a-soluble-tetrazolium-based-reduction-assay-to-evaluate-effect

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on <em>Candida tropicalis</em> Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Indian INstitute of Technology Roorkee

The rising incidence of non-albicans  species globally, along with the emergence of drug resistance, is a cause for concern. This study investigated the protective efficacy of secreted aspartyl proteinase 2 (Sap2) in systemic  infection. Vaccination with recombinant Sap2 (rSap2) protein from  enhanced survival of mice compared to rSap2 vaccinations from  ( = 0.02),  ( = 0.06), and sham immunization ( = 0.04). Compared to sham-immunized mice, the fungal CFU number was significantly reduced in organs of Sap2-parapsilosis-immunized mice. Histopathologically, increased neutrophilic recruitment was observed in Sap2-parapsilosis- and Sap2-tropicalis-immunized mice. Among different rSap2 proteins, Sap2-parapsilosis vaccination induced increased titers of Sap2-specific Ig, IgG, and IgM antibodies, which could bind whole fungus. Between different groups, sera from Sap2-parapsilosis-vaccinated mice exhibited increased  biofilm inhibition ability  and enhanced neutrophil-mediated fungal killing. Passive transfer of anti-Sap2-parapsilosis immune serum in naive mice significantly reduced fungal burdens compared to those in mice receiving anti-sham immune serum. Higher numbers of plasma cells and -binding B cells in Sap2-vaccinated mice suggest a role of B cells during early stages of Sap2-mediated immune response. Additionally, increased levels of Th1/Th2/Th17 cytokines observed in Sap2-parapsilosis-vaccinated mice indicate immunomodulatory properties of Sap2. Epitope analysis performed using identified B-cell epitopes provides a basis to understand differences in immunogenicity observed among Sap2-antigens and can aid the development of a multivalent or multiepitope anti- vaccine(s). In summary, our results suggest that Sap2-parapsilosis vaccination can improve mouse survival during  infection by inducing both humoral and cellular immunity, and higher titers of Sap2-induced antibodies are beneficial during systemic candidiasis.

Vaccination with Secreted Aspartyl Proteinase 2 Protein from  Can Enhance Survival of Mice during -Mediated Systemic Candidiasis.

The present work aims to synthesize the rifaximin loaded chitosan-alginate core-shell nanoparticles (Rif@CS/Alg-NPs) for antibacterial applications. The core-shell nanoparticles (Rif@CS/Alg-NPs) were characterized by Fourier Transform Infrared (FT-IR) spectroscopy, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-rays diffraction (XRD) and zeta analyzer. The antibacterial activities of Rif@CS/Alg-NPs were investigated against three species of bacteria namely Escherichia coli (E. coli), Pseudomonas aeruginosa (PA) and Bacillus haynesii (BH). Rif@CS/Alg-NPs exhibited outstanding antibacterial activities against E. coli, P. aeroginosa and Bacillus haynesii (BH) with 24 mm, 30 mm and 34 mm zone of inhibitions, respectively. Cytotoxicity of Rif@CS/Alg-NPs was also evaluated against human lung adenocarcinoma cell line A549 and found to be nontoxic. The drug release behavior of Rif@CS/Alg-NPs was investigated at different pH levels and maximum drug release (80%) was achieved at pH (7.2). The drug release kinetic data followed the Higuchi (R = 0.9963) kinetic model, indicating the drug release from Rif@CS/Alg-NPs as a square root of time-dependent process and diffusion controlled. Current research provides a cost-effective and green approach toward the synthesis of Rif@CS/Alg-NPs for its antibacterial applications.

Synthesis of rifaximin loaded chitosan-alginate core-shell nanoparticles (Rif@CS/Alg-NPs) for antibacterial applications.

Systemic candidiasis is an invasive fungal infection caused by members of the genus  The recent emergence of antifungal drug resistance and increased incidences of infections caused by non-albicans  species merit the need for developing immune therapies against  infections. Although the role of cellular immune responses in anti- immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. This review summarizes currently available information on humoral immune responses induced by several promising  vaccine candidates, which have been identified in the past few decades. The protective antibody and B-cell responses generated by polysaccharide antigens such as mannan, β-glucan, and laminarin, as well as protein antigens like agglutinin-like sequence gene (Als3), secreted aspartyl proteinase (Sap2), heat shock protein (Hsp90), hyphally-regulated protein (Hyr1), hyphal wall protein (Hwp1), enolase (Eno), phospholipase (PLB), pyruvate kinase (Pk), fructose bisphosphate aldolase (Fba1), superoxide dismutase gene (Sod5) and malate dehydrogenase (Mdh1), are outlined. As per studies reviewed, antibodies induced in response to leading  vaccine candidates contribute to protection against systemic candidiasis by utilizing a variety of mechanisms such as opsonization, complement fixation, neutralization, biofilm inhibition, direct candidacidal activity, etc. The contributions of B-cells in controlling fungal infections are also discussed. Promising results using anti- monoclonal antibodies for passive antibody therapy reinforces the need for developing antibody-based therapeutics including anti-idiotypic antibodies, single-chain variable fragments, peptide mimotopes, and antibody-derived peptides. Future research involving combinatorial immunotherapies using humanized monoclonal antibodies along with antifungal drugs/cytokines may prove beneficial for treating invasive fungal infections.

The Role of B-Cells and Antibodies against  Vaccine Antigens in Invasive Candidiasis.

Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although  is a predominant species causing systemic candidiasis, infections caused by non-albicans  (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from  for the detection of  infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an -based prokaryotic expression system, and refolded using a multi-step dialysis procedure. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized Balb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using proven candidiasis patient serum and controls. Immunoblotting results indicate that reactivity to rSap2 was specific to candidiasis patient sera with no cross reactivity observed in healthy controls. Increased levels of anti-Sap2-specific Ig, IgG and IgM antibodies were observed in candidiasis patients compared to controls and was similar in sensitivity obtained when whole  was used as coating antigen. In summary, the rSap2 protein from  has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy.

Expression and Purification along with Evaluation of Serological Response and Diagnostic Potential of Recombinant Sap2 Protein from  for Use in Systemic Candidiasis.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, which causes severe illness in humans and is responsible for epidemic outbreaks in Africa, Asia, North and South America, and Europe. Despite its increased global prevalence, no licensed vaccines are available to date for treating or preventing CHIKV infection. The envelope E2 protein is one of the promising subunit vaccine candidates against CHIKV. In this study, we describe successful cloning, expression, and purification of CHIKV E2 full-length (E2-FL) and truncated (E2-ΔC and E2-ΔNC) proteins in the  expression system. The recombinant E2 proteins were purified from inclusion bodies using Ni-NTA chromatography. Further, we describe a detailed refolding procedure for obtaining the CHIKV E2-FL protein in native conformation, which was confirmed using circular dichroism and Fourier transform infrared spectroscopy. BALB/c mice immunized with the three different E2 proteins exhibited increased E2-specific antibody titers compared to sham-immunized controls, suggesting induction of strong humoral immune response. On analyzing the E2-specific antibody response generated in immunized mice, the CHIKV E2-FL protein was observed to be the most immunogenic among the three different CHIKV E2 antigens used in the study. Our B-cell and T-cell epitope mapping results indicate that the presence of specific immunogenic peptides located in the N-terminal and C-terminal regions of the CHIKV E2-FL protein may contribute to its increased immunogenicity, compared to truncated CHIKV E2 proteins. In summary, our study provides a detailed protocol for expressing, purifying, and refolding of the CHIKV E2-FL protein and provides an understanding of its immunogenic epitopes, which can be exploited for the development of novel multiepitope-based anti-CHIKV vaccine strategies.

Expression, Purification, and Refolding of Chikungunya Virus Full-Length Envelope E2 Protein along with B-Cell and T-Cell Epitope Analyses Using Immuno-Informatics Approaches.

Diabetes mellitus has become a major public health concern all over the world. Vildagliptin is one of the antidiabeticdrug that can overcome the existing problem of this prevalent disease. Present study aims to synthesize and investigate the role of vildagliptin-loaded core-shell nanoparticle of grafted psyllium and alginate (VG@P/A-NPs) in anti-diabetes application. FTIR, SEM, XRD, CNMR and zeta analyzer were used for characterization of the core-shell nanoparticles (VG@P/A-NPs). The synthesized acrylamide-grafted-psyllium was also optimized through varying grafting parameters such as acrylamide and ceric ammonium nitrate (CAN) concentration, time and temperature to obtain the maximum yield of acrylamide-grafted-psyllium. Rheological analysis of pure psyllium, grafted psyllium and alginate were also performed. For biological studies, the first cytotoxicity of grafted psyllium and VG@P/A-NPs were examined on human lung adenocarcinoma cell line A549 in which it was observed that VG@P/A-NPs did not exhibited any toxicity. The antidiabetic potential of VG@P/A-NPs was investigated by glucose uptake assay, using TNF-α induced insulin resistance skeletal cell model using mouse muscle L6 cell line. The insulin signaling impaired cell line displayed a highly significant (p < 0.0001) dose-dependent increase in glucose uptake after treatment with increasing doses of VG@P/A-NPs.The drug release behavior of VG@P/A-NPs was examined at various pH and the highest drug release (98 %) was obtained at pH (7.4). The drug release kinetic data was following the Higuchi (R = 0.9848) kinetic model, suggesting the release of drug from vildagliptin-loaded grafted psyllium-alginate core-shell nanoparticles (VG@P/A-NPs) as a square root of time-dependent process and diffusion controlled. This study provides an economical and environment-friendly approach towards the synthesis of VG@P/A-NPs with antidiabetes applications.

Synthesis of vildagliptin loaded acrylamide-g-psyllium/alginate-based core-shell nanoparticles for diabetes treatment.

Mucormycosis is an invasive fungal infection caused by fungi belonging to order Mucorales. Recently, with the increase in COVID-19 infections, mucormycosis infections have become a matter of concern globally, because of the high morbidity and mortality rates associated with them. Due to the association of mucormycosis with COVID-19 disease, it has been termed COVID-19-associated mucormycosis (CAM). In the present review, we focus on mucormycosis incidence, pathophysiology, risk factors, immune dysfunction, interactions of Mucorales with endothelial cells, and the possible role of iron in Mucorales growth. We review the limitations associated with current diagnostic procedures and the requirement for more specific, cost-effective, convenient, and sensitive assays, such as PCR-based assays and monoclonal antibody-based assays for the effective diagnosis of mucormycosis. We discuss the current treatment options involving antifungal drug therapies, adjunctive therapy, surgical treatment, and their limitations. We also review the importance of nutraceuticals-based therapy for the prevention as well as treatment of mucormycosis. Our review also highlights the need to explore the potential of novel immunotherapeutics, which include antibody-based therapy, cytokine-based therapy, and combination/synergistic antifungal therapy, as treatment options for mucormycosis. In summary, this review provides a complete overview of COVID-19-associated mucormycosis, addressing the current research gaps and future developments required in the field.

Soma Rohatgi

Department of Biosciences and Bioengineering

Indian INstitute of Technology Roorkee

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms