Multimerin 1 aids in the progression of ovarian cancer possibly via modulation of DNA damage response and repair pathways.

Ovarian cancer is one of the leading causes of deaths among women. Despite advances in the treatment regimes, a high rate of diagnosis in the advanced stage makes it almost an incurable malignancy. Thus, more research efforts are required to identify potential molecular markers for early detection of the disease and therapeutic targets to augment the survival rate of ovarian cancer patients. Previously, in this context, we identified dysregulated expression of multimerin 1 (MMRN1) in ovarian cancer. To elucidate the relationship between MMRN1 expression and ovarian cancer progression, siRNA-based MMRN1 knockdown was employed and various cell assays were performed to study its effect on ovarian cancer cells. In addition, network of dysregulated proteins was identified by quantitative proteomics and associated pathways were explored by bioinformatics analysis. MMRN1 silencing showed a significant reduction in cell viability, adhesion, migration, and invasion and a high frequency of cell apoptosis. Label-free quantitative proteomics and in-depth statistical analysis identified 448 dysregulated proteins, majority of which were overexpressed in MMRN1 knockdown cells. The pathways overrepresented in ovarian cancer were DNA replication, mismatch repair, nucleotide excision repair, and cell cycle regulation. Conclusively, the findings of this study suggest that MMRN1 aids in the progression of ovarian cancer via modulation of DNA damage response and repair pathways.

Unveiling the Significance of FGF8 Overexpression in Orchestrating the Progression of Ovarian Cancer.

The asymptomatic nature, high rate of disease recurrence, and resistance to platinum-based chemotherapy highlight the need to identify and characterize novel target molecules for ovarian cancer. Fibroblast growth factor 8 (FGF8) aids in the development and metastasis of ovarian cancer; however, its definite role is not clear. We employed ELISA and IHC to examine the expression of FGF8 in the saliva and tissue samples of epithelial ovarian cancer (EOC) patients and controls. Furthermore, various cell assays were conducted to determine how FGF8 silencing influences ovarian cancer cell survival, adhesion, migration, and invasion to learn more about the functions of FGF8. In saliva samples, from controls through low-grade to high-grade EOC, a stepped overexpression of FGF8 was observed. Similar expression trends were seen in tissue samples, both at protein and mRNA levels. FGF8 gene silencing in SKOV3 cells adversely affected various cell properties essential for cancer cell survival and metastasis. A substantial reduction was observed in the cell survival, cell adhesion to the extracellular matrix, migration, and adhesion properties of SKOV3 cells, suggesting that FGF8 plays a crucial role in the development of EOC. Conclusively, this study suggests a pro-metastatic function of FGF8 in EOC.

Human Spermatozoa Quantitative Proteomic Signature Classifies Normo- and Asthenozoospermia.

Scarcely understood defects lead to asthenozoospermia, which results in poor fertility outcomes. Incomplete knowledge of these defects hinders the development of new therapies and reliance on interventional therapies, such as in vitro fertilization, increases. Sperm cells, being transcriptionally and translationally silent, necessitate the proteomic approach to study the sperm function. We have performed a differential proteomics analysis of human sperm and seminal plasma and identified and quantified 667 proteins in sperm and 429 proteins in seminal plasma data set, which were used for further analysis. Statistical and mathematical analysis combined with pathway analysis and self-organizing maps clustering and correlation was performed on the data set.It was found that sperm proteomic signature combined with statistical analysis as opposed to the seminal plasma proteomic signature can differentiate the normozoospermic versus the asthenozoospermic sperm samples. This is despite the results that some of the seminal plasma proteins have big fold changes among classes but they fall short of statistical significance. S-Plot of the sperm proteomic data set generated some high confidence targets, which might be implicated in sperm motility pathways. These proteins also had the area under the curve value of 0.9 or 1 in ROC curve analysis.Various pathways were either enriched in these proteomic data sets by pathway analysis or they were searched by their constituent proteins. Some of these pathways were axoneme activation and focal adhesion assembly, glycolysis, gluconeogenesis, cellular response to stress and nucleosome assembly among others. The mass spectrometric data is available via ProteomeXchange with identifier PXD004098.

