BackgroundWith the increased number of cholera outbreaks and emergence of multidrug resistance in Vibrio cholerae strains it has become necessary for the scientific community to devise and develop novel therapeutic approaches against cholera. Recent studies have indicated plausibility of therapeutic application of metal nano-materials. Among these, silver nanoparticles (AgNPs) have emerged as a potential antimicrobial agent to combat infectious diseases. At present nanoparticles are mostly produced using physical or chemical techniques which are toxic and hazardous. Thus exploitation of microbial systems could be a green eco-friendly approach for the synthesis of nanoparticles having similar or even better antimicrobial activity and biocompatibility. Hence, it would be worth to explore the possibility of utilization of microbial silver nanoparticles and their conjugates as potential novel therapeutic agent against infectious diseases like cholera.ResultsThe present study attempted utilization of Ochrobactrum rhizosphaerae for the production of AgNPs and focused on investigating their role as antimicrobial agents against cholera. Later the exopolymer, purified from the culture supernatant, was used for the synthesis of spherical shaped AgNPs of around 10 nm size. Further the exopolymer was characterized as glycolipoprotein (GLP). Antibacterial activity of the novel GLP–AgNPs conjugate was evaluated by minimum inhibitory concentration, XTT reduction assay, scanning electron microscopy (SEM) and growth curve analysis. SEM studies revealed that AgNPs treatment resulted in intracellular contents leakage and cell lysis.ConclusionThe potential of microbially synthesized nanoparticles, as novel therapeutic agents, is still relatively less explored. In fact, the present study first time demonstrated that a glycolipoprotein secreted by the O. rhizosphaerae strain can be exploited for production of AgNPs which can further be employed to treat infectious diseases. Although this type of polymer has been obtained earlier from marine fungi and bacteria, none of these reports have studied the role of this polymer in AgNPs synthesis and its application in cholera therapy. Interestingly, the microbial GLP-capped AgNPs exhibited antibacterial activity against V. cholerae comparable to ciprofloxacin. Thus the present study may open up new avenues for development of novel therapeutic agents for treatment of infectious diseases.Graphical abstractDevelopment of novel therapeutic agents for treatment of cholera

Microbes develop several strategies to survive in the adverse condition such as biofilm formation, attaining non-dividing state, altering drug target or drug, thereby increases the burden of drug dosage. to combat these issues, nanoparticles have shown an alternative approach for new treatment strategy but synthesis via chemical synthetic route limits their application in biomedical field. Here, green method for the synthesis of gold nanoparticles using sophorolipid (SL) is discussed that is characterized by various techniques. initially, the antimicrobial activity was checked against metabolically active state of microbes; Gram-positive Staphylococcus aureus and Gram-negative Vibrio cholerae using XTT assay and growth kinetics assay. Results suggested higher efficacy of nanoparticles for Gramnegative, therefore further analyzed against Escherichia coli that confirmed its potency for the same. AuNPs-SL also signifies its efficiency at least metabolically active state; non dividing cells and biofilm of these microbes. induced morphological changes were studied by SeM that revealed Aunps-SL led to disruption of cell membrane and leakage of intracellular fluid to the surroundings. Inhibition of respiratory enzymes activity also plays a crucial role in bactericidal action as indicated by LDH assay. Synergy of AuNPs-SL with different antibiotics was also analyzed using checkerboard assay. These results suggested the possible use of AuNPs-SL as an antimicrobial therapy in the field of nanomedicine. In recent years, the emergence of antimicrobial resistance has been recognized as one of the most compelling problem in the field of biomedical sciences. Development of resistance has been reported even against the newly invented drug candidates. The reasons behind the emergence of bacterial resistance are complicated, but it can be discriminated as, partly due to the selection of bacterial mutants by antibiotics and partly due to reduced dosage intake by patients that lead to reduced levels of antibiotics which in turn, trigger bacterial resistance. Multiplying (logarithmic phase) and non-multiplying (stationary phase, dormant or latent) are two different states of bacterial existence 1. Among different survival strategies under adverse conditions, formation of biofilm and achieving dormant or non dividing phase are the privilege one 2. A survey conducted by National Institutes of Health and Centre of Disease control suggested that most of the infection (nearly 65-80%) occurred by biofilm formation 3. Biofilms are complex mixtures of bacteria, containing both states of bacterial existence; multiplying and non-multiplying bacteria 4. In biofilm, cellular metabolic activity is reduced and cells are more resistant to drugs. A significant proportion of most human bacterial infectious disease (nearly 60%) 5 is encompassed by non-multiplying bacteria and these are not easily killed by antibiotics 6. Multiplying bacteria are killed easily in presence of antibiotics as compared to non-multiplying ones. Therefore, designing better t...

