Professional Education


  • Licenciado, Universidad De Buenos Aires (2007)
  • Doctor of Philosophy, Universidad De Buenos Aires (2013)

All Publications


  • Discovery of ciliary G protein-coupled receptors regulating pancreatic islet insulin and glucagon secretion. Genes & development Wu, C., Hilgendorf, K. I., Bevacqua, R. J., Hang, Y., Demeter, J., Kim, S. K., Jackson, P. K. 2021

    Abstract

    Multiple G protein-coupled receptors (GPCRs) are expressed in pancreatic islet cells, but the majority have unknown functions. We observed specific GPCRs localized to primary cilia, a prominent signaling organelle, in pancreatic alpha and beta cells. Loss of cilia disrupts beta-cell endocrine function, but the molecular drivers are unknown. Using functional expression, we identified multiple GPCRs localized to cilia in mouse and human islet alpha and beta cells, including FFAR4, PTGER4, ADRB2, KISS1R, and P2RY14. Free fatty acid receptor 4 (FFAR4) and prostaglandin E receptor 4 (PTGER4) agonists stimulate ciliary cAMP signaling and promote glucagon and insulin secretion by alpha- and beta-cell lines and by mouse and human islets. Transport of GPCRs to primary cilia requires TULP3, whose knockdown in primary human and mouse islets relocalized ciliary FFAR4 and PTGER4 and impaired regulated glucagon or insulin secretion, without affecting ciliary structure. Our findings provide index evidence that regulated hormone secretion by islet alpha and beta cells is controlled by ciliary GPCRs providing new targets for diabetes.

    View details for DOI 10.1101/gad.348261.121

    View details for PubMedID 34385262

  • SARS-CoV-2 infects human pancreatic beta cells and elicits beta cell impairment. Cell metabolism Wu, C., Lidsky, P. V., Xiao, Y., Lee, I. T., Cheng, R., Nakayama, T., Jiang, S., Demeter, J., Bevacqua, R. J., Chang, C. A., Whitener, R. L., Stalder, A. K., Zhu, B., Chen, H., Goltsev, Y., Tzankov, A., Nayak, J. V., Nolan, G. P., Matter, M. S., Andino, R., Jackson, P. K. 2021

    Abstract

    Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic beta cells can be infected by SARS-CoV-2 and cause beta cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in beta cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic beta cells in patients who succumbed to COVID-19 and selectively infects human islet beta cells invitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces beta cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic beta cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce beta cell killing.

    View details for DOI 10.1016/j.cmet.2021.05.013

    View details for PubMedID 34081912

  • Pancreatic Pseudoislets: An Organoid Archetype for Metabolism Research. Diabetes Friedlander, M. S., Nguyen, V. M., Kim, S. K., Bevacqua, R. J. 2021

    Abstract

    Pancreatic islets are vital endocrine regulators of systemic metabolism, and recent investigations have increasingly focused on understanding human islet biology. Studies of isolated human islets have advanced understanding of the development, function, and regulation of cells comprising islets, especially pancreatic alpha- and beta-cells. However, the multicellularity of the intact islet has stymied specific experimental approaches-particularly in genetics and cell signaling interrogation. This barrier has been circumvented by the observation that islet cells can survive dispersion and reaggregate to form "pseudoislets," organoids that retain crucial physiological functions, including regulated insulin and glucagon secretion. Recently, exciting advances in the use of pseudoislets for genetics, genomics, islet cell transplantation, and studies of intraislet signaling and islet cell interactions have been reported by investigators worldwide. Here we review molecular and cellular mechanisms thought to promote islet cell reaggregation, summarize methods that optimize pseudoislet development, and detail recent insights about human islet biology from genetic and transplantation-based pseudoislet experiments. Owing to robust, international programs for procuring primary human pancreata, pseudoislets should serve as both a durable paradigm for primary organoid studies and as an engine of discovery for islet biology, diabetes, and metabolism research.

