Differential host responses to COVID-19: Unraveling the complexity.

These diverse outcomes of Covid-19 are influenced by various factors including age, gender, underlying health conditions, immune responses, viral variants, external factors, and overall quality of life. Demographic analysis of patients aged 0-18 years experienced mild to moderate cases, above 55 years with co-morbidities, were more severely affected.COVID-19 incidence was higher in males (58 %) & (42 %) in females. The reduced expression of Toll-like receptors (TLR) in severe and critical patients is a crucial determinant. This reduced TLR expression is primarily attributed to the dominance of the PLpro viral protein of COVID-19. Disease enrichment analysis highlights the long-term impact of COVID-19, which can lead to post-recovery complications such as hypertension, diabetes, cardiac diseases, and brain ischemia in Covid-19 patients. In conclusion, a comprehensive strategy targeting key factors like PLpro, TLR, and inflammatory cytokines such as IL-1 and IL-6 could offer an effective approach to mitigate the devastating effects of COVID-19.

MicroRNA signatures differentiate types, grades, and stages of breast invasive ductal carcinoma (IDC): miRNA-target interacting signaling pathways.

BACKGROUND: Invasive ductal carcinoma (IDC) is the most common form of breast cancer which accounts for 85% of all breast cancer diagnoses. Non-invasive and early stages have a better prognosis than late-stage invasive cancer that has spread to lymph nodes. The involvement of microRNAs (miRNAs) in the initiation and progression of breast cancer holds great promise for the development of molecular tools for early diagnosis and prognosis. Therefore, developing a cost effective, quick and robust early detection protocol using miRNAs for breast cancer diagnosis is an imminent need that could strengthen the health care system to tackle this disease around the world. METHODS: We have analyzed putative miRNAs signatures in 100 breast cancer samples using two independent high fidelity array systems. Unique and common miRNA signatures from both array systems were validated using stringent double-blind individual TaqMan assays and their expression pattern was confirmed with tissue microarrays and northern analysis. In silico analysis were carried out to find miRNA targets and were validated with q-PCR and immunoblotting. In addition, functional validation using antibody arrays was also carried out to confirm the oncotargets and their networking in different pathways. Similar profiling was carried out in Brca2/p53 double knock out mice models using rodent miRNA microarrays that revealed common signatures with human arrays which could be used for future in vivo functional validation. RESULTS: Expression profile revealed 85% downregulated and 15% upregulated microRNAs in the patient samples of IDC. Among them, 439 miRNAs were associated with breast cancer, out of which 107 miRNAs qualified to be potential biomarkers for the stratification of different types, grades and stages of IDC after stringent validation. Functional validation of their putative targets revealed extensive miRNA network in different oncogenic pathways thus contributing to epithelial-mesenchymal transition (EMT) and cellular plasticity. CONCLUSION: This study revealed potential biomarkers for the robust classification as well as rapid, cost effective and early detection of IDC of breast cancer. It not only confirmed the role of these miRNAs in cancer development but also revealed the oncogenic pathways involved in different progressive grades and stages thus suggesting a role in EMT and cellular plasticity during breast tumorigenesis per se and IDC in particular. Thus, our findings have provided newer insights into the miRNA signatures for the classification and early detection of IDC.

MicroRNA Signatures in Blood or Bone Marrow Distinguish Subtypes of Pediatric Acute Lymphoblastic Leukemia.

OncomiRs are microRNAs that are associated with early onset of specific cancers. To identify microRNAs involved in pediatric acute lymphoblastic leukemia (ALL) subtypes T-ALL and B-ALL, peripheral blood and bone marrow samples were independently subjected to microarray analysis using two different high-fidelity array platforms. The unique and common gene signatures from both arrays were validated by TaqMan individual assays in 100 pediatric ALL samples. Survival studies were carried out in the test set and validation set with 50 randomly selected samples in each set. MicroRNA expression profile revealed characteristic signatures for distinguishing T and B lineages and identified 51 novel microRNAs in pediatric ALL. Interestingly, the present study also revealed endogenous similarities and differences between blood and bone marrow within each ALL subtype. When Cox regression analysis was carried out with these identified microRNAs, 11 of them exhibited expression levels significantly correlated with survival. Validation of some of the common and relevant microRNAs from both arrays showed that their targets are involved in key oncogenic signaling pathways. Thus, this study suggests that microRNAs have the potential to become important diagnostic tools for identification and monitoring clinical outcomes in ALL patients.

