A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.

Archaeal SRP RNA and SRP19 facilitate the assembly of SRP54-FtsY targeting complex.

The signal recognition particle (SRP) plays an essential role in protein translocation across biological membranes. Stable complexation of two GTPases in the signal recognition particle (SRP) and its receptor (SR) control the delivery of nascent polypeptide to the membrane translocon. In archaea, protein targeting is mediated by the SRP54/SRP19/7S RNA ribonucleoprotein complex (SRP) and the FtsY protein (SR). In the present study, using fluorescence resonance energy transfer (FRET), we demonstrate that archaeal 7S RNA stabilizes the SRP54·FtsY targeting complex (TC). Moreover, we show that archaeal SRP19 further assists 7S RNA in stabilizing the targeting complex (TC). These results suggest that archaeal 7S RNA and SRP19 modulate the conformation of the targeting complex and thereby reinforce TC to execute protein translocation via concomitant GTP hydrolysis.

Hydrocarbon degradation potential and competitive persistence of hydrocarbonoclastic bacterium Acinetobacter pittii strain ABC.

Acinetobacter pittii strain ABC was isolated from oily sludge sediments and characterized with regard to utilization/degradation of hydrocarbons and competitive persistence in hydrocarbon-amended media. The isolate grew in both aliphatic- and aromatic hydrocarbon-amended Bushnell-Haas medium (BHM). When incubated in 1% (v/v) Assam crude oil-amended BHM for 5 and 10 days, this strain was able to degrade 88% and 99.8% of the n-hexane extractable crude oil components, respectively. The isolate showed appreciable emulsification index (E24 65.26 ± 1.2%), hydrophobicity (60.88 ± 3.5%) and produced lipopeptide biosurfactant (0.57 g L-1). The isolate was able to tolerate heavy metal salts at concentrations reported in crude oil-polluted sediments from Assam. A 16S rDNA DGGE-based screening showed the persistence of A. pittii strain ABC in hydrocarbon-amended microcosms co-inoculated with other hydrocarbonoclastic bacterial strains (Pseudomonas aeruginosa AKS1, Bacillus sp. AKS2, Arthrobacter sp. BC1, and Novosphingobium panipatense P5:ABC), each isolated from the same oily sludge sediment. These findings indicate A. pittii strain ABC as a potential agent for the bioremediation of crude oil-polluted environment.

Wnt5a-Rac1-NF-κB homeostatic circuitry sustains innate immune functions in macrophages.

Macrophages play a critical role in innate immunity. Differentiation Ags present on macrophages such as CD14 orchestrate the first line of defense against infection. The basal/homeostatic signaling scheme that keeps macrophages thus groomed for innate immune functions remains unresolved. Wnt5a-Fz5 signaling being a primordial event during cell differentiation, we examined the involvement of Wnt5a-Fz5 signaling in the maintenance of innate immune functions. In this study, we demonstrate that innate immune functions of macrophages ensue at least partly through a homeostatic Wnt5a-Fz5-NF-κB (p65) circuit, which is Rac1 dependent. The autocrine/paracrine Wnt5a-Fz5-Rac1-p65 signaling cascade not only maintains basal levels of the immune defense modulating IFNs and CD14; it also supports macrophage survival. Wnt5a-Fz5-Rac1 signaling mediated p65 homeostasis in turn sustains Wnt5a expression in a feed-forward mode. The natural immune response of macrophages to Escherichia coli/LPS and virus is accordingly sustained. The depiction of sustenance of innate immune functions as an outcome of a homeostatic Wnt5a-p65 axis unfolds previously unidentified details of immune regulation and provides new insight into homeostatic cell signaling.

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.

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.

Improvement of degummed ramie fiber properties upon treatment with cellulase secreting immobilized A. larrymoorei A1.

Degummed ramie fiber is widely used in the textile industry. Cellulase enzyme can be effectively used for bio-polishing of the ramie fiber. We immobilized Agrobacterium larrymoorei A1, a potent extra-cellular cellulase producing bacteria, in Ca-alginate. The production of enzyme significantly increased with increasing alginate concentration and reached a maximum activity of 0.28 IU/ml at 20 g/l, which was 32% higher as compared to free cells. These immobilized cells were used on ramie fibers. Scanning electron micrograph (SEM) and differential interference contrast (DIC) studies showed increased smoothness and orientation of surface structure of the fibers after 19.5 h. The single fiber tenacity was almost same as compared to non-treated fiber and the initial modulus increased by 24.01%. The remarkable reusability of these immobilized cells provides a cost effective method for treatment of natural fibers containing cellulose.

