Neutrophils display distinct post-translational modifications in response to varied pathological stimuli.

Neutrophils play a vital role in the innate immunity by perform effector functions through phagocytosis, degranulation, and forming extracellular traps. However, over-functioning of neutrophils has been associated with sterile inflammation such as Type 2 Diabetes, atherosclerosis, cancer and autoimmune disorders. Neutrophils exhibiting phenotypical and functional heterogeneity in both homeostatic and pathological conditions suggests distinct signaling pathways are activated in disease-specific stimuli and alter neutrophil functions. Hence, we examined mass spectrometry based post-translational modifications (PTM) of neutrophil proteins in response to pathologically significant stimuli, including high glucose, homocysteine and bacterial lipopolysaccharides representing diabetes-indicator, an activator of thrombosis and pathogen-associated molecule, respectively. Our data revealed that these aforesaid stimulators differentially deamidate, citrullinate, acetylate and methylate neutrophil proteins and align to distinct biological functions associated with degranulation, platelet activation, innate immune responses and metabolic alterations. The PTM patterns in response to high glucose showed an association with neutrophils extracellular traps (NETs) formation, homocysteine induced proteins PTM associated with signaling of systemic lupus erythematosus and lipopolysaccharides induced PTMs were involved in pathways related to cardiomyopathies. Our study provides novel insights into neutrophil PTM patterns and functions in response to varied pathological stimuli, which may serve as a resource to design therapeutic strategies for the management of neutrophil-centred diseases.

Regulation and tumor-suppressive function of the miR-379/miR-656 (C14MC) cluster in cervical cancer.

Cervical cancer (CC) is a key contributor to cancer-related mortality in several countries. The identification of molecular markers and the underlying mechanism may help improve CC management. We studied the regulation and biological function of the chromosome 14 microRNA cluster (C14MC; miR-379/miR-656) in CC. Most C14MC members exhibited considerably lower expression in CC tissues and cell lines in The Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma patient cohorts. Bisulfite Sanger sequencing revealed hypermethylation of the C14MC promoter in CC tissues and cell lines. 5-aza-2 deoxy cytidine treatment reactivated expression of the C14MC members. We demonstrated that C14MC is a methylation-regulated miRNA cluster via artificial methylation and luciferase reporter assays. C14MC downregulation correlated with poor overall survival and may promote metastasis. C14MC activation via the lentiviral-based CRISPRa approach inhibited growth, proliferation, migration, and invasion; enhanced G2/M arrest; and induced senescence. Post-transcriptional regulatory network analysis of C14MC transcriptomic data revealed enrichment of key cancer-related pathways, such as metabolism, the cell cycle, and phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Reduced cell proliferation, growth, migration, invasion, and senescence correlated with the downregulation of active AKT, MYC, and cyclin E1 (CCNE1) and the overexpression of p16, p21, and p27. We showed that C14MC miRNA activation increases reactive oxygen species (ROS) levels, intracellular Ca2+ levels, and lipid peroxidation rates, and inhibits epithelial-mesenchymal transition (EMT). C14MC targets pyruvate dehydrogenase kinase-3 (PDK3) according to the luciferase reporter assay. PDK3 is overexpressed in CC and is inversely correlated with C14MC. Both miR-494-mimic transfection and C14MC activation inhibited PDK3 expression. Reduced glucose uptake and lactate production, and upregulation of PDK3 upon C14MC activation suggest the potential role of these proteins in metabolic reprogramming. Finally, we showed that C14MC activation may inhibit EMT signaling. Thus, C14MC is a tumor-suppressive and methylation-regulated miRNA cluster in CC. Reactivation of C14MC can be useful in the management of CC.

A Novel TPM1 Mutation Causes Familial Hypertrophic Cardiomyopathy in an Indian Family: Genetic and Clinical Correlation.

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder characterised by unexplained left ventricular hypertrophy in the absence of abnormal loading conditions. The global prevalence of HCM is estimated to be 1 in 250 in the general population. It is caused due to mutations in genes coding for sarcomeric proteins. α-tropomyosin (TPM1) is an important protein in the sarcomeric thin filament which regulates sarcomere contraction. Mutations in TPM1 are known to cause hypertrophic cardiomyopathy, dilated cardiomyopathy and left ventricular non-compaction. Mutations in TPM1 causing hypertrophic cardiomyopathy are < 1%. However, some high-risk mutations causing sudden cardiac death are also known in this gene. We present a case of a novel heterozygous TPM1 mutation, NM_001018005.2:c.203A>G, p.Gln68Arg; co-segregating in an Indian family with hypertrophic cardiomyopathy. Our report expands the mutational spectrum of HCM due to TPM1 and provides the correlated cardiac phenotype.

