Shruti Singh Kakan

All Publications

• Serum and tear autoantibodies from NOD and NOR mice as potential diagnostic indicators of local and systemic inflammation in Sjögren's disease. Frontiers in immunology Singh Kakan, S., Abdelhamid, S., Ju, Y., MacKay, J. A., Edman, M. C., Raman, I., Zhu, C., Raj, P., Hamm-Alvarez, S. F. 2024; 15: 1516330

  Abstract

  Sjögren's Disease (SjD) is an autoimmune disease characterized by lymphocytic infiltration of salivary and lacrimal glands (LG). The LG produces the protein-rich aqueous component of tears, and SjD-associated autoimmune dacryoadenitis (AD) may thus alter tear autoantibody composition.The presence of tertiary lymphoid structures (TLS) in LG from two murine models of SjD-associated AD, male non-obese diabetic (NOD) and male non-obese insulitis resistant (NOR) mice, were evaluated using immunofluorescence. IgG and IgA reactivity in serum and tears from these models were probed in three studies against a panel of 80-120 autoantigens using autoantibody microarrays relative to serum and tears from healthy male BALB/c mice. Sources of Ig in tears were investigated using scRNA-Seq of the LG (GSE132420). Data were analyzed by R package Limma and Seurat.Analysis of immunofluorescence in LG sections from both SjD models showed TLS. Only one autoantibody was significantly elevated in tears and serum in both SjD models across all studies. Three autoantibodies were significantly elevated in serum but not in tears in both SjD models across all studies. Conversely, six IgG and thirteen IgA autoantibodies (6 sharing the same autoantigen) were significantly elevated in tears but not serum in both SjD models. Igha and Ighg2b expressing cells were identified in the plasma cell cluster of NOD.H2b LG.NOD and NOR mice with SjD-associated AD have distinct autoantibody profiles in tears and serum. Tear IgA isotype autoantibodies showed a greater diversity than tear IgG autoantibodies. TLS observed in LG are a likely source of the tear autoantibodies.

  View details for DOI 10.3389/fimmu.2024.1516330

  View details for PubMedID 39936155

  View details for PubMedCentralID PMC11810956

• Tear Fluid as a Biomarker for Parkinson's Disease: Downregulation of DNA Repair Genes/Pathways via RNA-Seq Analysis Lew, M., Omidsalar, A., Kakan, S., Gerke, D., Tanveer, M., Feigenbaum, D., Tamadonfar, E., Hamm-Alvarez, S., Hjelm, B. LIPPINCOTT WILLIAMS & WILKINS. 2023

  View details for DOI 10.1212/WNL.0000000000202879

  View details for Web of Science ID 001053672107223

• The miRNA Landscape of Lacrimal Glands in a Murine Model of Autoimmune Dacryoadenitis. Investigative ophthalmology & visual science Singh Kakan, S., Li, X., Edman, M. C., Okamoto, C. T., Hjelm, B. E., Hamm-Alvarez, S. F. 2023; 64 (4): 1

  Abstract

  To analyze the changes in the lacrimal gland (LG) miRNAome from male nonobese diabetic (NOD) mice with autoimmune dacryoadenitis compared with LG from healthy male BALB/c and dacryoadenitis-free female NOD mice.LG from these mice were collected for small RNA sequencing to identify dysregulated miRNAs; hits were validated by RT-qPCR in male NOD and BALB/c LG. Dysregulation of validated species within immune cell-enriched cell fractions and epithelial-enriched cell fractions from LG was probed by RT-qPCR. Ingenuity pathway analysis identified putative miRNA targets, which were examined in publicly available mRNA-seq datasets. Western blotting and confocal imaging of immunofluorescence enabled validation of some molecular changes at the protein level.Male NOD LG exhibited 15 and 13 significantly up- and downregulated miRNAs, respectively. Dysregulated expression of 14 of these miRNAs (9 upregulated, 5 downregulated) was validated in male NOD versus BALB/c LG by RT-qPCR. Seven of the upregulated miRNAs were increased owing to their abundance in immune cell-enriched cell fractions, whereas four downregulated miRNAs were largely expressed in epithelial-enriched cell fractions. Ingenuity pathway analysis predicted the upregulation of IL-6 and IL-6-like pathways as an outcome of miRNA dysregulation. Increased expression of several genes in these pathways was confirmed by mRNA-seq analysis, whereas immunoblotting and immunofluorescence confirmed Ingenuity pathway analysis-predicted changes for IL-6Rα and gp130/IL-6st.Male NOD mouse LG exhibit multiple dysregulated miRNAs owing to the presence of infiltrating immune cells, and decreased acinar cell content. The observed dysregulation may increase IL-6Rα and gp130/IL-6st on acini and IL-6Rα on specific lymphocytes, enhancing IL-6 and IL-6-like cytokine signaling.

