Live-Cell Mitochondrial Targeted NIR Fluorescent Covalent Labeling of Specific Proteins Using a Dual Localization Effect.

Here, our designed water-soluble NIR fluorescent unsymmetrical Cy-5-Mal/TPP+ consists of a lipophilic cationic TPP+ subunit that can selectively target and accumulate in a live-cell inner mitochondrial matrix where a maleimide residue of the probe undergoes faster chemoselective and site-specific covalent attachment with the exposed Cys residue of mitochondrion-specific proteins. On the basis of this dual localization effect, Cy-5-Mal/TPP+ molecules remain for a longer time period even after membrane depolarization, enabling long-term live-cell mitochondrial imaging. Due to the adequate concentration of Cy-5-Mal/TPP+ reached in live-cell mitochondria, it facilitates site-selective NIR fluorescent covalent labeling with Cys-exposed proteins, which are identified by the in-gel fluorescence assay and LC-MS/MS-based proteomics and supported by a computational method. This dual targeting approach with admirable photostability, narrow NIR absorption/emission bands, bright emission, long fluorescence lifetime, and insignificant cytotoxicity has been shown to improve real-time live-cell mitochondrial tracking including dynamics and interorganelle crosstalk with multicolor imaging applications.

Host-Assisted Delivery of a Model Drug to Genomic DNA: Key Information from Ultrafast Spectroscopy and in silico Study.

Drug delivery to a target without adverse effects is one of the major criteria for clinical use. Herein, we have made an attempt to explore the delivery efficacy of SDS surfactant in a monomer and micellar stage during the delivery of the model drug, Toluidine Blue (TB) from the micellar cavity to DNA. Molecular recognition of pre-micellar SDS encapsulated TB with DNA occurs at a rate constant of k1 ∼652 s-1 . However, no significant release of encapsulated TB at micellar concentration was observed within the experimental time frame. This originated from the higher binding affinity of TB towards the nano-cavity of SDS at micellar concentration which does not allow the delivery of TB from the nano-cavity of SDS micelles to DNA. Thus, molecular recognition controls the extent of DNA recognition by TB which in turn modulates the rate of delivery of TB from SDS in a concentration-dependent manner.

A new ratiometric switch "two-way" detects hydrazine and hypochlorite via a "dye-release" mechanism with a PBMC bioimaging study.

A new ratiometric fluorescent probe (E)-2-(benzo[d]thiazol-2-yl)-3-(8-methoxyquinolin-2-yl)acrylonitrile (HQCN) was synthesised by the perfect blending of quinoline and a 2-benzothiazoleacetonitrile unit. In a mixed aqueous solution, HQCN reacts with hydrazine (N2H4) to give a new product 2-(hydrazonomethyl)-8-methoxyquinoline along with the liberation of the 2-benzothiazoleacetonitrile moiety. In contrast, the reaction of hypochlorite ions (OCl-) with the probe gives 8-methoxyquinoline-2-carbaldehyde. In both cases, the chemodosimetric approaches of hydrazine and hypochlorite selectively occur at the olefinic carbon but give two different products with two different outputs, as observed from the fluorescence study exhibiting signals at 455 nm and 500 nm for hydrazine and hypochlorite, respectively. A UV-vis spectroscopy study also depicts a distinct change in the spectrum of HQCN in the presence of hydrazine and hypochlorite. The hydrazinolysis of HQCN exhibits a prominent chromogenic as well as ratiometric fluorescence change with a 165 nm left-shift in the fluorescence spectrum. Similarly, the probe in hand (HQCN) can selectively detect hypochlorite in a ratiometric manner with a shift of 120 nm, as observed from the fluorescence emission spectra. HQCN can detect hydrazine and OCl- as low as 2.25 × 10-8 M and 3.46 × 10-8 M, respectively, as evaluated from the fluorescence experiments again. The excited state behaviour of the probe HQCN and the chemodosimetric products with hydrazine and hypochlorite are studied by the nanosecond time-resolved fluorescence technique. Computational studies (DFT and TDDFT) with the probe and the hydrazine and hypochlorite products were also performed. The observations made in the fluorescence imaging studies with human blood cells manifest that HQCN can be employed to monitor hydrazine and OCl- in human peripheral blood mononuclear cells (PBMCs). It is indeed a rare case that the single probe HQCN is found to be successfully able to detect hydrazine and hypochlorite in PBMCs, with two different outputs.

