Generation of a Live-Attenuated Strain of Chikungunya Virus from an Indian Isolate for Vaccine Development.

Chikungunya virus (CHIKV) re-emergence in the last decade has resulted in explosive epidemics. Along with the classical symptoms of fever and debilitating arthralgia, there were occurrences of unusual clinical presentations such as neurovirulence and mortality. These generated a renewed global interest to develop prophylactic vaccines. Here, using the classical approach of virus attenuation, we developed an attenuated CHIKV strain (RGCB355/KL08-p75) for the purpose. Repeated passaging (75 times) of a local clinical isolate of ECSA lineage virus in U-87 MG human astrocytoma cells, an interferon-response-deficient cell line, resulted in efficient adaptation and attenuation. While experimental infection of 3-day old CHIKV-susceptible BALB/c pups with the parent strain RGCB355/KL08-p4 resulted in death of all the animals, there was 100% survival in mice infected with the attenuated p75. In adult, immunocompetent, CHIKV-non-susceptible C57BL/6 mice, inoculation with p75 induced high antibody response without any signs of disease. Both p4 and p75 strains are uniformly lethal to interferon-response-deficient AG129 mice. Passive protection studies in AG129 mice using immune serum against p75 resulted in complete survival. Whole-genome sequencing identified novel mutations that might be responsible for virus attenuation. Our results establish the usefulness of RGCB355/KL08-p75 as a strain for vaccine development against chikungunya.

Design consideration and modelling studies of ultrasound and ultraviolet combined approach for shelf-life enhancement of pine apple juice.

Although both ultraviolet (UV) radiation and ultrasound (US) treatment have their capabilities in microbial inactivation, applying any one method alone may require a high dose for complete inactivation, which may affect the sensory and nutritional properties of pineapple juice. Hence, this study was intended to analyse and optimise the effect of combined US and UV treatments on microbial inactivation without affecting the selected quality parameters of pineapple juice. US treatment (33 kHz) was done at three different time intervals, viz. 10 min, 20 min and 30 min., after which, juice samples were subjected to UV treatment for 10 min at three UV dosage levels, viz. 1 J/cm2, 1.3 J/cm2, and 1.6 J/cm2. The samples were evaluated for total colour difference, pH, total soluble solids (TSS), titrable acidity (TA), and ascorbic acid content; total bacterial count and total yeast count; and the standardization of process parameters was done using Response Surface Methodology and Artificial Neural Network. The results showed that the individual, as well as combined treatments, did not significantly impact the physicochemical properties while retaining the quality characteristics. It was observed that combined treatment resulted in 5 log cycle reduction in bacterial and yeast populations while the individual treatment failed. From the optimization studies, it was found that combined US and UV treatments with 22.95 min and1.577 J/cm2 ensured a microbiologically safe product while retaining organoleptic quality close to that of fresh juice.

Enumerating the phytic acid content in maize germplasm and formulation of reference set to enhance the breeding for low phytic acid.

Phytic acid (Myoinositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is a ubiquitous compound present in plants. It is an important constituent in seed reducing the bioavailability of phosphorous and mineral nutrients when fed to monogastric animals like swine, poultry, fish etc. Hence, identification of maize germplasm with reduced phytic acid content is imperative to formulate the breeding programs to evolve low phytate lines. Towards this, three hundred and thirty-eight maize germplasm accessions available at Department of Millets, TNAU, were raised and screened for phytic acid content which varied from 2.77 to 16.70 mg/g of seed. Based on the variability present, a reference set with fifty-eight genotypes for phytic acid was formulated. The reference set was formed with random genotypes selected from the base population to follow a normal distribution (skewness; 0.17, kurtosis; 0.61 and K-S test for normality Dn = 0.70) for phytic acid. The non-significant difference between the means of the base and the reference ensured the entire representation of the base in the formulated reference for phytic acid. Among all the lines in the reference set, the lowest phytic acid content were observed in the lines UMI-113 (2.77 mg/g) followed by UMI-300-1 (3.17 mg/g), UMI-467 (5.50 mg/g) and UMI-158 (6.58 mg/g) could be used as donors for low phytic acid in breeding programs. The principal component analysis for studying the extent of variability in the reference, revealed six major principal components that exhibited 80.40% of variation with flowering traits, ear height and phytic acid as a major contributor for variability. The characters namely plant stand, germination percentage, kernel yield, ear length, ear diameter and number of kernels per row were found to be positively correlated with the phytic acid and this emphasizes the negative pleiotropic effects of low phytic acid lines in germination and seed set. Thus this formulated reference set enables the breeders to handle minimum population for further grouping the genotypes to analyse their heterotic potential combined with low phytic acid.