Structural insights of Mycobacterium GTPase-Obg and anti-sigma-F factor Usfx interaction.

An essential protein for bacterial growth, GTPase-Obg (Obg), is known to play an unknown but crucial role in stress response as its expression increases in Mycobacterium under stress conditions. It is well reported that Obg interacts with anti-sigma-F factor Usfx; however, a detailed analysis and structural characterization of their physical interaction remain undone. In view of above-mentioned points, this study was conceptualized for performing binding analysis and structural characterization of Obg-Usfx interaction. The binding studies were performed by surface plasmon resonance, while in silico docking analysis was done to identify crucial residues responsible for Obg-Usfx interaction. Surface plasmon resonance results clearly suggest that N-terminal and G domains of Obg mainly contribute to Usfx binding. Also, binding constants display strong affinity that was further evident by intermolecular hydrogen bonds and hydrophobic interactions in the predicted complex. Strong interaction between Obg and Usfx supports the view that Obg plays an important role in stress response, essentially required for Mycobacterium survival. As concluded by various studies that Obg is crucial for Mycobacterium survival under stress, this structural information may help us in designing novel and potential inhibitors against resistant Mycobacterium strains.

N-Glycoproteomics of Human Seminal Plasma Glycoproteins.

Seminal plasma aids sperm by inhibiting premature capacitation, helping in the intracervical transport and formation of an oviductal sperm reservoir, all of which appear to be important in the fertilization process. Epitopes such as Lewis x and y are known to be present on seminal plasma glycoproteins, which can modulate the maternal immune response. It is suggested by multiple studies that seminal plasma glycoproteins play, largely undiscovered, important roles in the process of fertilization. We have devised a strategy to analyze glycopeptides from a complex, unknown mixture of protease-digested proteins. This analysis provides identification of the glycoproteins, glycosylation sites, glycan compositions, and proposed structures from the original sample. This strategy has been applied to human seminal plasma total glycoproteins. We have elucidated glycan compositions and proposed structures for 243 glycopeptides belonging to 73 N-glycosylation sites on 50 glycoproteins. The majority of the proposed glycan structures were complex type (83%) followed by high-mannose (10%) and then hybrid (7%). Most of the glycoproteins were either sialylated, fucosylated, or both. Many Lewis x/a and y/b epitopes bearing glycans were found, suggesting immune-modulating epitopes on multiple seminal plasma glycoproteins. The study also shows that large scale N-glycosylation mapping is achievable with current techniques and the depth of the analysis is roughly proportional to the prefractionation and complexity of the sample.

Quantitative Proteomics of COVID-19 Recovered Patients Identifies Long-Term Changes in Sperm Proteins Leading to Cellular Stress in Spermatozoa.

Following an initial recovery, COVID-19 survivors struggle with a spectrum of persistent medical complications, including fatigue, breathlessness, weight loss, hair loss, and attention deficits. Additionally, there is growing evidence of adverse effects of COVID-19 on the male reproductive system. This investigation seeks to understand the long-term ramifications on male fertility by examining hormonal profiles, semen parameters, and sperm proteome of recovered COVID-19 patients compared to controls. The serum hormone profiles between the two groups showed minimal variations except for prolactin, cortisol, and testosterone levels. Testosterone levels were slightly lower, while prolactin and cortisol were elevated in COVID-19 cases compared to controls. Though semen parameters exhibited no significant disparities between the COVID-19 and control groups, quantitative proteomics analysis revealed changes in sperm proteins. It identified 190 differentially expressed proteins, of which 161 were upregulated and 29 downregulated in COVID-19 cases. Western blotting analysis validated the differential expression of serpin B4 and calpain 2. Bioinformatics analysis signifies cellular stress in the spermatozoa of COVID-19 recovered patients and thus, SOD and MDA levels in semen were measured. MDA levels were found to be significantly elevated, indicating lipid peroxidation in COVID-19 samples. While the effects of COVID-19 on semen parameters may exhibit a potential for reversal within a short duration, the alterations it inflicts on sperm proteome are persisting consequences on male fertility. This study paves the path for further research and emphasizes the significance of comprehending the complex molecular processes underlying the long-term consequences of COVID-19 on male reproductive health.

