High-throughput sequencing and in-silico analysis confirm pathogenicity of novel MSH3 variants in African American colorectal cancer.

The maintenance of DNA sequence integrity is critical to avoid accumulation of cancer-causing mutations. Inactivation of DNA Mismatch Repair (MMR) genes (e.g., MLH1 and MSH2) is common among many cancers, including colorectal cancer (CRC) and is the driver of classic microsatellite instability (MSI) in tumors. Somatic MSH3 alterations have been linked to a specific form of MSI called elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) that is associated with patient poor prognosis and elevated among African American (AA) rectal cancer patients. Genetic variants of MSH3 and their pathogenicity vary among different populations, such as among AA, which are not well-represented in publicly available databases. Targeted exome sequencing of MSH3 among AA CRC samples followed by computational bioinformatic pipeline and molecular dynamic simulation analysis approach confirmed six identified MSH3 variants (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, c.G3241A) that corresponded to MSH3 amino-acid changes (p.E413K; p.S466N; p.S920F; p.E976K; p.H1010Y; p.E1081K). All identified MSH3 variants were non-synonymous, novel, pathogenic, and show loss or gain of hydrogen bonding, ionic bonding, hydrophobic bonding, and disulfide bonding and have a deleterious effect on the structure of MSH3 protein. Some variants were located within the ATPase site of MSH3, affecting ATP hydrolysis that is critical for MSH3's function. Other variants were in the MSH3-MSH2 interacting domain, important for MSH3's binding to MSH2. Overall, our data suggest that these variants among AA CRC patients affect the function of MSH3 making them pathogenic and likely contributing to the development or advancement of CRC among AA. Further clarifying functional studies will be necessary to fully understand the impact of these variants on MSH3 function and CRC development in AA patients.

Gastrointestinal Manifestations and Their Association with Neurologic and Sleep Problems in Long COVID-19 Minority Patients: A Prospective Follow-Up Study.

BACKGROUND: Long-COVID is a condition post SARS-CoV-2 infection with persistent or recurring symptoms affecting multiple organs, and may involve viral persistence, changes to the microbiome, coagulopathies, and alterations to neuro-immune interactions. These factors can disrupt the Gut-Brain Axis, which is a complex system involving bidirectional communication between the central nervous system and the gastrointestinal (GI) system. As a result of these disruptions, individuals with long-COVID may develop post-infectious functional GI disorders, which can cause a range of symptoms affecting the digestive system. AIM: To understand frequency of GI manifestations of Long-COVID and to determine association with sleep or neurological symptoms in a predominantly minority population. METHODS: We included patients with positive SARS-CoV-2 PCR (n = 747) who were hospitalized from Feb. 2020 to May 2021 at Howard University Hospital and followed between 6 and 12 months from discharge. GI, sleep, and neurological symptoms (via the Montreal Cognitive Assessment (MoCA) scoring system) were assessed using a standardized questionnaire. Linear regression analysis, χ2 and Fisher's exact test were utilized to determine the statistical significance of correlations of GI/Neuro/COVID. RESULTS: The mean age of patients was 58, with 51.6% females and a predominant African American ethnicity (73.6%, n = 550). A total of 108 patients died during their initial hospital stay, with the remaining 639 patients followed-up. Three hundred fifty (350) patients responded to the questionnaire (57 patients died during the follow-up period). Overall, 39 (13.3%) patients reported GI-related symptoms, out of which 19 (6.4%) had persistent symptoms and 20 (6.8%) developed new onset GI symptoms. Nausea and vomiting were the most common 24/39 (61.5%), followed by abdominal pain 7/39 (18%), diarrhea 5/39 (12.8%), and others 3/39 (7.6%). Patients who presented with vomiting during acute SARS-CoV-2 infection were more likely to have Long-COVID GI manifestations (P = 0.023). Use of ACE inhibitors, abnormal lymphocyte count and elevated ferritin are other variables that showed significant associations with Long-COVID GI manifestations (P = 0.03, 0.006 and 0.03, respectively). During follow-up, a total of 28 (9.5%) patients reported difficulty with sleep and 79 (27%) patients had abnormal MoCA assessment. With further analysis, there was a trend between presentation of GI symptoms on admission with abnormal MoCA assessment, and an association between abnormal LFTs and history of liver disease during hospitalization with subsequent sleep problems. Baseline characteristics, clinical comorbidities, other laboratory values, hospital length of stay, mechanical ventilation, medications during hospitalization, re-admission and Flu or COVID-19 vaccination have not shown any association with Long-COVID GI symptoms in our cohort. CONCLUSION: Dyspeptic symptoms were common GI manifestations in the acute and post COVID periods. GI symptoms, abnormal LFTs and a history of liver disease during the acute infectious phase associates with abnormal MoCA and sleep problems during follow-up. Further large population studies are needed to determine if COVID-19 leads to a GI symptoms-associated Long-COVID phenotypes and other symptoms through the Gut-Brain-Axis.

