Correlation of hsa miR-101-5p and hsa miR-155-3p Expression With c-Fos in Patients of Oral Submucous Fibrosis (OSMF) and Oral Squamous Cell Carcinoma (OSCC).

Aim: MicroRNAs have been widely acknowledged as a diagnostic, prognostic, and/or therapeutic biomarker for the progression of OSCC, but the correlation of hsa-miR-101-5p and hsa-miR-155-3p is yet to be established with c-Fos in OSCC and OSMF. Methodology: An observational study enrolled 40 patients divided into 2 groups: Group I-21 OSMF patients without malignant transformation, Group II-19 patients with locally advanced, large-operable, or metastatic OSCC, after applying inclusion and exclusion criteria. Both miRNAs were extracted and analyzed from the tissue sample excised from the involved site. The linear regression analysis of the expression of hsa-miR-155-3p, hsa-miR-101-5p, and levels of c-fos in OSMF and OSCC patients and its correlation for habits, age, and gender were evaluated. Results: The expression of hsa-miR-101-5p was 0.81 times downregulated in OSCC tissue compared to OSMF, whereas hsa-miR-155-3p and c-fos were both upregulated 9.30 times and 1.75 times, respectively, in OSCC tissue. In Gutkha and tobacco chewers, the hsa-miR-155-3p expression could explain 12.3% (p = 0.031) for Gutkha chewers, whereas c-fos could explain 38.6% of the cases (p = 0.020) for tobacco chewers. The expression of hsa-miR-101-5p and hsa-miR-155-3p explained 43.7% and 59.5% of OSCC cases in alcoholics, respectively. Interestingly, in non-alcoholics, hsa-miR-155-3p and hsa-miR-101-5p were significant predictors of OSCC. Conclusion: Downregulation of tumor-suppressor hsa-miR-101-5p and upregulation of proto-onco hsa-miR-155-3p is responsible for intricate regulation of the progression of OSMF to OSCC via deregulated expression of c-Fos and tobacco chewing and advancing age is significant contributors for OSCC.

The role of nanocomposites against biofilm infections in humans.

The use of nanomaterials in several fields of science has undergone a revolution in the last few decades. It has been reported by the National Institutes of Health (NIH) that 65% and 80% of infections are accountable for at least 65% of human bacterial infections. One of their important applications in healthcare is the use of nanoparticles (NPs) to eradicate free-floating bacteria and those that form biofilms. A nanocomposite (NC) is a multiphase stable fabric with one or three dimensions that are much smaller than 100 nm, or systems with nanoscale repeat distances between the unique phases that make up the material. Using NC materials to get rid of germs is a more sophisticated and effective technique to destroy bacterial biofilms. These biofilms are refractory to standard antibiotics, mainly to chronic infections and non-healing wounds. Materials like graphene and chitosan can be utilized to make several forms of NCs, in addition to different metal oxides. The ability of NCs to address the issue of bacterial resistance is its main advantage over antibiotics. This review highlights the synthesis, characterization, and mechanism through which NCs disrupt Gram-positive and Gram-negative bacterial biofilms, and their relative benefits and drawbacks. There is an urgent need to develop materials like NCs with a larger spectrum of action due to the rising prevalence of human bacterial diseases that are multidrug-resistant and form biofilms.

Analyzing the Association of Visceral Adipose Tissue Growth Differentiation Factor-15 and MicroRNA in Type 2 Diabetes Mellitus.