  View details for DOI 10.1167/iovs.64.4.1

  View details for PubMedID 37010857

  View details for PubMedCentralID PMC10080918

• Serum and Tear Autoantibodies from nor Mice as Potential Diagnostic Indicators of Local and Systemic Inflammation in Sjogren's Syndrome Kakan, S., Ju, Y., Edman, M., Hamm-Alvarez, S. WILEY. 2022: 3992-3994

  View details for Web of Science ID 000877386506003

• Tear miRNAs Identified in a Murine Model of Sjögren's Syndrome as Potential Diagnostic Biomarkers and Indicators of Disease Mechanism. Frontiers in immunology Kakan, S. S., Edman, M. C., Yao, A., Okamoto, C. T., Nguyen, A., Hjelm, B. E., Hamm-Alvarez, S. F. 2022; 13: 833254

  Abstract

  The tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjögren's syndrome (SS), was analyzed to identify unique miRNAs.Male NOD mice, aged 12-14 weeks, were used to identify tear miRNAs associated with development of autoimmune dacryoadenitis. Age- and sex-matched male BALB/c mice served as healthy controls while age-matched female NOD mice that do not develop the autoimmune dacryoadenitis characteristic of SS were used as additional controls. Total RNA was isolated from stimulated tears pooled from 5 mice per sample and tear miRNAs were sequenced and analyzed. Putative miRNA hits were validated in additional mouse cohorts as well as in tears of SS patients versus patients with another form of dry eye disease, meibomian gland disease (MGD) using qRT-PCR. The pathways influenced by the validated hits were identified using Ingenuity Pathway Analysis.In comparison to tears from both healthy (male BALB/c) and additional control (female NOD) mice, initial analy1sis identified 7 upregulated and 7 downregulated miRNAs in male NOD mouse tears. Of these, 8 were validated by RT-qPCR in tears from additional mouse cohorts. miRNAs previously implicated in SS pathology included mmu-miR-146a/b-5p, which were significantly downregulated, as well as mmu-miR-150-5p and mmu-miR-181a-5p, which were upregulated in male NOD mouse tears. All other validated hits including the upregulated miR-181b-5p and mmu-miR-203-3p, as well as the downregulated mmu-miR-322-5p and mmu-miR-503-5p, represent novel putative indicators of autoimmune dacryoadenitis in SS. When compared to tears from patients with MGD, miRNAs hsa-miR-203a-3p, hsa-miR-181a-5p and hsa-miR-181b-5p were also significantly increased in tears of SS patients.A panel of differentially expressed miRNAs were identified in tears of male NOD mice, with some preliminary validation in SS patients, including some never previously linked to SS. These may have potential utility as indicators of ocular symptoms of SS; evaluation of the pathways influenced by these dysregulated miRNAs may also provide further insights into SS pathogenesis.

  View details for DOI 10.3389/fimmu.2022.833254

  View details for PubMedID 35309364

  View details for PubMedCentralID PMC8931289

• Identification of miRNAs in tears of a murine model of Sjogren's syndrome that may represent putative diagnostic biomarkers Kakan, S., Edman, M., Hjelm, B., Okamoto, C. T., Hamm-Alvarez, S. F. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2021

  View details for Web of Science ID 000690761600712

• Small RNA Deep Sequencing Identifies a Unique miRNA Signature Released in Serum Exosomes in a Mouse Model of Sjögren's Syndrome. Frontiers in immunology Kakan, S. S., Janga, S. R., Cooperman, B., Craig, D. W., Edman, M. C., Okamoto, C. T., Hamm-Alvarez, S. F. 2020; 11: 1475

