley, T. M., Gilchrist, C. A., Mann, B. J., Singh, U., Ackers, J. P., Bhattacharya, S., Bhattacharya, A., Lohia, A., Guillen, N., Duchene, M., Nozaki, T., Hall, N. 2007; 65: 51-190

  Abstract

  The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

  View details for DOI 10.1016/S0065-308X(07)65002-7

  View details for Web of Science ID 000252405100002

  View details for PubMedID 18063096

• Impact of intestinal colonization and invasion on the Entamoeba histolytica transcriptome MOLECULAR AND BIOCHEMICAL PARASITOLOGY Gilchrist, C. A., Houpt, E., Trapaidze, N., Fei, Z., Crasta, O., Asgharpour, A., Evans, C., Martino-Catt, S., Baba, D. J., Stroup, S., Hamano, S., Ehrenkaufer, G., Okada, M., Singh, U., Nozaki, T., Mann, B. J., Petri, W. A. 2006; 147 (2): 163-176

  Abstract

  A genome-wide transcriptional analysis of Entamoeba histolytica was performed on trophozoites isolated from the colon of six infected mice and from in vitro culture. An Affymetrix platform gene expression array was designed for this analysis that included probe sets for 9435 open reading frames (ORFs) and 9066 5' and 3' flanking regions. Transcripts were detected for > 80% of all ORFs. A total of 523 transcripts (5.2% of all E. histolytica genes) were significantly changed in amebae isolated from the intestine on Days 1 and 29 after infection: 326 and 109 solely on Days 1 and 29, and 88 on both days. Quantitative real-time reverse transcriptase PCR confirmed these changes in 11/12 genes tested using mRNA isolated from an additional six mice. Adaptation to the intestinal environment was accompanied by increases in a subset of cell signaling genes including transmembrane kinases, ras and rho family GTPases, and calcium binding proteins. Significant decreases in mRNA abundance for genes involved in glycolysis and concomitant increases in lipases were consistent with a change in energy metabolism. Defense against bacteria present in the intestine (but lacking from in vitro culture) was suggested by alterations in mRNA levels of genes similar to the AIG1 plant antibacterial proteins. Decreases in oxygen detoxification pathways were observed as expected in the anaerobic colonic lumen. Of the known virulence factors the most remarkable changes were a 20-35-fold increase in a cysteine proteinase four-like gene, and a 2-3-fold decrease in two members of the Gal/GalNAc lectin light subunit family. Control of the observed changes in mRNA abundance in the intestine might potentially rest with four related proteins with DNA binding domains that were down-regulated 6-16-fold in the intestinal environment. In conclusion, the first genome-wide analysis of the transcriptome of E. histolytica demonstrated that the vast majority of genes are transcribed in trophozoites, and that in the host intestine trophozoites altered the expression of mRNAs for genes implicated in metabolism, oxygen defense, cell signaling, virulence, antibacterial activity, and DNA binding.

  View details for DOI 10.1016/j.molbiopara.2006.02.007

  View details for Web of Science ID 000237525600003

  View details for PubMedID 16569449

• Identification of differentially expressed genes in virulent and nonvirulent Entamoeba species: Potential implications for amebic pathogenesis INFECTION AND IMMUNITY MacFarlane, R. C., Singh, U. 2006; 74 (1): 340-351

  Abstract

  Entamoeba histolytica is a protozoan parasite that causes colitis and liver abscesses. Several Entamoeba species and strains with differing levels of virulence have been identified. E. histolytica HM-1:IMSS is a virulent strain, E. histolytica Rahman is a nonvirulent strain, and Entamoeba dispar is a nonvirulent species. We used an E. histolytica DNA microarray consisting of 2,110 genes to assess the transcriptional differences between these species/strains with the goal of identifying genes whose expression correlated with a virulence phenotype. We found 415 genes expressed at lower levels in E. dispar and 32 genes with lower expression in E. histolytica Rahman than in E. histolytica HM-1:IMSS. Overall, 29 genes had decreased expression in both the nonvirulent species/strains than the virulent E. histolytica HM-1:IMSS. Interestingly, a number of genes with potential roles in stress response and virulence had decreased expression in either one or both nonvirulent Entamoeba species/strains. These included genes encoding Fe hydrogenase (9.m00419), peroxiredoxin (176.m00112), type A flavoprotein (6.m00467), lysozyme (6.m00454), sphingomyelinase C (29.m00231), and a hypothetical protein with homology to both a Plasmodium sporozoite threonine-asparagine-rich protein (STARP) and a streptococcal hemagglutinin (238.m00054). The function of these genes in Entamoeba and their specific roles in parasite virulence need to be determined. We also found that a number of the non-long-terminal-repeat retrotransposons (EhLINEs and EhSINEs), which have been shown to modulate gene expression and genomic evolution, had lower expression in the nonvirulent species/strains than in E. histolytica HM-1:IMSS. Our results, identifying expression profiles and patterns indicative of a virulence phenotype, may be useful in characterizing the transcriptional framework of virulence.