    View details for DOI 10.2337/db20-1115

    View details for PubMedID 33947722

  • CRISPR-based genome editing in primary human pancreatic islet cells. Nature communications Bevacqua, R. J., Dai, X., Lam, J. Y., Gu, X., Friedlander, M. S., Tellez, K., Miguel-Escalada, I., Bonas-Guarch, S., Atla, G., Zhao, W., Kim, S. H., Dominguez, A. A., Qi, L. S., Ferrer, J., MacDonald, P. E., Kim, S. K. 2021; 12 (1): 2397

    Abstract

    Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet beta-cell regulators, like the transcription factor PDX1 and the KATP channel subunit KIR6.2, accompanied by impaired beta-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes in ABCC8, SIX2 and SIX3 expression, and impaired beta-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.

    View details for DOI 10.1038/s41467-021-22651-w

    View details for PubMedID 33893274

  • SIX2 and SIX3 coordinately regulate functional maturity and fate of human pancreatic β cells. Genes & development Bevacqua, R. J., Lam, J. Y., Peiris, H. n., Whitener, R. L., Kim, S. n., Gu, X. n., Friedlander, M. S., Kim, S. K. 2021

    Abstract

    The physiological functions of many vital tissues and organs continue to mature after birth, but the genetic mechanisms governing this postnatal maturation remain an unsolved mystery. Human pancreatic β cells produce and secrete insulin in response to physiological cues like glucose, and these hallmark functions improve in the years after birth. This coincides with expression of the transcription factors SIX2 and SIX3, whose functions in native human β cells remain unknown. Here, we show that shRNA-mediated SIX2 or SIX3 suppression in human pancreatic adult islets impairs insulin secretion. However, transcriptome studies revealed that SIX2 and SIX3 regulate distinct targets. Loss of SIX2 markedly impaired expression of genes governing β-cell insulin processing and output, glucose sensing, and electrophysiology, while SIX3 loss led to inappropriate expression of genes normally expressed in fetal β cells, adult α cells, and other non-β cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, β cells from diabetic humans with impaired insulin secretion also had reduced SIX2 transcript levels. Revealing how SIX2 and SIX3 govern functional maturation and maintain developmental fate in native human β cells should advance β-cell replacement and other therapeutic strategies for diabetes.

    View details for DOI 10.1101/gad.342378.120

    View details for PubMedID 33446570

  • Baculovirus transduction in mammalian cells is affected by the production of type I and III IFNs, which is mainly mediated by the cGAS-STING pathway. Journal of virology Amalfi, S., Molina, G. N., Bevacqua, R., Lopez, M. G., Taboga, O., Alfonso, V. 2020

    Abstract

    The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of human epithelial cells HEK293 and HEK293 T, deficient in cGAS or in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted more relevant in the production of interferon (IFN)-beta and IFN-lambda1 in response to baculovirus infection. In human epithelial cells, IFN-lambda1 production was also induced in a cGAS-independent and DNA-PK-dependent manner. Finally, we demonstrated that these cellular responses towards baculovirus infection affect the efficiency of transduction of baculovirus vectors.IMPORTANCE Baculoviruses are non-pathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.

    View details for DOI 10.1128/JVI.01555-20

    View details for PubMedID 32796076

  • Molecular and genetic regulation of pig pancreatic islet cell development. Development (Cambridge, England) Kim, S. n., Whitener, R. L., Peiris, H. n., Gu, X. n., Chang, C. A., Lam, J. Y., Camunas-Soler, J. n., Park, I. n., Bevacqua, R. J., Tellez, K. n., Quake, S. R., Lakey, J. R., Bottino, R. n., Ross, P. J., Kim, S. K. 2020

    Abstract

    Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases like diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α- and δ-cells followed by transcriptome analysis (RNA-Seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet β-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to β-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.

    View details for DOI 10.1242/dev.186213

    View details for PubMedID 32108026

  • Molecular and genetic regulation of pig pancreatic islet cell development. Development (Cambridge, England) Kim, S. n., Whitener, R. L., Peiris, H. n., Gu, X. n., Chang, C. A., Lam, J. Y., Camunas-Soler, J. n., Park, I. n., Bevacqua, R. J., Tellez, K. n., Quake, S. R., Lakey, J. R., Bottino, R. n., Ross, P. J., Kim, S. K. 2020

    Abstract

    Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases like diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α- and δ-cells followed by transcriptome analysis (RNA-Seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet β-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to β-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.