Exploring the Incidence and Associated Risk Factors of Barrett's Esophagus in African Americans: A Retrospective Study.

Background: The prevalence of Barrett's esophagus (BE) in African Americans (AA) is uncertain. However, several potential risk factors, includes family history, male sex, ethnicity, chronic heartburn and acid reflux, age over 60, current or past smoking, and obesity are associated with BE. The aim of this study is to determine the incidence of BE in AA patients who underwent Esophageal Gastro Endoscopy (EGD). Methods: Medical and demographic data of 1,253 AA patients with various symptoms, including BE, Esophageal adenocarcinoma (EAC), esophageal squamous adenocarcinoma (SCC), hiatal hernia, H. pylori infection, Gastro-Esophageal Reflux Disease (GERD), Gastritis, and esophagitis, were collected from January 2004 to December 2014 at Howard University Hospital. Results: Among the 1,253 patients, the median age was 61 and 49% were male out of the total. The frequencies of EAC (p= 0.05), and SCC (p= 0.002) were significantly high in males, along with SCC frequency significantly increased with older age (p<0.001). Furthermore, esophageal polyps with hiatal hernia (p=0.008) and H. pylori (p=<0.001) were found to be associated with esophagitis, and its presence may contribute to the development of BE. Conclusion: The findings highlighted the high prevalence of GERD symptoms and pathologic gastritis along with EAC was less common than SCC and both types of esophageal cancer were associated with male gender and older age whereas, H. pylori infection was identified as a risk factor for pathologic gastritis in AA. Overall data emphasize the need for extensive research, increased awareness, diagnosis, and management of GERD, gastritis, and related conditions to uncover the underlying mechanisms and factors contributing to these conditions in the AA population.

Management of tibial plateau fractures: a fresh review.

Tibial plateau fractures are complex injuries which carry significant morbidity and economic burden. They can present complex geometry depending upon the direction of the force and position of the limb and are often associated with significant soft-tissue injury. While the goals of adequate reduction, stable fixation, and early mobilization remain unchanged, the management of these injuries can be challenging even to experienced orthopaedic surgeons. Lacking consensus, this review aims to provide a summary of current state of orthopaedic practice in the face of tibial plateau fractures. A PubMed search for relevant recent articles as well as a reading of classical articles on tibial plateau fractures was carried out. The focus remained on articles concerned with management modalities and recent advances. A review of some classification systems was also done and included. A great majority of these fractures need operative fixation while respecting the soft tissues. Numerous methods have been reported in the literature including but not limited to plates, screws, external fixators, arthroscopy assisted methods, balloon-cement tibioplasty, or a combination thereof. There is a shortage of randomized controlled trials comparing various operative methods. This article provides a review of various techniques and latest advances made in the management of tibial plateau fractures. The key to achieving optimal functional outcome is using a tailored approach to the individual patient accounting for factors related to the injury pattern, type of host, surgical skills and experience, and local availability of implant devices while taking care of soft tissue. While there is no gold standard, a staged procedure is recommended with early spanning and definitive fixation at later stage by any appropriate methods while respecting the soft tissue, achieving anatomical reduction and adequate fixation and, early rehabilitation.

Microbiomic and Metabolomic Analyses Unveil the Protective Effect of Saffron in a Mouse Colitis Model.