Exploring Differentially Expressed Sperm miRNAs in Idiopathic Recurrent Pregnancy Loss and Their Association with Early Embryonic Development.

The high incidence of idiopathic recurrent pregnancy loss (iRPL) may stem from the limited research on male contributory factors. Many studies suggest that sperm DNA fragmentation and oxidative stress contribute to iRPL, but their roles are still debated. MicroRNAs (miRNAs) are short non-coding RNAs that regulate various biological processes by modulating gene expression. While differential expression of specific miRNAs has been observed in women suffering from recurrent miscarriages, paternal miRNAs remain unexplored. We hypothesize that analyzing sperm miRNAs can provide crucial insights into the pathophysiology of iRPL. Therefore, this study aims to identify dysregulated miRNAs in the spermatozoa of male partners of iRPL patients. Total mRNA was extracted from sperm samples of iRPL and control groups, followed by miRNA library preparation and high-output miRNA sequencing. Subsequently, raw sequence reads were processed for differential expression analysis, target prediction, and bioinformatics analysis. Twelve differentially expressed miRNAs were identified in the iRPL group, with eight miRNAs upregulated (hsa-miR-4454, hsa-miR-142-3p, hsa-miR-145-5p, hsa-miR-1290, hsa-miR-1246, hsa-miR-7977, hsa-miR-449c-5p, and hsa-miR-92b-3p) and four downregulated (hsa-miR-29c-3p, hsa-miR-30b-5p, hsa-miR-519a-2-5p, and hsa-miR-520b-5p). Functional enrichment analysis revealed that gene targets of the upregulated miRNAs are involved in various biological processes closely associated with sperm quality and embryonic development.

Male Contributory Factors in Recurrent Pregnancy Loss.

With 40% of idiopathic cases, recurrent pregnancy loss (RPL) is a problem of great concern for patients and clinicians. In addition to financial burden, it causes a lot of frustration and anxiety in affected couples. The primary objective of this review was to gain knowledge of recent advances in the field of recurrent pregnancy losses and to understand the role of male contributory factors in idiopathic cases. For a long time, researchers and clinicians were seeking an explanation for idiopathic RPL (iRPL) in females only; however, with recent advances in reproductive biology, the role of spermatozoa in early embryonic development has caught the attention of researchers. Clinically, only routine semen parameters and karyotyping are investigated in iRPL male partners, which seem to be insufficient in the present scenario, and thus, more information at the molecular level is required for a comprehensive understanding of iRPL. In concluding remarks, we suggest targeted multi-omics investigations in a large cohort to improve our understanding of the role of male contributory factors in iRPL.

Proteomic changes in human spermatozoa during in vitro capacitation and acrosome reaction in normozoospermia and asthenozoospermia.