    View details for DOI 10.1242/dev.186213

    View details for PubMedID 34004773

  • CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos. Reproduction (Cambridge, England) Savy, V. n., Alberio, V. n., Canel, N. G., Ratner, L. D., Gismondi, M. I., Ferraris, S. F., Fernandez-Martín, R. n., Knott, J. G., Bevacqua, R. J., Salamone, D. F. 2020; 159 (6): 767–78

    Abstract

    CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.

    View details for DOI 10.1530/REP-19-0517

    View details for PubMedID 32240977

  • Improved embryo development using high cysteamine concentration during IVM and sperm co-culture with COCs previous to ICSI in bovine THERIOGENOLOGY Gabriela Canel, N., Suva, M., Jimena Bevacqua, R., Elena Arias, M., Felmer, R., Felipe Salamone, D. 2018; 117: 26–33

    Abstract

    In contrast to other species, intracytoplasmic sperm injection (ICSI) in bovine remains inefficient, resulting in low embryo developmental rates. It is unclear whether such inefficiency is due to the poor response of bovine ooplasms to the injection stimulus, or to the inability of bull sperm to induce oocyte activation. In order to facilitate these events, two strategies were assessed: the use of high concentration of cysteamine [Cys] during IVM; and the selection of sperm attached to cumulus cells after incubation with COCs for ICSI. First, COCs were IVM with increasing [Cys] and subjected to IVF. Zygotes from all groups were cultured under different O2 tensions and development to blastocyst was evaluated. In a second experiment, sperm were co-cultured for 3 h with COCs and acrosome reaction was studied. Afterwards, the best IVM and IVC conditions determined on Experiment 1 were used for ICSI assay. COCs were matured for 21 h with 1 (Cys 1) or 0.1 mM Cys (Cys 0.1 groups, standard condition). In addition, COCs were incubated for ≥3 h with 16 × 106 sperm/ml and only sperm attached to cumulus cells were selected for ICSI (ICSI + Co-cult groups). After chemical activation, embryos were cultured in SOF medium under low O2 tension. Cleavage and blastocyst rates were evaluated at days 2 and 7 of IVC, respectively. Finally, the relative expression of eight genes indicators of embryo quality was compared between ICSI and IVF control blastocysts by qPCR. Cleavage rates were higher for Cys 0.1 ICSI + Co-cult and Cys 1 ICSI + Co-cult groups (n = 117, 92% and n = 116, 79%, respectively) compared to their controls (n = 132, 60% for Cys 0.1 ICSI and n = 108, 52% for Cys 1 ICSI) (p ≤ 0.05). Interestingly, the combined treatment (Cys 1 ICSI + Co-cult) showed higher blastocyst rates than all other ICSI groups (23 vs. 11, 18 and 14% for Cys 0.1 ICSI + Co-cult, Cys 1 ICSI, and Cys 0.1 ICSI, respectively) (p ≤ 0.05). Moreover, incubation with COCs increased the rates of live acrosome reacted sperm (p ≤ 0.05). The relative abundance of mRNAs coding for INFτ, CAT, DNMT1, OCT4, and HDAC3 did not differ between treatments (p ≤ 0.05). SOD2, HADC1 and HADC2 expression was higher for Cys 0.1 ICSI than for IVF embryos (p ≤ 0.05). Group Cys 1 ICSI did not differ from IVF for those three genes, neither did Cys 1 ICSI + Co-cult, except for HDAC1 (p ≤ 0.05). In conclusion, the use of 1 mM Cys during IVM and of sperm incubated with mature COCs might be a good strategy to improve ICSI outcomes in cattle.

    View details for DOI 10.1016/j.theriogenology.2018.05.017

    View details for Web of Science ID 000436055900004

    View details for PubMedID 29807255