Low-dose exposure to malathion and radiation results in the dysregulation of multiple neuronal processes, inducing neurotoxicity and neurodegeneration in mouse.

Neurodegenerative disorders are a debilitating and persistent threat to the global elderly population, carrying grim outcomes. Their genesis is often multifactorial, with a history of prior exposure to xenobiotics such as pesticides, heavy metals, enviornmental pollutants, ionizing radiation etc,. A holistic molecular insight into their mechanistic induction upon single or combinatorial exposure to different toxicants is still unclear. In the present study, one-month-old C57BL/6 male mice were administered orally with malathion (50 mg/kg body wt. for 14 days) and single whole-body radiation (0.5 Gy) on the 8th day. Post-treatment, behavioural assays for exploratory behaviour, memory, and learning were performed. After sacrifice, brains were collected for histology, biochemical assays, and transcriptomic analysis. Transcriptomic analysis revealed several altered processes like synaptic transmission and plasticity, neuronal survival, proliferation, and death. Signalling pathways like MAPK, PI3K-Akt, Apelin, NF-κB, cAMP, Notch etc., and pathways related to neurodegenerative diseases were altered. Increased astrogliosis was observed in the radiation and coexposure groups, with significant neuronal cell death and a reduction in the expression of NeuN. Sholl analysis, dendritic arborization and spine density studies revealed decreased total apical neuronal path length and dendritic spine density. Reduced levels of the antioxidants GST and GSH and acetylcholinesterase enzyme activity were also detected. However, no changes were seen in exploratory behaviour or learning and memory post-treatment. Thus, explicating the molecular mechanisms behind malathion and radiation can provide novel insights into external factor-driven neurotoxicity and neurodegenerative pathogenesis.

Moonlighting functions of the ubiquitin-like protein, Hub1/UBL-5.

The faithful splicing of pre-mRNA is critical for accurate gene expression. Dysregulation of pre-mRNA splicing has been associated with several human diseases including cancer. The ubiquitin-like protein Hub1/UBL5 binds to the substrates non-covalently and promotes pre-mRNA splicing. Additionally, UBL5 promotes the common fragile sites stability and the Fanconi anemia pathway of DNA damage repair. These functions strongly suggests that UBL5 could potentially be implicated in cancer. Therefore, we analyzed the UBL5 expression in TCGA tumor sample datasets and observed the differences between tumor and normal tissues among different tumor subtypes. We have noticed the alteration frequency of UBL5 in TCGA tumor samples. Altogether, this review summarizes the UBL5 functions and discusses its putative role in tumorigenesis.

Integrated Bioinformatics Analysis Identifies Crucial Biochemical Processes Shared between Pancreatitis and Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy associated with rapid progression and an abysmal prognosis. Previous research has shown that chronic pancreatitis can significantly increase the risk of developing PDAC. The overarching hypothesis is that some of the biological processes disrupted during the inflammatory stage tend to show significant dysregulation, even in cancer. This might explain why chronic inflammation increases the risk of carcinogenesis and uncontrolled proliferation. Here, we try to pinpoint such complex processes by comparing the expression profiles of pancreatitis and PDAC tissues. METHODS: We analyzed a total of six gene expression datasets retrieved from the EMBL-EBI ArrayExpress and NCBI GEO databases, which included 306 PDAC, 68 pancreatitis and 172 normal pancreatic samples. The disrupted genes identified were used to perform downstream analysis for ontology, interaction, enriched pathways, potential druggability, promoter methylation, and the associated prognostic value. Further, we performed expression analysis based on gender, patient's drinking habit, race, and pancreatitis status. RESULTS: Our study identified 45 genes with altered expression levels shared between PDAC and pancreatitis. Over-representation analysis revealed that protein digestion and absorption, ECM-receptor interaction, PI3k-Akt signaling, and proteoglycans in cancer pathways as significantly enriched. Module analysis identified 15 hub genes, of which 14 were found to be in the druggable genome category. CONCLUSION: In summary, we have identified critical genes and various biochemical processes disrupted at a molecular level. These results can provide valuable insights into certain events leading to carcinogenesis, and therefore help identify novel therapeutic targets to improve PDAC treatment in the future.