BACKGROUND: The cellular and molecular mechanisms of the events that help spermatozoa acquire their fertilizing capability during capacitation and acrosome reaction are not completely understood. OBJECTIVE: This study was performed with a postulation that the identification of sperm proteins and their changes during in vitro capacitation and acrosome reaction will unravel unknown molecular aspects of fertilization that impact male fertility. MATERIALS AND METHODS: Spermatozoa collected from sequential conditions, that is, separation of ejaculated spermatozoa by Percoll gradient centrifugation, in vitro capacitation, and acrosome reaction were processed for tandem mass spectrometric analysis, followed by protein identification, label-free quantitation, and statistical analysis. RESULTS AND DISCUSSION: Collectively, a total of 1088 sperm proteins were identified. In comparison to ejaculated spermatozoa, 44 and 141 proteins were differentially expressed in capacitated and acrosome reacted spermatozoa, respectively. A large number of proteins were found downregulated, including clusterin, pyruvate dehydrogenase E1 component, semenogelin-1 and 2, heat shock protein 90, beta-microseminoprotein, and keratin. It was expected as sperm-membrane-associated proteins are removed during capacitation. There were significant proteomic alterations in asthenozoospermia compared to normozoospermia; however, variation was more noticeable among proteins of acrosome reacted spermatozoa and those released during the acrosome reaction. The processes enriched among downregulated proteins in asthenozoospermia included acrosome assembly, binding of spermatozoa to zona pellucida, nucleosome assembly, flagellated sperm motility, protein folding, oxidative phosphorylation, tricarboxylic acid cycle, chromatin silencing, gluconeogenesis, glycolytic process, and glycolysis. CONCLUSION: The dynamic information generated about proteomic alterations in spermatozoa during capacitation and acrosome reaction and their variability in asthenozoospermia will contribute not only to enhancing our understanding of processes that prepare spermatozoa to acquire fertilization capability but also help in deciphering novel factors of male infertility.

Differential Sperm Proteomics Reveals the Significance of Fatty Acid Synthase and Clusterin in Idiopathic Recurrent Pregnancy Loss.

Recurrent pregnancy loss (RPL) is a pervasive health issue affecting a large number of couples globally, which leads to increased emotional and financial strain on the affected families. While female factors have been extensively studied and are well known, the contribution of male factors to RPL remains largely unknown. As high as 40% of RPL cases are unexplained, which are termed as idiopathic RPL (iRPL), necessitating the investigation of male factors. The role of spermatozoa in early embryonic development is now well established, and recent research studies have shown that oxidative stress and DNA fragmentation in sperm cells are linked to RPL. The aim of this study was to identify proteomic markers of iRPL in human spermatozoa using tandem mass spectrometry. A label-free method quantified a total of 1820 proteins, and statistical analysis identified 359 differentially expressed proteins, the majority of which were downregulated in iRPL samples (344). Bioinformatics analysis revealed that proteomic alterations were mainly associated with biological processes such as response to stress, protein folding, chromatin organization, DNA conformation change, oxidative phosphorylation, and electron transport chain. In coherence with past studies, we determined fatty acid synthase (FASN) and clusterin (CLU) to be the most potential sperm markers for iRPL and confirmed their expression changes in iRPL by western blotting. Conclusively, we believe that FASN and CLU might serve as potential markers of iRPL and suggest exploratory functional studies to identify their specific role in pregnancy loss.

Quantification studies in human seminal plasma samples identify prolactin inducible protein as a plausible marker of azoospermia.

BACKGROUND: Prolactin inducible protein (PIP) is a ~17 kDa protein, which is known to play vital roles in immunoregulation, fertility, antimicrobial activity, apoptosis and tumour progression. OBJECTIVES: This study reports quantification of PIP concentration in human seminal plasma (SP) samples. METHODOLOGY: PIP was purified by immunoprecipitation and its concentration in human SP samples was quantified by ELISA method. RESULTS: Average concentration of PIP in normozoospermia, oligozoospermia and azoospermia was 290.3 ± 71.5 µg/mL, 306.4 ± 71.2 µg/mL and 60.5 ± 23.6 µg/mL respectively. CONCLUSION: There was no significant variation in PIP levels in normozoospermia and oligozoospermia while its expression was down-regulated in azoospermia, indicating that PIP may be a plausible marker of azoospermia.