Among various techniques of immobilization, EDC/NHS cross linking is a simple and single step process for covalent coupling between enzymes and nanoparticles. Here we describe immobilization of lipase on amine functionalized gold nanoparticles (AuNPs-NH 2 ) to attain enhanced activity and stability. To achieve a suitable orientation, it is necessary to understand the contribution of different functional groups on the enzyme's surface. Therefore, the crystal structure of lipase was analyzed using a computational method (PyMOL) to find the exposed acidic amino acid residues that can be exploited for conjugation. Confirmation of conjugation (AuNP-NH 2 -lipase) was determined by various techniques such as agarose gel electrophoresis, zeta measurement, FTIR-spectroscopy and TEM. Further, catalytic parameters (V max , K M,app , K cat , and K cat /K M,app ) have been studied to establish activity enhancement upon immobilization. The data also suggested that, AuNP-NH 2 -lipase has desirable improved parameters such as temperature and storage stability. The thermodynamic parameters for the kinetics of deactivation IntroductionEnzymes have wide application in food, detergent, medicine, diagnostics, energy and many other industrial sectors due to their catalytic activity under mild conditions, specicity, costeffectiveness and greener production conditions. 1,2 However industrial application of free enzymes is restricted due to high lability, poor stability and problems in their recovery and reuse. 3Immobilization is the effective way to overcome these limitations and extend the horizon of their applications.4 Various methods of immobilization include physical adsorption, ionic interaction, covalent bonding between support and enzyme or entrapment within the different matrices.5 Hydrophobic interactions, ionic interactions, hydrogen bonds, van der Waals interactions and solvation are the major forces responsible for non-covalent interactions. Except for covalent attachment, the above mentioned techniques mainly rely on weak interactions, therefore enzymes are prone to detachment from the support surface with time. Covalent bonding mediated immobilization helps in improving enzyme activity at the cost of loss of conformational integrity.6 A very simple method of crosslinking biomolecules to nanoparticles relies on EDC/NHS coupling in which carbodiimides mediate formation of amide bond between carboxy and amine groups.7 High solubility in water, ease to remove byproducts, zero length cross linking molecule and single step reaction provides an edge over other methods in EDC/NHS coupling.8 Several enzymes have been conjugated using this strategy, but they lack in their structure based consideration prior to immobilization. Improper orientation aer immobilization ultimately affects activity; therefore, it is highly essential to understand the structure of protein before immobilization. One of the rationale approaches is to exploit specic surface amino acids for conjugating enzymes with the nanosurface so that the catalytic p...

One of the most prevalent gynaecologic malignancies with the greatest fatality rate is ovarian cancer. By recognizing the potential of immune system, immunotherapies have brought about a revolution in the treatment of cancer. However, interactions of malignant, non-malignant, and immune cells in the peritoneal cavity form a unique TME which is less explored in terms of immunosuppression. Less tumour mutational burden and strong immunosuppression in peritoneal TME leads to poor outcome of immune checkpoint inhibition (ICI) and CART cell therapy in ovarian cancer. Therefore, alternative immunotherapeutic strategies are of utmost importance to achieve sound clinical success. The development of peptide vaccines based on tumor-associated antigens (TTAs) displayed in ovarian cancer cells can be a potential target to provoke an anti-tumor immune response and subsequent clearance of tumour cells. This insilico study was conducted to find out potential epitopes for a peptide vaccine construct using the immunopeptidomics landscape of ovarian carcinoma. In this investigation, we have selected MUC16, IDO1, FOLR1, and DDX5 for epitope prediction. Potential epitopes for B-cells, Helper T-lymphocytes (HTL) and Cytotoxic T-lymphocytes (CTL) were predicted on the basis of antigenic, allergenic, and toxic properties to design a multiepitope construct. The insilico evaluation of physicochemical properties and higher order structural analyses of the final construct revealed a potential vaccine candidate. The designed vaccine construct may be employed as a therapeutic immunization candidate for ovarian malignancies either alone or in combination with ICI. However, further in vitro and animal experimentation is required to establish the efficacy of the vaccine candidate.