Exome sequencing of choreoacanthocytosis reveals novel mutations in VPS13A and co-mutation in modifier gene(s).

Choreoacanthocytosis, one of the forms of neuroacanthocytosis, is caused by mutations in vacuolar protein sorting-associated protein A (VPS13A), and is often misdiagnosed with other form of neuroacanthocytosis with discrete genetic defects. The phenotypic variations among the patients with VPS13A mutations significantly obfuscates the understanding of the disease and treatment strategies. In this study, two unrelated cases were identified, exhibiting the core phenotype of neuroacanthocytosis but with considerable clinical heterogeneity. Case 1 presented with an additional Parkinsonism phenotype, whereas seizures were evident in case 2. To decipher the genetic basis, whole exome sequencing followed by validation with Sanger sequencing was performed. A known homozygous pathogenic nonsense mutation (c.799C > T; p.R267X) in exon 11 of the VPS13A gene was identified in case 1 that resulted in a truncated protein. A novel missense mutation (c.9263T > G; p.M3088R) in exon 69 of VPS13A identified in case 2 was predicted as pathogenic. In silico analysis of the p.M3088R mutation at the C-terminus of VPS13A suggests a loss of interaction with TOMM40 and may disrupt mitochondrial localization. We also observed an increase in mitochondrial DNA copy numbers in case 2. Mutation analysis revealed benign heterozygous variants in interacting partners of VPS13A such as VAPA in case 1. Our study confirmed the cases as ChAc and identified the novel homozygous variant of VPS13A (c.9263T > G; p.M3088R) within the mutation spectrum of VPS13A-associated ChAc. Furthermore, mutations in VPS13A and co-mutations in its potential interacting partner(s) might contribute to the diverse clinical manifestations of ChAc, which requires further study.

MiR-4521 perturbs FOXM1-mediated DNA damage response in breast cancer.

Introduction: Forkhead (FOX) transcription factors are involved in cell cycle control, cellular differentiation, maintenance of tissues, and aging. Mutation or aberrant expression of FOX proteins is associated with developmental disorders and cancers. FOXM1, an oncogenic transcription factor, is a promoter of cell proliferation and accelerated development of breast adenocarcinomas, squamous carcinoma of the head, neck, and cervix, and nasopharyngeal carcinoma. High FOXM1 expression is correlated with chemoresistance in patients treated with doxorubicin and Epirubicin by enhancing the DNA repair in breast cancer cells. Method: miRNA-seq identified downregulation of miR-4521 in breast cancer cell lines. Stable miR-4521 overexpressing breast cancer cell lines (MCF-7, MDA-MB-468) were developed to identify miR-4521 target gene and function in breast cancer. Results: Here, we showed that FOXM1 is a direct target of miR-4521 in breast cancer. Overexpression of miR-4521 significantly downregulated FOXM1 expression in breast cancer cells. FOXM1 regulates cell cycle progression and DNA damage response in breast cancer. We showed that miR-4521 expression leads to increased ROS levels and DNA damage in breast cancer cells. FOXM1 plays a critical role in ROS scavenging and promotes stemness which contributes to drug resistance in breast cancer. We observed that breast cancer cells stably expressing miR-4521 lead to cell cycle arrest, impaired FOXM1 mediated DNA damage response leading to increased cell death in breast cancer cells. Additionally, miR-4521-mediated FOXM1 downregulation perturbs cell proliferation, invasion, cell cycle progression, and epithelial-to-mesenchymal progression (EMT) in breast cancer. Discussion: High FOXM1 expression has been associated with radio and chemoresistance contributing to poor patient survival in multiple cancers, including breast cancer. Our study showed that FOXM1 mediated DNA damage response could be targeted using miR-4521 mimics as a novel therapeutic for breast cancer.

Bioinformatic Analysis of miR-200b/429 and Hub Gene Network in Cervical Cancer.