Label-free proteomics of spermatozoa identifies candidate protein markers of idiopathic recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) affects a large number of couples worldwide, increasing their mental and financial burdens. While female factors that contribute to RPL have been studied extensively, the role of male factors is largely unknown, and approximately 40 % RPL cases remain unexplained despite thorough clinical examinations. These cases are clinically termed as idiopathic RPL (iRPL). Several studies have recently found that spermatozoa play an important role, beyond fertilization, in iRPL, specifically in early embryonic development. Consequently, scientists explored spermatozoa to understand iRPL and revealed that both oxidative stress and DNA fragmentation contribute to RPL. In this study, we analyzed sperm samples from male partners of iRPL patients and fertile men who recently fathered a child by LC-MS/MS to identify proteomic markers of iRPL. A total of 1,988 proteins were quantified by a label-free method, and stringent statistical analysis was performed for the selection of candidate biomarkers of iRPL. Out of 1,647 proteins quantified, only 7 proteins qualified the selection criteria, which are lactotransferrin, ATP synthase subunit beta mitochondrial, fatty acid synthase, anterior gradient protein 2 homolog, hemoglobin subunit beta, short-chain specific acyl-CoA dehydrogenase mitochondrial, cytoplasmic dynein 1 heavy chain, and 14-3-3 protein sigma. We then performed gene annotations, pathways, and network analyses to gain more biological insights, identifying an association between oxidative stress and iRPL.

Sub-fertility in crossbred bulls: Identification of proteomic alterations in spermatogenic cells using high throughput comparative proteomics approach.

The present study was carried out to compare the proteomic profiles of spermatogenic cells of crossbred and zebu cattle in an effort to understand the possible reasons for a higher incidence of sub-fertility in crossbred bulls. The spermatogenic cells collected from the testes of pre-pubertal (6 mo) and adult (24 mo) crossbred and zebu males through fine needle aspiration were proliferated in vitro, and proteomic profiling was done using a shotgun proteomics approach. The age- and species-specific variations in the expression level of proteins were identified in spermatogenic cells. The number of differentially expressed proteins (DEPs) identified in pre-pubertal zebu and crossbred was 546, while 579 DEPs were identified between adult zebu and crossbred bulls. Out of these, 194 DEPS were common to these groups and 40 DEPs displayed a fold change ≥2. However, only 20 proteins exhibited similar expression variation trends (upregulated or downregulated) among pre-pubertal as well as adult zebu and crossbred bulls. Out of these 20 DEPs, 13 proteins were upregulated, and 7 proteins were downregulated in spermatogenic cells of zebu compared to crossbred bulls. Among the upregulated proteins were RPLP2, PAXIP1, calumenin, prosaposin, GTF2F1, TMP2, ubiquitin conjugation factor E4A, COL1A2, vimentin, protein FAM13A, peripherin, GFPT2, and GRP78. Seven proteins that were downregulated in zebu bulls compared to crossbred included APOA1, G patch domain-containing protein 1, NAD P transhydrogenase mitochondrial, glutamyl aminopeptidase, synaptojanin 1 fragment, Arf GAP with SH3 domain ANK repeat and PH domain-containing protein 1, and protein transport protein sec16B. It was inferred that the proteins associated with sperm function and fertilization processes, such as calumenin, prosaposin, vimentin, GRP78, and APOA1 could be studied further to understand the precise cause of subfertility in crossbred bulls.

Sample complexity reduction aids efficient detection of low-abundant proteins from human amniotic fluid.

Working with biological fluids poses a challenge of visualizing proteins present in lower concentrations. This study describes a batch-mode chromatographic method for the fractionation of human amniotic fluid (AF). This method is easy to use with minimal sample quantity, resin volume and sample processing time. For albumin depletion, two methods were evaluated. The results demonstrated that specific depletion of albumin, using affinity-ligand-based resin, is more efficient than the conventional dye-based method. The albumin-depleted human AF was fractionated by strong anion-exchange resin in spin devices, for sample, complexity reduction and enrichment of low-abundant proteins. Analysis of four eluate fractions generated after this step shows enrichment of few low-abundant proteins. Two novel low-abundant proteins, Rab GDP dissociation inhibitor beta and peptide methionine sulfoxide reductase, were identified from human AF. Alpha-1-B glycoprotein was successfully identified by this strategy, whereas the published literature reports that it was not identified by strong anion-exchange FPLC followed by SDS-PAGE. Therefore, the current method has distinct advantages over the conventional column-based chromatography. This study also reports altered expression of some proteins in Rh-isoimmunized AF samples in comparison with normal AF.

