Recombinant YopE and LcrV vaccine candidates protect mice against plague and yersiniosis.

No licensed vaccine exists for the lethal plague and yersiniosis. Therefore, a combination of recombinant YopE and LcrV antigens of Yersinia pestis was evaluated for its vaccine potential in a mouse model. YopE and LcrV in formulation with alum imparted a robust humoral immune response, with isotyping profiles leaning towards the IgG1 and IgG2b subclasses. It was also observed that a significantly enhanced expression of IFN-γ, TNF-α, IL-6, IL-2, and IL-1ß from the splenic cells of vaccinated mice, as well as YopE and LcrV-explicit IFN-γ eliciting T-cells. The cocktail of YopE + LcrV formulation conferred complete protection against 100 LD50Y. pestis infection, while individually, LcrV and YopE provided 80 % and 60 % protection, respectively. Similarly, the YopE + LcrV vaccinated animal group had significantly lower colony forming unit (CFU) counts in the spleen and blood compared to the groups administered with YopE or LcrV alone when challenged with Yersinia pseudotuberculosis and Yersinia enterocolitica. Histopathologic evidence reinforces these results, indicating the YopE + LcrV formulation provided superior protection against acute lung injury as early as day 3 post-challenge. In conclusion, the alum-adjuvanted YopE + LcrV is a promising vaccine formulation, eliciting a robust antibody response including a milieu of pro-inflammatory cytokines and T-cell effector functions that contribute to the protective immunity against Yersinia infections. YopE and LcrV, conserved across all three human-pathogenic Yersinia species, provide cross-protection. Therefore, our current vaccine (YopE + LcrV) targets all three pathogens: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. However, the efficacy should be tested in other higher mammalian models.

Anti-Depressant Like Effects of Aethoscytus Foveolus Oil by Improving Stress-mediated Alterations of Monoamine Oxidase, Oxidative Stress, and Neuroinflammation In-vivo.

Depression is a neuropsychological disorder with a complex pathophysiology and its pharmacotherapy is compromised by adverse side effects. Addressing the need for effective treatment for depression, the current study aims to characterize the antidepressant activity of oil extract derived from Aethoscytus foveolus, bugs that are widely available in India, in a mice model of stress-induced depression. Chemical moieties characterized by GC-MS of A. foveolus oil extract have shown good affinity for monoamine oxidase A (MAO-A) in-silico. In-vitro MAO-inhibitory assay using mouse brain homogenates also showed similar results at IC50 1.363 nM (R2 = 0.981, SD ± 0.05, n = 3) of it. These results encouraged us to investigate the antidepressant potential of this oil extract in vivo. Stress-exposed mice (Swiss Albino, either sex, 25-30 gm) were administered 5 and 10 mg/kg doses of oil extract and classified as separate groups (N = 6 per group). Behavioral tests like the forced-swim test, tail-suspension test, and open-field test demonstrated significant attenuation of stress-induced depressive-like behavior of mice by both doses (p < 0.0001 with positive control group i.e., stress group), while biochemical tests on mice brain tissues showed amelioration of stress-induced hyperactivation of MAO (p < 0.0001) and oxidative stress (by increasing Superoxide dismutase and catalase, while reducing lipid peroxidase and nitric oxide) (p < 0.0001). The altered mRNA expression of proinflammatory cytokines (NF-κB, IL-6, IL-12, and TNF-α) (p < 0.015) was also improved by this oil extract. In addition, histopathology of hippocampus tissues of mice supports that this oil recovers stress-mediated structural changes of the brain. In conclusion our findings suggest that oil derived from A. foveolus could be beneficial in the alleviation of stress-mediated depressive-like behavior of mice, and in our knowledge, this is the first report identifying anti-neurodegenerative potential of A. foveolus.

Validating the Nutraceutical Significance of Minor Millets by Employing Nutritional-Antinutritional Profiling.