Background: Growth differentiation factor-15 (GDF-15) is involved in insulin resistance and diabetes. In this study, we determine the associations of GDF-15 with miR-181b-5p, miR-330-3p, mothers against decapentaplegic homolog 7 (SMAD7), and insulin resistance in visceral adipose tissue (VAT) and peripheral blood mononuclear cells (PBMCs) in type 2 diabetes mellitus (T2DM) patients. Methods: Sixty patients, equally divided into those with T2DM and non-diabetic controls, were recruited for gene expression analysis. Protein-protein interaction (STRING), target prediction (miRNet), and functional enrichment were conducted accordingly. Results: Our study showed that VAT and PBMCs had similar expression profiles, where GDF-15 and miR-181b-5p were upregulated, whereas SMAD7 and miR-330-3p were downregulated. Serum GDF-15 could differentiate between T2DM and non-diabetic patients (P<0.001). Target prediction revealed a microRNA (miRNA)-messenger RNA regulatory network, transcription factors, and functional enrichment for the miRNA that suggested involvement in T2DM pathogenesis. Conclusion: VAT GDF-15 is associated with insulin resistance and is possibly regulated by miR-181b-5p, miR-330-3p, and SMAD7 in T2DM.

Acute kidney injury in COVID 19 - an update on pathophysiology and management modalities.

Acute kidney injury (AKI), characterised by fluid imbalance and overload, is prevalent in severe disease phenotypes of coronavirus disease 2019 (COVID-19). The elderly immunocompromised patients with pre-existing comorbidities being more risk-prone to severe COVID-19, the importance of early diagnosis and intervention in AKI is imperative. Histopathological examination of COVID-19 patients with AKI reveals viral invasion of the renal parenchyma and evidence of AKI. The definitive treatment for AKI includes renal replacement therapy and renal transplant. Immunosuppressant regimens and its interactions with COVID-19 have to be further explored to devise effective treatment strategies in COVID-19 transplant patients. Other supportive strategies for AKI patients include hemodynamic monitoring and maintenance of fluid balance. Antiviral drugs should be meticulously monitored in the management of these high-risk patients. We have focussed on the development of renal injury provoked by the SARS-CoV-2, the varying clinical characteristics, and employment of different management strategies, including renal replacement therapy, alongside the emerging cytokine lowering approaches.

A clinical and in-silico study of microRNA-21 and growth differentiation factor-15 expression in pre-diabetes, type 2 diabetes and diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN), a microvascular complication associated with long-standing diabetes, is a major cause of the end-stage renal disease (ESRD). Our in-silico analysis indicates several enrichment analyses involved in glucose metabolism to be affected by GDF15 transcription factors. METHODS: In-silico analysis was used to identify GDF15 and Insulin related protein-protein interaction (PPI) network and a common set of GDF15 regulating transcription factors by various databases. Common targeting miRNA of GDF15 regulating transcription factors were investigated in miRNet and TargetScan. Further, healthy controls (N.=30) and patients with pre-type-2 diabetes mellitus (pre-diabetes) (N.=30), T2DM (N.=30) and DN (N.=30) were included for analysis of routine biochemical tests, serum GDF15 levels by ELISA and to evaluate the Fold change expression (FCE) of circulating hsa-miR-21 by RT-PCR. RESULTS: MicroRNA-21 was found to directly target GDF15 downregulating transcription factors KLF4, TP53, and CEBPB. A significant difference in the levels of serum GDF15 was observed in Pre-diabetes (708.56±76.37), T2DM (1528.87±140.75) and DN patients (10-fold higher; 5507.90±503.88) when compared to healthy controls (567.36±69.99). The FCE of circulating hsa-miR-21 was 6.19 (pre-diabetes), 8.22 (T2DM), 9.19 (DN), folds higher in cases as compared to controls, reflecting an increasing trend and several folds higher levels of hsa-miR-21 in patients. CONCLUSIONS: We suggest the potential of serum GDF15 and circulating-hsa-miR-21 to serve as clinically important biomarkers and therapeutic targets for controlling advancement of diabetes to DN.

Expression of human cardiac-specific genes: a novel method for post-mortem interval estimation.