Effect of Added Alpha 2 Agonists with Local Anaesthetic in Infraclavicular Brachial Plexus Block: A Comparative Study between Dexmedetomidine and Clonidine.

BACKGROUND: Many adjuvant drugs are added with local anesthetics to increase the quality of regional blocks. AIM: To compare the effects of dexmedetomidine and clonidine added to bupivacaine in infraclavicular brachial plexus block in prolonging the duration of analgesia in patients undergoing orthopedic surgery of forearm or hand and also to compare the duration of sensory and motor block, sedation, and hemodynamic changes like bradycardia and hypotension in two groups. SETTINGS AND DESIGN: This was an observational study conducted in a tertiary care hospital. MATERIALS AND METHODS: A study was conducted among 60 patients admitted for elective upper limb surgeries under ultrasound-guided infraclavicular block. Patients who received bupivacaine 0.5% (20 mL) + Clonidine 1 µg.kg-1 were classified as Group A and those received bupivacaine 0.5% (20 mL) + dexmedetomidine 1 µg.kg-1 were classified as Group B. STATISTICAL ANALYSIS: Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) software version 25. RESULTS: Duration of analgesia was significantly higher in Group B as compared to Group A (mean + standard deviation = 764 ± 17.573 min vs. 526 ± 9.958 min, respectively, P = 0.001). The mean time for onset of a sensory block as well as motor block was significantly less in Group B when compared to Group A (P = 0.001). The mean duration of both sensory block and motor block was higher in Group B as compared to Group A (P = 0.001). CONCLUSIONS: The dexmedetomidine group (Group B) provides a quicker and prolonged analgesic action without major adverse effects.

Homologous Recombination DNA Repair Pathway Disruption and Retinoblastoma Protein Loss Are Associated with Exceptional Survival in High-Grade Serous Ovarian Cancer.

Purpose: Women with epithelial ovarian cancer generally have a poor prognosis; however, a subset of patients has an unexpected dramatic and durable response to treatment. We sought to identify clinical, pathological, and molecular determinants of exceptional survival in women with high-grade serous cancer (HGSC), a disease associated with the majority of ovarian cancer deaths.Experimental Design: We evaluated the histories of 2,283 ovarian cancer patients and, after applying stringent clinical and pathological selection criteria, identified 96 with HGSC that represented significant outliers in terms of treatment response and overall survival. Patient samples were characterized immunohistochemically and by genome sequencing.Results: Different patterns of clinical response were seen: long progression-free survival (Long-PFS), multiple objective responses to chemotherapy (Multiple Responder), and/or greater than 10-year overall survival (Long-Term Survivors). Pathogenic germline and somatic mutations in genes involved in homologous recombination (HR) repair were enriched in all three groups relative to a population-based series. However, 29% of 10-year survivors lacked an identifiable HR pathway alteration, and tumors from these patients had increased Ki-67 staining. CD8+ tumor-infiltrating lymphocytes were more commonly present in Long-Term Survivors. RB1 loss was associated with long progression-free and overall survival. HR deficiency and RB1 loss were correlated, and co-occurrence was significantly associated with prolonged survival.Conclusions: There was diversity in the clinical trajectory of exceptional survivors associated with multiple molecular determinants of exceptional outcome in HGSC patients. Concurrent HR deficiency and RB1 loss were associated with favorable outcomes, suggesting that co-occurrence of specific mutations might mediate durable responses in such patients. Clin Cancer Res; 24(3); 569-80. ©2017 AACRSee related commentary by Peng and Mills, p. 508.

Interferon regulated gene (IRG) expression-signature in a mouse model of chikungunya virus neurovirulence.

Interferon regulated genes (IRGs) are critical in controlling virus infections. Here, we analyzed the expression profile of IRGs in the brain tissue in a mouse model of chikungunya virus (CHIKV) neurovirulence. Neurovirulence is one of the newer complications identified in disease caused by re-emerging strains of CHIKV, an alphavirus with positive-strand RNA in the Togaviridae family. In microarray analysis, we identified significant upregulation of 269 genes, out of which a predominant percentage (76%) was IRGs. The highly modulated IRGs included Ifit1, Ifi44, Ddx60, Usp18, Stat1, Rtp4, Mnda, Gbp3, Gbp4, Gbp7, Oasl2, Oas1g, Ly6a, Igtp, and Gbp10, along with many others exhibiting lesser changes in expression levels. We found that these IRG mRNA transcripts are modulated in parallel across CHIKV-infected mouse brain tissues, human neuronal cell line IMR-32 and hepatic cell line Huh-7. The genes identified to be highly modulated both in mouse brain and human neuronal cells were Ifit1, Ifi44, Ddx60, Usp18, and Mnda. In Huh-7 cells, however, only two IRGs (Gbp4 and Gbp7) showed a similar level of upregulation. Concordant modulation of IRGs in both mice and human cells indicates that they might play important roles in regulating CHIKV replication in the central nervous system (CNS). The induction of several IRGs in CNS during infection underscores the robustness of IRG-mediated innate immune response in CHIKV restriction. Further studies on these IRGs would help in evolving possibilities for their targeting in host-directed therapeutic interventions against CHIKV.