  View details for DOI 10.1128/IAI.74.1.340-351.2006

  View details for Web of Science ID 000234276400037

  View details for PubMedID 16368989

  View details for PubMedCentralID PMC1346599

• Disruption of a locus encoding a nucleolar zinc finger protein decreases tachyzoite-to-bradyzoite differentiation in Toxoplasma gondii INFECTION AND IMMUNITY Vanchinathan, P., Brewer, J. L., Harb, O. S., Boothroyd, J. C., Singh, U. 2005; 73 (10): 6680-6688

  Abstract

  During its life cycle in intermediate hosts, Toxoplasma gondii exists in two interconverting developmental stages: tachyzoites and bradyzoites. This interconversion is essential for the survival and pathogenicity of the parasite, but little is known about the genetic mechanisms that control this process. We have previously generated tachyzoite-to-bradyzoite differentiation (Tbd(-)) mutants using chemical mutagenesis and a green fluorescent protein-based selection strategy. The genetic loci responsible for the Tbd(-) phenotype, however, could not be identified. We have now used an insertional mutagenesis strategy to generate two differentiation mutants: TBD-5 and TBD-6 that switch to bradyzoites at 10 and 50% of wild-type levels, respectively. In TBD-6 there is a single insertion of the mutagenesis vector 164 bp upstream of the transcription start site of a gene encoding a zinc finger protein (ZFP1). Disruption of this locus in wild-type parasites reproduces the decreased stage conversion phenotype. ZFP1 is targeted to the parasite nucleolus by CCHC motifs and significantly altered expression levels are toxic to the parasites. This represents the first identification of a gene necessary for efficient conversion of tachyzoites to bradyzoites.

  View details for DOI 10.1128/IAI.73.10.6680-6688.2005

  View details for Web of Science ID 000232087600055

  View details for PubMedID 16177345

  View details for PubMedCentralID PMC1230886

• Coding and noncoding genomic regions of Entamoeba histolytica have significantly different rates of sequence polymorphisms: Implications for epidemiological studies JOURNAL OF CLINICAL MICROBIOLOGY Bhattacharya, D., Haque, R., Singh, U. 2005; 43 (9): 4815-4819

  Abstract

  To evaluate genetic variability among Entamoeba histolytica strains, we sequenced 9,077 bp from each of 14 isolates. The polymorphism rates from coding and noncoding regions were significantly different (0.07% and 0.37%, respectively), indicating that these regions are subject to different selection pressures. Additionally, single nucleotide polymorphisms (SNPs) potentially associated with specific clinical outcomes were identified.

  View details for DOI 10.1128/JCM.43.9.4815-4819.2005

  View details for Web of Science ID 000232020400078

  View details for PubMedID 16145147

  View details for PubMedCentralID PMC1234137

• Genomic DNA microarrays for Entamoeba histolytica: Applications for use in expression profiling and strain genotyping 2nd EMBO Workshop on Pathogenesis and Amoebiasis Macfarlane, R., Bhattacharya, D., Singh, U. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2005: 196–202

  Abstract

  The parasite Entamoeba histolytica is a causative agent of dysentery and liver abscesses. Found predominantly in developing countries, this parasitic infection is responsible for significant morbidity and mortality. We have developed a genomic DNA microarray for E. histolytica. The array composed of 11,328 clones contains >2000 unique genes and was utilized for expression profiling and comparative genomic hybridizations of Entamoeba strains. We present a synopsis of our results to date and potential future applications of microarray technology for the study of Entamoeba biology.