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.

First evidence of pathogenicity of V234I mutation of hVAPB found in Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis is a motor neurodegenerative disease which is characterized by progressive loss of motor neurons followed by paralysis and eventually death. In human, VAMP-associated protein B (VAPB) is the causative gene of the familial form of ALS8. Previous studies have shown that P56S and T46I point mutations of hVAPB are present in this form of ALS. Recently, another mutation, V234I of hVAPB was found in one familial case of ALS. This is the first study where we have shown that V234I-VAPB does not form aggregate like other two mutants of VAPB and localizes differently than the wild type VAPB. It induces Ubiquitin aggregation followed by cell death. We propose that V234I-VAPB exhibits the characteristics of ALS in spite of not having the typical aggregation property of different mutations in various neurodegenerative diseases.

Host proteins Alpha-2-Macroglobulin and LRP1 associate with Chandipura virus.

Chandipura Virus is an emerging tropical pathogen with a high mortality rate among children. No mode of treatment or antivirals exists against CHPV infection, due to little information regarding its host interaction. Studying viral pathogen interaction with its host can not only provide valuable information regarding its propagation strategy, but also on which host proteins interact with the virus. Identifying these proteins and understanding their role in the infection process can provide more stable anti-viral targets. In this study, we focused on identifying host factors that interact with CHPV and may play a critical role in CHPV infection. We are the first to report the successful identification of Alpha-2-Macroglobulin (A2M), a secretory protein of the host that interacts with CHPV. We also established that LRP1 (Low-density lipoprotein receptor-related protein 1) and GRP78 (Glucose regulated protein 78), receptors of A2M, also interact with CHPV. Furthermore, we could also demonstrate that knocking out A2M has a severe effect on viral infection. We conclusively show the interaction of these host proteins with CHPV. Our findings also indicate that these host proteins could play a role in viral entry into the host cell.

Glutamate induces neutrophil cell migration by activating class I metabotropic glutamate receptors.

Leukocytes are recruited at the site of infection or injury as a part of the innate immune system, and play a very critical role in fighting the invading microorganisms and/or healing wounds. Neutrophils are the most abundant leukocytes in healthy humans and are the principal cell types that arrive at the target site in the initial phase of this process. Previous studies from our laboratory have shown that the amino acid glutamate is a novel chemotaxis-inducing factor for human neutrophils. In this report, we provide evidences that clearly demonstrate that the glutamate-induced neutrophil cell migration activity is mediated by the class I metabotropic glutamate receptors. Our results further show that a specific integrin ß2 (ITG ß2) receptor, namely LFA1 (α(L)ß(2)) is activated upon glutamate treatment and is required for further downstream signaling events leading to increased migration of human neutrophil cells. Following glutamate stimulation, LFA1 is phosphorylated by the Src Kinase Lck at the Y735 residue, which triggers a downstream signaling cascade leading to activation of PI3K, Syk, Vav and finally the Rho family GTPase, Rac2. Interestingly, glutamate was previously found to be present in elevated levels in wound fluid. Furthermore, glutamate level was also found to go up following inflammation. Taken together, our study suggests a novel mode of neutrophil recruitment to the target site following an infection or injury.

Immune Response to COVID-19 in India through Vaccination and Natural Infection.

In India, COVID-19 (Corona Virus Disease-2019) continues to this day, although with subdued intensity, following two major waves of viral infection. Despite ongoing vaccination drives to curb the spread of COVID-19, the relative potential of the administered vaccines to render immune protection to the general population and their advantage over natural infection remain undocumented. In this study, we examined the humoral and cell-mediated immune responses induced by the two vaccines Covishield and Covaxin, in individuals living in and around Kolkata, India. We also compared the immune responses induced separately by vaccination and natural infection. Our results indicate that although Covishield generates a better humoral immune response toward SARS-CoV-2, both vaccines are almost equivalent in terms of cell-mediated immune response to the virus. Both Covishield and Covaxin, however, are more effective toward the wild-type virus than the Delta variant. Additionally, the overall immune response resulting from natural infection in and around Kolkata is not only similar to that generated by vaccination but the cell-mediated immune response to SARS-CoV-2 also lasts for at least ten months in some individuals after the viral infection.