PCR Amplifiable DNA from Breast Disease FFPE Section for Mutational Analysis.

Formalin-fixed paraffin-embedded (FFPE) tissue specimens have been a staple of research, providing precious resources for molecular and genomic studies. However, the biggest challenge is the extraction of high-quality DNA from FFPE tissues, given that the integrity of DNA is critically affected by formalin fixation. Formaldehyde induces crosslinks in DNA that renders single or double-stranded DNA breaks. Such breaks cause extensive fragmentation that directly influences the quality of DNA purified and the number of templates available for PCR amplification. Thus, protocol for DNA purification from FFPE tissues must effectively extract highly fragmented DNA and reverse cross-linking caused by formalin fixation. DNA extraction methods available in the literature were selected and modified at different stages to optimize a protocol that extracts DNA of sufficient quality and fragment size to be detectable by PCR. Archived FFPE tissues belonged to patients with triple negative breast cancer (TNBC) and benign breast disease were used for the protocol optimization. The best optimized protocol was then used to amplify Exon 4 region of Proviral integration site for Moloney murine leukemia virus1 (Pim1) kinase gene to analyze any probable somatic mutations both in TNBCs and benign breast diseases. Of the 12 different protocols developed, best quality DNA in terms of fragment size and purity was obtained when Tween20 lysis buffer was used for both deparaffinization and overnight digestion along with high salt precipitation. Optimized protocol was then validated by extracting DNAs from 10 TNBCs and 5 benign breast disease specimens with consistent purity and fragment size. PCR amplification and subsequent Sanger's sequencing revealed the presence of mutations in the Exon 4 region of Pim1 kinase. Deparaffinization and overnight digestion in Tween20 lysis buffer along with high salt precipitation yielded the best quality PCR amplifiable DNA for mutational analysis.

Biodrug Suppresses Breast and Colorectal Cancer in Murine Models.

RNA interference (RNAi) remains one of the most promising and emerging strategies for the effective cancer treatment due to its high target specificity and greater potency. However, it is hindered due to lack of appropriate targeting technologies. Therefore, there is an imminent need to develop specific and robust delivery systems for successful gene silencing. Nanotechnology-based strategies have been in place to combat the shortcomings associated with viral-based delivery systems. Herein we describe protocols for successful in vitro and in vivo delivery of gene-specific nucleic acids such as siRNAs and shRNAs using PEI-PGMA nanoparticles for efficient cancer therapy.

Environmental impact on the onset of hypertension-induced end-stage renal disease.

This study intends to assess the impact of environmental factors on the onset of hypertension-induced end-stage renal disease (ESRD) and to compare the level of oxidative stress with nonhypertensive diabetic ESRD. ESRD patients were evaluated along with healthy controls through questionnaire for demographic, nutritional and lifestyle variables. Oxidants were measured along with antioxidants. Multiple linear regression (MLR) models were applied to analyze association of studied variables with oxidants and antioxidants. Most of the hypertensive nephrosclerotic patients were residing in locality that was either closer to industrial belt or polluting water bodies, belonging to low socioeconomic status that invariably affected their lifestyle and nutritional status. Hypertensive ESRD patients showed more pronounced oxidative stress than diabetic ESRD. Several of the studied variables were significantly associated with oxidants and antioxidants. Demographic, nutritional and lifestyle variables appeared to have suggestive effect on the onset of hypertensive nephrosclerosis.

Clinical efficacy of platelet rich plasma in combination with methotrexate in chronic plaque psoriatic patients.

Psoriasis affects up to 3% of the world's population or more than 125 million people. There is an urgent need for new treatment strategy, as up to 50% of patients are not satisfied with current therapies. We evaluated the combined efficiency of platelet rich plasma (PRP) and methotrexate (MTX) in the management of patients with plaque psoriasis. Twenty-one patients with chronic plaque psoriasis were recruited in the study. Sixteen patients were assigned into combinational treatment group (PRP + MTX) and monotherapy group (MTX alone) consisted of five patients. All patients in combinational therapy received autologous PRP in their first sitting and subsequently followed with folitrax-15 for 4 weeks, while patients in monotherapy group received only folitrax-15, all patients received intra-lesional injections. Digital photograph, Psoriasis Area Severity Index (PASI) score and adverse events were recorded at weeks 0, 4, 8, 12 and 16 and were evaluated by three investigators independently. Patients treated with PRP/MTX showed substantial improvement in term of reduction in erythema, induration and desquamation at each visit and was effectively cleared off psoriasis at week 16. Combination treatment of PRP with MTX was well tolerated by all patients without any serious adverse events.

