Molecular cloning, expression, and purification, along with in silico epitope analysis of recombinant enolase proteins (a potential vaccine candidate) from Candida albicans and Candida auris.

Candida albicans is the predominant cause of systemic candidiasis, although other non albicans Candida species are progressively becoming more widespread nowadays. Candida auris has emerged as a deadly multidrug-resistant fungal pathogen, posing a significant threat to global public health. In the absence of effective antifungal therapies, the development of a vaccine against C. auris infections is imperative. Enolase, a key glycolytic enzyme, has emerged as a promising vaccine candidate due to its immunogenic properties and essential role in fungal virulence. Herein, full-length Enolase gene sequences from C. albicans and C. auris were cloned into suitable expression vector and transformed into Escherichia coli expression hosts. Recombinant Enolase proteins were successfully expressed and purified using affinity chromatography under native conditions, followed by SDS-PAGE characterization and Western blot analysis. CD spectroscopy verified the existence of expressed proteins in soluble native conformation. Preliminary in silico studies verified the immunogenicity of recombinant Enolase proteins isolated from both C. albicans and C. auris. Furthermore, bioinformatics analysis revealed conserved B-cell and T-cell epitopes across C. albicans and C. auris Enolase proteins, suggesting potential cross-reactivity and broad-spectrum vaccine efficacy. Our findings are anticipated to play a role in advancing therapeutic as well as diagnostic strategies against systemic candidiasis.

2-D NMR studies of geneoside, a novel pregnane pentaglycoside of 2-deoxy and 2, 6 dideoxy monosaccharides from 'Wattakaka lanceolata'.

Plants of Asclepiadaceae and Apocynaceae family are a rich source of pregnane and pregnane glycosides. They are found in nature either in free state or as their glycosides. They have shown antitumor, anticancer, and hypoglycaemic, antioxidant and antimicrobial activities. In our continued studies on the isolation of pregnane glycosides we have isolated a novel pregnane pentaglycoside comprised of 2-deoxy and 2, 6-dideoxy monosaccharides from Wattakaka lanceolata (Asclepiadaceae). The structure of the new glycoside, Geneoside was established as11α, 12ß-O-diacetyl-drevogenin-P-3-O-ß-D-cymaropyaranosyl (1→4)-ß-D-cymaropyranosyl (1→4) -ß-D-Oleandropyranosyl (1→4)-ß-D-digitalopyranosyl (1→4)-ß-D-digitalopyranoside. The stereoscopic structure was established by chemical degradation, chemical transformation and recent physicochemical techniques viz 1H,13C, 2-D NMR (COSY, TOCSY, HSQC and HMBC) and Mass spectrometry.

Isolation of a novel pentasaccharide Ebaliose from immunostimulant fraction of buffalo milk oligosaccharide.

Buffalo milk processed by method of Kobata and Ginsburg for isolation of its oligosaccharide contents showed significant stimulation of anti-body, delayed type hypersensitivity response to sheep red blood cells in BALB/c mice. This oligosaccharide mixture also stimulated non-specific immune response in terms of MMI. In continuation to our previous studies of oligosaccharide contents of buffalo milk another novel pentasaccharide Ebaliose was isolated from immunostimulant oligosaccharide fraction of buffalo milk. Ebaliose was procured in its purest form, by combining the different chromatographic techniques like Gel filtration, Silica Gel column chromatography, and HPLC. The structure of novel oligosaccharide was determined by 1H,13C, HSQC, TOCSY, COSY, HMBC, ESI-MS, chemical transformation, and chemical degradation. The stereoscopic structure of this novel pentasaccharide was established as under.

Isolation and characterization of emerging Mpox virus from India.

Mpox (previously known as Monkeypox) has recently re-emerged, primarily through human-to-human transmission in non-endemic countries including India. Virus isolation is still considered as the gold standard for diagnosis of viral infections. Here, the qPCR positive skin lesion sample from a patient was inoculated in Vero E6 cell monolayer. Characteristic cytopathic effect exhibiting typical cell rounding and detachment was observed at passage-02. The virus isolation was confirmed by qPCR. The replication kinetics of the isolate was determined that revealed maximum viral titre of log 6.3 PFU/mL at 72 h postinfection. Further, whole genome analysis through next generation sequencing revealed that the Mpox virus (MPXV) isolate is characterized by several unique SNPs and INDELs. Phylogenetically, it belonged to A.2 lineage of clade IIb, forming a close group with all other Indian MPXV along with few from USA, UK, Portugal, Thailand and Nigeria. This study reports the first successful isolation and phenotypic and genotypic characterization of MPXV from India.

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.

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

Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although C. albicans is a predominant species causing systemic candidiasis, infections caused by non-albicans Candida (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 C. parapsilosis for the detection of Candida infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an E. coli-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 Candida was used as coating antigen. In summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy.

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

Systemic candidiasis is an invasive fungal infection caused by members of the genus Candida. The recent emergence of antifungal drug resistance and increased incidences of infections caused by non-albicans Candida species merit the need for developing immune therapies against Candida infections. Although the role of cellular immune responses in anti-Candida 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 Candida 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, ß-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 Candida 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-Candida 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.

Coleus blumei viroid 7: a novel viroid resulting from genome recombination between Coleus blumei viroids 1 and 5.