Supramolecular Dipeptide-Based Near-Infrared Fluorescent Nanotubes for Cellular Mitochondria Targeted Imaging and Early Apoptosis.

Herein, we have designed and synthesized unsymmetrical visible Cy-3 and near-infrared (NIR) Cy-5 chromophores anchoring mitochondria targeting functional group conjugated with a Phe-Phe dipeptide by a microwave-assisted Fmoc solid phase peptide synthesis method on Wang resin. These dipeptide-based Cy-3-TPP/FF as well as Cy-5-TPP/FF molecules self-assemble to form fluorescent nanotubes in solution, and it has been confirmed by TEM, SEM, and AFM. The Cy-3-TPP/FF and Cy-5-TPP/FF molecules in solution exhibit narrow excitation as well as emission bands in the visible and NIR region, respectively. These lipophilic cationic fluorescent peptide molecules spontaneously and selectively accumulate inside the mitochondria of human carcinoma cells that have been experimentally validated by live cell confocal laser scanning microscopy and display a high Pearson's correlation coefficient in a colocalization assay. Live cell multicolor confocal imaging using the NIR Cy-5-TPP/FF in combination with other organelle specific dye is also accomplished. Moreover, these lipophilic dipeptide-based cationic molecules reach the critical aggregation concentration inside the mitochondria because of the extremely negative inner mitochondrial membrane potential [(ΔΨm)cancer ≈ -220 mV] and form supramolecular nanotubes which are accountable for malignant mitochondria targeted early apoptosis. The early apoptosis is arrested using Cy-5-TPP/FF and confirmed by annexin V-FITC/PI apoptosis detection assay.

The drug resistance mechanisms in Leishmania donovani are independent of immunosuppression.

The protozoan parasite L. donovani resides inside macrophages as amastigotes and inflicts a potentially lethal disease visceral leishmaniasis (VL). Due to absence of a vaccine, chemotherapy with antimonials, amphotericin B, miltefosine or paromomycin remains the only option for treating VL. Prolonged treatment with a single drug resulted in parasite strains resistant to each of these drugs. As immuno-suppression characterizes the disease, we examined whether eliciting immunosuppressive cytokines is a mechanism of manifestation of drug-resistance. We infected BALB/c mice with the clinical isolates of L. donovani- BHU1066 (sensitive), NS2 (antimony-resistant), BHU1064 (miltefosine-resistant), BHU919 (Amphotericin B-resistant) and BHU1020 (paromomycin-resistant)- from the respective drug-unresponsive patients and assessed splenic parasite load and production of pro-inflammatory and anti-inflammatory cytokines. Although the splenic parasite loads in the drug-resistant L. donovani-infected BALB/c mice were higher than that observed in the drug-sensitive parasites-infected mice, the cytokine profiles were not significantly different between these two sets of mice. The drug-resistance in L. donovani results from innate drug modulation but perhaps not from host immune-suppressive cytokines.

Adiponectin Genetic Variant and Expression Coupled with Lipid Peroxidation Reveal New Signatures in Diabetic Dyslipidemia.