  View details for DOI 10.1016/j.exppara.2005.03.006

  View details for Web of Science ID 000230067400007

  View details for PubMedID 15955312

• Transcriptional profiling of Entamoeba histolytica trophozoites INTERNATIONAL JOURNAL FOR PARASITOLOGY MacFarlane, R. C., Shah, P. H., Singh, U. 2005; 35 (5): 533-542

  Abstract

  We have developed an Entamoeba histolytica genomic DNA microarray and used it to develop a transcriptional profile of 1,971 E. histolytica (HM-1:IMSS) genes. The arrays accurately detected message abundance and 31-47% of amebic genes were expressed under standard tissue culture conditions (levels detectable by Northern blot analysis or RT-PCR respectively). Genes expressed at high levels ( approximately 2% of total) included actin (8.m00351), and ribosomal genes (20.m00312). Moderately expressed genes ( approximately 14% of total) included cysteine proteinase (191.m00117), profilin (156.m00098), and an Argonaute family member (11.m00378). Genes with low-level expression ( approximately 15% of total) included Ariel1 (160.m00087). Genes with very low expression ( approximately 16% of total) and those not expressed ( approximately 52% of total) included encystation-specific genes such as Jacob cyst wall glycoprotein (33.m00261), chitin synthase (3.m00544), and chitinase (22.m00311). Transcriptional modulation could be detected using the arrays with 17% of genes upregulated at least two-fold in response to heat shock. These included heat shock proteins (119.m00119 and 279.m00091), cyst wall glycoprotein Jacob (33.m00261), and ubiquitin-associated proteins (16.m00343; 195.m00092). Using Caco-2 cells to model the host-parasite interaction, we verified that host cell killing was dependent on live ameba. However, surprisingly these events did not appear to induce major transcriptional changes in the parasites.

  View details for DOI 10.1016/j.ijpara.2005.02.006

  View details for Web of Science ID 000228765900007

  View details for PubMedID 15826645

• Comparative genomic hybridizations of Entamoeba strains reveal unique genetic fingerprints that correlate with virulence EUKARYOTIC CELL Shah, P. H., MacFarlane, R. C., Bhattacharya, D., Matese, J. C., Demeter, J., Stroup, S. E., Singh, U. 2005; 4 (3): 504-515

  Abstract

  Variable phenotypes have been identified for Entamoeba species. Entamoeba histolytica is invasive and causes colitis and liver abscesses but only in approximately 10% of infected individuals; 90% remain asymptomatically colonized. Entamoeba dispar, a closely related species, is avirulent. To determine the extent of genetic diversity among Entamoeba isolates and potential genotype-phenotype correlations, we have developed an E. histolytica genomic DNA microarray and used it to genotype strains of E. histolytica and E. dispar. On the basis of the identification of divergent genetic loci, all strains had unique genetic fingerprints. Comparison of divergent genetic regions allowed us to distinguish between E. histolytica and E. dispar, identify novel genetic regions usable for strain and species typing, and identify a number of genes restricted to virulent strains. Among the four E. histolytica strains, a strain with attenuated virulence was the most divergent and phylogenetically distinct strain, raising the intriguing possibility that genetic subtypes of E. histolytica may be partially responsible for the observed variability in clinical outcomes. This microarray-based genotyping assay can readily be applied to the study of E. histolytica clinical isolates to determine genetic diversity and potential genotypic-phenotypic associations.

  View details for DOI 10.1128/EC.4.3.504-515.2005

  View details for Web of Science ID 000227781300002

  View details for PubMedID 15755913

  View details for PubMedCentralID PMC1087797

• The genome of the protist parasite Entamoeba histolytica NATURE Loftus, B., Anderson, I., Davies, R., Alsmark, U. C., Samuelson, J., Amedeo, P., Roncaglia, P., Berriman, M., Hirt, R. P., Mann, B. J., Nozaki, T., Suh, B., Pop, M., Duchene, M., Ackers, J., Tannich, E., Leippe, M., Hofer, M., Bruchhaus, I., Willhoeft, U., Bhattacharya, A., Chillingworth, T., Churcher, C., Hance, Z., Harris, B., Harris, D., Jagels, K., Moule, S., Mungall, K., Ormond, D., Squares, R., Whitehead, S., Quail, M. A., Rabbinowitsch, E., Norbertczak, H., Price, C., Wang, Z., Guillen, N., Gilchrist, C., Stroup, S. E., Bhattacharya, S., Lohia, A., Foster, P. G., Sicheritz-Ponten, T., Weber, C., Singh, U., Mukherjee, C., El-Sayed, N. M., Petri, W. A., Clark, C. G., Embley, T. M., Barrell, B., Fraser, C. M., Hall, N. 2005; 433 (7028): 865-868

  Abstract

  Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

  View details for DOI 10.1038/nature03291

  View details for Web of Science ID 000227174600042

  View details for PubMedID 15729342

• DNA content analysis on microarrays. Methods in molecular biology (Clifton, N.J.) Singh, U., Shah, P. H., MacFarlane, R. C. 2004; 270: 237-248