Arg²³5 is an essential catalytic residue of Bacillus pumilus DKS1 pectate lyase to degum ramie fibre.

After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of 35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5-9.0. Both Ca²(+) and Mn²(+) ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn²(+) and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1 pectate lyase.

N-protein-RNA interaction is a drug target in a negative strand RNA virus.

The negative strand RNA virus family contains many human pathogens. Finding new antiviral drug targets against this class of human pathogens is one of the significant healthcare needs. Nucleocapsid proteins of negative strand RNA viruses wrap the viral genomic RNA and play essential roles in gene transcription and genome replication. Chandipura virus, a member of the Rhabdoviridae family, has a negative strand RNA genome. In addition to wrapping the genomic RNA, its nucleocapsid protein interacts with the positive strand leader RNA and plays a vital role in the virus life-cycle. We have designed a peptide, based on prior knowledge and demonstrated that the peptide is capable of binding specifically to the positive strand leader RNA. When the peptide was transported inside the cell, it inhibited viral growth with IC50 values in the low micromolar range. Given the widespread occurrence of leader RNAs in negative strand RNA viruses and its interaction with the nucleocapsid protein, it is likely that this interaction could be a valid drug target for other negative strand RNA viruses.

Protein-stabilized silver nanoparticles encapsulating gentamycin for the therapy of bacterial biofilm infections.

Aim: An antibiotic-conjugated protein-stabilized nanoparticle hybrid system was developed to combat the challenges faced during the treatment of drug-resistant bacterial biofilm-associated infections. Materials & methods: Biocompatible silver nanoparticles were synthesized using intracellular protein and gentamycin was attached. The resulting nanohybrid was characterized and its antibacterial efficiency was assessed against Gram-positive, Gram-negative and drug-resistant bacteria. Results: Spectroscopic and electron microscopic analysis revealed that the nanoparticles were spherical with a diameter of 2-6 nm. Red-shifting of the surface plasmon peak and an increase in hydrodynamic diameter confirmed attachment of gentamycin. The nanohybrid exhibited antibacterial efficiency against a range of bacteria with the ability to inhibit and disrupt bacterial biofilm. Conclusion: A unique nanohybrid was designed that has potential to be used to control drug-resistant bacterial infections in the future.

Characterization of microbial response to petroleum hydrocarbon contamination in a lacustrine ecosystem.

Microbiomes of freshwater basins intended for human use remain poorly studied, with very little known about the microbial response to in situ oil spills. Lake Pertusillo is an artificial freshwater reservoir in Basilicata, Italy, and serves as the primary source of drinking water for more than one and a half million people in the region. Notably, it is located in close proximity to one of the largest oil extraction plants in Europe. The lake suffered a major oil spill in 2017, where approximately 400 tons of crude oil spilled into the lake; importantly, the pollution event provided a rare opportunity to study how the lacustrine microbiome responds to petroleum hydrocarbon contamination. Water samples were collected from Lake Pertusillo 10 months prior to and 3 months after the accident. The presence of hydrocarbons was verified and the taxonomic and functional aspects of the lake microbiome were assessed. The analysis revealed specialized successional patterns of lake microbial communities that were potentially capable of degrading complex, recalcitrant hydrocarbons, including aromatic, chloroaromatic, nitroaromatic, and sulfur containing aromatic hydrocarbons. Our findings indicated that changes in the freshwater microbial community were associated with the oil pollution event, where microbial patterns identified in the lacustrine microbiome 3 months after the oil spill were representative of its hydrocarbonoclastic potential and may serve as effective proxies for lacustrine oil pollution.

Protein assembled nano-vehicle entrapping photosensitizer molecules for efficient lung carcinoma therapy.