Fluorescent magnetic iron oxide nanoparticles for cardiac precursor cell selection from stromal vascular fraction and optimization for magnetic resonance imaging.

Fluorescent magnetic iron oxide nanoparticles have been used to label cells for imaging as well as for therapeutic purposes. The purpose of this study was to modify the approach to develop a nanoprobe for cell selection and imaging with a direct therapeutic translational focus. The approach involves physical coincubation and adsorption of superparamagnetic iron oxide nanoparticle-polyethylene glycol (SPION-PEG) complexes with a monoclonal antibody (mAb) or a set of antibodies. Flow cytometry, confocal laser scanning microscopy, transmission electron microscopy, iron staining, and magnetic resonance imaging were used to assess cell viability, function, and labeling efficiency. This process has been validated by selecting adipose tissue-derived cardiac progenitor cells from the stromal vascular fraction using signal regulatory protein alpha (SIRPA)/kinase domain receptor (KDR) mAbs. These markers were chosen because of their sustained expression during cardiomyocyte differentiation. Sorting of cells positive for SIRPA and KDR allowed the enrichment of cardiac progenitors with 90% troponin-I positivity in differentiation cultures. SPION labeled cardiac progenitor cells (1×10(5) cells) was mixed with gel and used for 3T magnetic resonance imaging at a concentration, as low as 12.5 µg of iron. The toxicity assays, at cellular and molecular levels, did not show any detrimental effects of SPION. Our study has the potential to achieve moderate to high specific cell selection for the dual purpose of imaging and therapy.

Evaluation of antibody response in mice against avian influenza A (H5N1) strain neuraminidase expressed in yeast Pichia pastoris.

Avian influenza has raised many apprehension in the recent years because of its potential transmitability to humans. With the increasing emergence of drug-resistant avian influenza strains, development of potential vaccines are imperative to manage this disease. Two structural antigens, haemagglutinin and neuraminidase, have been the target candidates for the development of subunit vaccine against influenza. In an effort to develop a faster and economically beneficial vaccine, the neuraminidase gene of a highly pathogenic avian influenza isolate was cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant neuraminidase (rNA) antigen was purified, and its bioactivity was analysed. The rNA was found to be functional, as determined by the neuraminidase assay. Four groups of mice were immunized with different concentrations of purified rNA antigen, which were adjuvanted with aluminium hydroxide. The immune response against rNA was analysed by enzyme-linked immunosorbent assay (ELISA) and neuraminidase inhibition assay. The mice groups immunized with 25 (mu) g and 10 (mu) g of antigen had a significant immune response against rNA. This method can be utilized for faster and cost-effective development of vaccines for a circulating and newer strain of avian influenza, and would aid in combating the disease in a pandemic situation, in which production time matters greatly.

Polyphenolics profile and antioxidant properties of Raphanus sativus L.

Raphanus sativus, a common cruciferous vegetable has been attributed to possess a number of pharmacological properties. Antioxidant and radical scavenging activity of R. sativus root extracted with solvents of varying polarity were evaluated using different model systems. Polyphenolic content was estimated to be in the range 13.18-63.54 mg g⁻¹ dry weight, with a considerable amount being obtained with polar solvents. High-performance liquid chromatography analysis indicated the presence of an array of polyphenolics. Catechin was found to be the most abundant phenolic compound in water extract and sinapic acid, the predominant phenolic compound in methanolic, ethyl acetate and hexane extracts. The methanolic extract showed significant ferric reducing ability, moderate metal chelating activity and strong radical scavenging activity. The methanolic extract could be successfully utilised as an ingredient in functional foods. However, water extract could be more pertinent to human nutrition as it contained a significant amount of catechin, which was comparable to traditional sources like green and black tea.