Despite the existence of effective drugs used to treat inflammatory bowel disease (IBD), many patients fail to respond or lose response over time. Further, many drugs can carry serious adverse effects, including increased risk of infections and malignancies. Saffron (Crocus sativus) has been reported to have anti-inflammatory properties. Its protective role in IBD and how the microbiome and metabolome play a role has not been explored extensively. We aimed to establish whether saffron treatment modulates the host microbiome and metabolic profile in experimental colitis. Colitis was induced in C57BL/6 mice with 3% DSS and treated with either saffron in a dose of 20 mg/kg body weight or vehicle through daily gavage. On day 10, stool pellets from mice were collected and analyzed to assess saffron's effect on fecal microbiota and metabolites through 16S rRNA sequencing and untargeted primary metabolite analysis. Saffron treatment maintained gut microbiota homeostasis by counter-selecting pro-inflammatory bacteria and maintained Firmicutes/Bacteroides ratio, which was otherwise disturbed by DSS treatment. Several metabolites (uric acid, cholesterol, 2 hydroxyglutaric acid, allantoic acid, 2 hydroxyhexanoic acid) were altered significantly with saffron treatment in DSS-treated mice, and this might play a role in mediating saffron's colitis-mitigating effects. These data demonstrate saffron's therapeutic potential, and its protective role is modulated by gut microbiota, potentially acting through changes in metabolites.

High Prevalence of Diabetes Among Hospitalized COVID-19 Minority Patients: Data from a Single Tertiary Hospital.

BACKGROUND AND AIM: Type 2 diabetes mellitus (DM) is a common comorbidity in the minority population and is associated with poor outcomes in COVID-19 patients. We hypothesized that COVID-19 patients with pre-existing diabetes mellitus are prone to fatal outcomes compared to non-diabetic patients. We aimed to illustrate the characteristics and outcomes and identify the risk factors for in-hospital mortality of COVID-19 patients with DM. METHODS: In this single-center retrospective study, electronic medical records of hospitalized patients with confirmed COVID-19 diagnosis at Howard University Hospital (HUH) from March 2020 to Dec 2021 were analyzed. Clinical, demographic, and serological information, as well as outcomes, were recorded and analyzed. RESULTS: Among 463 COVID-19 patients, 66.3% (n = 307) were African Americans (AA) and 35.9% (n = 166) had diabetes, with a mean age of 64 years. The majority of the diabetic patients were AA (n = 123, 74.1%) and had a higher mortality rate (n = 26, 74.3%) compared to others. Length of stay in the hospital is significantly more for the diabetic than for the non-diabetic patients (11.3 vs. 8.3 days, p = 0.03). A higher proportion of ICU admission (32.3% vs. 17.9%, p = < 0.001), intubation (17% vs. 11.7%, p = 0.04), and increased mortality (21.1% vs. 12.2%, p = 0.01) were identified in COVID-19 patients with DM than in those with no DM. Among DM patients, non-survivors were older (69.9 vs. 62.9 years). DM patients were more likely to have underlying hypertension (72.3% vs. 43.3%, p = < 0.001), obesity (44.8% vs. 32.1%, p = 0.007), chronic kidney disease (23.6 vs. 11.8%, p = 0.001), and cardiovascular disease (29.5% vs. 14.3%, p = 0.001) than the non-DM patients. HbA1C above 9%, indicating poorly controlled hyperglycemia, was associated with poor outcome among the DM subjects. AST (23.5% vs. 31.3%) and creatinine (61.4% vs. 37.9%) were significantly more elevated in DM COVID-19 patients (all p-values < 0.05). The levels of serum troponin (42.5% vs. 30.9%, p = 0.03), interleukin-6 (67.2 vs. 50%, p = 0.04), ferritin (65.6% vs. 44.6%, p = 0.03), procalcitonin (58.1% vs. 46.1, p = 0.03), and D-dimers (92.8% vs. 86.5%, p = 0.04) were significantly higher in DM patients as compared to those in non-DM COVID-19 patients, indicating more susceptibility of diabetic COVID-19 patients to coagulation dysfunction and inflammatory storm. CONCLUSION: The prevalence of DM is high among hospitalized COVID-19 patients in our cohort. While DM patients have a higher mortality rate and ICU admission than non-DM patients, other factors such as underlying comorbidities, old age, elevated creatinine, AST, serum inflammatory markers, and D-dimer are more significant predictors of fatal outcomes. DM patients had higher metabolic derangements, hypercoagulability, and severe inflammatory response. No significant difference of outcome was noted between DM patients of different races in our cohort. In the diabetic group, it appears that race may not significantly contribute to the observed mortality disparity. This could be attributed to the significant influence of diabetes, which acts as a major effector, potentially overshadowing the significance of race in this context.