The efficiency of drug depends not only on its potency but also on its ability to reach the target sites in preference to non-target sites. In this regard, protein assembled nanocarrier is the most promising strategy for intracellular anti-cancer drug delivery. The key motive of this study is to fabricate biocompatible protein assembled nanocarrier conjugated photosensitizer system for stimuli-responsive treatment of lung carcinoma. Here, we have synthesized a unique nanohybrid of protein assembled gold nanoparticles (AuNPs), attaching a model photosensitizer, Protoporphyrin IX (PpIX) to the protein shell of the nanoparticles (NPs) imparting an ideal drug-carrier nature. Photo-induced alteration in hydrodynamic diameter suggests structural perturbation of the nanohybrid which in terms signifies on-demand drug delivery. The drug release profile has been further confirmed by using steady-state fluorescence experiments. AuNP-PpIX showed excellent anti-cancer efficiency upon green light irradiation on lung adenocarcinoma cell line (A549) through intracellular reactive oxygen species (ROS) generation. The cellular morphological changes upon PDT and internalization of nanohybrid were monitored using confocal laser scanning microscope. This anti-cancer effect of nanohybrid was associated with apoptotic pathway which was confirmed in the flow cytometric platform. The developed nanomedicine is expected to find relevance in clinical anti-cancer PDT models in the near future.

Glutamate is the chemotaxis-inducing factor in placental extracts.

Human placental extracts are known to help wound healing. Rapid migration of neutrophils to the wound site is a prerequisite to the wound healing process. Gel filtration analysis of heat-treated placental extract gave the initial cue to the small nature of the migration promoting factor of the extract. HPLC analysis of the extract revealed glutamate to be the predominant free amino acid. Our studies show that glutamate at an optimum concentration of 8 muM induced phenotypic neutrophil chemotaxis, as seen in the time lapse- and transwell assays. Glutamate was also found to induce chemokinesis of the neutrophil, though the stimulation of chemotaxis was more pronounced. The glutamate induced chemotaxis was accompanied by polarization of the actin cytoskeleton, and by polymerization of F-actin. These data indicate that glutamate has a strong chemotactic functionality in the neutrophil, which could be of interest both therapeutically and in further investigation of the molecular basis of chemotaxis.

Glutamate is the chemotaxis-inducing factor in placental extracts.

Human placental extracts are known to help wound healing. Rapid migration of neutrophils to the wound site is a prerequisite to the wound healing process. Gel filtration analysis of heat-treated placental extract gave the initial cue to the small nature of the migration promoting factor of the extract. HPLC analysis of the extract revealed glutamate to be the predominant free amino acid. Our studies show that glutamate at an optimum concentration of 8 microM induced phenotypic neutrophil chemotaxis, as seen in the time lapse and transwell assays. Glutamate was also found to induce chemokinesis of the neutrophil, though the stimulation of chemotaxis was more pronounced. The glutamate induced chemotaxis was accompanied by polarization of the actin cytoskeleton, and by polymerization of F-actin. These data indicate that glutamate has a strong chemotactic functionality in the neutrophil, which could be of interest both therapeutically and in further investigation of the molecular basis of chemotaxis.

Large-scale degumming of ramie fibre using a newly isolated Bacillus pumilus DKS1 with high pectate lyase activity.

A combined (enzymatic and chemical) process using a Bacillus pumilus strain (DKS1), isolated from the soil, was used to degum ramie bast fibres. After 24 h of incubation with the isolated pectinolytic strain using a low-cost medium, the weight loss of the ramie fibre was found to be 25% under small scale. High activity of pectate lyase was detected in the culture supernatants; 400 kg of ramie fibres was degummed with 24% weight loss in large-scale degumming under field conditions. No cellulase activity was found. Microbial intervention followed by mild (0.1%) alkali treatment showed high percentage of weight loss from the ramie fibre. Bacterial degumming followed by chemical treatment resulted in an increase of single fibre tenacity (cN/tex) by more than 20.81% as compared to non-degummed (decorticated) fibre samples. Scanning electron micrographs (SEM) and fluorescence microscope showed that after Bacillus pumilus DKS1 treatment the surface of the decorticated ramie fibre becomes very smooth. These results indicate the process provides an economical and eco-friendly method for the small scale as well as large-scale degumming of decorticated ramie fibre. This study has great relevance to the textile as well as paper industry.