In legal medicine, the determination of post-mortem interval (PMI) is not only an important but also one of the most difficult aspects. Several methods are used to estimate PMI such as physicochemical, entomological, biochemical, metabolic, autolytic, and physical methods. These methods provide a wide range of PMI as they are affected by different factors. The approach behind the present study is to calculate an accurate PMI by using mRNA degradation and fold change expression (FCE) of cardiac-specific genes viz. N-terminal pro-B-type natriuretic peptide (NPPB) and cardiac troponin I (TNNI3). Seventeen cadaver heart tissues were analysed within a time frame of up to 12 hours from the time since death, at different time intervals at room temperature. Gene expression was determined and the data were analysed using the value of average delta Ct (ΔCt) value of the assessed gene and housekeeping gene. Delta delta Ct (ΔΔCt) method was used to calculate the FCE at the different 7-time groups. The FCE of TNNI3 was almost stable till 15 hours of PMI and then after 15 hours, expression shows a decrease up to 24 hours after death; whereas, NPPB shows that FCE was stable till 12 hours of PMI and then after 12 hours, expression shows a decrease up to 24 hours after death. The FCE of NPPB and TNNI3 was almost stable till 12 hours. Thus, the estimation of PMI by analysis of the FCE of cardiac-specific genes can be a new promising method in forensic medicine.

Growth Differentiation Factor-15 as a Candidate Biomarker in Gynecologic Malignancies: A Meta-analysis.

Growth differentiation factor-15 (GDF-15), though emerged as a novel marker in gynecological cancers, is yet to be recognized in clinical diagnostics. Eligible studies were sorted from multiple online databases, namely PubMed, Cochrane, ClinicalTrials.gov, Google Scholar, Web of Science, Embase, Scopus, LILACS, and Opengrey. From six studies, histopathologically diagnosed cases without prior treatment, and with diagnostic accuracy data for GDF-15 in gynecological cancers, were included. Our meta-analysis shows that GDF-15 has pooled diagnostic odds ratio of 12.74 at 80.5% sensitivity and 74.1% specificity, and an area under the curve of 0.84. Hence, GDF-15 is a potential marker to differentiate gynecological malignancy from non-malignant tumors.

Visceral Adipose Tissue Molecular Networks and Regulatory microRNA in Pediatric Obesity: An In Silico Approach.

Childhood obesity carries an increased risk of metabolic complications, sleep disturbances, and cancer. Visceral adiposity is independently associated with inflammation and insulin resistance in obese children. However, the underlying pathogenic mechanisms are still unclear. We aimed to detect the gene expression pattern and its regulatory network in the visceral adipose tissue of obese pediatric individuals. Using differentially-expressed genes (DEGs) identified from two publicly available datasets, GSE9624 and GSE88837, we performed functional enrichment, protein-protein interaction, and network analyses to identify pathways, targeting transcription factors (TFs), microRNA (miRNA), and regulatory networks. There were 184 overlapping DEGs with six significant clusters and 19 candidate hub genes. Furthermore, 24 TFs targeted these hub genes. The genes were regulated by miR-16-5p, miR-124-3p, miR-103a-3p, and miR-107, the top miRNA, according to a maximum number of miRNA-mRNA interaction pairs. The miRNA were significantly enriched in several pathways, including lipid metabolism, immune response, vascular inflammation, and brain development, and were associated with prediabetes, diabetic nephropathy, depression, solid tumors, and multiple sclerosis. The genes and miRNA detected in this study involve pathways and diseases related to obesity and obesity-associated complications. The results emphasize the importance of the TGF-ß signaling pathway and its regulatory molecules, the immune system, and the adipocytic apoptotic pathway in pediatric obesity. The networks associated with this condition and the molecular mechanisms through which the potential regulators contribute to pathogenesis are open to investigation.

ABC transporters in breast cancer: their roles in multidrug resistance and beyond.