Increasing burden of non-communicable diseases like diabetes and cardiovascular disorders has made the global health scenario more challenging. Dyslipidemia in diabetes is a compounding risk factor for cardiovascular diseases, but there is dearth of identifying appropriate signatures to address this issue. The protein, adiponectin, is actively involved in regulating glucose levels as well as fatty acid breakdown playing crucial role in dyslipidemia and vascular complications. To identify the underlying genetic and molecular profile of adiponectin metabolic pathway in diabetic dyslipidemia and to correlate it with known biochemical and oxidative stress parameters of T2DM, we performed a case-control study in a total 264 individuals belonging to three categories such as diabetes with dyslipidemia (n = 88), diabetes without dyslipidemia (n = 86) and normal healthy controls (n = 90). Expression of adiponectin (ADIPOQ) and its receptors (ADIPOR1 and ADIPOR2) were measured in visceral and subcutaneous adipose tissues. A significant downregulated expression of ADIPOQ and its receptors in adipose tissues and PBMCs were linked with diabetic dyslipidemic condition. A multiple linear regression followed by MDR analysis implicated the elevated plasma malondialdehyde and decreased adiponectin level to be correlated with diabetic dyslipidemia. More interestingly, two single nucleotide polymorphisms of ADIPOQ (rs2241766 and rs1501299) were genetically associated with the risk of developing dyslipidemia. Other important biochemical factors found to be increased in diabetic dyslipidemic conditions included plasma C-reactive protein and 4-hydroxynonenal adducts. Our results explore, a complex interplay of genetic and biochemical parameters in diabetic dyslipidemia which is significant from the perspective of risk stratification and novel therapeutic strategy development.

Nuclear factor NF-κB1 functional promoter polymorphism and its expression conferring the risk of Type 2 diabetes-associated dyslipidemia.

Type 2 diabetes mellitus (T2DM) accompanied by hyperlipidemia confers higher risk for diabetes as well as cardiovascular diseases. NF-κB is actively involved in generating low-grade inflammation and oxidative stress triggering the development of diabetic complications. In this study, we have attempted to investigate the association between NF-κB1 functional promoter polymorphism-94 ATTG insertion/deletion (rs28362491) with inflammatory markers in developing diabetes-linked dyslipidemia. We performed a case-control study in a total of 401 individuals belonging to three categories such as Type 2 diabetes with dyslipidemia, Type 2 diabetes without dyslipidemia, and normal healthy controls. Experiments were carried out using genotyping, real-time PCR, and western blot. Pearson's correlation, analysis of variance, and logistic regression were utilized for statistical analysis. As per genetic association conducted in this study the SNP rs28362491 showed significant allelic and genotypic associations (Allelic: OR = 1.374, CI 0.9797-1.927, p = 0.003, and Genotypic in dominant model: OR = 1.77, CI 1.04-2.99, p = 0.002) with the risk of diabetes and associated dyslipidemia. The -94 ATTG insertion/insertion (ins/ins) genotype was associated with significantly increased level of serum TNF-α (p = 0.002), serum IL-6 (p = 0.067) in diabetes-induced dyslipidemia. Multiple linear regression analysis identifies independent correlation of Total cholesterol, HDL, LDL, TNF-α, and rs28362491 ATTG ins/ins with triglyceride in diabetic dyslipidemic condition. T2DM with dyslipidemia having ins/ins genotype showed significant increased expression of pro-inflammatory cytokines such as TNF-α, IL-6, and activation of NF-κB. Our study reports that individuals with ATTG insertion allele and ATTG ins/ins genotype at NF-κB1 promoter regulatory gene predicts the risk and severity of T2DM-linked dyslipidemia.

Supramolecular ß-Sheet Forming Peptide Conjugated with Near-Infrared Chromophore for Selective Targeting, Imaging, and Dysfunction of Mitochondria.