  Abstract

  Sjögren's Syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and loss of function of moisture-producing exocrine glands as well as systemic inflammation. SS diagnosis is cumbersome, subjective and complicated by manifestation of symptoms that overlap with those of other rheumatic and ocular diseases. Definitive diagnosis averages 4-5 years and this delay may lead to irreversible tissue damage. Thus, there is an urgent need for diagnostic biomarkers for earlier detection of SS. Extracellular vesicles called exosomes carry functional small non-coding RNAs which play a critical role in maintaining cellular homeostasis via transcriptional and translational regulation of mRNA. Alterations in levels of specific exosomal miRNAs may be predictive of disease status. Here, we have assessed serum exosomal RNA using next generation sequencing in a discovery cohort of the NOD mouse, a model of early-intermediate SS, to identify dysregulated miRNAs that may be indicative of SS. We found five miRNAs upregulated in serum exosomes of NOD mice with an adjusted p < 0.05-miRNA-127-3p, miRNA-409-3p, miRNA-410-3p, miRNA-541-5p, and miRNA-540-5p. miRNAs 127-3p and 541-5p were also statistically significantly upregulated in a validation cohort of NOD mice. Pathway analysis and existing literature indicates that differential expression of these miRNAs may dysregulate pathways involved in inflammation. Future studies will apply these findings in a human cohort to understand how they are correlated with manifestations of SS as well as understanding their functional role in systemic autoimmunity specific to SS.

  View details for DOI 10.3389/fimmu.2020.01475

  View details for PubMedID 32849505

  View details for PubMedCentralID PMC7396589

• Tears - more to them than meets the eye: why tears are a good source of biomarkers in Parkinson's disease. Biomarkers in medicine Edman, M. C., Janga, S. R., Kakan, S. S., Okamoto, C. T., Freire, D., Feigenbaum, D., Lew, M., Hamm-Alvarez, S. F. 2020; 14 (2): 151-163

  Abstract

  Tears are a known source of biomarkers for both ocular and systemic diseases with particular advantages; specifically, the noninvasiveness of sample collection and a unique and increasingly better-defined protein composition. Here, we discuss our rationale for use of tears for discovery of biomarkers for Parkinson's disease (PD). These reasons include literature supporting changes in tear flow and composition in PD, and the interconnections between the ocular surface system and neurons affected in PD. We highlight recent data on the identification of tear biomarkers including oligomeric α-synuclein, associated with neuronal degeneration in PD, in tears of PD patients and discuss possible sources for its release into tears. Challenges and next steps for advancing such biomarkers to clinical usage are highlighted.

  View details for DOI 10.2217/bmm-2019-0364

  View details for PubMedID 32064896

• Berunda Polypeptides: Multi-Headed Fusion Proteins Promote Subcutaneous Administration of Rapamycin to Breast Cancer In Vivo. Theranostics Dhandhukia, J. P., Li, Z., Peddi, S., Kakan, S., Mehta, A., Tyrpak, D., Despanie, J., MacKay, J. A. 2017; 7 (16): 3856-3872

  Abstract

  Recombinant Elastin-Like Polypeptides (ELPs) serve as attractive scaffolds for nanoformulations because they can be charge-neutral, water soluble, high molecular weight, monodisperse, biodegradable, and decorated with functional proteins. We recently reported that fusion of the FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) reduces rapamycin (Rapa) toxicity and enables intravenous (IV) therapy in both a xenograft breast cancer model and a murine autoimmune disease model. Rapa has poor solubility, which leads to variable oral bioavailability or drug precipitation following parenteral administration. While IV administration is routine during chemotherapy, cytostatic molecules like Rapa would require repeat administrations in clinical settings. To optimize FKBP/Rapa for subcutaneous (SC) administration, this manuscript expands upon first-generation FSI nanoparticles (Rh ~ 25 nm) and compares them with two second-generation carriers (FA and FAF) that: i) do not self-assemble; ii) retain a hydrodynamic radius (Rh ~ 7 nm) above the renal filtration cutoff; iii) increase tumor accumulation; and iv) have either one (FA) or two (FAF) drug-binding FKBP domains per ELP protein. Methods: The carriers were compared and evaluated for temperature-concentration phase behavior by UV-Vis spectrophotometry; equilibrium binding and thermodynamics by Isothermal Titration Calorimetry; drug retention and formulation stability by Dialysis and Dynamic Light Scattering; in vitro efficacy using a cell proliferation assay; in vivo efficacy in human MDA-MB-468 orthotopic breast cancer xenografts; downstream target inhibition using western blot; tissue histopathology; and bio-distribution via optical imaging in the orthotopic xenograft mouse model. Results: Named after the two-headed bird in Hindu mythology, the 'Berunda polypeptide' FAF with molecular weight of 97 kDa and particle size, Rh ~ 7 nm demonstrated polypeptide conformation of a soluble hydrated coiled polymer, retained formulation stability for one month post Rapa loading, eliminated toxicity observed with free Rapa after SC administration, suppressed tumor growth, decreased phosphorylation of a downstream target, and increased tumor accumulation in orthotopic breast tumor xenografts. Conclusion: This comprehensive manuscript demonstrates the versatility of recombinant protein-polymers to investigate drug carrier architectures. Furthermore, their facilitation of SC administration of poorly soluble drugs, like Rapa, may enable chronic self-administration in patients.