The miR-200b/429 located at 1p36 is a highly conserved miRNA cluster emerging as a critical regulator of cervical cancer. Using publicly available miRNA expression data from TCGA and GEO followed by independent validation, we aimed to identify the association between miR-200b/429 expression and cervical cancer. miR-200b/429 cluster was significantly overexpressed in cancer samples compared to normal samples. miR-200b/429 expression did not correlate with patient survival; however, its overexpression correlated with histological type. Protein-protein interaction analysis of 90 target genes of miR-200b/429 identified EZH2, FLT1, IGF2, IRS1, JUN, KDR, SOX2, MYB, ZEB1, and TIMP2 as the top ten hub genes. PI3K-AKT and MAPK signaling pathways emerged as major target pathways of miR-200b/429 and their hub genes. Kaplan-Meier survival analysis showed the expression of seven miR-200b/429 target genes (EZH2, FLT1, IGF2, IRS1, JUN, SOX2, and TIMP2) to influence the overall survival of patients. The miR-200a-3p and miR-200b-5p could help predict cervical cancer with metastatic potential. The cancer hallmark enrichment analysis showed hub genes to promote growth, sustained proliferation, resistance to apoptosis, induction of angiogenesis, activation of invasion, and metastasis, enabling replicative immortality, evading immune destruction, and tumor-promoting inflammation. The drug-gene interaction analysis identified 182 potential drugs to interact with 27 target genes of miR-200b/429 with paclitaxel, doxorubicin, dabrafenib, bortezomib, docetaxel, ABT-199, eribulin, vorinostat, etoposide, and mitoxantrone emerging as the top ten best candidate drugs. Taken together, miR-200b/429 and associated hub genes can be helpful for prognostic application and clinical management of cervical cancer.

Severe acute respiratory syndrome coronaviruses contributing to mitochondrial dysfunction: Implications for post-COVID complications.

Mitochondria play a central role in oxidative phosphorylation (OXPHOS), bioenergetics linked with ATP production, fatty acids biosynthesis, calcium signaling, cell cycle regulation, apoptosis, and innate immune response. Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection manipulates the host cellular machinery for its survival and replication in the host cell. The infectiaon causes perturbed the cellular metabolism that favours viral replication leading to mitochondrial dysfunction and chronic inflammation. By localizing to the mitochondria, SARS CoV proteins increase reactive oxygen species (ROS) levels, perturbation of Ca2+ signaling, changes in mtDNA copy number, mitochondrial membrane potential (MMP), mitochondrial mass, and induction of mitophagy. These proteins also influence the fusion and fission kinetics, size, structure, and distribution of mitochondria in the infected host cells. This results in compromised bioenergetics, altered metabolism, and innate immune signaling, and hence can be a key player in determining the outcome of SARS-CoV infection. SARS-CoV infection contributes to stress and activates apoptotic pathways. This review summarizes how mitochondrial function and dynamics are affected by SARS-CoV and how the mitochondria-SARS-CoV interaction benefits viral survival and growth by evading innate host immunity. We also highlight how the SARS-CoV-mediated mitochondrial dysfunction contributes to post-COVID complications. Besides, a discussion on targeting virus-mitochondria interactions as a therapeutic strategy is presented.

Salivary DNA methylation markers for cancer of oral cavity.

PURPOSE: Aberrant DNA methylation plays a crucial role in oral carcinogenesis. Our previous study demonstrated hypermethylation of DAPK1, LRPPRC, RAB6C, and ZNF471 promoters in patients with tongue squamous cell carcinoma compared with normal samples. Methylation profiling using salivary DNA is considered a non-invasive alternative to tissue samples. Hence, the present study tested the DNA methylation status of these four promoters as indicators of oral cancer progression. METHODS: We performed the bisulfite-based targeted next-generation sequencing of four candidate genes in saliva and tissue DNA from normal, premalignant, and squamous cell carcinoma subjects. The clinicopathological association, diagnostic, and prognostic utility of aberrant DNA methylation were evaluated using the TCGA-HNSCC dataset. Using the Xgboost algorithm and logistic regression, CpG sites were prioritized, and Receiver Operating Characteristic was generated. By Log-rank test and Kaplan-Meier (KM) curves, an association between methylation and overall survival (OS), disease-free interval (DFI), and progression-free interval (PFI) were computed. RESULTS: We identified all four genes as significantly hypermethylated in premalignant and malignant samples compared with normal samples. The methylation levels were comparable between saliva and tissue samples with an r-value of 0.6297 to 0.8023 and 0.7823 to 0.9419 between premalignant tissue vs. saliva and OC vs. saliva, respectively. We identified an inverse correlation between DAPK1, LRPPRC, RAB6C, and ZNF471 promoter methylation with their expression. A classifier of 8 differentially methylated CpG sites belonging to DAPK1, RAB6C, and ZNF471 promoters was constructed, showing an AUC of 0.984 to differentiate tumors from normal samples. The differential methylation status of DAPK1, LRPPRC, and ZNF71 promoters was prognostically important. Abnormal expression of all four genes was associated with immune infiltration. CONCLUSIONS: Thus, methylation analysis of these candidate CpG sites from saliva can be helpful as a non-invasive tool for the clinical management of OC.