ATP-binding cassette (ABC) transporters are membrane-spanning proteins involved in cholesterol homeostasis, transport of various molecules in and out of cells and organelles, oxidative stress, immune recognition, and drug efflux. They are long implicated in the development of multidrug resistance in cancer chemotherapy. Existing clinical and molecular evidence has also linked ABC transporters with cancer pathogenesis, prognostics, and therapy. In this review, we aim to provide a comprehensive update on all ABC transporters and their roles in drug resistance in breast cancer (BC). For solid tumours such as BC, various ABC transporters are highly expressed in less differentiated subtypes and metastases. ABCA1, ABCB1 and ABCG2 are key players in BC chemoresistance. Restraining these transporters has evolved as a possible mechanism to reverse this phenomenon. Further, ABCB1 and ABCC1 are important in BC prognosis. Newer therapeutic approaches have been developed to target all these molecules to dysregulate their effect, reduce cell viability, induce apoptosis, and increase drug sensitivity. In the future, targeted therapy for specific genetic variations and upstream or downstream molecules can help improve patient prognosis.

Serial estimation of gene expression of cardiac troponin I (cTnI) and autophagy gene HMGB1 to determine postmortem interval.

The estimation of accurate post mortem interval (PMI) is a crucial question in forensic medicine. Several approaches have been used to determine the PMI including physical, metabolic, autolytic, entomological, physiochemical and biochemical methods over time. For estimation of PMI, RNA degradation after death is reported to be an important tool. This study aimed to analyse the pattern of gene expression by serial estimation of cardiac specific cardiac troponin I (cTnI) gene and autophagy gene HMGB1 for determining PMI at room temperature by using housekeeping gene GAPDH. Right ventricular heart tissue weighing 10 g was collected and harvested from 17 medico-legal autopsies. The tissue was homogenized in phosphate-buffered saline (PBS) on ice. Further, homogenate of cardiac tissue was analysed by quantitative Real time polymerase chain reaction (qRtPCR) for gene amplification and gene expression of cTnI, HMGB1 gene and GAPDH, at different time intervals (0,6,12 h) at room temperature. The result revealed ∆Ct value of cTnI gene of the cardiac muscle showing almost equal degradation at equal time interval correlated with PMI within 0-12 h at room temperature, and the ∆Ct value of HMGB1 degraded to half in every subsequent 6-hour interval at room temperature. In conclusion, the estimation of PMI by analysis of serial estimation of gene expression is a decent new tool in forensic medicine. The study shows an equal degradation of cTnI gene at equal time interval and HMGB1 degrades to half at six-hour interval. Therefore, these can be useful for estimation for PMI.

FOXM1 mediates GDF-15 dependent stemness and intrinsic drug resistance in breast cancer.

BACKGROUND: Stemness, a key component of breast cancer (BC) heterogeneity, is responsible for chemoresistance. Growth differentiation factor-15 (GDF-15) induces drug resistance and stemness in BC cells. In this study, the expressions and interactions of GDF-15, FOXM1, and stemness (OCT4 and SOX2), and drug resistance (ABCC5) markers were evaluated in BC. METHODS AND RESULTS: 40 diagnosed BC patients and 40 healthy controls were included in this study. Serum GDF-15 was significantly raised (p < 0.001) in BC patients. Expressions of GDF-15, OCT4, SOX2, and FOXM1 in BC tissue and cell lines (MCF-7 and MDA-MB-231) were determined by RT-PCR, while phosphorylated AKT (p-AKT) was analyzed by Western blot. Not only were the fold change expressions higher in cancer tissue as compared to surrounding control tissue, but a higher expression was observed for all the genes along with p-AKT in MDA-MB-231 cells compared to MCF-7. Tissue GDF-15 was significantly associated with ABCC5 (p < 0.001), OCT4 (p = 0.002), SOX2 (p < 0.001), and FOXM1 (p < 0.001). To further analyze the signaling pathway involved in stemness and drug resistance in BC, GDF-15 knockdown was performed, which reduced the expression of p-AKT, FOXM1, OCT4 and SOX2, and ABCC5, whereas recombinant GDF-15 treatment reversed the same. In silico analyses in UALCAN revealed a similar picture for these genes to that of BC tissue expression. CONCLUSIONS: GDF-15 promotes stemness and intrinsic drug resistance in BC, possibly mediated by the p-AKT/FOXM1 axis.

