In silico identification and characterization of the SNPs in the human ASTL gene and their probable role in female infertility.

Ovastacin (ASTL), a zinc metalloprotease, is released from a fertilized egg during exocytosis of cortical granules which occurs minutes after the sperm and egg fuse. ASTL cleaves ZP2, one of the four primary glycoproteins of human zona pellucida, and this cleavage prevents polyspermy, causes zona pellucida hardening, and also protects the pre-implantation embryo. Any perturbation in the activity of ASTL can thus disturb this process and may lead to infertility without changing the gross morphology of the oocyte. A small amount of ASTL is also released by unfertilized oocytes but its catalytic activity is absent as it is bound by its inhibitor, Fetuin-B (FETUB). Pre-mature release of ASTL when FETUB is absent also causes infertility. To identify and understand the structural and functional effects of deleterious SNPs of ASTL on its interaction with ZP2 and FETUB and hence on fertility, a total of 4,748 SNPs from the dbSNP database were evaluated using a variety of in silico tools. All of the 40 shortlisted nsSNPs were present in the catalytic domain of the protein. Comparison of the wild type with mutants using MutPred2 suggests an alteration in the catalytic activity/zinc binding site in many SNPs. Docking studies show the involvement of hydrophobic interactions and H bonding between ASTL and ZP2 and also between ASTL and FETUB. Four positions in ASTL involved in the hydrophobic interactions (P105 and D200 between ASTL and ZP2; D198 and L278 between ASTL and FETUB) and 5 in H bonding (E75 and R159 between ASTL and ZP2; and K93, R159, and C281 between ASTL and FETUB) have SNP's associated with them validating their importance. Interestingly, a cluster of multiple SNPs was found in the motif 198DRD200, which is also a well-conserved region among several species. Statistical Coupling Analysis (SCA) suggested that the deleterious SNPs were present in the functionally important amino acid positions of ASTL and are evolutionarily coupled. Thus, these results attempt to identify the regions in ASTL, mutations in which can affect its binding with ZP2 or FETUB and cause female infertility.

Immune Factors Drive Expression of SARS-CoV-2 Receptor Genes Amid Sexual Disparity.

The emergence of COVID-19 has led to significant morbidity and mortality, with around seven million deaths worldwide as of February 2023. There are several risk factors such as age and sex that are associated with the development of severe symptoms due to COVID-19. There have been limited studies that have explored the role of sex differences in SARS-CoV-2 infection. As a result, there is an urgent need to identify molecular features associated with sex and COVID-19 pathogenesis to develop more effective interventions to combat the ongoing pandemic. To address this gap, we explored sex-specific molecular factors in both mouse and human datasets. The host immune targets such as TLR7, IRF7, IRF5, and IL6, which are involved in the immune response against viral infections, and the sex-specific targets such as AR and ESSR were taken to investigate any possible link with the SARS-CoV-2 host receptors ACE2 and TMPRSS2. For the mouse analysis, a single-cell RNA sequencing dataset was used, while bulk RNA-Seq datasets were used to analyze the human clinical data. Additional databases such as the Database of Transcription Start Sites (DBTS), STRING-DB, and the Swiss Regulon Portal were used for further analysis. We identified a 6-gene signature that showed differential expression in males and females. Additionally, this gene signature showed potential prognostic utility by differentiating ICU patients from non-ICU patients due to COVID-19. Our study highlights the importance of assessing sex differences in SARS-CoV-2 infection, which can assist in the optimal treatment and better vaccination strategies.

Clinical and molecular assessment of an onco-immune signature with prognostic significance in patients with colorectal cancer.

Understanding the complex tumor microenvironment is key to the development of personalized therapies for the treatment of cancer including colorectal cancer (CRC). In the past decade, significant advances in the field of immunotherapy have changed the paradigm of cancer treatment. Despite significant improvements, tumor heterogeneity and lack of appropriate classification tools for CRC have prevented accurate risk stratification and identification of a wider patient population that may potentially benefit from targeted therapies. To identify novel signatures for accurate prognostication of CRC, we quantified gene expression of 12 immune-related genes using a medium-throughput NanoString quantification platform in 93 CRC patients. Multivariate prognostic analysis identified a combined four-gene prognostic signature (TGFB1, PTK2, RORC, and SOCS1) (HR: 1.76, 95% CI: 1.05-2.95, *p < 0.02). The survival trend was captured in an independent gene expression data set: GSE17536 (177 patients; HR: 3.31, 95% CI: 1.99-5.55, *p < 0.01) and GSE14333 (226 patients; HR: 2.47, 95% CI: 1.35-4.53, *p < 0.01). Further, gene set enrichment analysis of the TCGA data set associated higher prognostic scores with epithelial-mesenchymal transition (EMT) and inflammatory pathways. Comparatively, a lower prognostic score was correlated with oxidative phosphorylation and MYC and E2F targets. Analysis of immune parameters identified infiltration of T-reg cells, CD8+ T cells, M2 macrophages, and B cells in high-risk patient groups along with upregulation of immune exhaustion genes. This molecular study has identified a novel prognostic gene signature with clinical utility in CRC. Therefore, along with prognostic features, characterization of immune cell infiltrates and immunosuppression provides actionable information that should be considered while employing personalized medicine.