Expression analysis and function of mitochondrial genome-encoded microRNAs.

MicroRNAs (miRNAs) play a significant role in nuclear and mitochondrial anterograde and retrograde signaling. Most of the miRNAs found inside mitochondria are encoded in the nuclear genome, with a few mitochondrial genome-encoded non-coding RNAs having been reported. In this study, we have identified 13 mitochondrial genome-encoded microRNAs (mitomiRs), which were differentially expressed in breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231), non-malignant breast epithelial cell line (MCF-10A), and normal and breast cancer tissue specimens. We found that mitochondrial DNA (mtDNA) depletion and inhibition of mitochondrial transcription led to reduced expression of mitomiRs in breast cancer cells. MitomiRs physically interacted with Ago2, an RNA-induced silencing complex (RISC) protein, in the cytoplasm and inside mitochondria. MitomiRs regulate the expression of both nuclear and mitochondrial transcripts in breast cancer cells. We showed that mitomiR-5 targets the PPARGC1A gene and regulates mtDNA copy number in breast cancer cells. MitomiRs identified in the present study may be a promising tool for expression and functional analysis in patients with a defective mitochondrial phenotype, including cancer and metabolic syndromes. This article has an associated First Person interview with the first author of the paper.

A Microfluidic Cancer-on-Chip Platform Predicts Drug Response Using Organotypic Tumor Slice Culture.

Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies. SIGNIFICANCE: The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.

Analysis of Nuclear Encoded Mitochondrial Gene Networks in Cervical Cancer.

BACKGROUND: Cervical cancer (CC) is one of the most common female cancers in many developing and underdeveloped countries. High incidence, late presentation, and mortality suggested the need for molecular markers. Mitochondrial defects due to abnormal expression of nuclear-encoded mitochondrial genes (NEMG) have been reported during cancer progression. Nevertheless, the application of NEMG for the prognosis of CC is still elusive. Herein, we aimed to investigate the associations between NEMG and CC prognosis. MATERIALS AND METHODS: The differentially expressed genes (DEG) in the TCGA-CESC dataset and NEMGs were retrieved from TACCO and Mitocarta2.0 databases, respectively. The impact of methylation on NEMG expression were predicted using DNMIVD and UALCAN tools. HCMDB tool was used to predict genes having metastatic potential. The prognostic models were constructed using DNMIVD, TACCO, GEPIA2, and SurvExpress. The functional enrichment analysis (FEA) was performed using clusterProfiler. The protein-protein interaction network (PPIN) was constructed to identify the hub genes (HG) using String and CytoHubba tools. Independent validation of the HG was performed using Oncomine and Human Protein Atlas databases. The druggable genes were predicted using DGIdb. RESULTS: Among the 52 differentially expressed NEMG, 15 were regulated by DNA methylation. The expression level of 16, 10, and 7 has the potential for CC staging, prediction of metastasis, and prognosis. Moreover, 1 driver gene and 16 druggable genes were also identified. The FEA identified the enrichment of cancer-related pathways, including AMPK and carbon metabolism in cancer. The combined expression of 10 HG has been shown to affect patient survival. CONCLUSION: Our findings suggest that the abnormal expression of NEMGs may play a critical role in CC development and progression. The genes identified in our study may serve as a prognostic indicator and therapeutic target in CC.
.

Contribution of nuclear and mitochondrial gene mutations in mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome.