The genus Coleviroid, family Pospiviroidae, comprises six known viroids, all infecting Plectranthus scutellarioides (Coleus blumei; coleus). In 2017, a novel viroid-like RNA sequence that shares ca. 65% identity with Coleus blumei viroid 1 (CbVd-1) was identified in a coleus cultivar infected by multiple coleviroids. Further sequence and secondary structure analyses are consistent with the discovery of a seventh viroid in the genus Coleviroid: tentatively named "Coleus blumei viroid 7" (CbVd-7). The viroid appears to be the product of a natural recombination event between CbVd-1 and Coleus blumei viroid 5. We prove CbVd-7 to be infectious and in turn demonstrate the ability of all known coleviroid left- and right-arm segments to recombine. With a length of 234 nucleotides, this is the smallest viroid described to date.

Development of High-Resolution DNA Melting Analysis for Simultaneous Detection of Potato Mop-Top Virus and Its Vector, Spongospora subterranea, in Soil.

In this study, a set of duplex reverse transcription PCR (RT-PCR)-mediated high-resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector, Spongospora subterranea f. sp. subterranea (Sss), was developed. The infestation of soil by PMTV was detected with a tobacco-based baiting system. Total RNA extracted from the soil led to successful RT-PCR gel electrophoresis detection of both PMTV and Sss. To facilitate more efficient detection, newly designed primer pairs for PMTV RNA species (i.e., RNA-Rep, RNA-CP, and RNA-TGB) were analyzed together with the existing Sss primers via real-time RT-PCR. The resulting amplicons exhibited melting profiles that could be readily differentiated. Under duplex RT-PCR format, all PMTV and Sss primer combinations led to successful detection of respective PMTV RNA species and Sss in the samples by HRM analyses. When the duplex HRM assay was applied to soil samples collected from six fields at four different sites in New Brunswick, Canada, positive detection of PMTV or Sss was found in 63 to 100% samples collected from fields in which PMTV-infected tubers had been observed. In contrast, the samples from fields where neither PMTV- nor Sss-infected tubers had been observed resulted in negative detection by the assay. Bait tobacco bioassay for PMTV and Sss produced similar results. Of the soil samples collected from PMTV-infested fields, 63 to 83% and 100% led to PMTV and Sss infections in the bait tobacco plants, respectively, whereas no PMTV- or Sss-infected plants were obtained from soil samples collected from PMTV- and Sss-free fields.

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

The rising incidence of non-albicans Candida 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 C. tropicalis infection. Vaccination with recombinant Sap2 (rSap2) protein from C. parapsilosis enhanced survival of mice compared to rSap2 vaccinations from C. albicans (P = 0.02), C. tropicalis (P = 0.06), and sham immunization (P = 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 C. tropicalis biofilm inhibition ability in vitro 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 Candida-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-Candida vaccine(s). In summary, our results suggest that Sap2-parapsilosis vaccination can improve mouse survival during C. tropicalis infection by inducing both humoral and cellular immunity, and higher titers of Sap2-induced antibodies are beneficial during systemic candidiasis.

Evaluation of preparedness for medical emergencies at dental offices: A survey.

BACKGROUND: The best way to handle an emergency is to be prepared in advance. The purpose of this study was to assess the availability of medical emergency drugs at dental offices and to determine the level of knowledge and preparedness of dentists to manage medical emergencies at their dental offices. MATERIALS AND METHODS: A descriptive cross-sectional study was conducted from January to June 2014 with 250 dental graduates to determine their knowledge and ability in the management of medical emergencies and assess availability of emergency drugs and equipments in dental offices in the Ahmedabad and Udaipur areas of India. The questionnaire consisted of mainly objective questions, requiring a simple yes or no reply. Mann-Whitney test was used for the analysis. A 95% confidence level was used and a P value of less than or equal to 0.05 was considered statistically significant. RESULTS: The results of our study showed that almost all the surveyed dentists (98%) enquired about medical history, but only 12% obtained filled health history proforma from patients regarding the above. Only 38.4% participants recorded the vital signs of patients before commencing any treatment. Also, 7.6% reported about having attended any workshops on emergency training or management programs. Emergency kits were available with only 24% participants and the available kits were assessed for the availability of emergency drugs. Also, 34% were confident about giving intramuscular injection and only 6.6% were sure about giving intravenous injections. The P value was found to be highly statistically significant. CONCLUSION: The results of our study reflect an alarming situation about the capability of dentists to deal with such conditions.

Microtensile dentin bond strength of fifth with five seventh-generation dentin bonding agents after thermocycling: An in vitro study.

OBJECTIVES: The objective of this in vitro study was to compare the microtensile dentin bond strength (µTBS) of five seventh-generation dentin bonding agents (DBA) with fifth-generation DBA before and after thermocycling. MATERIALS AND METHODS: Ten extracted teeth were assigned to fifth generation control group (optibond solo) and each of the five experimental groups namely, Group I (G-Bond) ,Group II (S(3) Clearfil), Group III (One Coat 7.0), Group IV (Xeno V), and Group V (Optibond all in one). The crown portions of the teeth were horizontally sectioned below the central groove to expose the dentin. The adhesive resins from all groups were bonded to the teeth with their respective composites. Specimens of sizes 1 × 1 × 6 mm(3) were obtained. Fifty specimens that bonded to dentin from each group were selected. Twenty-five of the specimens were tested for debonding without thermocycling and the remaining were subjected to thermocycling followed by µTBS testing. The data were analyzed with one-way ANOVA and Dunnett's-test for comparison with the reference group(Vth Generation). RESULTS: There was no significant difference (P > 0.05) between the fifth- and seventh-generation adhesives before and after thermocycling. The results of our study showed significantly higher value (P < 0.05) of µTBS of seventh-generation Group II (Clearfil S(3)) compared to the fifth-generation before and after thermocycling. CONCLUSION: The study demonstrated that the Clearfil S(3) bond had the highest µTBS values. In addition, of the five tested seventh-generation adhesive resins were comparable to the fifth-generation DBA.