Saffron, Its Active Components, and Their Association with DNA and Histone Modification: A Narrative Review of Current Knowledge.

Intensive screening for better and safer medications to treat diseases such as cancer and inflammatory diseases continue, and some phytochemicals have been discovered to have anti-cancer and many therapeutical activities. Among the traditionally used spices, Crocus sativus (saffron) and its principal bioactive constituents have anti-inflammatory, antioxidant, and chemopreventive properties against multiple malignancies. Early reports have shown that the epigenetic profiles of healthy and tumor cells vary significantly in the context of different epigenetic factors. Multiple components, such as carotenoids as bioactive dietary phytochemicals, can directly or indirectly regulate epigenetic factors and alter gene expression profiles. Previous reports have shown the interaction between active saffron compounds with linker histone H1. Other reports have shown that high concentrations of saffron bind to the minor groove of calf thymus DNA, resulting in specific structural changes from B- to C-form of DNA. Moreover, the interaction of crocin G-quadruplex was reported. A recent in silico study has shown that residues of SIRT1 interact with saffron bio-active compounds and might enhance SIRT1 activation. Other reports have shown that the treatment of Saffron bio-active compounds increases γH2AX, decreases HDAC1 and phosphorylated histone H3 (p-H3). However, the question that still remains to be addressed how saffron triggers various epigenetic changes? Therefore, this review discusses the literature published till 2022 regarding saffron as dietary components and its impact on epigenetic mechanisms. Novel bioactive compounds such as saffron components that lead to epigenetic alterations might be a valuable strategy as an adjuvant therapeutic drug.

A Phagosomally Expressed Gene, rv0428c, of Mycobacterium tuberculosis Demonstrates Acetyl Transferase Activity and Plays a Protective Role Under Stress Conditions.

Mycobacterium tuberculosis genome is composed of several hypothetical gene products that need to be characterized for understanding the physiology of bacteria. Rv0428c was one of the 11 proteins exclusively identified within the phagosomal compartment of macrophages infected with mycobacteria and marked as hypothetical. The expression of rv0428c gene was upregulated under acidic and nutritive stress conditions in M. tuberculosis H37Ra, which was supported by potential sigma factor binding sites in the region upstream to the rv0428c gene. The bioinformatics analysis predicted it to be a GCN5- acetyl transferase, belonging to the Histone acetyl transferase (HAT) family. The docking analysis predicted formation of hydrogen bonds and hydrophobic interactions between donor acetyl-co-A and histone H3 tail region. rv0428c gene was cloned and expressed in E. coli. The protein was purified to homogeneity and was fairly stable over a wide range of pH 5.0-9.0 and temperature up to 40 °C. The HAT activity of purified Rv0428c was confirmed by in vitro acetylation assay using recombinant H3 histone expressed in bacteria as substrate, which increased in time dependent manner. The results suggested that it is the second confirmed acetyl transferase in M. tuberculosis H37Rv. Furthermore, rv0428c was over expressed in surrogate host M. smegmatis, which led to enhanced growth rate and altered colony morphology. The expression of rv0428c in M. smegmatis promoted the survival of bacteria under acidic and nutritive stress conditions. In conclusion, Rv0428c, a phagosomal acetyl transferase of M. tuberculosis, might be involved in survival under stress conditions.

DNA methylation mediated downregulation of histone H3 variant H3.3 affects cell proliferation contributing to the development of HCC.