BACKGROUND: Mitochondrial disorders are clinically complex and have highly variable phenotypes among all inherited disorders. Mutations in mitochon drial DNA (mtDNA) and nuclear genome or both have been reported in mitochondrial diseases suggesting common pathophysiological pathways. Considering the clinical heterogeneity of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) phenotype including focal neurological deficits, it is important to look beyond mitochondrial gene mutation. METHODS: The clinical, histopathological, biochemical analysis for OXPHOS enzyme activity, and electron microscopic, and neuroimaging analysis was performed to diagnose 11 patients with MELAS syndrome with a multisystem presentation. In addition, whole exome sequencing (WES) and whole mitochondrial genome sequencing were performed to identify nuclear and mitochondrial mutations. RESULTS: Analysis of whole mtDNA sequence identified classical pathogenic mutation m.3243A > G in seven out of 11 patients. Exome sequencing identified pathogenic mutation in several nuclear genes associated with mitochondrial encephalopathy, sensorineural hearing loss, diabetes, epilepsy, seizure and cardiomyopathy (POLG, DGUOK, SUCLG2, TRNT1, LOXHD1, KCNQ1, KCNQ2, NEUROD1, MYH7) that may contribute to classical mitochondrial disease phenotype alone or in combination with m.3243A > G mutation. CONCLUSION: Individuals with MELAS exhibit clinical phenotypes with varying degree of severity affecting multiple systems including auditory, visual, cardiovascular, endocrine, and nervous system. This is the first report to show that nuclear genetic factors influence the clinical outcomes/manifestations of MELAS subjects alone or in combination with m.3243A > G mutation.

DINAX- a comprehensive database of inherited ataxias.

BACKGROUND: Neurodegenerative disorders such as hereditary ataxia often manifest overlapping symptoms and are likely to be misdiagnosed based on clinical phenotypes. To identify the genes associated with such disorders for diagnostic purposes, geneticists often use high throughput technologies which generate an enormous amount of data on variants whose relevance can be unclear. Besides, analysis and interpretation of high throughput data require gleaning of several web-based resources which can be laborious and time-consuming. To overcome these, we have created a Database for Inherited Ataxia (DINAX), a repository of gene variants from publicly available information. METHODS: DINAX is implemented as a MySQL relational database using the PHP scripting language. Web interfaces were developed using HTML, CSS, and JavaScript. Variant and phenotype information was collected and manually curated from published literature and primary databases such as OMIM and ClinVar. These were further analyzed to decipher expression and pathway analysis. RESULTS: DINAX is an inventory of 7166 genomic variants (single nucleotide polymorphisms, deletions, insertions, and translocations) reported till date among the 185 genes associated with different subtypes of inherited ataxia. DINAX implements a dual search methodology for genes and phenotypes linking to ataxia associated genes, variants, and their source. Pathway analysis confirmed their association with ataxia. CONCLUSION: The database is created to provide a single web source for obtaining information about ataxia related genes. Besides, the database facilitates easy identification of known and reported variants as well as the novel or unreported variants. DINAX is freely available at http://slsdb.manipal.edu/dinax.

Hypoxia increases mutational load of breast cancer cells through frameshift mutations.

Tumor hypoxia-induced downregulation of DNA repair pathways and enhanced replication stress are potential sources of genomic instability. A plethora of genetic changes such as point mutations, large deletions and duplications, microsatellite and chromosomal instability have been discovered in cells under hypoxic stress. However, the influence of hypoxia on the mutational burden of the genome is not fully understood. Here, we attempted to elucidate the DNA damage response and repair patterns under different types of hypoxic stress. In addition, we examined the pattern of mutations exclusively induced under chronic and intermittent hypoxic conditions in two breast cancer cell lines using exome sequencing. Our data indicated that hypoxic stress resulted in transcriptional downregulation of DNA repair genes which can impact the DNA repair induced during anoxic as well as reoxygenated conditions. In addition, our findings demonstrate that hypoxic conditions increased the mutational burden, characterized by an increase in frameshift insertions and deletions. The somatic mutations were random and non-recurring, as huge variations within the technical duplicates were recognized. Hypoxia also resulted in an increase in the formation of potential neoantigens in both cell lines. More importantly, these data indicate that hypoxic stress mitigates DNA damage repair pathways and causes an increase in the mutational burden of tumor cells, thereby interfering with hypoxic cancer cell immunogenicity.