Herein, conjugation of the amyloid-ß (Aß) peptide fragment, Lys-Leu-Val-Phe-Phe (KLVFF, fragment of Aß16-20), with an unsymmetrical near-infrared (NIR) cyanine-5 (Cy-5) chromophore is achieved using microwave-assisted solid phase synthesis on 2-chlorotrityl chloride resin. Selective mitochondria tracking and staining in human carcinoma cells are accomplished by the KLVFF/Cy-5 conjugate containing triphenylphosphonium functionality, and this is compared to a control molecule KLVFF/Cy-5c. Mitochondrial target specificity of KLVFF/Cy-5 is established by the colocalization assay using mitochondria selective probe MitoTracker Red, which is monitored by confocal laser scanning microscope and shows a high Pearson's correlation coefficient. The KLVFF/Cy-5 conjugate has high photostability, NIR absorption/emission, high molar extinction coefficient, narrow absorption/emission band, high fluorescence lifetime, and high fluorescence quantum yield. Moreover, mitochondria targeting KLVFF/Cy-5 conjugate reaches the critical aggregation concentration inside the mitochondria of cancer cells due to the strong negative inner mitochondrial membrane potential [(ΔΨm)cancer -220 mV] and self-assembles to form amyloid fibrils at the target site, which is responsible for the mitochondrial dysfunction and cytotoxicity. Annexin V-FITC/PI apoptosis detection assay is used to determine the signal pathway of mitochondria targeted cellular dysfunction.

The protective role of metformin in autophagic status in peripheral blood mononuclear cells of type 2 diabetic patients.

Autophagy plays an important role in the pathophysiology of type 2 diabetes (T2D). Metformin is the most common antidiabetic drug. The main objective of this study was to explore the molecular mechanism of metformin in starvation-induced autophagy in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients. PBMCs were isolated from 10 diabetic patients and 7 non-diabetic healthy volunteers. The autophagic puncta and markers were measured with the help of monodansylcadaverine staining and western blot. Additionally, transmission electron microscopy was also performed. No significant changes were observed in the initial autophagy marker protein levels in PBMCs of T2D after metformin treatment though diabetic PBMCs showed a high level of phospho-mammalian target of rapamycin, p62 and reduced expression of phospho-AMP-activated protein kinase and lysosomal membrane-associated protein 2, indicating a defect in autophagy. Also, induction of autophagy by tunicamycin resulted in apoptosis in diabetic PBMCs as observed by caspase-3 cleavage and reduced expression of Bcl2. Inhibition of autophagy by bafilomycin rendered consistent expression of p62 indicating a defect in the final process of autophagy. Further, electron microscopic studies also confirmed massive vacuole overload and a sign of apoptotic cell death in PBMCs of diabetic patients, whereas metformin treatment reduced the number of autophagic vacuoles perhaps by lysosomal fusion. Thus, our results indicate that defective autophagy in T2D is associated with the fusion process of lysosomes which could be overcome by metformin.

Inbred mouse strains differentially susceptible to Leishmania donovani infection differ in their immune cell metabolism.

Leishmania donovani is a protozoan parasite that infects mammalian macrophages, wherein the parasite resides and replicates as amastigotes, inflicting the potentially fatal disease visceral leishmaniasis. The disease is characterized by severe immunosuppression and hypocholesterolemia implying metabolic changes in L. donovani infection; whether such metabolic changes are also linked to susceptibility to the infection is not known. Herein, four inbred mouse strains were first characterized for their resistance or susceptibility profile to L. donovani infection. It was observed that these four mouse strains were differentially susceptible to L. donovani infection. Splenic expression of four key cytokines- IL-10, IL-12, IFN-γ and IL-4- revealed that the differential susceptibility of these four mouse strains to L. donovani was partially associated with these cytokines. The association was further correlated with the expression of different enzymes of the glycolytic pathway in the spleen of these L. donovani-infected mice. Thus, the observations reported here suggest an association between host metabolism, cytokine secretion profile and L. donovani susceptibility. As the chemotherapeutic choices are extremely limited and a vaccine for human use is yet to be discovered for the neglected tropical disease that is prevalent in 88 countries affecting 320 million people, this metabolic study is a significant research area that has potentials to develop a new target for anti-leishmanial chemotherapy.

Changes in the Nuclear Envelope in Laminopathies.