In-Silico Analysis of Differentially Expressed Genes and Their Regulating microRNA Involved in Lymph Node Metastasis in Invasive Breast Carcinoma.

Axillary nodal metastasis is related to poor prognosis in breast cancer (BC). Key candidate genes in BC lymph node metastasis have been identified from Gene Expression Omnibus datasets and explored through functional enrichment database for annotation, visualization and integrated discovery (DAVID) , protein-protein interaction by Search Tool for the Retrieval of Interacting Genes and proteins (STRING), network visualization (Cytoscape), survival analysis (GEPIA, KM Plotter), and target prediction (miRNet). A total of 102 overlapping differentially expressed genes were found. In-silico survival and expression analyses revealed six candidate hub genes, Desmocollin 3 (DSC3), KRT5, KRT6B, KRT17, KRT81, and SERPINB5, to be significantly associated with nodal metastasis and overall survival, and 83 MicroRNA (miRNAs), which may be potential diagnostic markers and therapeutic targets in BC patients.

Growth Differentiation Factor-15 as a Biomarker of Obese Pre-diabetes and Type 2 Diabetes Mellitus in Indian Subjects: A Case-control Study.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is an ever-growing epidemic in India and poses significant morbidity, mortality, and socioeconomic burden. INTRODUCTION: Growth differentiation factor-15 (GDF15) is a stress-responsive cytokine, increased in T2DM patients compared to control subjects without the disease. We aimed to assess whether serum GDF15 and adipose tissue GDF15 expression can differentiate between obese pre-diabetes and T2DM and control populations. METHODOLOGY: We recruited 156 individuals including 73 type 2 diabetes, 30 pre-diabetes, and 53 healthy controls. Clinical history, anthropometric measurements and biochemical profiling were taken. Insulin resistance indices were calculated following HOMA models. Serum GDF15 was measured by sandwich ELISA. Visceral adipose tissue (VAT) expression of GDF15 was observed in 17 T2DM patients and 29 controls using SYBR Green chemistry in RT-PCR using GAPDH as the housekeeping gene. The data were analyzed on R programming platform using RStudio. RESULTS: Serum GDF15 was significantly higher (p<0.001) in T2DM subjects (median 1445.47 pg/mL) compared to pre-diabetes (627.85 pg/mL) and healthy controls (609.01 pg/mL). Using the ΔΔCt method, the VAT GDF15 expression was 1.54 fold and 1.57 fold upregulated in T2DM (n=17) compared to control subjects (n=29), and obese (n=12) compared to non-obese (n=34)subjects, respectively. The optimal cut-off point following Youden's index method was found to be 868.09 pg/mL. ROC curve analysis revealed that serum GDF15 had a sensitivity, specificity, and area under the curve (AUC) of 90.41%, 79.52%, and 0.892 respectively. GDF15 levels were significantly associated with age, BMI, HbA1c, fasting blood sugar, and insulin resistance indices. CONCLUSION: Hence, serum GDF15 is a biomarker for T2DM patients in our study population from Western India. However, larger prospective cohorts are necessary to validate this claim.

Critical appraisal of epigenetic regulation of galectins in cancer.

Galectins are defined as the glycan-binding protein containing either one or two carbohydrate-binding domains and participate in various biological functions such as developmental processes, vascularisation programs, cell migration, and immune-regulation and apoptosis. Galectins are also linked to many diseases, including cancer. They are widely spread in extracellular and intracellular spaces, and their altered expression in cancer leads to tumor progression, metastasis, angiogenesis and stemness through different signalling pathways. Promoter methylation, microRNA, and histone modification constitute the epigenetic changes that regulate galectin activity in cancer. Our review discusses the concept of epigenetics in cancer and how the aforementioned factors i.e., promoter methylation, histone modification, change in miRNAs expression affect the glycomic changes in malignancies.