Millets are group of underutilized cereal crops with higher nutritional values. The present investigation used different classes of minor millets, including barnyard (sava), little (kutki), finger (ragi), kodo and foxtail millets, for evaluation of their nutritional parameters, i.e., the content of proteins, total amino acids, total sugars, insoluble fibers, soluble fibers, total dietary fibers, iron (Fe) and zinc (Zn), along with antinutritional and antioxidant parameters, viz., tannic acid, phytic acid, phenol, flavonoid, proline and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Alpha amylase and alpha glucosidase activity were also thought to elevate millets as a viable staple meal. Foxtail millet showed the maximum inhibition, with an IC50 value of 20.46 ± 1.80 µg mL-1 with respect to α-amylase. The coefficient of correlation between nutritional and antinutritional compositions showed that the starch content was significantly and positively correlated with insoluble fiber (r = 0.465) and dietary fiber (r = 0.487). Moreover, sugar was positively correlated with the phytic acid (r = 0.707), Fe and Zn (r = 0.681) contents. To determine the peptides responsible for anticancer activity, the foxtail protein was subjected to ultrafiltration; it was found that the 3 kDa fraction retained the greatest anticancer activity. Selected millet germplasm line(s) that have the best nutraceutical properties could be used in millet improvement programs.

Natural Biosurfactant as Antimicrobial Agent: Strategy to Action Against Fungal and Bacterial Activities.

Natural surfactants have gained importance as the usage of synthetic surfactants shows economical aspects, health, and environmental effect. This study examined the anti-microbial activity of safflower seed waste (Ssw) isolated surfactant against dandruff-causing Malassezia furfur and skin diseases causing bacterial strains. Saponin was the major component and non-ionic surfactants derived from plants, which have a special molecular structure with hydrophilic glycoside backbone and lipophilic triterpene derivative. The antimicrobial activity of isolated surfactants was confirmed by the MIC and kill-time assays. Our results showed that the isolated saponin may interact with the cell wall and membrane first and destroy the cell wall and membranes, which finally results in bacterial death. Besides, isolated saponin penetrates the cytoplasmic membrane or enters inside the cell after the destruction of cell structure, and then inhibits the normal synthesis of DNA and proteins that are required for bacterial growth. These results suggested that the effects of the Ssw isolated saponin on the growth inhibition of selected bacterial strains may be at the molecular level rather than only physical damage. Extraction of Biosurfactant (saponin) from Safflower seed waste and its antimicrobial activity.

Synthesis and Antibacterial Screening of Some Pyrazole Derivatives Catalyzed by Cetyltrimethylammoniumbromide (CTAB).

AIMS: In this article, we have developed an eco-friendly one-pot multi-component reaction methodology employed for the green synthesis of functionalized pyrazole derivatives viz cyclo-condensation of aromatic aldehydes, ethyl acetoacetate and phenyl hydrazine and/or hydrazine hydrate in the presence of cetyltrimethylammoniumbromide (CTAB) at 90°C temperature in an aqueous medium. MATERIALS AND METHODS: In the present protocol, we developed a green method for the synthesis of functionalized pyrazole derivatives through one-pot, multi-component cyclo-condensation of aromatic aldehydes, phenyl hydrazine or hydrazine hydrate and ethyl acetoacetate using cetyltrimethylammoniumbromide (CTAB) as a catalyst in water as a solvent. Our methodology confers advantages such as short reaction time, atom economy, purification of the product without using column chromatographic and hazardous solvent. The reaction is being catalyzed by cetyltrimethylammoniumbromide (CTAB) and thus, products are formed under the green reaction conditions. RESULTS AND DISCUSSION: Initially, the reaction of benzaldehyde and phenylhydrazine with ethyl acetoacetate was carried out in water at room temperature in the absence of the catalyst; no product was obtained after 24 h (Table 1 entry 1). When the reaction was carried out using L-proline as a catalyst in ethanol at 70°C, the yield of the product was 20%. CONCLUSION: This research not only provides a green and efficient method for the synthesis of sulfinic esters but also shows new applications of electrochemistry in organic synthesis. We consider that this green and efficient synthetic protocol used to prepare sulfinic esters will have good applications in the future. In conclusion, we have developed successfully a green and efficient one-pot multi-component methodology for the synthesis of substituted pyrazoles using CTAB as a catalyst in water as a solvent with excellent yields. Purifications of compounds were achieved without the use of traditional chromatographic procedures. This methodology has advantages of operational simplicity, clean reaction profiles and relatively broad scope, which make it more attractive for the diversity oriented synthesis of these heterocyclic libraries. In this methodology, we suggest a further alternative possibility for the formation of substituted pyrazoles. The compound 7h can be used as an anticancer drug in the pharma industry.

Development of a Rapid and Sensitive Colorimetric Loop-Mediated Isothermal Amplification Assay: A Novel Technology for the Detection of Coxiella burnetii From Minimally Processed Clinical Samples.

