Toll-like receptor (TLR) gene expression and immunostimulatory effect of CpG oligonucleotides in hormone receptor positive cell line T47D and triple negative breast cancer cell line MDA-MB-468.

BACKGROUND: We investigated the expression of TLR genes and the effects of CpG ODN in Estrogen Receptor positive, Progesterone Receptor positive breast cancer cell line (T47D) and a triple-negative breast cancer cell line (MDA-MB-468) followed by studying the immunostimulatory activity of CpG oligonucleotides in breast cancer cell lines T47D and MDA-MB-468. MATERIALS AND METHODS: We evaluated the expression pattern of TLR genes (TLR1 to TLR9) in T47D and MDA-MB-468 cells using Real-time qPCR analysis. The intracellular TLR9 protein expression was studied by flow cytometry. The effect of CpG ODN on cell viability was tested using MTT assay. The relative expression of pro-inflammatory (IL6 and TNFα) and anti- inflammatory/immunosuppressive cytokines genes (IL10 and TGF beta1) were examined by Real-time qPCR. RESULTS: We found that MDA-MB-468 cells expressed TLR2, TLR3, TLR6, TLR8, and TLR9 genes and T47D cells expressed TLR3, TRL5, TLR8, and TLR9 genes. Stimulation of TLR9 in vitro with CpG significantly reduced the cell viability of T47D and MDA-MB-468 cells. IL6 cytokine gene expression was significantly reduced in both CpG treated T47D cells and MDA-MB-468 cells. TNFα gene expression was significantly reduced after treatment with CpG in MDA-MB-468 cells but not in T47D cells. IL10 and TGFß1 expression were downregulated in CpG treated T47D cells. Whereas, IL10 and TGFß1 were elevated in CpG treated MDA-MB-468 cells. CONCLUSION: Our in vitro finding gives preliminary evidence that triggering TLR9 using CpG ODN decreases the cell proliferation and alters the pro-inflammatory cytokines in favor of inhibition of hormone receptor positive breast cancer cells T47D and triple negative breast cancer cells MDA-MB-468.

Comparison of Real-time RT-PCR cycle threshold (Ct) values with clinical features and severity of COVID-19 disease among hospitalized patients in the first and second waves of COVID-19 pandemic in Chennai, India.

Introduction: Real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) of nasopharyngeal/ oropharyngeal swab has been the gold standard test for detection of SARS-CoV-2 infection The relationship between cycle threshold (Ct) values of rRT-PCR and severity of disease remain disputable and not clearly defined in COVID-19. Methodology: This is a single-centered retrospective observational study conducted at Government Corona Hospital (GCH), Guindy, Chennai. In the present study, we compared the Ct value of rRT-PCR from nasopharyngeal swab specimens with a diverse range of symptoms and disease severity among 240 individuals who were hospitalized with COVID-19, viz., mild cases (MC; n = 160), moderately severe cases (MSC; n = 46) and severe cases (SC; n = 34) in the first and second waves of COVID-19 pandemic. Results: The study included 240 hospitalized COVID-19 patients with a median age of 52 years (range 21 to 90 years). MC, MSC, and SC all had median Ct values of 25.0 (interquartile range - IQR 20.0 to 30.5), 29.5 (IQR 23.0 to 34.0), and 29.0 (IQR 24 to 37.5) for the ORF1ab gene. The Ct value differed significantly between mild vs moderate, and mild vs severe cases. The Ct value of SC group with co-morbidity of type 2 diabetes have a significant difference compared to non-diabetes group (p value <0.05). There was a significant difference in the median Ct value of ORF1ab gene among the MSC group and MC but not in the SC group in the first and second waves of the pandemic (p<0.05). Conclusion: We conclude that SARS-CoV-2 Ct values of rRT-PCR alone does not have a role in aiding severity stratification among patients with COVID-19 since the viral dynamics and Ct value may vary due to the emerging variants that occur in different waves of the pandemic.