  Abstract

  The genome sequencing of protozoan parasites has facilitated the development of powerful postgenomics tools such as DNA microarrays and revolutionized the study of parasite biology. Large-scale genomic comparisons are useful in identifying the extent of genomic variability among related strains and isolates. Identification of deletions between geographically diverse clinical isolates is important in understanding parasite biology and the "fitness" of a given strain in dissemination. Additionally, the development of reliable diagnostic tests or identification of potential vaccine candidates is predicated on the large-scale conservation of the candidate genes. Parasites with variable virulence phenotypes (vaccine strain vs virulent strain) can also be studied for their genomic variability and provide further insights into the potential role of genotypic variability and its relationship to virulence. This chapter outlines the utilization of DNA microarrays to study genomic content.

  View details for PubMedID 15153631

• DNA microarrays in parasitology: strengths and limitations TRENDS IN PARASITOLOGY Boothroyd, J. C., Blader, I., Cleary, M., Singh, U. 2003; 19 (10): 470-476

  Abstract

  Genome sequencing efforts have provided a wealth of new biological information that promises to have a major impact on our understanding of parasites. Microarrays provide one of the major high-throughput platforms by which this information can be exploited in the laboratory. Many excellent reviews and technique articles have recently been published on applying microarrays to organisms for which fully annotated genomes are at hand. However, many parasitologists work on organisms whose genomes have been only partially sequenced and where little, if any, annotation is available. The focus of this review is on how to use and apply microarrays to these situations.

  View details for DOI 10.1016/j.pt.2003.08.002

  View details for Web of Science ID 000186058400012

  View details for PubMedID 14519585

• Investigating amoebic pathogenesis using Entamoeba histolytica DNA microarrays JOURNAL OF BIOSCIENCES Singh, U., Shah, P. 2002; 27 (6): 595-601

  View details for Web of Science ID 000179604900008

• Toxoplasma gondii asexual development: Identification of developmentally regulated genes and distinct patterns of gene expression EUKARYOTIC CELL Cleary, M. D., Singh, U., Blader, I. J., Brewer, J. L., Boothroyd, J. C. 2002; 1 (3): 329-340

  Abstract

  Asexual development in Toxoplasma gondii is a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of approximately 4,400 Toxoplasma cDNAs, representing a minimum of approximately 600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.

  View details for DOI 10.1128/EC.1.3.329-340.2002

  View details for Web of Science ID 000178729300002

  View details for PubMedID 12455982

  View details for PubMedCentralID PMC118016

• Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction MOLECULAR MICROBIOLOGY Singh, U., Brewer, J. L., Boothroyd, J. C. 2002; 44 (3): 721-733

  Abstract

  Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi.

  View details for Web of Science ID 000175466900011

  View details for PubMedID 11994153

• Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii MOLECULAR MICROBIOLOGY Matrajt, M., Donald, R. G., Singh, U., Roos, D. S. 2002; 44 (3): 735–47

  Abstract

  Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite-specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage-specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by bradyzoite induction in vitro and selection in 6-thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions.

  View details for DOI 10.1046/j.1365-2958.2002.02904.x

  View details for Web of Science ID 000175466900012

  View details for PubMedID 11994154

• Context-dependent roles of the Entamoeba histolytica core promoter element GAAC in transcriptional activation and protein complex assembly MOLECULAR AND BIOCHEMICAL PARASITOLOGY Singh, U., Gilchrist, C. A., Schaenman, J. M., Rogers, J. B., Hockensmith, J. W., Mann, B. J., Petri, W. A. 2002; 120 (1): 107-116

  Abstract

  Transcriptional control of the hgl5 gene of Entamoeba histolytica is mediated through an unusual core promoter composed of TATA, GAAC and Initiator elements. In the hgl5 promoter the GAAC element (AATGAACT) determines the site and rate of transcription initiation. Here we tested the role of the GAAC element in transcription activation from upstream regulatory elements (UREs) in the hgl5 promoter. We also examined the function of the GAAC element in the ferredoxin (fdx) promoter and characterized the protein binding to the GAAC element. Electrophoretic mobility shift assays (EMSA) demonstrated that the GAAC region is necessary for higher-order nuclear protein complex assembly. The function of the GAAC element in transcription activation mediated by UREs revealed that mutation of the GAAC element did not affect transcription activation mediated by the hgl5 URE4 but abrogated activation by the hgl5 URE3. We compared the role of the GAAC elements in the hgl5 and fdx promoters. Competitive gel shift assays were consistent with the same nuclear protein binding to the GAAC elements in both genes. Mutation of the GAAC element in the fdx gene decreased reporter gene expression, however, in contrast to hgl5 gene, had no effect on the site of transcription initiation. These results support a role for the GAAC element in assembly of nuclear proteins at the core promoter and in transcription activation mediated by URE3. The differing effect on transcription initiation in the hgl5 and fdx genes upon mutation of the GAAC element suggests a context-dependence of the GAAC-binding protein in gene expression.

