Identification of suitable reference genes in blood samples of carcinoma lung patients using quantitative real-time polymerase chain reaction.

INTRODUCTION: Lung cancer (LC), among all other cancers, is the leading cause of death worldwide, while the third most common cancer-causing mortality in India. Several techniques of the assay for early detection of cancer that improve survival rates have been employed in tissues and cell lines. Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) is one of the most common techniques employed for gene expression studies for the normalization of a target gene using a reference gene (RG). The present study used the three most common RGs: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-Actin, and 18s ribosomal ribonucleic acid (18s rRNA), which were assessed by qPCR to validate, as of which is a more effective RG in blood samples of LC patients. MATERIALS AND METHODS: A total of thirty participants with LC of non-small cell and small cell type were included along with twenty healthy controls. Ribonucleic acid (RNA) isolated from peripheral blood mononuclear cells was quantified, prepared for complementary deoxyribose nucleic acid synthesis, and analyzed for expression of three RG on RTqPCR. RESULTS: Expression levels as Ct values of studied RG were reported as mean ± standard deviation for GAPDH (26.97 ± 5.107), ß-actin (20.5 ± 2.3), and 18s rRNA (25.10 ± 4.075). GAPDH showed the lowest expression, whereas ß-actin showed the highest expression among the studied RG in subjects of LC. The expression of GAPDH and 18s rRNA were statistically significantly lower than ß-actin (p < 0.0001), whereas expression levels of GAPDH and 18s rRNA were comparable. However, the expression level of only ß-actin in LC patients was comparable with healthy controls with P < 0.1611 at 95% confidence interval. CONCLUSION: It is concluded that ß -actin may be considered the most suitable RG isolated and studied from peripheral blood mononuclear cells using RT qPCR in LC.

Association of antibiotics and heavy metal arsenic to horizontal gene transfer from multidrug-resistant clinical strains to antibiotic-sensitive environmental strains.

The emergence of multidrug-resistant bacteria is currently posing a significant threat to global public health. By testing for resistance to different antibiotic classes, we discovered that the majority of clinical bacteria are multidrug-resistant. These clinical multidrug-resistant species have antibiotic resistance genes on their plasmids that can be horizontally transferred to various antibiotic susceptible environmental bacterial species, resulting in antibiotic-resistant transconjugates. Furthermore, we discovered that the presence of an optimal concentration of antibiotics or heavy metal (arsenic) facilitates horizontal gene transfer through the formation of transconjugants. Notably, the addition of a conjugation inhibitor (2-hexadecynoic acid, a synthetic fatty acid) completely blocked the formation of antibiotic or arsenic-induced transconjugants. We discovered a high level of arsenic in water from the Shukratal region, Uttarakhand, India, which corresponded to a high serum level of arsenic in clinically infected individuals from the Shukratal region compared to other locations in Uttarakhand. Importantly, bacterial strains isolated from infected people who drink water from the Shukratal region with high arsenic levels were found to be more antibiotic-resistant than strains isolated from other sites. We discovered that bacterial strains isolated from individuals with high serum arsenic levels are significantly more resistant to antibiotics than individuals with low serum arsenic levels within the Shurkratal. This research sheds light on imminent threats to global health in which improper clinical, industrial, and other waste disposal, increased antibiotic concentrations in the environment, and increased human interference can easily transform commensal and pathogenic bacteria found in environmental niches into life-threatening multidrug-resistant superbugs.

Genetic Variations in IL-1ß, TNF-α, and TGF-ß Associated with the Severity of Chronic Cervical Spondylitis in Patients.

Chronic cervical spondylitis (CCS), a degenerative disorder of the spine, is known for causing disability among old and young people. Single-nucleotide polymorphisms (SNPs) in various cytokine genes have demonstrated an impactful association with several inflammatory disorders. In the present study, we have investigated the SNPs and allelic distribution of the three most prevalent cytokines genes, IL-1ß (-511C/T), TNF-α (-308G/A), and TGF-ß (-509C/T), along with serum levels of these cytokines in 252 subjects. SNPs were analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and digested fragments were separated and visualized using agarose gel electrophoresis and Native Polyacrylamide gel electrophoresis (PAGE). The serum cytokine levels were analyzed with a flow cytometer using a customized multiplex bead-based assay. It was observed that these SNPs did not reflect the susceptibility to CCS but were associated with susceptibility to CCS. We found a significant association between the C/C and G/G genotypes and the C and G alleles of IL-1ß and TNF-α, respectively, suggesting a lower risk of CCS. The frequency distribution of risk alleles (-511T) and (-308A) were simultaneously higher in CCS compared to the control, reflecting the susceptibility to CCS. TGF-ß showed a significant association with disease susceptibility, along with a significant correlation between age and the chronicity of CCS. The serum cytokine levels were significantly different in CCS and controls.

Efficacy of Young Cinnamomum zeylanicum Blume Bark on Hyperglycemia and PTPase Activity in Type 2 Diabetes.