Q fever is an important zoonotic disease caused by the bacterium Coxiella burnetii. The agent is considered as a potential agent for bioterrorism because of its low infectious dose, aerial route of transmission, resistance to drying, and many commonly used disinfectants. Humans are largely infected by the inhalation of aerosols that are contaminated with parturition products of infected animals as well as by the consumption of unpasteurized milk products. Thus, rapid and accurate detection of C. burnetii in shedders, especially those that are asymptomatic, is important for early warning, which allows controlling its spread among animals and animal-to-human transmission. In the present study, a colorimetric loop-mediated isothermal amplification (LAMP) assay was developed to confirm the presence of IS1111a gene of C. burnetii in sheep vaginal swabs. The sensitivity of this assay was found to be very comparable to the quantitative PCR (qPCR) assay, which could detect three copies of the gene, which corresponds to a single cell of C. burnetii. The applicability of the colorimetric LAMP assay in the disease diagnosis was assessed by evaluating 145 vaginal swab samples collected from the sheep breeding farms with a history of stillbirth and repeated abortions. Compared to qPCR, colorimetric LAMP had a sensitivity of 93.75% (CI, 69.77-99.84%) and specificity of 100% (CI, 97.20-100%), with a positive (PPV) and negative predictive value (NPV) of 100 and 99.24%, respectively. A very high level of agreement was observed between both colorimetric LAMP and reference qPCR assay. The colorimetric LAMP assay reported here is a rapid and simple test without extensive sample preparation and has a short turnaround time of <45 min. To the best of our understanding, it is the very first study describing the use of colorimetric LAMP assay that detects C. burnetii in vaginal swab samples with minimal sample processing for DNA extraction.

Anticancer Activity of Lectins from Bauhinia purpurea and Wisteria floribunda on Breast Cancer MCF-7 Cell Lines.

BACKGROUND: Individual and collaborative efforts are being made worldwide in search of effective chemical or natural drugs with less severe side-effects for treatment of cancer. Due to the specificity and selectivity properties of lectins for saccharides, several plant lectins are known to induce cytotoxicity into tumor cells. OBJECTIVE: To study the antiproliferative activity of two N-acetyl galactosamine specific plant lectins from seeds of Bauhinia purpurea and Wisteria floribunda against MCF-7 Breast cancer cell lines. METHODS: MTT, lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS), and caspase- 3 assays and flow cytometry for cell cycle analysis were performed. RESULTS: The agglutinins BPL and WFL; 446 µgml-1 (2.2 µM) and 329 µgml-1 (2.8 µM), respectively caused remarkable concentration-dependent antiproliferative effect on MCF-7. The effect was seen to be a consequence of binding of the lectin to the cell surface and triggering S and G2 phase arrest. Apoptosis induced was found to be associated with LDH leakage, cell cycle arrest and ROS generation. The apoptotic signal was observed to be amplified by activation of caspase-3 resulting in cell death. CONCLUSION: The study provides a base for detailed investigation and further use of lectins in cancer studies.

Vector derived artificial miRNA mediated inhibition of West Nile virus replication and protein expression.

West Nile virus (WNV) has been found to be a common cause of neuroinvasive arboviral disease worldwide in human and horses. The process of RNA interference induced by small RNA molecules, like small interfering RNA (siRNA) and microRNA (miRNA), proved to be a novel approach for preventing viral infections. So far there is no published data for inhibition of West Nile virus by vector delivered artificial miRNA which believed to have more inhibitory potential than small interfering (siRNA). In the present study, we designed two artificial miRNA (amiRNAs) targeting the conserved NS5 and NS2A genomic regions of West Nile virus. These amiRNAs oligos were cloned in to miRNA based vector having murine miR-155 backbone which allows the high expression of amiRNAs in green fluorescent protein (GFP) tagged form. Vero cells were transiently transfected by cytomegalovirus (CMV) promoter derived vector expressing amiRNAs transcribed by RNA Pol II. Efficacy of amiRNA targeting the NS5 and NS2A regions of WNV was determined in highly virulent WNV Eg101 strain in Vero cells. The result indicated that both amiRNA effectively inhibit West Nile virus replication. The concatenated amiRNA having dual pre-amiRNA expression cassette showed better efficacy. amiRNA targeting NS5 showed best protection against WNV infection and percentage reduction of WNV titer was observed at 96 hpi is 97.11%. Further study for inhibition of WNV replication was assessed by plaque assay, quantitative reverse transcriptase PCR (qRT-PCR) assay, Immunofluorescence assay and Western blot analysis. Present study concludes that amiRNA (NS5) targeting conserved region of gene significantly reduced the virus replication as determined by plaque assay. Similarly, reduction was also observed at RNA and protein level through real-time RT-PCR and Western blot analysis directly correlate with the inhibition of WNV replication. Here, we describe our current understanding of the role of miRNAs in host defense response against West Nile virus, as well as their potential as new therapeutic approaches.