Double-membrane-bound nucleus is the major organelle of every metazoan cell, which controls various nuclear processes like chromatin maintenance, DNA replication, transcription and nucleoskeleton-cytoskeleton coupling. Nuclear homeostasis depends on the integrity of nuclear membrane and associated proteins. Lamins, underlying the inner nuclear membrane (INM), play a crucial role in maintaining nuclear homeostasis. In this review, we have focussed on the disruption of nuclear homeostasis due to lamin A/C mutation which produces a plethora of diseases, termed as laminopathies.

Dielectric properties of plasma membrane: A signature for dyslipidemia in diabetes mellitus.

Dielectric properties of a living biological membrane play crucial role indicating the status of the cell in pathogenic or healthy condition. A distinct variation in membrane capacitance and impedance was observed for peripheral blood mononuclear cell (PBMC) suspensions for diabetic and diabetic-dyslipidemic subjects compared to healthy control. Low frequency region were explicitly considered in electrical analysis to address complex membrane dielectric factors that alter the system capacitance of a PBMC suspension. Such variation was marked in size, morphology and membrane function of PBMCs for control and diseased cases. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies reveal significant alteration in surface morphology of PBMCs in diseased condition. Side scatter of flow cytometry reveals complexity of PBMCs in diseased condition. Changes in size between groups were not found by SEM and forward scatter. Functional alteration in PBMCs was manifested by significant changes in cell membrane properties like Na+, K+ ATPase and Ca2+, Mg2+ ATPase activity, reduced plasma membrane fluidity and changes in intracellular Ca2+ content, which bear significant correlation in diabetic and diabetic dyslipidemic subjects. Therefore, dielectric parameters of PBMCs in diabetic-dyslipidemic challenges may led to interesting correlation opening the possibility of identifying crucial signature biomarkers.

Isoniazid induces apoptosis of activated CD4+ T cells: implications for post-therapy tuberculosis reactivation and reinfection.

Tuberculosis (TB) remains the second highest killer from a single infectious disease worldwide. Current therapy of TB is lengthy and consists of multiple expensive antibiotics, in a strategy referred to as Directly Observed Treatment, Short Course (DOTS). Although this therapy is effective, it has serious disadvantages. These therapeutic agents are toxic and are associated with the development of a variety of drug-resistant TB strains. Furthermore, patients treated with DOTS exhibit enhanced post-treatment susceptibility to TB reactivation and reinfection, suggesting therapy-related immune impairment. Here we show that Isoniazid (INH) treatment dramatically reduces Mycobacterium tuberculosis antigen-specific immune responses, induces apoptosis in activated CD4(+) T cells, and renders treated animals vulnerable to TB reactivation and reinfection. Consequently, our findings suggest that TB treatment is associated with immune impairment.

Diversity and Distribution of Archaea in the Mangrove Sediment of Sundarbans.

Mangroves are among the most diverse and productive coastal ecosystems in the tropical and subtropical regions. Environmental conditions particular to this biome make mangroves hotspots for microbial diversity, and the resident microbial communities play essential roles in maintenance of the ecosystem. Recently, there has been increasing interest to understand the composition and contribution of microorganisms in mangroves. In the present study, we have analyzed the diversity and distribution of archaea in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. The extraction of DNA from sediment samples and the direct application of 16S rRNA gene amplicon sequencing resulted in approximately 142 Mb of data from three distinct mangrove areas (Godkhali, Bonnie camp, and Dhulibhashani). The taxonomic analysis revealed the dominance of phyla Euryarchaeota and Thaumarchaeota (Marine Group I) within our dataset. The distribution of different archaeal taxa and respective statistical analysis (SIMPER, NMDS) revealed a clear community shift along the sampling stations. The sampling stations (Godkhali and Bonnie camp) with history of higher hydrocarbon/oil pollution showed different archaeal community pattern (dominated by haloarchaea) compared to station (Dhulibhashani) with nearly pristine environment (dominated by methanogens). It is indicated that sediment archaeal community patterns were influenced by environmental conditions.

Antimicrobial activities of actinomycetes isolated from unexplored regions of Sundarbans mangrove ecosystem.