Transcriptional Profiling and Deriving a Seven-Gene Signature That Discriminates Active and Latent Tuberculosis: An Integrative Bioinformatics Approach.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M.tb.). Our integrative analysis aims to identify the transcriptional profiling and gene expression signature that distinguish individuals with active TB (ATB) disease, and those with latent tuberculosis infection (LTBI). In the present study, we reanalyzed a microarray dataset (GSE37250) from GEO database and explored the data for differential gene expression analysis between those with ATB and LTBI derived from Malawi and South African cohorts. We used BRB array tool to distinguish DEGs (differentially expressed genes) between ATB and LTBI. Pathway enrichment analysis of DEGs was performed using DAVID bioinformatics tool. The protein-protein interaction (PPI) network of most upregulated genes was constructed using STRING analysis. We have identified 375 upregulated genes and 152 downregulated genes differentially expressed between ATB and LTBI samples commonly shared among Malawi and South African cohorts. The constructed PPI network was significantly enriched with 76 nodes connected to 151 edges. The enriched GO term/pathways were mainly related to expression of IFN stimulated genes, interleukin-1 production, and NOD-like receptor signaling pathway. Downregulated genes were significantly enriched in the Wnt signaling, B cell development, and B cell receptor signaling pathways. The short-listed DEGs were validated in a microarray data from an independent cohort (GSE19491). ROC curve analysis was done to assess the diagnostic accuracy of the gene signature in discrimination of active and latent tuberculosis. Thus, we have derived a seven-gene signature, which included five upregulated genes FCGR1B, ANKRD22, CARD17, IFITM3, TNFAIP6 and two downregulated genes FCGBP and KLF12, as a biomarker for discrimination of active and latent tuberculosis. The identified genes have a sensitivity of 80-100% and specificity of 80-95%. Area under the curve (AUC) value of the genes ranged from 0.84 to 1. This seven-gene signature has a high diagnostic accuracy in discrimination of active and latent tuberculosis.

Gene expressions and their significance in organoid cultures obtained from breast cancer patient-derived biopsies.

Gene expression changes are one of the hallmarks of malignant cells and such changes in specific genes have been identified for a variety of human cancers. Such an association in gene expression changes becomes very significant for breast cancers due to the genetic heterogeneity seen in such cancers. It is due to such genetic implication that breast cancers are classified into several subtypes; based on the expression and the magnitude of expression of estrogen and progesterone receptor genes. Changes in the expression of ERBB2, ESR1, PLAU, MUC1, PGR, and TP53 are implicated in breast cancers. Of the various models available for cancer research, organoid cultures from patient-derived biopsies are being considered as the most relevant for invitro testing. Organoid cultures derived from patient biopsies mitigate several limitations of other commonly available models such as cancer cell lines. Such organoids retain the functional physiology of solid tumors which include gene expression. Also, utilizing patient derived organoids for in vitro testing paves way for personalized medicine which greatly enhances the effectiveness of cancer therapy for individuals. We present the genes implicated in breast cancers, the ways in which organoids can be derived from breast cancer biopsies and their applications for gene expression studies.

Microarray and pattern miner analysis of AXL and VIM gene networks in MDA­MB­231 cells.

MDA­MB­231 cells represent malignant triple­negative breast cancer, which overexpress epidermal growth factor receptor (EGFR) and two genes (AXL and VIM) associated with poor prognosis. The present study aimed to identify novel therapeutic targets and elucidate the functional networks for the AXL and VIM genes in MDA­MB­231 cells. We identified 71 genes upregulated in MDA­MB­231 vs. MCF7 cells using BRB­Array tool to re­analyse microarray data from six GEO datasets. Gene ontology and STRING analysis showed that 43/71 genes upregulated in MDA­MB­231 compared with MCF7 cells, regulate cell survival and migration. Another 19 novel genes regulate migration, metastases, senescence, autophagy and chemoresistance. The Pattern Miner systems biology tool uses specific genes as inputs or 'baits' to identify outputs from the NCI­60 database. Using five genes regulating cancer cell migration (AXL, VIM, EGFR, CAPN2, and COL4A1) as input 'baits', we used pattern miner to identify statistically significant, co­expressed genes from the list of 71 genes upregulated in MDA­MB­231 compared with MCF7 cells. Outputs were subsets of the 71 genes, which showed significant co­expression with one or more of the five input genes. These outputs were used to develop functional networks for AXL and VIM. Analysis of these networks verified known properties of AXL and VIM, and suggested novel functions for these two genes. Thus, genes in the AXL network promote migration, metastasis and chemoresistance, whereas the VIM gene network regulates novel tumorigenic processes, such as lipogenesis, senescence and autophagy. Notably, these two networks contain 12 genes not reported for TNBC.

Role of nanoparticle size in self-assemble processes of collagen for tissue engineering application.

Nanoparticle mediated extracellular matrix may offer new and improved biomaterial to wound healing and tissue engineering applications. However, influence of nanoparticle size in extracellular matrix is still unclear. In this work, we synthesized different size of silver nanoparticles (AgNPs) comprising of 10nm, 35nm and 55nm using nutraceuticals (pectin) as reducing as well as stabilization agents through microwave irradiation method. Synthesized Ag-pectin nanoparticles were assimilated in the self-assemble process of collagen leading to fabricated collagen-Ag-pectin nanoparticle based scaffolds. Physico-chemical properties and biocompatibility of scaffolds were analyzed through FT-IR, SEM, DSC, mechanical strength analyzer, antibacterial activity and MTT assay. Our results suggested that 10nm sized Ag-pectin nanoparticles significantly increased the denaturation temperature (57.83°C) and mechanical strength (0.045MPa) in comparison with native collagen (50.29°C and 0.011MPa). The in vitro biocompatibility assay reveals that, collagen-Ag-pectin nanoparticle based scaffold provided higher antibacterial activity against to Gram positive and Gram negative as well as enhanced cell viability toward keratinocytes. This work opens up a possibility of employing the pectin caged silver nanoparticles to develop collagen-based nanoconstructs for biomedical applications.