Development of nsP2 protease based cell free high throughput screening assay for evaluation of inhibitors against emerging Chikungunya virus.

Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z' value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.

Biochemical and functional properties of a lectin purified from the seeds of Cicer arietinum L.

A 35 kDa rabbit erythrocyte agglutinating lectin from the seeds of Cicer arietinum was purified and designated as CAL. The lectin was inhibited by fetuin and N-acetyl-d-galactosamine at a concentration of 20 and 50 mM respectively, but not by simple mono or oligosaccharides. CAL is active between pH 5 and 10 presented thermo stability up to 50 °C and demonstrated DNA damage inhibition at 30 µg concentration. The lectin elicited maximum mitogenic activity towards mice splenocytes at 7.5 µg ml- 1. CAL exerted an inhibitory activity on HIV-1 reverse transcriptase with IC50 of 180 µM. CAL abilities in animal bioassay resulted decreased levels of total triglyceride and creatinine. In vitro and in vivo studies revealed that CAL may constitute an important role impending biomedical applications.

Characterization of chickpea (Cicer arietinum L.) lectin for biological activity.

Lectins are proteins that are subject of intense investigations. Information on lectin from chickpea (Cicer arietinum L.) with respect to its biological activities are very limited. In this study, we purified lectin from the seeds of chickpea employing DEAE-cellulose and SP-Sephadex ion exchange chromatography and identified its molecular subunit mass as 35 kDa. The free radical scavenging activity of lectin measured by the DPPH assay has IC50 of 0.88 µg/mL. Lectin exerted antifungal activity against Candida krusei, Fusarium oxysporium oxysporium, Saccharomyces cerevisiae and Candida albicans, while antibacterial activity against E. coli, B. subtilis, S. marcescens and P. aeruginosa. The minimum inhibitory concentrations were 200, 240, 160 and 140 µg for C. krusei, F. oxysporium, S. cerevisiae and C. albicans respectively. Lectin was further examined for its antiproliferative potential against cancerous cell line. The cell viability assay indicated a high inhibition activity on Ishikawa, HepG2, MCF-7 and MDA-MB-231 with IC50 value of 46.67, 44.20, 53.58 and 37.46 µg/mL respectively. These results can provide a background for future research into the benefits of chickpea lectin to pharmacological perspective.

Vicilin-A major storage protein of mungbean exhibits antioxidative potential, antiproliferative effects and ACE inhibitory activity.

Enzymatic hydrolysates of different food proteins demonstrate health benefits. Search for diet related food protein hydrolysates is therefore of interest within the scope of functional foods. Mungbean is one of the popular foods in India because of rich protein source. In this study, mungbean vicilin protein (MBVP) was enzymatically hydrolysed by alcalase and trypsin under optimal conditions. We have studied the antioxidant, antiproliferative and angiotensin-converting enzyme (ACE) inhibitory activities of mungbean vicilin protein hydrolysate (MBVPH) vis-a-vis alcalase-generated mungbean vicilin protein hydrolysate (AMBVPH) and trypsin-generated mungbean vicilin protein hydrolysate (TMBVPH). The results showed that MBVPH exhibited higher antioxidant potential, ACE inhibitory and antiproliferative activities than MBVP. The alcalase treated hydrolysate displayed highest ACE inhibitory activity with IC50 value of 0.32 mg protein/ml. The MBVP showed significant antiproliferative activity against both MCF-7 and MDA-MB-231 breast cancer cells at the doses between 0.2-1.0 mg/ml. The data suggested that MBVPH can be utilized as physiologically active functional foods with sufficient antihypertensive activity. The results indicate that mungbean can be utilized as a rich resource of functional foods.