Enumeration of deregulated miRNAs in liquid and tissue biopsies of cervical cancer.

OBJECTIVE: The altered miRNAs expression in cervical cancer tissue can be a critical player during tumorigenesis, may contribute to tumor cell heterogeneity and may determine distinct phenotypes within the tumor. Recent studies have highlighted the role of circulating miRNAs as a minimally-invasive biomarker and its potential as biosignature to complement routine tissue-based procedures. METHODS: In order to determine whether miRNAs in serum can indicate changes in cervical tissue specimens, we performed small RNA sequencing and selected miRNAs were validated using qRT-PCR in serum and tissue specimens (n = 115). Further, luciferase assay were performed to investigate the interactions between hsa-miR-409-3p and hsa-miR-454-3p binding sites on 3'UTR region of MTF2 and ST18 respectively. RESULTS: We have identified a total of 14 differentially expressed miRNAs common in serum and tissue specimens. Among them, hsa-miR-17-5p, hsa-miR-32-5p and hsa-miR-454-3p were upregulated while, hsa-miR-409-3p was downregulated in serum and tissue of cervical cancer subjects. Our in-silico small RNA sequencing data analysis identified isomiRs and classified miRNA into clusters and subtypes (exonic, intronic and intergenic) with respect to the expression status in serum and tissue specimens. Expression level of hsa-miR-409-3p and hsa-miR-454-3p were inversely correlated with their target genes MTF2 and ST18 levels respectively in human cervical cancer specimens. Luciferase assay demonstrated that hsa-miR-409-3p and hsa-miR-454-3p functionally interacts with 3'-UTR of MTF2 and ST18 respectively to decrease their activity. CONCLUSION: Our results support the significant role of circulating miRNAs in disease dissemination and their potential utility as biosignatures of clinical relevance.

Host and MTB genome encoded miRNA markers for diagnosis of tuberculosis.

MicroRNAs (miRNAs) are a class of noncoding RNA molecules which are involved in various cellular and physiological processes. Previously, studies have identified several miRNAs that are potential diagnostic biomarkers for various infectious diseases including tuberculosis. We have performed small RNA sequencing using the Ion Torrent PGM platform in extra pulmonary tuberculosis (EPTB) subject's serum samples to identify circulating miRNAs during mycobacterium tuberculosis (MTB) infection. Our analysis identified 20 circulating miRNAs upregulated and 5 miRNAs downregulated during MTB infection in patient's serum. In addition, we have identified 6 MTB genome encoded miRNAs upregulated in EPTB patient's serum samples. Taqman based qRT-PCR analysis of host-genome encoded (hsa-miR-146a-5p and hsa-miR-125b-5p) and MTB genome encoded (MTB-miR5) miRNAs showed increased expression in a cohort of 52 healthy, pulmonary tuberculosis (PTB) and extra pulmonary tuberculosis (EPTB) patients serum samples. Our study identified for the first time a panel of host and MTB genome specific differentially expressed circulating miRNAs in serum samples of an Indian patient cohort with tuberculosis infection with a potential as a non-invasive diagnostic biomarker for tuberculosis infection.

Whole mitochondria genome mutational spectrum in occupationally exposed lead subjects.

Lead is a public health hazard substance affecting millions of people worldwide especially those who are occupationally exposed. Our study aimed to investigate the effect of occupational lead exposure on mitochondria DNA (mtDNA). By sequencing the whole mitochondria genome, we identified 25 unique variants in lead exposed subjects affecting 10 protein coding genes in the order of MT-ND1, MT-ND2, MT-CO2, MT-ATP8, MT-ATP6, MT-CO3, MT-ND3, MT-ND4, MT-ND5, and MT-CYB. Mitochondria functional analysis revealed that exposure to lead can reduce reactive oxygen species (ROS) levels, alter mitochondria membrane potential (MMP) and increase mitochondrial mass (MM). This was further supported by mtDNA copy number analysis which was increased in lead exposed individuals compared to unexposed control group indicating the compensatory mechanism that lead has in stabilizing the mitochondria. This is the first report of mtDNA mutation and copy number analysis in occupationally lead exposed subjects where we identified mtDNA mutation signature associated with lead exposure thus providing evidence for altered molecular mechanism to compensate mitochondrial oxidative stress.