Improved design and development of a functional moulded prosthetic foot.

PURPOSE: In the Indian scenario, the Jaipur foot is a low-cost breakthrough that enabled the disabled person to adapt to the Indian environment. The aim of this study is to modify the present foot in terms of ankle support design and method of fabrication, foot moulds profile and the inner core material in order to improve the performance and durability. METHOD: The optimized design of ankle support and flat foot profile moulds suitable for both left and right foot were developed through CAD/CAM and prosthetic feet were fabricated using ethylene vinyl acetate (EVA) foam as an appropriate alternative core material for microcellular rubber (MCR). The developed prosthetic feet were tested for rigidity by load-deflection analysis in universal testing machine. RESULT: EVA foot had shown better rigidity than conventional MCR foot, which will help in weight transfer during walking and increase the durability. The CAD modeled ankle support and single block EVA had made the manufacturing process easy and reduced the weight of foot and improved Gait to the person fitted with it due to improved flat foot profile. CONCLUSION: The new artificial foot had proven to be efficacious technically as well as functionally, which is clearly borne out from the extremely positive feedback given by the amputees. Implications of Rehabilitation Persons with below knee amputation are usually provided with transtibial prosthesis, which allows for easier ambulation and helps them to get back to their normal life. Transtibial prosthesis is an artificial limb that replaces a lower limb that is amputated below the knee. In our study, a new prosthetic foot with a modified ankle support and flat foot profile using better inner-core material than the conventional Jaipur foot was developed and the process was also optimized for mass production. The developed prosthetic foot can be fitted with both above and below knee exoskeleton type of prosthesis.

Neural and fuzzy computation techniques for playout delay adaptation in VoIP networks.

Playout delay adaptation algorithms are often used in real time voice communication over packet-switched networks to counteract the effects of network jitter at the receiver. Whilst the conventional algorithms developed for silence-suppressed speech transmission focused on preserving the relative temporal structure of speech frames/packets within a talkspurt (intertalkspurt adaptation), more recently developed algorithms strive to achieve better quality by allowing for playout delay adaptation within a talkspurt (intratalkspurt adaptation). The adaptation algorithms, both intertalkspurt and intratalkspurt based, rely on short term estimations of the characteristics of network delay that would be experienced by up-coming voice packets. The use of novel neural networks and fuzzy systems as estimators of network delay characteristics are presented in this paper. Their performance is analyzed in comparison with a number of traditional techniques for both inter and intratalkspurt adaptation paradigms. The design of a novel fuzzy trend analyzer system (FTAS) for network delay trend analysis and its usage in intratalkspurt playout delay adaptation are presented in greater detail. The performance of the proposed mechanism is analyzed based on measured Internet delays. Index Terms-Fuzzy delay trend analysis, intertalkspurt, intratalkspurt, multilayer perceptrons (MLPs), network delay estimation, playout buffering, playout delay adaptation, time delay neural networks (TDNNs), voice over Internet protocol (VoIP).

Surface modified and medicated polyurethane materials capable of controlling microorganisms causing foot skin infection in athletes.

Foot odor and foot infection are major problems of athletes and persons with hyperhidrosis. Many shoes especially sports shoes have removable cushion insoles/foot beds for foot comfort. Polyurethane (PU) foam and elastomer have been used as cushion insole in shoes. In the present work, new insole materials based on porous viscoelastic PU sheets having hydrophilic property and antimicrobial drug coating to control foot infection and odor were developed. Bacteria and fungus that are causing infection and bad odor of the foot of athletes were isolated by microbial cell culturing of foot sweat. The surface of porous viscoelastic PU sheets was modified using hydrophilic polymers and coated with antimicrobial agent, silver sulfadiazine (SS). The surface modified PU sheets were characterized using ATR-FTIR, TGA, DSC, SEM, contact angle measurement and water absorption study. Results had shown that modified PU sheets have hydrophilicity greater than that of original PU sheet. FTIR spectra and SEM pictures confirmed modification of PU surface with hydrophilic polymers and coating with SS. Minimum inhibitory concentration studies indicated that SS has activity on all isolated bacteria of athletic foot sweat. The maximum inhibition was found for Pseudomonas (20mm) followed by Micrococci (17 mm), Diphtheroids (16 mm) and Staphylococci (12 mm). During perspiration of foot the hydrophilic polymers on PU surface will swell and release SS. Future work will confirm the application of these materials as inserts in athletic shoes.