Diabetes is a major public health concern and natural easy-going remedies are being searched. Since Cinnamomum zeylanicum Blume has a low coumarin concentration and possible insulin-enhancing properties, it is preferred over all other cinnamon species. Although similar research has been done on humans, there have been very few studies on this particular species, and none among South Asians. Moreover, no human trial that properly described their intervening agent (C. zeylanicum) and checked its efficacy at the molecular level along with clinical variables was conducted. Therefore, the current research aimed to explore the effects of C. zeylanicum on the glycemic index, lipid profile, and expression of the protein tyrosine phosphatase 1 B (PTP1B) enzyme in the peripheral blood mononuclear cells (PBMC) in type 2 diabetes. We examined the presence of bioactive compounds in young C. zeylanicum bark (Alba grade) from native Sri Lanka using gas chromatography-mass spectrometry, high-performance thin-layer chromatography, and thin-layer chromatography before introducing it in the clinical study where trans-Cinnamaldehyde was found to be a major chemical constituent (>60%). Then, from January 2020 to March 2022, a randomized double-blinded placebo-controlled trial was carried out in the Diabetic Clinic at AIIMS Rishikesh. A total of 154 diabetic patients were enrolled and were taken either cinnamon or placebo capsules (1.5 g/day) for 120 days on an empty stomach with warm water along with their conventional treatment. Reduction in fasting blood glucose levels in the cinnamon group was found -35.50% (95% CI, -173 to 58.4), whereas in the placebo group change was 5.00% (95% CI, -165 to 224). For glycosylated hemoglobin, it differed -0.85% (95% CI, -8.2 to 1.6) in the cinnamon group compared to the placebo where it was found 0.15% (95% CI, -6.1 to 5.5). PTP1B expression in PBMC was determined from pre- and post-trial blood samples using the Western Blot, and significant inhibition was also observed (p=0.039). The study result depicts, C. zeylanicum is emerging as a beneficial plant for type 2 diabetes in Northern India and could be used as an adjunctive treatment rather than as a standalone managerial remedy.

Derivation of a Modified Friedewald's equation for LDL cholesterol estimation in Sub-Himalayan population: A comparative study.

Background: Despite the availability of alternative homogenous assays for LDL-C measurement, most of the laboratories still use Friedewald Equation (FE). However, various novel equations have shown better performance than FE specific to a particular population. Besides, no equation has been devised for use in Sub-Himalayan population. Methods: A cross-sectional laboratory data-based study was conducted by recruiting lipid profiles of 1851 samples to validate 10 different equations for calculating LDL and to devise a novel Modified Friedewald Equation (MFE) specific for Sub-Himalayan population. Results: The novel MFE is presented as: LDL-C = -2.421 + (0.752 × TC) - (0.047 × TG) - (0.350 × HDL). A significant difference was observed between direct LDL-C (118.84 ± 40.39 mg/dL) and all other equations except MFE (118.84 ± 37.96 mg/dl, P > 0.999) and Puavilai Equation (117.99 ± 49.05 mg/dL, P = 0.138). Additionally, MFE showed lowest mean percentage bias of 0.14% with 95% limits of agreement within ± 2SD on Bland-Altman analysis. On ROC analysis at cut-offs of clinical decision limits of 100 mg/dl, 130 mg/dl, 160 mg/dl, and 190 mg/dl, MFE outperformed all other equations with highest AUC (0.974, 0.978, 0.982, and 0.995) respectively with specificity >95% at higher levels. MFE also showed highest correlation (r = 0.954, P < 0.001) and least rMSE (13.8) with direct LDL although all the equations showed significant positive correlation with direct LDL (p < 0.001). Conclusion: MFE derived in this study showed a better diagnostic performance as compared to other 10 equations taking Direct LDL-C as gold standard for Sub-Himalayan Population and may be used as a substitute for FE in the study population.

Diagnostic, prognostic, and therapeutic significance of long non-coding RNA MALAT1 in cancer.

Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a widely studied lncRNA in cancer. Although dispensable for normal physiology, MALAT1 is important for cancer-related pathways regulation. It is localized in the nuclear speckles periphery along with centrally located pre-RNA splicing factors. MALAT1 associated cancer signaling pathways include MAPK/ERK, PI3K/AKT, ß-catenin/Wnt, Hippo, VEGF, YAP, etc. Molecular tools such as immunoprecipitation, RNA pull-down, reporter assay, Northern blotting, microarray, and q-RT-PCR has been used to elucidate MALAT1's function in cancer pathogenesis. MALAT1 can regulate multiple steps in the development of tumours. The diagnostic and prognostic significance of MALAT1 has been demonstrated in cancers of the breast, cervix, colorectum, gallbladder, lung, ovary, pancreas, prostate, glioma, hepatocellular carcinoma, and multiple myeloma. MALAT1 has also emerged as a novel therapeutic target for solid as well as hematological malignancies. In experimental models, siRNA and antisense oligonucleotide (ASO) based strategy has been used for targeting MALAT1. The lncRNA has also been targeted for the chemosensitization and radiosensitization of cancer cells. However, most studies have been performed in preclinical models. How the cross-talk of MALAT1 with other signaling pathways affect cancer pathogenesis is the focus of this article. The diagnostic, prognostic, and therapeutic significance of MALAT1 in multiple cancer types are discussed.