  View details for DOI 10.7150/thno.19981

  View details for PubMedID 29109782

  View details for PubMedCentralID PMC5667409

• Discovery of novel lysine ɛ-aminotransferase inhibitors: An intriguing potential target for latent tuberculosis. Tuberculosis (Edinburgh, Scotland) Devi, P. B., Sridevi, J. P., Kakan, S. S., Saxena, S., Jeankumar, V. U., Soni, V., Anantaraju, H. S., Yogeeswari, P., Sriram, D. 2015; 95 (6): 786-794

  Abstract

  Mycobacterium tuberculosis (MTB) has remarkable ability to persist in the human host and causes latent infection in one third of the world population. Currently available tuberculosis (TB) drugs while effective in killing actively growing MTB, is largely ineffective in killing persistent or latent MTB. Lysine-ɛ aminotransferase (LAT) enzyme is reported to be highly up-regulated (41.86 times) in in vitro models of TB designed to mimic the latent stage. Hence inhibition of this MTB LAT seems attractive for developing novel drugs against latent TB. In the present study, crystal structure of the MTB LAT bound to substrate was used as a framework for structure-based design utilizing database compounds to identify novel thiazole derivative as LAT inhibitors. Thirty six compounds were synthesized and evaluated in vitro for their ability to inhibit LAT, in vitro activity against latent MTB, in vivo activity using Mycobacterium marinum infected zebra fish and cytotoxicity as steps toward the derivation of structure-activity relationship (SAR) for lead optimization. Compound 4-methoxy-2-(pyridin-4-yl)thiazole-5-carboxylic acid (24) emerged as the most promising lead with an IC50 of 1.22 ± 0.85 μM against LAT and showed 2.8 log reduction against nutrient starved MTB, with little cytotoxicity at a higher concentration (>50 μM). It also exhibited 1.5 log reduction of M. marinum load in in vivo zebra fish model at 10 mg/kg.

  View details for DOI 10.1016/j.tube.2015.04.010

  View details for PubMedID 26299907

• Extending the N-linked aminopiperidine class to the mycobacterial gyrase domain: pharmacophore mapping from known antibacterial leads. European journal of medicinal chemistry Bobesh, K. A., Renuka, J., Jeankumar, V. U., Shruti, S. K., Sridevi, J. P., Yogeeswari, P., Sriram, D. 2014; 85: 593-604

  Abstract

  Bacterial DNA gyrase is a well-established and clinically validated target to develop novel antibacterial. Our effort was designated to search for synthetically better compounds with possibility of hit to lead development. With this as objective, a series of 1-(2-(4-aminopiperidin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one derivatives were designed by molecular hybridization strategy and synthesized following nine step reaction to yield activity in low nanomolar range and commendable antibacterial activities. Compound 1-(4-fluorophenyl)-3-(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)piperidin-4-yl)urea (35) emerged as the most promising inhibitor with an IC50 of 78 nM against Mycobacterium tuberculosis DNA gyrase enzyme, with MTB MIC of 0.62 μM, and not cytotoxic at 50 μM in eukaryotic cell line.