  View details for Web of Science ID 000174312300010

  View details for PubMedID 11849710

• Diagnosis and management of amebiasis CLINICAL INFECTIOUS DISEASES Petri, W. A., Singh, U. 1999; 29 (5): 1117-1125

  View details for Web of Science ID 000083512600001

  View details for PubMedID 10524950

• Diagnosis and management of amebiasis. Clinical Infectious Diseases. Singh, U., Petri, Jr, WA 1999: 1117-25
• The novel core promoter element GAAC in the hgl5 gene of Entamoeba histolytica is able to direct a transcription start site independent of TATA or initiator regions JOURNAL OF BIOLOGICAL CHEMISTRY Singh, U., Rogers, J. B. 1998; 273 (34): 21663-21668

  Abstract

  Entamoeba histolytica, an enteric protozoa, is the third leading parasitic cause of death worldwide. Investigation of the transcriptional machinery of this eukaryotic pathogen has revealed an unusual core promoter structure that consists of nonconsensus TATA and initiator regions and a novel third conserved core promoter sequence, the GAAC element. Mutation of this region in the hgl5 promoter decreases reporter gene expression and alters the transcription start site. Using positional analysis of this element, we have now demonstrated that it is able to direct a new transcription start site, 2-7 bases downstream of itself, independent of TATA and Inr regions. The GAAC region was also shown to control the rate of transcription via nuclear run on analysis and an amebic nuclear protein was demonstrated to specifically interact with this sequence. This is the first description in the eukaryotic literature of a third conserved core promoter element, distinct from TATA or initiator regions, that is able to direct a transcription start site. We have formulated two models for the role of the GAAC region: (i) the GAAC-binding protein is a part of the TFIID complex and (ii) the GAAC-binding protein functions to "tether" TATA-binding protein to the TATA box.

  View details for Web of Science ID 000075492600034

  View details for PubMedID 9705300

• The novel core promoter element GAAC in the hgl5 gene of Entamoeba histolytica is able to direct a transcription start site independent of TATA or Inr regions. Journal of Biological Chemistry. Singh U, Rogers J. 1998: 21663-21668
• Transcription initiation is controlled by three core promoter elements in the hgl5 gene of the protozoan parasite Entamoeba histolytica PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Singh, U., Rogers, J. B., Mann, B. J., Petri, W. A. 1997; 94 (16): 8812-8817

  Abstract

  Entamoeba histolytica is a single cell eukaryote that is the etiologic agent of amoebic colitis. Core promoter elements of E. histolytica protein encoding genes include a TATA-like sequence (GTATTTAAAG/C) at -30, a novel element designated GAAC (GAACT) that has a variable location between TATA and the site of transcription initiation, and a putative initiator (Inr) element (AAAAATTCA) overlying the site of transcription initiation. The presence of three separate conserved sequences in a eukaryotic core promoter is unprecedented and prompted examination of their roles in regulating transcription initiation. Alterations of all three regions in the hgl5 gene decreased reporter gene activity with the greatest effect seen by mutation of the GAAC element. Positional analysis of the TATA box demonstrated that transcription initiated consistently 30-31 bases downstream of the TATA region. Mutation of either the TATA or GAAC elements resulted in the appearance of new transcription start sites upstream of +1 in the promoter of the hgl5 gene. Mutation of the Inr element resulted in no change in the site of transcription initiation; however, in the presence of a mutated TATA and GAAC regions, the Inr element controlled the site of transcription initiation. We conclude that all three elements play a role in determining the site of transcription initiation. The variable position of the GAAC element relative to the site of transcription initiation, and the multiple transcription initiations that resulted from its mutation, indicate that the GAAC element has an important and apparently novel role in transcriptional control in E. histolytica.

  View details for Web of Science ID A1997XQ12400093

  View details for PubMedID 9238060

• Three conserved cis-acting sequences in the core promoter control gene expression in the protozoan parasite Entamoeba histolytica. Archives of medical research Singh, U., Purdy, J., Mann, B. J., Petri, W. A. 1997; 28: 41-42

  View details for PubMedID 9033006