Dual-Mode Virus Detection: Combining Electrochemical and Fluorescence Modalities for Enhanced Sensitivity and Reliability.

This study introduces a dual-mode biosensor specifically designed for the quantitative detection of viruses in rapid analysis. The biosensor is unique in its use of both optical (fluorescence) and electrochemical (impedance) detection methods using the same nanocomposites, providing a dual confirmation system for virus (norovirus-like particles) quantification. The system is based on using two antibody-conjugated nanocomposites: CdSeS quantum dots and Au-N,S-GQD nanocomposites. For optical detection, the principle relies on the fluorescence quenching of CdSeS by Au-N,S-GQD in a sandwich structure with the target. Conversely, electrochemical detection is based on the change in impedance caused by the formation of the same sandwich structure. The biosensor demonstrated exceptional sensitivity, capable of detecting norovirus at concentrations of as low as femtomolar in the electrochemical method and picomolar in the optical method. In the dual-responsive concentration range from 10-13 to 10-10 M, the sensor is highly sensitive in both methods, creating significant changes in fluorescence intensity and impedance in the presence of virus. Furthermore, the biosensor exhibits a high degree of specificity, with a negligible response to nontarget proteins, even within complex test solutions. This work represents a significant advancement in the field of biosensor technology, offering a fast, accurate, and reliable method for diagnosing viral infections and diseases.

Evaluation of antioxidant potential of pigments extracted from Bacillus spp. and Halomonas spp. isolated from mangrove rhizosphere.

The present study aimed to isolate different pigment-producing bacteria from the mangrove rhizosphere habitat and to extract their pigments for evaluating their antioxidant and sun-protective properties. Three pigment-producing bacterial cultures were isolated from soil samples and were identified by morphological analysis and 16S rDNA sequencing. The pigments were isolated by the solvent extraction method and named as MZ (Pink), Orange, and Yellow. They were characterized by Fourier Transform Infrared (FTIR) and UV-Vis spectroscopy. The sun protection factor (SPF) values of these pigments were then determined using the Mansur equation. The total polyphenol content was estimated by the Folin-Ciocâlteu method, and the antioxidant activity of the pigments was determined using DPPH (2,2-diphenyl-1-picrylhydrazyl), FRAP (ferric reducing antioxidant power), and ABTS (2,2-azinobis-3-ethyl-enzothiazoline-6-sulfonic acid) assays. The in vitro antioxidant potential of the pigments in the presence of oxidative stress (H2O2) was confirmed in the mouse macrophage cell line RAW264.7 by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The pigment-producing bacterial isolates were identified as Bacillus infantis (MZ), Halomonas spp. (Orange), and Bacillus spp. (Yellow). The pigments were found to be carotenoid in nature, and the SPF values were in the range of 3.99 to 5.22. All three pigments had high polyphenol content (22 to 48 µg tannic acid equivalent) and showed significant antioxidant properties in both chemical and cell line-based studies. The results of this study indicate that these pigments have the potential to be used as an antioxidant agent and can be further developed as a pharmaceutical compound.

Detection of Beijing strains of MDR M. tuberculosis and their association with drug resistance mutations in katG, rpoB, and embB genes.

BACKGROUND: Molecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients. METHODS: In this study 381 M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n = 131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene. RESULTS: The sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p = 0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p = < 0.0001, < 0.0001 & 0.0014 respectively) was also found. CONCLUSIONS: This rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains.

Mycobacterium tuberculosis strains from ancient and modern lineages induce distinct patterns of immune responses.