Most Variable Genes and Transcription Factors in Acute Lymphoblastic Leukemia Patients.

Acute lymphoblastic leukemia (ALL) is a hematologic tumor caused by cell cycle aberrations due to accumulating genetic disturbances in the expression of transcription factors (TFs), signaling oncogenes and tumor suppressors. Though survival rate in childhood ALL patients is increased up to 80% with recent medical advances, treatment of adults and childhood relapse cases still remains challenging. Here, we have performed bioinformatics analysis of 207 ALL patients' mRNA expression data retrieved from the ICGC data portal with an objective to mark out the decisive genes and pathways responsible for ALL pathogenesis and aggression. For analysis, 3361 most variable genes, including 276 transcription factors (out of 16,807 genes) were sorted based on the coefficient of variance. Silhouette width analysis classified 207 ALL patients into 6 subtypes and heat map analysis suggests a need of large and multicenter dataset for non-overlapping subtype classification. Overall, 265 GO terms and 32 KEGG pathways were enriched. The lists were dominated by cancer-associated entries and highlight crucial genes and pathways that can be targeted for designing more specific ALL therapeutics. Differential gene expression analysis identified upregulation of two important genes, JCHAIN and CRLF2 in dead patients' cohort suggesting their possible involvement in different clinical outcomes in ALL patients undergoing the same treatment.

Exploring the aggregation-prone regions from structural domains of human TDP-43.

TDP-43 (transactive- response DNA binding protein) amazes structural biologist as its aberrant ubiquitinated cytosolic inclusions is largely involved in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). An important question in TDP-43 research is to identify the structural region mediating the formation of cytoplasmic pathological aggregates. In this study, we attempted to delineate the aggregation-prone sequences of the structural domain of TDP-43. Here, we investigated the self-assembly of peptides of TDP-43 using aggregation prediction algorithms, Zipper DB and AMYLPRED2. The three aggregation-prone peptides identified were from N-terminal domain (24GTVLLSTV31), and RNA recognition motifs, RRM1 (128GEVLMVQV135) and RRM2 (247DLIIKGIS254). Furthermore, the amyloid fibril forming propensities of these peptides were analyzed through different biophysical techniques and molecular dynamics simulation. Our study shows the different aggregation ability of conserved stretches in structural domain of TDP-43 that will possibly induce full-length aggregation of TDP-43 in vivo. The peptide form RRM2 demonstrates the higher intrinsic amyloid forming propensity and suggests that RRM2 might form the structural core of TDP-43 aggregation seen in vivo. The results of this study would help in designing peptide based inhibitors of TDP-43 aggregation.

Identification of functional interactome of a key cell division regulatory protein CedA of E.coli.

Cell division is compromised in DnaAcos mutant Escherichia coli cells that results in filamentous cell morphology. This is countered by over-expression of CedA protein that induces cytokinesis and thus, regular cell morphology is regained; however via an unknown mechanism. To understand the process systematically, exact role of CedA should be deciphered. Protein interactions are crucial for functional organization of a cell and their identification helps in revealing exact function(s) of a protein and its binding partners. Thus, this study was intended to identify CedA binding proteins (CBPs) to gain more clues of CedA function. We isolated CBPs by pull down assay using purified recombinant CedA and identified nine CBPs by mass spectrometric analysis (MALDI-TOF MS and LC-MS/MS), viz. PDHA1, RL2, DNAK, LPP, RPOB, G6PD, GLMS, RL3 and YBCJ. Based on CBPs identified, we hypothesize that CedA plays a crucial and multifaceted role in cell cycle regulation and specific pathways in which CedA participates may include transcription and energy metabolism. However, further validation through in-vitro and in-vivo experiments is necessary. In conclusion, identification of CBPs may help us in deciphering mechanism of CedA mediated cell division during chromosomal DNA over-replication.

Interplay between CedA, rpoB and double stranded DNA: A step towards understanding CedA mediated cell division in E. coli.