A clinical and in-silico study exploring the association of CASP-3, NF-kB, miR-187, and miR-146 in pre-eclampsia.

INTRODUCTION: Apoptosis is involved in pathogenesis of Pre-eclampsia (PE), further research is needed to determine its molecular mechanism. METHODS: The study recruited two groups (controls; 09, PE; 11). Biochemical tests, RT-PCR and ELISA were employed for analysis of genes and MicroRNAs (miRNA). Bioinformatics tools were employed for interactomics analysis. RESULTS: There was increased apoptosis in maternal placental tissue (MPT) and Maternal Blood Cells (MBC) as demonstrated by expression of CASP3 and NF-κB1. miR-146-5p and 187-5p were downregulated in MBC and MPT but upregulated in fetal placental tissue (FPT).. DISCUSSION: An increased apoptosis in MBC and MPT is a significant contributory factor for PE in pregnancy, while FPT is immune to the aforementioned effects.

A Combined Analysis of Serum Growth Differentiation Factor-15 and Cancer Antigen 15-3 Enhances the Diagnostic Efficiency in Breast Cancer.

INTRODUCTION: Existing diagnostic biomarkers of breast cancer (BC) are limited by poor sensitivity. In this study, we evaluated the role of serum GDF-15 in early BC diagnosis, independently and in combination with CA15-3, a known blood biomarker of BC. MATERIAL AND METHODS: A total of 113 diagnosed, pre-therapy BC patients and 54 healthy controls were recruited. Clinical characteristics, TNM staging, and hormone receptor status of the patients were recorded. Serum GDF-15 and serum CA15-3 were measured by sandwich ELISA and chemiluminescence assay, respectively. RESULTS: The serum GDF-15 levels were significantly (p<0.001) elevated in BC patients compared to healthy controls and in patients with larger tumor size, advanced disease stage, and distant metastasis. ROC analysis revealed that at the cut-off of 525.77 pg/mL, GDF-15 had greater sensitivity than CA15-3. GDF-15 and CA15-3 performed better in combination than individually, with the combined test having an AUC of 0.85 and sensitivity and specificity of 0.63 and 0.98, respectively.Further, serum GDF-15 had a better predictive ability for early-stage BC compared to CA15-3. GDF-15 could independently diagnose BC patients after adjusting for age. CONCLUSION: We conclude that serum GDF-15 is a promising, robust marker for detecting early-stage BC. However, larger prospective studies are necessary to validate this claim.

Leptin DNA Methylation and Its Association with Metabolic Risk Factors in a Northwest Indian Obese Population.

BACKGROUND: It is well established that obesity is a major health risk in diabetes and associated diseases. Epigenetic changes, specially DNA methylation, play an important role in regulation of adipokines. The objective of the present study was to evaluate the DNA methylation status at the promoter region of the leptin gene in obese individuals and its association with metabolic risk factors. METHODS: The study included obese (n=100) and non-obese (n=75) individuals aged 25-45 years, and measured their physical, biochemical parameters (glucose, insulin, and lipid profiles) and leptin, DNA methyltransferase 1 (DNMT1), and DNA methyltransferase 3 beta (DNMT3b) mRNA expressions with real-time reverse transcription-polymerase chain reaction (qRT-PCR). DNA methylation of the leptin gene at the promoter region was analyzed by methyl-specific qPCR . RESULTS: The study found that the DNA methylation level at the promoter area of the leptin gene was negatively associated with weight in obese subjects. Furthermore, study findings showed that the DNA methylation level was negatively associated with fasting insulin, glucose, homeostatic model assessment for insulin resistance, and total cholesterol. There was also a higher expression of DNMT1 and DNMT-3b in obese subjects as compared with non-obese subjects. CONCLUSION: The leptin epigenetic profile may be associated with obesity and its associated metabolic risk factors.