Hexane extract of Raphanus sativus L. roots inhibits cell proliferation and induces apoptosis in human cancer cells by modulating genes related to apoptotic pathway.

Raphanus sativus, a common cruciferous vegetable has been attributed to possess a number of pharmacological and therapeutic properties. It has been used in indigenous system of medicine for the treatment of various human ailments in India. This present study evaluated the chemopreventive efficacy of different parts of R. sativus such as root, stem and leaves, extracted with solvents of varying polarity and investigated the molecular mechanism leading to growth arrest and apoptotic cell death in human cancer cell lines. Of the different parts, significant growth inhibitory effect was observed with hexane extract of R. sativus root. Analysis of hexane extract by GC-MS revealed the presence of several isothiocyanates (ITCs) such as 4-(methylthio)-3-butenyl isothiocyanate (MTBITC), 4-(methylthio)-3-butyl isothiocyanate (erucin), 4-methylpentyl isothiocyanate, 4-pentenyl isothiocyanate and sulforaphene. R. sativus root extract induced cell death both in p53 proficient and p53 deficient cell lines through induction of apoptotic signaling pathway regardless of the p53 status of cells. The molecular mechanisms underlying R. sativus-induced apoptosis may involve interactions among Bcl(2) family genes, as evidenced by up-regulation of pro-apoptotic genes and down-regulation of anti-apoptotic genes along with activation of Caspase-3. Our findings present the first evidence that hexane extract of R. sativus root exerts potential chemopreventive efficacy and induces apoptosis in cancer cell lines through modulation of genes involved in apoptotic signaling pathway.

Polyphenolics profile, antioxidant and radical scavenging activity of leaves and stem of Raphanus sativus L.

Aerial parts (leaves and stem) of Raphanus sativus, which are usually discarded were found to possess potent antioxidant and radical scavenging activity, as measured by standard antioxidant assays. Methanolic and acetone extracts of R. sativus leaves had total polyphenolic content of 86.16 and 78.77 mg/g dry extract, which were comparable to the traditional rich sources such as green tea and black tea. HPLC identification of polyphenolics indicated the presence of catechin, protocatechuic acid, syringic acid, vanillic acid, ferulic acid, sinapic acid, o-coumaric acid, myricetin, and quercetin in leaves and stem. Among the different extraction solvents, methanolic extract of leaves and stem showed potent reductive capacity, significantly inhibited linoleic acid peroxidation and displayed metal chelating activity. Further, they scavenged free radicals effectively with IC50 (half maximal inhibitory concentration) of 31 and 42 microg/ml for DPPH radical, 23 and 52 microg/ml for superoxide radical, 67 and 197 microg/ml for hydrogen peroxide,and 56 and 62 microg/ml for nitric oxide, respectively. Leaves showed most potent antioxidant and radical scavenging activity as compared to stem, which may be accounted for the high polyphenolic content. Leaves and stem of R. sativus,often under-utilized part of this vegetable, thus possessed considerable amount of polyphenolics. Hence, it should be egarded as a potential source of natural antioxidants and could be effectively employed as an ingredient in health or in functional food.

Isothiocyanate profile and selective antibacterial activity of root, stem, and leaf extracts derived from Raphanus sativus L.

Acetone and hexane extracts derived from the root, stem, and leaf of Raphanus sativus were investigated for their antibacterial activity against foodborne and resistant pathogens, such as Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Salmonella typhimurium, Enterobacter aerogenes, Enterobacter cloacae, and Escherichia coli. Total and individual isothiocyanate (ITC) components and their relationship with the antibacterial activity of R. sativus were also evaluated. Both acetone and hexane fractions of root, stem, and leaf exhibited selective antibacterial activity against the organisms tested. Antibacterial activity was strongest in the acetone fraction of root with larger zone of inhibition and lower minimum inhibitory concentration. The results obtained were comparable to that seen with standard antibiotics. Of the different parts of R. sativus studied, root tended to be more active than the stem and leaf extracts in inhibiting the bacterial growth. Gas chromatographic analysis revealed the presence of variable amounts of five different ITCs such as allyl isothiocyanate (AITC), phenyl isothiocyanate (PITC), benzyl isothiocyanate (BITC), phenethyl isothiocyanate, and 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) in different parts of the plant. The low linear correlation between the total ITC content and antibacterial activity implied that bacterial growth inhibitory ability of R. sativus was not dependent on the total ITC content. However, the antibacterial activity of R. sativus was well correlated with AITC, PITC, and BITC for all organisms except for Enteroc. faecalis, whose inhibitory effect was more related to MTBITC.

