Tear miRNAs Identified in a Murine Model of Sjögren's Syndrome as Potential Diagnostic Biomarkers and Indicators of Disease Mechanism.

Objective: The tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjögren's syndrome (SS), was analyzed to identify unique miRNAs. Methods: 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. Results: 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. Conclusions: 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.

Small RNA Deep Sequencing Identifies a Unique miRNA Signature Released in Serum Exosomes in a Mouse Model of Sjögren's Syndrome.

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.

Tears - more to them than meets the eye: why tears are a good source of biomarkers in Parkinson's disease.

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.

Discovery of novel lysine ɛ-aminotransferase inhibitors: An intriguing potential target for latent tuberculosis.

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.

Gyrase ATPase domain as an antitubercular drug discovery platform: structure-based design and lead optimization of nitrothiazolyl carboxamide analogues.

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.

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].

BACKGROUND AND OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Effective adsorption of hexavalent chromium through a three center (3c) co-operative interaction with an ionic liquid and biopolymer.

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.