Cell division is compromised in DnaAcos mutant E. coli cells due to chromosome over-replication. In these cells, CedA acts as a regulatory protein and initiates cell division by a hitherto unknown mechanism. CedA, a double stranded DNA binding protein, interacts with various subunits of RNA polymerase complex, including rpoB. To reveal how this concert between CedA, rpoB and DNA brings about cell division in E. coli, we performed biophysical and in silico analysis and obtained mechanistic insights. Interaction between CedA and rpoB was shown by circular dichroism spectrometry and in silico docking experiments. Further, CedA and rpoB were allowed to interact individually to a selected DNA and their binding was monitored by fluorescence spectroscopy. The binding constants of these interactions as determined by BioLayer Interferometry clearly show that rpoB binds to DNA with higher affinity (KD2=<1.0E-12M) as compared to CedA (KD2=9.58E-09M). These findings were supported by docking analysis where 12 intermolecular H-bonds were formed in rpoB-DNA complex as compared to 4 in CedA-DNA complex. Based on our data we propose that in E. coli cells chromosome over-replication signals CedA to recruit rpoB to specific DNA site(s), which initiates transcription of cell division regulatory elements.

Gene expression profiling, pathway analysis and subtype classification reveal molecular heterogeneity in hepatocellular carcinoma and suggest subtype specific therapeutic targets.

A very low 5-year survival rate among hepatocellular carcinoma (HCC) patients is mainly due to lack of early stage diagnosis, distant metastasis and high risk of postoperative recurrence. Hence ascertaining novel biomarkers for early diagnosis and patient specific therapeutics is crucial and urgent. Here, we have performed a comprehensive analysis of the expression data of 423 HCC patients (373 tumors and 50 controls) downloaded from The Cancer Genome Atlas (TCGA) followed by pathway enrichment by gene ontology annotations, subtype classification and overall survival analysis. The differential gene expression analysis using non-parametric Wilcoxon test revealed a total of 479 up-regulated and 91 down-regulated genes in HCC compared to controls. The list of top differentially expressed genes mainly consists of tumor/cancer associated genes, such as AFP, THBS4, LCN2, GPC3, NUF2, etc. The genes over-expressed in HCC were mainly associated with cell cycle pathways. In total, 59 kinases associated genes were found over-expressed in HCC, including TTK, MELK, BUB1, NEK2, BUB1B, AURKB, PLK1, CDK1, PKMYT1, PBK, etc. Overall four distinct HCC subtypes were predicted using consensus clustering method. Each subtype was unique in terms of gene expression, pathway enrichment and median survival. Conclusively, this study has exposed a number of interesting genes which can be exploited in future as potential markers of HCC, diagnostic as well as prognostic and subtype classification may guide for improved and specific therapy.

Heparin binding carboxypeptidase E protein exhibits antibacterial activity in human semen.

Carboxypeptidase E (CPE) cleaves basic amino acid residues at the C-terminal end and involves in the biosynthesis of numerous peptide hormones and neurotransmitters. It was purified from human seminal plasma by ion exchange, heparin affinity and gel filtration chromatography followed by identification through SDS-PAGE and MALDI-TOF/MS analysis, which was further confirmed by western blotting. CPE was characterized as glycoprotein by Periodic Acid Schiff (PAS) staining and treating with deglycosylating enzyme N-glycosidase F. The interaction of CPE with heparin was illustrated by surface plasmon resonance (SPR) and in silico interaction analysis. The association constant (KA) and dissociation constant (KD) of CPE with heparin was determined by SPR and found to be 1.06 × 10(5)M and 9.46 × 10(-6)M, respectively. It was detected in human spermatozoa also by western blotting using mouse anti-CPE primary antibody. 20-100 µg/ml concentration of CPE was observed as highly effective in killing Escherichia coli by colony forming unit (CFU) assay. We suggest that CPE might act not only in the innate immunity of male reproductive tract but also regulate sperm fertilization process by interacting heparin.