BACKGROUND: New broad spectrum antimicrobial agents are urgently needed to combat frequently emerging multi drug resistant pathogens. Actinomycetes, the most talented group of microorganisms isolated from unexplored regions of the world may be the ultimate solution to this problem. Thus the aim of this study was to isolate several bioactive actinomycetes strains capable of producing antimicrobial secondary metabolite from Sundarbans, the only mangrove tiger land of the world. RESULTS: Fifty four actinomycetes were isolated and analyzed for antimicrobial activity against fifteen test organisms including three phytopathogens. Nine morphologically distinct and biologically active isolates were subjected to polyphasic identification study.16 s rDNA sequencing indicated eight isolates to reveal maximum similarity to the genus streptomyces, whereas one isolate presented only 93.57% similarity with Streptomyces albogriseolus NRRL B-1305(T). Seventy-one carbon sources and twenty-three chemical sources utilization assay revealed their metabolic relatedness. Among these nine isolates three specific strains were found to have notably higher degree of antimicrobial potential effective in a broader range including phyto-pathogenic fungus. Finally the strain SMS_SU21, which showed antimicrobial activity with MIC value of 0.05 mg ml(-1) and antioxidant activity with IC50 value of 0.242 ± 0.33 mg ml(-1) was detected to be the most potential one. True prospective of this strain was evaluated utilizing GC-MS and the bioactive compound responsible for antimicrobial activity was purified. CONCLUSION: Rare bioactive actinomycetes were isolated from unexplored heritage site. Antimicrobial compound has also been identified and purified which is active against a broad range of pathogens.

Spatiotemporal analysis of bacterial diversity in sediments of Sundarbans using parallel 16S rRNA gene tag sequencing.

The influence of temporal and spatial variations on the microbial community composition was assessed in the unique coastal mangrove of Sundarbans using parallel 16S rRNA gene pyrosequencing. The total sediment DNA was extracted and subjected to the 16S rRNA gene pyrosequencing, which resulted in 117 Mbp of data from three experimental stations. The taxonomic analysis of the pyrosequencing data was grouped into 24 different phyla. In general, Proteobacteria were the most dominant phyla with predominance of Deltaproteobacteria, Alphaproteobacteria, and Gammaproteobacteria within the sediments. Besides Proteobacteria, there are a number of sequences affiliated to the following major phyla detected in all three stations in both the sampling seasons: Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Chloroflexi, Cyanobacteria, Nitrospira, and Firmicutes. Further taxonomic analysis revealed abundance of micro-aerophilic and anaerobic microbial population in the surface layers, suggesting anaerobic nature of the sediments in Sundarbans. The results of this study add valuable information about the composition of microbial communities in Sundarbans mangrove and shed light on possible transformations promoted by bacterial communities in the sediments.

Denaturation properties and folding transition states of leghemoglobin and other heme proteins.

This work reports unfolding transitions of monomeric heme proteins leghemoglobin (Lb), myoglobin (Mb), and cytochrome c (Cyt c) utilizing UV-Vis spectral and steady-state and time-resolved fluorescence methods. Conformational stabilities of the native "folded" state of the proteins and their "unfolded" states were investigated in the light of a two-state transition model. Two-state transition values for ΔGD (298K) were obtained by denaturation with the chaotropic agents urea and guanidium hydrochloride (GdnHCl). The free energy value of Lb is the lowest compared to Cyt c and Mb along the denaturation pathway. The m value is also the lowest for Lb compared to Cyt c and Mb. The m value (a measure of dependence of ΔGD on denaturant concentration) for Cyt c and Mb is lower when it is denatured with urea compared to GdnHCl. The UV-Vis absorbance maximum and steady state fluorescence emission maximum were drastically red shifted in the presence of a certain denaturant concentration both in cases of Mb and Lb, but the scenario is different for Cyt c. The results are analyzed using a two-state transition model. The lifetime data clearly indicate the presence of an intermediate state during denaturation. The unfolding transition can modulate the conformation, stability, and surface exposure of these biologically important proteins.