Chromatin alterations brought by histone variants and modifications potentially regulate gene transcription from tumor initiation to progression. Histone H3.3 variant is one such epigenetic player important for disease progression and development. Though many studies have implicated H3.3 role in cancer progression and metastasis, its regulation, importance of specific modifications and chaperones have been not understood yet. We report DNA methylation mediated downregulation of histone H3 variant H3.3 in HCC and a concomitant increase in the level of the H3.2 variant. The loss of H3.3 in cancer tissues correlates with a decrease in the histone modifications associated with active transcription like H3K9/K14/K27Ac and H3K4Me3. The ectopic overexpression of H3.3 and H3.2 did not affect global PTMs and cell physiology, probably owing to the deregulation of specific histone chaperones CAF-1 (for H3.2) and HIRA (for H3.3) as observed in HCC tissues. Notably, knockdown of P150, a subunit of CAF-1 leads to a cell cycle arrest in S-phase in a neoplastic rat liver cell line, possibly due to the decrease in the histone levels necessary for DNA packaging. Remarkably, modulation of H3.3 in pre-neoplastic rat liver cells lead to an increase in cell proliferation and a decreased transcription of tumor suppressor genes, recapitulating the tumor cell phenotype. Our data suggests, inhibition of DNA methylation and histone deacetylation leads to the restoration of histone H3 variant expression in tumor cells.

In-Silico Analysis of Chromatin Modifiers and Profiling of Histone Deacetylases (HDAC's) in Human Oral Cancer.

Histone modifications have been demonstrated to play a significant role in oral squamous cell carcinoma (OSCC) epigenetic regulation. An in-silico analysis of The Cancer Genome Atlas (TCGA) of various histone acetyl transferases (HATs) and histone deacetylases (HDACs) suggested that HATs do not differ between normal and tumor samples whereas HDAC2 and HDAC1 change maximally and marginally respectively between normal and tumor patients with no change being noted in HDAC6 expression. Hence, this investigation was carried out to validate the expression states of HDAC 1, 2 and 6 mRNAs in buccal mucosa and tongue SCC samples in an Indian cohort. Buccal mucosa and tongue squamous cell carcinoma tissues with intact histopathology were processed for RNA isolation followed by cDNA synthesis which was then subjected to q-PCR for HDACs. The average RNA yield of the tongue tissue sample was ∼2 µg/mg of tissue and the A260/280 ratios were between 2.03 and 2.06. The average RNA yield of buccal mucosa tissue sample was ∼1 µg/mg of tissue and the A260/280 ratio were between 2.00 and 2.08. We have demonstrated that HDAC2 was overexpressed in tongue and buccal mucosa samples. Over-expression of HDAC2 imply potential use of HDACi along with standard chemotherapeutic drug in oral cancer treatment.

RPS13, a potential universal reference gene for normalisation of gene expression in multiple human normal and cancer tissue samples.

BACKGROUND: Reference genes are considered stable genes and are used for normalizing the gene expression profile across different cell types; as well as, in normal and diseased samples. However, these gene associates with different biological processes, and hence expression vary in different pathological conditions. Therefore, in the present study, eight different reference genes were used and compared to identify common reference gene usable for an array of different cell types and human cancers. METHODS AND RESULTS: The expression stability of the eight reference genes across eleven normal and cancerous tissues was confirmed through real time-qPCR. Ribosomal protein S13 (RPS13) was found to be a common and stable reference gene across intra- and inter-comparison between various normal and tumor tissue types. Further, TCGA data analysis across and between normal and tumor tissue types also showed minimum deviation in expression of RPS13 gene out of eight routinely used reference genes. CONCLUSION: RPS13 is the common stable reference gene in normalization for gene expression based analysis in cancer research.

Tumor-specific overexpression of histone gene, H3C14 in gastric cancer is mediated through EGFR-FOXC1 axis.

Incorporation of different H3 histone isoforms/variants have been reported to differentially regulate gene expression via alteration in chromatin organization during diverse cellular processes. However, the differential expression of highly conserved histone H3.2 genes, H3C14 and H3C13 in human cancer has not been delineated. In this study, we investigated the expression of H3.2 genes in primary human gastric, brain, breast, colon, liver, and head and neck cancer tissues and tumor cell lines. The data showed overexpression of H3.2 transcripts in tumor samples and cell lines with respect to normal counterparts. Furthermore, TCGA data of individual and TCGA PANCAN cohort also showed significant up-regulation of H3.2 genes. Further, overexpressed H3C14 gene coding for H3.2 protein was regulated by FOXC1 transcription factor and G4-cassette in gastric cancer cell lines. Elevated expression of FOXC1 protein and transcripts were also observed in human gastric cancer samples and cell lines. Further, FOXC1 protein was predominantly localized in the nuclei of neoplastic gastric cells compared to normal counterpart. In continuation, studies with EGF induction, FOXC1 knockdown, and ChIP-qPCR for the first time identified a novel axis, EGFR-FOXC1-H3C14 for regulation of H3C14 gene overexpression in gastric cancer. Therefore, the changes the epigenomic landscape due to incorporation of differential expression H3 variant contributes to change in gene expression pattern and thereby contributing to pathogenesis of cancer.

HISTome2: a database of histone proteins, modifiers for multiple organisms and epidrugs.

BACKGROUND: Epigenetics research is progressing in basic, pre-clinical and clinical studies using various model systems. Hence, updating the knowledge and integration of biological data emerging from in silico, in vitro and in vivo studies for different epigenetic factors is essential. Moreover, new drugs are being discovered which target various epigenetic proteins, tested in pre-clinical studies, clinical trials and approved by the FDA. It brings distinct challenges as well as opportunities to update the existing HIstome database for implementing and applying enormous data for biomedical research. RESULTS: HISTome2 focuses on the sub-classification of histone proteins as variants and isoforms, post-translational modifications (PTMs) and modifying enzymes for humans (Homo sapiens), rat (Rattus norvegicus) and mouse (Mus musculus) on one interface for integrative analysis. It contains 232, 267 and 350 entries for histone proteins (non-canonical/variants and canonical/isoforms), PTMs and modifying enzymes respectively for human, rat, and mouse. Around 200 EpiDrugs for various classes of epigenetic modifiers, their clinical trial status, and pharmacological relevance have been provided in HISTome2. The additional features like 'Clustal omega' for multiple sequence alignment, link to 'FireBrowse' to visualize TCGA expression data and 'TargetScanHuman' for miRNA targets have been included in the database. CONCLUSION: The information for multiple organisms and EpiDrugs on a common platform will accelerate the understanding and future development of drugs. Overall, HISTome2 has significantly increased the extent and diversity of its content which will serve as a 'knowledge Infobase' for biologists, pharmacologists, and clinicians. HISTome2: The HISTone Infobase is freely available on http://www.actrec.gov.in/histome2/ .

Histone deacetylase inhibitor pre-treatment enhances the efficacy of DNA-interacting chemotherapeutic drugs in gastric cancer.

BACKGROUND: The prognosis of gastric cancer continues to remain poor, and epigenetic drugs like histone deacetylase inhibitors (HDACi) have been envisaged as potential therapeutic agents. Nevertheless, clinical trials are facing issues with toxicity and efficacy against solid tumors, which may be partly due to the lack of patient stratification for effective treatments. AIM: To study the need of patient stratification before HDACi treatment, and the efficacy of pre-treatment of HDACi as a chemotherapeutic drug sensitizer. METHODS: The expression activity of class 1 HDACs and histone acetylation was examined in human gastric cancer cells and tissues. The potential combinatorial regime of HDACi and chemotherapy drugs was defined on the basis of observed drug binding assays, chromatin remodeling and cell death. RESULTS: In the present study, the data suggest that the differential increase in HDAC activity and the expression of class 1 HDACs are associated with hypo-acetylation of histone proteins in tumors compared to normal adjacent mucosa tissue samples of gastric cancer. The data highlights for the first time that pre-treatment of HDACi results in an increased amount of DNA-bound drugs associated with enhanced histone acetylation, chromatin relaxation and cell cycle arrest. Fraction-affected plots and combination index-based analysis show that pre-HDACi chemo drug combinatorial regimes, including valproic acid with cisplatin or oxaliplatin and trichostatin A with epirubicin, exhibit synergism with maximum cytotoxic potential due to higher cell death at low combined doses in gastric cancer cell lines. CONCLUSION: Expression or activity of class 1 HDACs among gastric cancer patients present an effective approach for patient stratification. Furthermore, HDACi therapy in pre-treatment regimes is more effective with chemotherapy drugs, and may aid in predicting individual patient prognosis.