  View details for DOI 10.1016/j.ejmech.2014.08.018

  View details for PubMedID 25127151

• Gyrase ATPase domain as an antitubercular drug discovery platform: structure-based design and lead optimization of nitrothiazolyl carboxamide analogues. ChemMedChem Jeankumar, V. U., Renuka, J., Kotagiri, S., Saxena, S., Kakan, S. S., Sridevi, J. P., Yellanki, S., Kulkarni, P., Yogeeswari, P., Sriram, D. 2014; 9 (8): 1850-9

  Abstract

  In this study, we explored the pharmaceutically underexploited mycobacterial gyrase ATPase (GyrB) domain as a template for a structure-based virtual screening of our in-house (BITS Pilani) compound collection to discover new inhibitors targeting Mycobacterium tuberculosis (M.tb.) The hit identified was further customized by using a combination of molecular docking and medicinal chemistry strategies to obtain an optimized analogue displaying considerable in vitro enzyme efficacy and bactericidal properties against the M.tb. H37 Rv strain. The binding affinity of the ligand toward the GyrB domain was reascertained by differential scanning fluorimetry experiments. Further evaluation of the hERG toxicity (a major limitation among the previously reported N-linked aminopiperidine analogues) indicated these molecules to be completely devoid of cardiotoxicity, a significant achievement within this class.

  View details for DOI 10.1002/cmdc.201402035

  View details for PubMedID 24962352

• Investigating structure-activity relationship and mechanism of action of antitubercular 1-(4-chlorophenyl)-4-(4-hydroxy-3-methoxy-5-nitrobenzylidene) pyrazolidine-3,5-dione [CD59]. International journal of mycobacteriology Samala, G., Kakan, S. S., Nallangi, R., Devi, P. B., Sridevi, J. P., Saxena, S., Yogeeswari, P., Sriram, D. 2014; 3 (2): 117-26

  Abstract

  The objective of this study is to synthesize and evaluate 1-(4-chlorophenyl)-4-(4-hydroxy-3-methoxy-5-nitrobenzylidene) pyrazolidine-3,5-dione (CD59) analogues to establish structure-activity relationship and mechanism of action.Thirty analogues of reported antitubercular CD59 were prepared by two-step synthetic protocols and characterized. The compounds were evaluated for in vitro activities against Mycobacterium tuberculosis (MTB), cytotoxicity against RAW 264.7 cells. The molecules were also evaluated for three mycobacterial enzymes to study the mechanism of action.Among the compounds, 4-(2-bromobenzylidene)-1-(4-chlorophenyl)pyrazolidine-3,5-dione (4k) was found to be the most active compound in vitro with MICs of 4.13μM against log-phase culture of MTB and also non-toxic up to 50μM.Amongst all, the compounds 4g, 3i and 3n were most active against the enzymes MTB Pantothenate synthetase, lysine amino transferase and Alanine dehydrogenase, respectively. Further screening of these molecules was required in the in vitro dormant MTB models.

  View details for DOI 10.1016/j.ijmyco.2014.02.006

  View details for PubMedID 26786333

• A novel amine impregnated graphene oxide adsorbent for the removal of hexavalent chromium CHEMICAL ENGINEERING JOURNAL Kumar, A., Kakan, S., Rajesh, N. 2013; 230: 328-337

  View details for DOI 10.1016/j.cej.2013.06.089

  View details for Web of Science ID 000324663100040

• Effective adsorption of hexavalent chromium through a three center (3c) co-operative interaction with an ionic liquid and biopolymer. Journal of hazardous materials Krishna Kumar, A. S., Gupta, T., Kakan, S. S., Kalidhasan, S., Rajesh, V., Rajesh, N. 2012; 239-240: 213-24

  Abstract

  Biopolymers as well as ionic liquids are known for their potential applications. In this work, we report the utility of chitosan as an excellent platform for impregnating the ionic liquid, tetraoctylammonium bromide by ultrasonication and its subsequent adsorption for chromium(VI). The effective mass transfer due to sonication coupled with the hydrogen bonding interaction between chitosan-ionic liquid and the electrostatic interaction involving the amino groups in chitosan and hexavalent chromium governs this three center (3c) co-operative mechanism. The adsorption followed a pseudo second order kinetics with a Langmuir adsorption capacity of 63.69 mg g(-1). Various isotherm models were used to correlate the experimental data and the adsorption process is exothermic with a decreased randomness at the solid-solution interface. The thermodynamics of the spontaneous adsorption process could be explained through a positive co-operative effect between the host (chitosan) and the guest (ionic liquid). The adsorbed chromium(VI) could be converted to ammonium chromate using ammonium hydroxide, thereby regenerating the adsorbent. The method could be translated into action in the form of practical application to a real sample containing chromium.

  View details for DOI 10.1016/j.jhazmat.2012.08.065

  View details for PubMedID 23009794