MicroRNA 21 Emerging Role in Diabetic Complications: A Critical Update.

BACKGROUND: Diabetes Mellitus is a multifactorial disease encompassing various pathogenic pathways. To avoid morbidity and mortality related to diabetic complications, early detection of disease complications as well as targeted therapeutic strategies are essential. INTRODUCTION: MicroRNAs (miRs) are short non-coding RNA molecules that regulate eukaryotic posttranscriptional gene expression. MicroRNA-21 has diverse gene regulatory functions and plays a significant role in various complications of Type 2 diabetes mellitus (T2DM). METHODS: The study included electronic database searches on Pubmed, Embase, and Web of Science with the search items MicroRNA21 and each of the diabetic complications. The search was carried out up to November, 2019. RESULTS: MicroRNA-21 modulates diabetic cardiomyopathy by affecting vascular smooth muscle cell proliferation and apoptosis, cardiac cell growth and death, and cardiac fibroblast functions. At the renal tubules, miR-21 can regulate the mesangial expansion, interstitial fibrosis, macrophage infiltration, podocyte loss, albuminuria and fibrotic and inflammatory gene expression related to diabetic nephropathy. Overexpression of miR-21 has been seen to play a pivotal role in the pathogenesis of diabetic retinopathy by contributing to diabetes-induced endothelial dysfunction as well as low-grade inflammation. CONCLUSION: Considering the raised levels of miR-21 in various diabetic complications, it may prove to be a candidate biomarker for diabetic complications. Further, miR-21 antagonists have shown great potential in the treatment of diabetic cardiomyopathy, diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy related complications in the future. The current review is the first of its kind encompassing the roles miR-21 plays in various diabetic complications, with a critical discussion of its future potential role as a biomarker and therapeutic target.

Optimising total RNA quality and quantity by phenol-chloroform extraction method from human visceral adipose tissue: A standardisation study.

A multitude of challenges is faced during RNA extraction from human visceral adipose tissue (VAT) due to its atypical nature and a dearth of existing literature. Our study provides a convenient and inexpensive manual method using TRIzol reagent for the reproducible recovery of intact RNA from sparse human VAT samples. Fifty-two (52) samples were grouped and tested for the effect of different factors viz. initial VAT amount, TRIzol volume per unit tissue mass, residual fat following homogenisation and first centrifugation, an additional chloroform wash, and an additional ethanol wash on the extraction process. We found that increasing initial tissue mass and decreasing TRIzol volume simultaneously improved RNA yield and purity. A fat layer removal step and additional ethanol wash further propel the A260/280 and A260/230 to their desired values. Our modifications in the isolation protocol were combined and tested through reverse transcriptase quantitative PCR, which yielded consistent results, upholding our optimisation.

Perspectives on the role of PTEN in diabetic nephropathy: an update.

Phosphatase and tensin homolog (PTEN) is a potent tumor suppressor gene that antagonizes the proto-oncogenic phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway and governs basic cellular metabolic processes. Recently, its role in cell growth, metabolism, architecture, and motility as an intramolecular and regulatory mediator has gained widespread research interest as it applies to non-tumorous diseases, such as insulin resistance (IR) and diabetic nephropathy (DN). DN is characterized by renal tubulointerstitial fibrosis (TIF) and epithelial-mesenchymal transition (EMT), and PTEN plays a significant role in the regulation of both. Epigenetics and microRNAs (miRNAs) are novel players in post-transcriptional regulation and research evidence demonstrates that they reduce the expression of PTEN by acting as key regulators of autophagy and TIF through activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway. These regulatory processes might play an important role in solving the complexities of DN pathogenesis and IR, as well as the therapeutic management of DN with the help of PTEN K27-linked polyubiquitination. Currently, there are no comprehensive reviews citing the role PTEN plays in the development of DN and its regulation via miRNA and epigenetic modifications. The present review explores these facets of PTEN in the pathogenesis of IR and DN.