Discovery of novel inhibitors targeting the Mycobacterium tuberculosis O-acetylserine sulfhydrylase (CysK1) using virtual high-throughput screening

Cysteine biosynthesis in Mycobacterium tuberculosis (MTB) is crucial for this pathogen to combat oxidative stress and for long term survival in the host. Hence inhibition of this pathway is attractive for developing novel drugs against tuberculosis. In the present study, the crystal structure of the mycobacterial enzyme O-acetylserine sulfhydrylase CysK1 bound to an oligopeptide inhibitor was used as a framework for virtual screening of the BITS-Pilani in-house database to identify new scaffolds as CysK1 inhibitors. Thirty compounds were synthesized and evaluated in vitro for their ability to inhibit CysK1, activity against M. tuberculosis and cytotoxicity as steps towards the derivation of structure–activity relationships (SAR) and lead optimization. Compound 8-nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione (4n) emerged as the most promising lead with an IC₅₀ of 17.7 μM for purified CysK1 and MIC of 7.6 μM for M. tuberculosis, with little or no cytotoxicity (>50 μM).     

Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482

The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.     

Disorders related to sexuality and gender identity in the ICD‐11: revising the ICD‐10 classification based on current scientific evidence,best clinical practices,and human rights considerations

In the World Health Organization's forthcoming eleventh revision of the International Classification of Diseases and Related Health Problems (ICD‐11), substantial changes have been proposed to the ICD‐10 classification of mental and behavioural disorders related to sexuality and gender identity. These concern the following ICD‐10 disorder groupings: F52 Sexual dysfunctions, not caused by organic disorder or disease; F64 Gender identity disorders; F65 Disorders of sexual preference; and F66 Psychological and behavioural disorders associated with sexual development and orientation. Changes have been proposed based on advances in research and clinical practice, and major shifts in social attitudes and in relevant policies, laws, and human rights standards. This paper describes the main recommended changes, the rationale and evidence considered, and important differences from the DSM‐5. An integrated classification of sexual dysfunctions has been proposed for a new chapter on Conditions Related to Sexual Health, overcoming the mind/body separation that is inherent in ICD‐10. Gender identity disorders in ICD‐10 have been reconceptualized as Gender incongruence, and also proposed to be moved to the new chapter on sexual health. The proposed classification of Paraphilic disorders distinguishes between conditions that are relevant to public health and clinical psychopathology and those that merely reflect private behaviour. ICD‐10 categories related to sexual orientation have been recommended for deletion from the ICD‐11.     

Possible role of differentially expressing novel protein markers (ligatin and fibulin‐7) in human aqueous humor and trabecular meshwork tissue in glaucoma progression

The pathological mechanism underlying glaucoma has always been a complex aspect of this permanently blinding disease but proteomic studies have been helpful in elucidating it to a great extent in several studies. This study was designed to evaluate the expression and to get an idea about the function of two novel markers (ligatin and fibulin‐7) identified in human aqueous humor (hAH) in relation to glaucomatous progression. A significant increase in the protein content of glaucomatous hAH compared to that of non‐glaucomatous controls (NG‐Ctrls) was observed. Ligatin, fibulin‐7, and its proteolysis were revealed in hAH of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG) and NG‐Ctrls. Quantification confirmed no significant difference in expression of ligatin, whereas fibulin‐7 was significantly (P < 0.05) low in hAH of PACG in comparison to NG‐Ctrls and POAG. Importantly the immunohistochemical assay for both indicated their possible involvement in the maintenance of the appropriate structure of TM in vivo. Since oxidative stress is a major contributor to glaucomatous pathogenesis, in vitro analysis of nuclear and cytoplasmic fractions indicated intracellular changes in localization and expression of ligatin upon oxidative insult of human trabecular meshwork (TM) cells. While no such changes were found for fibulin‐7 expression. This was also corroborated with the immunocytochemical assay. Though a study with a small sample size, this is the first report which confirms the presence of ligatin and fibulin‐7 in hAH, quantified their differential expression, and indicated the possibility of their involvement in the maintenance of the TM structure.     

Induction of biochemical stress markers and apoptosis in transgenic Drosophila melanogaster against complex chemical mixtures: role of reactive oxygen species

The study was aimed to investigate the effect of leachates of solid waste from a flashlight battery factory and a pigment plant on 70 kDa heat shock protein (Hsp70) expression, generation of reactive oxygen species (ROS), antioxidant enzymes activities and apoptosis in Drosophila. Third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) were fed on diet mixed with leachates of solid wastes (0.05-2.0%, v/v) released from two industrial plants at three different pHs (7.00, 4.93 and 2.88) for 2-48 h. A concentration- and time-dependent significant change in Hsp70 expression, ROS generation, antioxidant enzymes activities and MDA content was observed in the exposed larvae preceding the antioxidant enzymes activities. Mitochondria-mediated, caspase-dependent apoptotic cell death in the larvae exposed to 1.0 and 2.0% leachates of flashlight battery factory was concurrent with a significant regression in Hsp70 expression and a higher ROS generation. A positive correlation drawn between ROS generation and apoptotic markers and a negative correlation between apoptotic markers and Hsp70 expression in these groups indicated the important role of ROS in the leachate-induced cellular damage. Hsp70 along with antioxidant enzymes offered protection to the organisms exposed to all the tested concentrations of the leachates of pigment plant waste and 0.5% leachate of flashlight battery factory in a cooperative manner when ROS generation was less induced. Conversely, higher levels of ROS generation in the organisms treated with 1.0 and 2.0% leachate of flashlight battery factory after 24 and 48 h resulted in regression of Hsp70 expression in them leading to cell death. The study suggests that (1) leachates of flashlight battery factory waste more adversely affected the organisms in comparison to the leachates of pigment plant waste. (2) Hsp70 may be used as a biomarker of cellular damage in organisms exposed to leachates. (3) Cell based assays using D. melanogaster as an in vivo model may provide important mechanistic information about the adverse effect of xenobiotics.     

J-domain protein, Jac1p, of yeast mitochondria required for iron homeostasis and activity of Fe-S cluster proteins

J-proteins are molecular chaperones with a characteristic domain predicted to mediate interaction with Hsp70 proteins. We have previously isolated yeast mutants of the mitochondrial Hsp70, Ssq1p, in a genetic screen for mutants with altered iron homeostasis. Here we describe the isolation of mutants of the J-domain protein, Jac1p, using the same screen. Mutant jac1 alleles predicted to encode severely truncated proteins (lacking 70 or 152 amino acids) were associated with phenotypes strikingly similar to the phenotypes of ssq1 mutants. These phenotypes include activation of the high affinity cellular iron uptake system and iron accumulation in mitochondria. In contrast to iron accumulation, Fe-S proteins of mitochondria were specifically deficient. In jac1 mutants, like in ssq1 mutants, processing of the Yfh1p precursor protein from intermediate to mature forms was delayed. In the genetic backgrounds used in this study, jac1 null mutants were found to be viable, permitting analysis of genetic interactions. The Deltajac1 Deltassq1 double mutant was more severely compromised for growth than either single mutant, suggesting a synthetic or additive effect of these mutations. Overexpression of Jac1p partially suppressed ssq1 slow growth and vice versa. Similar mitochondrial localization and similar mutant phenotypes suggest that Ssq1p and Jac1p are functional partners in iron homeostasis.