Alleviation of oxidative and nitrosative stress following curative resection in patient with oral cavity cancer.

BACKGROUND: Oxidative stress by reactive oxygen species (ROS) and nitrosative stress by reactive nitrogen species (RNS) are proven initiators and promoters in carcinogenesis. Elevated ROS/RNS with lowered antioxidants occur in patients with squamous cell carcinoma of oral cavity. Ours is the first study to evaluate the effect of curative resection on both oxidative and nitrosative stress in such patients. METHODS: This study was conducted on 24 cancer patients and with age- and sex-matched healthy controls. Lipid peroxidation products, nitric oxide (NO) products and ceruloplasmin (CPL) in plasma were measured before and after surgery. Similarly enzymatic antioxidants in erythrocytes and non-enzymatic antioxidants in plasma were measured. RESULTS: Statistically significant elevation of lipid peroxidation, NO products and CPL and depletion of antioxidants were found in cancer patients compared with controls. After curative surgical resection there was a statistically significant fall in oxidants and CPL coupled with a rise in antioxidants. CONCLUSIONS: Our results suggest that oxidative/nitrosative stress could play a significant role in oral cavity cancer (OCC) and that curative resection is effective in alleviating this oxidative/nitrosative burden. Significant mitigation of oxidative/nitrosative stress could indicate the completeness of resection since tumor forms the major source of oxidants.

Evidence of oxidative and nitrosative stress in patients with cervical squamous cell carcinoma.

BACKGROUND: We conducted a study to evaluate reactive oxygen species (ROS) and reactive nitrogen species (RNS) simultaneously together with the antioxidant status in patients with cervical carcinoma. METHODS: We measured lipid peroxidation product, including malondialdehye (MDA), nitric oxide (NO) products, including nitrite (NO(2)(-)), nitrate (NO(3)(-)) and total nitrite (TNO(2)(-)) and antioxidant enzymes in 45 patients with cervical cancer and compared them against 30 healthy controls. RESULTS: Plasma as well as erythrocyte MDA and plasma NO levels was higher (p<0.001) in cervical cancer as compared to healthy controls. Antioxidant enzymes, Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, were decreased (p<0.001) whereas glutathione S-transferase (GST) activity was increased (p<0.001) in cervical cancer patients. Lipid peroxidation and NO products accumulation correlated significantly with a deranged antioxidant system. CONCLUSIONS: We demonstrated a possible involvement of both oxidative and nitrosative stress, as evidenced by increased lipid peroxidation and NO levels with altered antioxidant defense system in the pathogenesis of cervical cancer.

Enhanced lipid peroxidation and nitric oxide products with deranged antioxidant status in patients with head and neck squamous cell carcinoma.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can function both as initiators and promoters in carcinogenesis. Antioxidants provide protection against cellular and molecular damage caused by ROS and RNS. We conducted a study to evaluate the levels of lipid peroxidation products, nitric oxide (NO) products and antioxidants in patients with head and neck squamous cell carcinoma (HNSCC). Fifty one HNSCC patients, 33 healthy tobacco smokers/chewers as tobacco user controls, and 37 non-smokers/chewers as normal controls were recruited for this study. Lipid peroxidation products, NO products and antioxidants were measured using spectrophotometric methods. Lipid peroxidation products, including lipid hydroperoxide (LHP) and malondialdehyde (MDA), nitric oxide (NO) products, including nitrite (NO(2)(-)), nitrate (NO(3)(-)), and total nitrite (TNO(2)(-)) were found to be significantly elevated with a concomitant depletion of antioxidants in HNSCC patients as compared to tobacco users and normal controls. These derangements were also evident albeit to a lesser degree in tobacco users as compared to normal controls. Results from this study demonstrate a potential involvement of both ROS and RNS in the pathogenesis of HNSCC and also illustrate the risk of ROS/RNS induced damage healthy tobacco users are exposed to, implicating their higher risk for upper aerodigestive tract cancer.