The clinical relevance of gene expression based prognostic signatures in colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent cancers, with more than one million new cases every year. In the last few decades, several advancements in therapeutic and preventative levels have reduced the mortality rate, but new biomarkers are required for improved prognosis. The alterations at the genetic and epigenetic level have been recognized as major players in tumorigenesis. The products of gene expression in the form of mRNA, microRNA, and long-noncoding RNA, have started to emerge as important regulatory molecules, playing an important role in cancer. Gene-expression based prognostic risk scores, which quantify and compare their expression, have emerged as promising biomarkers with enormous clinical value. These composite multi-gene models in which more than one gene is used to predict prognosis have been shown to be significantly effective in identifying patients with multiple clinico-pathological risks like overall mortality, response to chemotherapy, risk of metastasis, etc. The advent of microarray and advanced sequencing technologies have led to the generation of large datasets like TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus), which have fueled the search for new biomarkers. Continuous evaluation of these candidate biomarkers in clinical settings is promising to improve the management of CRC. These composite gene signatures provide potential in identifying high-risk patients, which might help clinicians to better manage these patients and design appropriate personalized therapeutic interventions. In this review, we emphasize on composite prognostic scores from diverse resources with clinical utility in CRC.

Identification and Clinical Validation of a Novel 4 Gene-Signature with Prognostic Utility in Colorectal Cancer.

Colorectal cancer (CRC) is a high burden disease with several genes involved in tumor progression. The aim of the present study was to identify, generate and clinically validate a novel gene signature to improve prediction of overall survival (OS) to effectively manage colorectal cancer. We explored The Cancer Genome Atlas (TCGA), COAD and READ datasets (597 samples) from The Protein Atlas (TPA) database to extract a total of 595 candidate genes. In parallel, we identified 29 genes with perturbations in > 6 cancers which are also affected in CRC. These genes were entered in cBioportal to generate a 17 gene panel with highest perturbations. For clinical validation, this gene panel was tested on the FFPE tissues of colorectal cancer patients (88 patients) using Nanostring analysis. Using multivariate analysis, a high prognostic score (composite 4 gene signature-DPP7/2, YWHAB, MCM4 and FBXO46) was found to be a significant predictor of poor prognosis in CRC patients (HR: 3.42, 95% CI: 1.71-7.94, p < 0.001 *) along with stage (HR: 4.56, 95% CI: 1.35-19.15, p = 0.01 *). The Kaplan-Meier analysis also segregated patients on the basis of prognostic score (log-rank test, p = 0.001 *). The external validation using GEO dataset (GSE38832, 122 patients) corroborated the prognostic score (HR: 2.7, 95% CI: 1.99-3.73, p < 0.001 *). Additionally, higher score was able to differentiate stage II and III patients (130 patients) on the basis of OS (HR: 2.5, 95% CI: 1.78-3.63, p < 0.001 *). Overall, our results identify a novel 4 gene prognostic signature that has clinical utility in colorectal cancer.

NMR characterization of conformational fluctuations and noncovalent interactions of SUMO protein from Drosophila melanogaster (dSmt3).

Structural heterogeneity in the native-state ensemble of dSmt3, the only small ubiquitin-like modifier (SUMO) in Drosophila melanogaster, was investigated and compared with its human homologue SUMO1. Temperature dependence of amide proton's chemical shift was studied to identify amino acids possessing alternative structural conformations in the native state. Effect of small concentration of denaturant (1M urea) on this population was also monitored to assess the ruggedness of near-native energy landscape. Owing to presence of many such amino acids, especially in the ß2 -loop-α region, the native state of dSmt3 seems more flexible in comparison to SUMO1. Information about backbone dynamics in ns-ps timescale was quantified from the measurement of 15 N-relaxation experiments. Furthermore, the noncovalent interaction of dSmt3 and SUMO1 with Daxx12 (Daxx729 DPEEIIVLSDSD740 ), a [V/I]-X-[V/I]-[V/I]-based SUMO interaction motif, was characterized using Bio-layer Interferometery and NMR spectroscopy. Daxx12 fits itself in the groove formed by ß2 -loop-α structural region in both dSmt3 and SUMO1, but the binding is stronger with the former. Flexibility of ß2 -loop-α region in dSmt3 is suspected to assist its interaction with Daxx12. Our results highlight the role of native-state flexibility in assisting noncovalent interactions of SUMO proteins especially in organisms where a single SUMO isoform has to tackle multiple substrates single handedly.

The lectin domain of the polypeptide GalNAc transferase family of glycosyltransferases (ppGalNAc Ts) acts as a switch directing glycopeptide substrate glycosylation in an N- or C-terminal direction, further controlling mucin type O-glycosylation.

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding.

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.

Primate recombinant zona pellucida proteins expressed in Escherichia coli bind to spermatozoa.

To delineate the role of individual zona pellucida (ZP) glycoproteins during sperm-oocyte interaction, bonnet monkey (bm; Macaca radiata) ZPA (bmZPA), ZPB (bmZPB), and ZPC (bmZPC) have been cloned without native signal sequence and transmembrane-like domain, and expressed in Escherichia coli. Recombinant proteins have been purified from the inclusion bodies in presence of low concentration of chaotropic agent (2 M urea) and high pH (pH 12), and subsequently refolded in presence of oxidized and reduced glutathione. Binding of the recombinant refolded zona proteins to bonnet monkey spermatozoa in an indirect immunofluorescence assay revealed that recombinant bmZPC binds to the head region of the capacitated spermatozoa but does not bind to the acrosome reacted spermatozoa. Recombinant bmZPB binds to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. After induction of acrosome reaction by calcium ionophore A23187, the binding of recombinant bmZPB shifts to the equator, post-acrosome and midpiece of the spermatozoa. bmZPA binds to the principal segment of capacitated spermatozoa but the binding shifts to the equatorial segment, tip of the inner acrosomal membrane and midpiece in acrosome reacted spermatozoa. These studies suggest that polypeptide backbone is sufficient for the binding of ZPA, ZPB and ZPC to spermatozoa in non-human primates. Further studies with recombinant glycosylated zona proteins will help in delineating the role of carbohydrate moieties for higher affinity binding of the ligand to spermatozoa and subsequent signal transduction pathways.