INTRODUCTION: It is possible that the difference in virulence and prevalence of different strains of Mycobacterium tuberculosis is related to the diverse immune response they evoke in the host. Outbreak strains have been shown to subvert the innate immune response as a potential host evasion mechanism. However, the immunological outcome of the interactions of different clinical strains with different host cells is still not understood. METHODOLOGY: Extremely Drug Resistant (XDR) Beijing, a modern lineage clinical strain and a comparator ancient lineage strain, EAI-5, were selected for the present study. The early induction of proinflammatory cytokines in human peripheral blood monocyte derived macrophages (MDM), monocyte derived dendritic cells (MDDC) and whole blood (WB) infected by selected clinical isolates and laboratory strains H37Rv and BCG were assessed. RESULTS: The two clinical strains exhibited distinct patterns of cytokine induction. The ancient lineage strain induced substantially higher expression of all proinflammatory cytokines like TNF-α, IL-1ß, IL-12 and chemokines like MCP-1, IL-8. However, the modern lineage strain exhibited suppressed response for the same. Further, the immune responses to two strains were conserved in infected MDM, MDDC and WB i.e. showing similar patterns of response across multiple human hosts. However, the differential response pattern was not observed when bacterial sonicates were used instead of live mycobacteria. CONCLUSIONS: The lineage specific patterns in induction of proinflammatory cytokines and chemokines by different M. tuberculosis strains remain similar in macrophage and dendritic cells isolated from different individuals. The present study also confirms that whole cell sonicates of different lineages of M. tuberculosis fail to induce such lineage specific response.

G1-4A, a Polysaccharide from Tinospora cordifolia Inhibits the Survival of Mycobacterium tuberculosis by Modulating Host Immune Responses in TLR4 Dependent Manner.

Rapid emergence of drug resistance in Mycobacterium tuberculosis (MTB) is a major health concern and demands the development of novel adjunct immunotherapeutic agents capable of modulating the host immune responses in order to control the pathogen. In the present study, we sought to investigate the immunomodulatory effects of G1-4A, a polysaccharide derived from the Indian medicinal plant Tinospora cordifolia, in in-vitro and aerosol mouse models of MTB infection. G1-4A treatment of MTB infected RAW264.7 macrophages significantly induced the surface expression of MHC-II and CD-86 molecules, secretion of proinflammatory cytokines (TNF-α, IL-ß, IL-6, IL-12, IFN-γ) and nitric oxide leading to reduced intracellular survival of both drug sensitive (H37Rv) as well as multi drug resistant strains (Beijing and LAM) of MTB, which was partially attributed to G1-4A induced NO production in TLR4-MyD88 dependent manner. Similarly, bacillary burden was significantly reduced in the lungs of MTB infected BALB/c mice treated with G1-4A, with simultaneous up-regulation of the expression of TNF-α, INF-γ and NOS2 in the mouse lung along with increased levels of Th1 cytokines like IFN-γ, IL-12 and decreased levels of Th2 cytokine like IL-4 in the serum. Furthermore, combination of G1-4A with Isoniazid (INH) exhibited better protection against MTB compared to that due to INH or G1-4A alone, suggesting its potential as adjunct therapy. Our results demonstrate that modulation of host immune responses by G1-4A might improve the therapeutic efficacy of existing anti-tubercular drugs and provide an attractive strategy for the development of alternative therapies to control tuberculosis.

Drug resistant clinical isolates of Mycobacterium tuberculosis from different genotypes exhibit differential host responses in THP-1 cells.

Mycobacterium tuberculosis (MTB) persistently infects and survives within the host macrophages. Substantial genotypic variation exists among MTB strains which correlate with their interactions with the host. The present study was designed to establish a correlation, if any, between infection and induction of innate immune response by genetically diverse drug resistant MTB isolates from India. For this purpose, three clinical isolates from ancient and modern lineages, along with H37Ra and H37Rv were evaluated for intracellular growth, phagocytic index, induction of proinflammatory cytokines and apoptosis following infection in THP-1 cell line. A wide variation in the induction of cytokines was revealed subsequent to infection with different strains. EAI-5 strain from ancient lineage 1, induced higher proinflammatory responses, higher apoptosis and moderate intracellular growth compared to other strains, in contrast, for Beijing strain of modern lineage 2, all three parameters were lowest among the clinical isolates. Further, the responses induced by LAM-6 from modern lineage 4 were at a moderate level, similar to the laboratory strain H37Rv which also belongs to lineage 4. Thus, these profiles were specific to their respective lineages and/or genotypes and independent of their drug resistance status. Further, a positive correlation, among TNF-α, IL-1ß, IL-6 and IL-12 induced in infected THP-1 cells was demonstrated. In addition, induction of all pro-inflammatory cytokines correlated well with the host cell apoptosis. A positive correlation was observed between phagocytic index in the category of '>10 bacilli/cell' and induction of apoptosis, only for virulent strains, indicating that initial accumulation of MTB strains inside the host cell may be an important determining factor for different innate responses.