Association of Nitric Oxide Synthase2 gene polymorphisms with leprosy reactions in northern Indian population.

The pathogen Mycobacterium leprae causes leprosy that affects mainly skin and nerves. Polymorphisms of certain genes are substantiated to be associated with the susceptibility/resistance to leprosy. The present investigation addressed the association of Nitric Oxide Synthase2 gene polymorphisms and leprosy in a population from northern part of India. A total of 323 leprosy cases and 288 healthy controls were genotyped for four NOS2 promoter variants (rs1800482, rs2779249, rs8078340 and rs2301369) using FRET technology in Real Time PCR. None of these SNPs in promoter sites was associated with susceptibility/resistance to leprosy. NOS2 rs1800482 was found to be monomorphic with GG genotype. However, NOS2-1026T allele was observed to be in higher frequency with leprosy cases (BL and LL) who were not suffering from any reactional episodes compared to cases with ENL reaction {OR=0.30, 95% CI (0.10-0.86), p=0.024}. NOS2-1026GT genotype was more prevalent in cases without reaction (BT, BB and BL) compared to RR reactional patients {OR=0.38, 95% CI (0.17-0.86), p=0.02}. Although haplotype analysis revealed that no haplotype was associated with leprosy susceptibility/resistance with statistical significance, GTG haplotype was noted to be more frequent in healthy controls. These SNPs are observed to be in linkage disequilibrium. Although, these SNPs are not likely to influence leprosy vulnerability, -1026G>T SNP was indicated to have noteworthy role in leprosy reactions.

Vector-delivered artificial miRNA effectively inhibited replication of Chikungunya virus.

Chikungunya virus (CHIKV) has emerged as one of the most significant arboviral threats in many parts of the world. In spite of large scale morbidity, and long lasting polyarthralgia, no licensed vaccine or antivirals are available for the clinical management of CHIKV infection. In this study, a novel RNA interference based strategy has been adopted for effective inhibition of CHIKV. Four artificial microRNAs (amiRNAs) were designed to target different regions of CHIKV genome. These amiRNAs significantly inhibited CHIKV replication in Vero cells at both RNA and protein levels as assessed by qRT-PCR, immunoblotting and immunofluorescence techniques. Further inhibition of the infectious CHIKV up to 99.8% was demonstrated by plaque reduction assay. Concatemerization of amiRNA resulted in higher inhibition of CHIKV than individual amiRNAs. In addition, we studied the effect of combination of RNAi based therapy with other classical antivirals like chloroquine, ribavirin and mycophenolic acid, that helped in understanding the rational selection of RNAi based combination therapy. These findings provide a promising avenue for the development of novel amiRNA or combination based therapeutics against emerging CHIKV.

Phytochemical Evaluation of Moth Bean (Vigna aconitifolia L.) Seeds and Their Divergence.

In the present study, phytochemical contents of 25 moth bean (Vigna aconitifolia) seed accessions were evaluated. This includes protease inhibitors, phytic acid, radical scavenging activity, and tannins. The studies revealed significant variation in the contents of theses phytochemicals. Presence of photochemical composition was correlated with seed storage proteins like albumin and globulin. Qualitative identification of total seed storage protein abundance across two related moth bean accessions using two-dimensional gel electrophoresis (2D-GE) was performed. Over 20 individual protein fractions were distributed over the gel as a series of spots in two moth bean accessions. Seed proteome accumulated spots of high intensity over a broad range of pI values of 3-10 in a molecular weight range of 11-170 kDa. In both seed accessions maximum protein spots are seen in the pI range of 6-8.

Characterization of Seed Storage Proteins from Chickpea Using 2D Electrophoresis Coupled with Mass Spectrometry.

Proteomic analysis was employed to map the seed storage protein network in landrace and cultivated chickpea accessions. Protein extracts were separated by two-dimensional gel electrophoresis (2D-GE) across a broad range 3.0-10.0 immobilized pH gradient (IPG) strips. Comparative elucidation of differentially expressed proteins between two diverse geographically originated chickpea accessions was carried out using 2D-GE coupled with mass spectrometry. A total of 600 protein spots were detected in these accessions. In-gel protein expression patterns revealed three protein spots as upregulated and three other as downregulated. Using trypsin in-gel digestion, these differentially expressed proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) which showed 45% amino acid homology of chickpea seed storage proteins with Arabidopsis thaliana.