Histone H2A isoforms: Potential implications in epigenome plasticity and diseases in eukaryotes.

Epigenetic mechanisms including the post-translational modifications of histones, incorporation of histone variants and DNA methylation have been suggested to play an important role in genome plasticity by allowing the cellular environment to define gene expression and the phenotype of an organism. Studies over the past decade have elucidated how these epigenetic mechanisms are significant in orchestrating various biological processes and contribute to different pathophysiological states. However, the role of histone isoforms and their impact on different phenotypes and physiological processes associated with diseases are not fully clear. This review is focussed on the recent advances in our understanding of the complexity of eukaryotic H2A isoforms and their roles in defining nucleosome organization. We elaborate on their potential roles in genomic complexity and regulation of gene expression, and thereby on their overall contribution towards cellular phenotype and development of diseases.

Elevated HDAC activity and altered histone phospho-acetylation confer acquired radio-resistant phenotype to breast cancer cells.

BACKGROUND: Poor-responsiveness of tumors to radiotherapy is a major clinical problem. Owing to the dynamic nature of the epigenome, the identification and targeting of potential epigenetic modifiers may be helpful to curb radio-resistance. This requires a detailed exploration of the epigenetic changes that occur during the acquirement of radio-resistance. Such an understanding can be applied for effective utilization of treatment adjuncts to enhance the efficacy of radiotherapy and reduce the incidence of tumor recurrence. RESULTS: This study explored the epigenetic alterations that occur during the acquirement of radio-resistance. Sequential irradiation of MCF7 breast cancer cell line up to 20 Gy generated a radio-resistant model. Micrococcal nuclease digestion demonstrated the presence of compact chromatin architecture coupled with decreased levels of histone PTMs H3K9ac, H3K27 ac, and H3S10pK14ac in the G0/G1 and mitotic cell cycle phases of the radio-resistant cells. Further investigation revealed that the radio-resistant population possessed high HDAC and low HAT activity, thus making them suitable candidates for HDAC inhibitor-based radio-sensitization. Treatment of radio-resistant cells with HDAC inhibitor valproic acid led to the retention of γH2AX and decreased H3S10p after irradiation. Additionally, an analysis of 38 human patient samples obtained from 8 different tumor types showed variable tumor HDAC activity, thus demonstrating inter-tumoral epigenetic heterogeneity in a patient population. CONCLUSION: The study revealed that an imbalance of HAT and HDAC activities led to the loss of site-specific histone acetylation and chromatin compaction as breast cancer cells acquired radio-resistance. Due to variation in the tumor HDAC activity among patients, our report suggests performing a prior assessment of the tumor epigenome to maximize the benefit of HDAC inhibitor-based radio-sensitization.

Establishing a correlation between RIN and A260/280 along with the multivariate evaluation of factors affecting the quality of RNA in cryopreserved cancer bio-specimen.

Biomarkers are biological characteristic to measure and evaluate normal and pathological states. To define expression-based molecular biomarkers, high-quality tissue samples are a prerequisite for the preparation of standard RNA. It is already known that RIN number defines the RNA quality, however its relation with A260/280 ratio and Ct value is not defined clearly; therefore, understanding an association will provide a reliable method for describing RNA quality. Multiple cryopreserved human tumor tissue types from ACTREC Biorepository and TMH-INTTR were analyzed for the effect of storage time on RNA quality. The RNA from tumor samples were isolated and analyzed by RIN, A260/280 ratio, and Ct value to establish inter-relationships. Around 50% samples had a RIN of ≥ 6.9 and A260/280 ≤ 2.04; 27% had a RIN ≥ 5 and A260/280 ≤ 2.08, and remaining 23% displayed RIN < 5 and A260/280 > 2.08. However, the RNA quality has no association with the storage period of tissue samples. Moreover, all samples which had A260/280 ≤ 2.08 showed acceptable Ct values of 17-24. The data clearly suggests that the A260/280 ratio is able to predict RNA quality. To the best of our knowledge, this is the first Indian report analyzing the labile nucleic acid-RNA quality from different cancer tissue types cryopreserved for diverse time periods. In conclusion, RIN and A260/280 ratio can help in predicting the quality of RNA independently; however, both together provide better assurance for further downstream processing.