Oxidative stress-related genes in type 2 diabetes: association analysis and their clinical impact.

Worldwide prevalence of diabetes mellitus motivates a number of association studies to be conducted throughout the world. Eleven polymorphisms from nine candidate genes in oxidative stress pathway have been analyzed in eastern Indian type 2 diabetic patients (n = 145) and healthy controls (n = 100). Different biochemical parameters were also analyzed for their association with the disease. Significant associations were observed for rs2070424 A>G SOD1 (OR 3.91, 95% CI 2.265-8.142, P < 0.001), rs854573 A>G PON1 (OR 3.415, 95% CI 2.116-5.512, P < 0.001), rs6954345 G>C PON2 (OR 3.208, 95% CI 2.071-4.969, P < 0.001), RAGE rs1800624 -374 T>A (OR 3.58, 95% CI 2.218-5.766, P < 0.001), and NOS3 -786 T>C (OR 3.75, 95% CI 2.225-6.666, P < 0.001). Haplotype containing two risk alleles of PON1 and PON2 genes was significantly associated with disease (OR 8.34, 95% CI 1.554-44.804, P < 0.002). Our results suggest that carriers of major and efficient alleles of oxidative stress genes are more likely to survive the comorbid complications and single copy of risk allele is sufficient for developing the disease.

Changing bacterial profile of Sundarbans, the world heritage mangrove: impact of anthropogenic interventions.

Mangrove microbial communities and their associated activities have profound impact on biogeochemical cycles. Although microbial composition and structure are known to be influenced by biotic and abiotic factors in the mangrove sediments, finding direct correlations between them remains a challenge. In this study we have explored sediment bacterial diversity of the Sundarbans, a world heritage site using a culture-independent molecular approach. Bacterial diversity was analyzed from three different locations with a history of exposure to differential anthropogenic activities. 16S rRNA gene libraries were constructed and partial sequencing of the clones was performed to identify the microbial strains. We identified bacterial strains known to be involved in a variety of biodegradation/biotransformation processes including hydrocarbon degradation, and heavy metal resistance. Canonical Correspondence Analysis of the environmental and exploratory datasets revealed correlations between the ecological indices associated with pollutant levels and bacterial diversity across the sites. Our results indicate that sites with similar exposure of anthropogenic intervention reflect similar patterns of microbial diversity besides spatial commonalities.

A peptide fragment from the human COX3 protein disrupts association of Mycobacterium tuberculosis virulence proteins ESAT-6 and CFP10, inhibits mycobacterial growth and mounts protective immune response.

BACKGROUND: Tuberculosis (TB) is one of the most prevalent infectious diseases affecting millions worldwide. The currently available anti-TB drugs and vaccines have proved insufficient to contain this scourge, necessitating an urgent need for identification of novel drug targets and therapeutic strategies. The disruption of crucial protein-protein interactions, especially those that are responsible for virulence in Mycobacterium tuberculosis - for example the ESAT-6:CFP10 complex - are a worthy pursuit in this direction. METHODS: We therefore sought to improvise a method to attenuate M. tuberculosis while retaining the latter's antigenic properties. We screened peptide libraries for potent ESAT-6 binders capable of dissociating CFP10 from ESAT-6. We assessed the disruption by a peptide named HCL2, of the ESAT-6:CFP10 complex and studied its effects on mycobacterial survival and virulence. RESULTS: We found that HCL2, derived from the human cytochrome c oxidase subunit 3 (COX3) protein, disrupts ESAT-6:CFP10 complex, binds ESAT-6 potently, disintegrates bacterial cell wall and inhibits extracellular as well as intracellular mycobacterial growth. In addition, an HCL2 expressing M. tuberculosis strain induces both Th1 and Th17 host protective responses. CONCLUSIONS: Disruption of ESAT-6:CFP10 association could, therefore, be an alternate method for attenuating M. tuberculosis, and a possible route towards future vaccine generation.