Nucleophosmin (NPM1)/B23 in the Proteome of Human Astrocytic Cells Restricts Chikungunya Virus Replication.

Chikungunya virus (CHIKV), a positive-stranded RNA virus, can cause neurological complications by infecting the major parenchymal cells of the brain such as neurons and astrocytes. A proteomic analysis of CHIKV-infected human astrocytic cell line U-87 MG revealed tight functional associations among the modulated proteins. The predominant cellular pathways involved were of transcription-translation machinery, cytoskeletol reorganization, apoptosis, ubiquitination, and metabolism. In the proteome, we could also identify a few proteins that are reported to be involved in host-virus interactions. One such protein, Nucleophosmin (NPM1)/B23, a nucleolar protein, showed enhanced cytoplasmic aggregation in CHIKV-infected cells. NPM1 aggregation was predominantly localized in areas wherein CHIKV antigen could be detected. Furthermore, we observed that inhibition of this aggregation using a specific NPM1 oligomerization inhibitor, NSC348884, caused a significant dose-dependent enhancement in virus replication. There was a marked increase in the amount of intracellular viral RNA, and ∼105-fold increase in progeny virions in infected cells. Our proteomic analysis provides a comprehensive spectrum of host proteins modulated in response to CHIKV infection in astrocytic cells. Our results also show that NPM1/B23, a multifunctional chaperone, plays a critical role in restricting CHIKV replication and is a possible target for antiviral strategies.

Selective enrichment of STRs for applications in forensic human identification.

Forensic human identification (HID) is currently based on determining repeat length polymorphisms located in short tandem repeat regions in the human genome. Despite the great progress made in the area of multiplex PCR-based approaches, limitations associated with challenging forensic samples such as DNA degradation, cooccurrence of inhabited microbial DNA and PCR inhibitors significantly affect the success rate of human DNA profiling. We have developed a sequence-specific pre-PCR STR enrichment method and evaluated its efficacy using DNA samples doped with various contaminants in view of its application on compromised forensic samples. This strategy has enabled us to generate complete and reproducible DNA profiles from samples doped with fivefold excess of nonhuman DNA and three to fourfold excess of various potent PCR inhibitors than that is claimed to be tolerated by some of the widely used commercial multiplex STR kits, from as little as two nanograms of degraded human DNA. The "hybrid capture"-based STR enrichment strategy described in this study is easily adaptable and offers a sensitive, efficient, and economical approach for successful human DNA profiling from compromised and recalcitrant forensic samples that are usually encountered in mass disaster incidents and missing persons' identifications.

Utility of a rapid immunochromatographic strip test in detecting canine parvovirus infection compared with polymerase chain reaction.

AIM: The present study was undertaken to detect the presence of canine parvovirus (CPV) in fecal samples of diarrheic dogs by conventional polymerase chain reaction (PCR) and immunochromatographic (IC) strip test and to compare the diagnostic potential of these tests. MATERIALS AND METHODS: A total of 50 fecal samples collected from diarrheic dogs suspected for CPV infection were subjected to PCR using CPV-555 primer amplifying the gene coding for the VP1 protein. These samples were also tested by IC strip test using a commercial rapid Ag test kit. The results were statistically analyzed using McNemar test. RESULTS: A total of 22 samples (44%) were detected as positive by PCR, which yielded a specific amplicon of 583 bp. In IC strip test, 18 (36%) samples were found to be positive. The sensitivity of the test as compared to PCR was found to be 72.22% and specificity was 92.86%. Positive predictive value and negative predictive value of IC strip test was found to be 88.89% and 81.25%, respectively. Statistical analysis of the results of PCR and IC assay using McNemar test revealed no significant difference (p>0.05). CONCLUSION: The IC strip test could be employed as a rapid field level diagnostic tool for the diagnosis of canine parvoviral diarrhea.