Analysis, residue behaviour and risk assessment of combination product of iprovalicarb + copper oxychloride in representative fruiting vegetables, cucurbit and in soil using LC-MS/MS and ICP-MS.

Combination product of two herbicides, i.e. iprovalicarb and copper oxychloride, is a new formulation. There is paucity of data on the dissipation pattern and risk assessment of this combination product in crops. To understand the dissipation behaviour/kinetics of this product, a supervised field trial was undertaken on cucumber and tomato. Method validation for a QuEChERS-based method for analysis of these pesticides from cucumber and tomato matrices reveals that all the parameters were within the acceptance range in accordance with SANTE. The limit of quantitation (LOQ) for iprovalicarb in cucumber and tomato fruits, and in soil matrices when analysed on LC-MS/MS was established at 0.01 mg kg-1. Similarly, the LOQ for copper oxychloride (as copper) on ICP-MS was established at 0.5 mg kg-1 in cucumber and tomato fruits and 5.0 mg kg-1 in soil. Dissipation of iprovalicarb was slower in tomato fruits as compared to cucumber fruits. The initial accumulation of the residues of iprovalicarb was 0.073 and 0.243 mg kg-1 in cucumber and 0.214 and 0.432 mg kg-1 in tomato fruits at standard and double dose, respectively. Similarly, copper oxychloride residues were 3.51 and 6.45 mg kg-1 in cucumber and 1.26 and 2.56 mg kg-1 in tomato fruits at standard and double dose, respectively. The residues were below LOQ in cucumber fruits, tomato fruits and soil at the time of harvest. The residues of copper oxychloride persisted till harvest time in cucumber fruits and in soil. A preharvest interval (PHI) of 3 day is recommended on safer side for the combination product of iprovalicarb + copper oxychloride. Theoretical maximum daily intake (TMDI) is less than maximum permissible intake (MPI) for iprovalicarb and copper oxychloride at both the doses from 0 day and onward. The results from the present study can be of immense importance for establishing label claims, maximum residue limits (MRLs) and risk assessment by national and international regulatory agencies.

Dengue virus induced autophagy is mediated by HMGB1 and promotes viral propagation.

Dengue virus (DENV) exploits various cellular pathways including autophagy to assure enhanced virus propagation. The mechanisms of DENV mediated control of autophagy pathway are largely unknown. Our investigations have revealed a novel role for high-mobility group box1 protein (HMGB1) in regulation of cellular autophagy process in DENV-2 infected A549 cell line. While induction of autophagy by rapamycin treatment resulted in enhanced DENV-2 propagation, the blockade of autophagy flux with bafilomycin A1 suppressed viral replication. Furthermore, siRNA-mediated silencing of HMGB1 significantly abrogated dengue induced autophagy, while LPS induced HMGB1 expression counteracted these effects. Interestingly, silencing of HMGB1 showed reduction of BECN1 and stabilization of BCL-2 protein. On the contrary, LPS induction of HMGB1 resulted in enhanced BECN1 and reduction in BCL-2 levels. This study shows that the modulation of autophagy by DENV-2 is HMGB1/BECN1 dependent. In addition, glycyrrhizic acid (GA), a potent HMGB1 inhibitor suppressed autophagy as well as DENV-2 replication. Altogether, our data suggests that HMGB1 induces BECN1 dependent autophagy to promote DENV-2 replication.

STAT3 inhibition mediated upregulation of multiple immune response pathways in dengue infection.

Dengue infection is a world-wide public health threat infecting millions of people annually. Till date no specific antiviral or vaccine is available against dengue virus. Recent evidence indicates that targeting host STAT3 could prove to be an effective antiviral therapy against dengue infection. To explore the potential of STAT3 inhibition as an antiviral strategy, we utilized a STAT3 inhibitor stattic as antiviral agent and performed whole proteome analysis of mammalian cells by mass spectrometry. Differentially expressed proteins among the infected and stattic treated groups were sorted based on their fold change expression and their functional annotation studies were carried out to establish their biological networks. The results presented in the current study indicated that treatment with stattic induces several antiviral pathways to counteract dengue infection. Together with this, we also observed that treatment with stattic downregulates pathways involved in viral transcription and translation thus establishing STAT3 as a suitable target for the development of antiviral interventions. This study establishes the role of STAT3 inhibition as an alternative strategy to counteract DENV pathogenesis. Targeting STAT3 by stattic or similar molecules may help in identifying novel therapeutic interventions against DENV and probably other flaviviruses.

High-mobility group box 1 protein promotes dengue virus replication by interacting with untranslated regions of viral genome.

Dengue virus (DENV) is most prevalent arthropod-borne human pathogen belongs to Flaviviridae family causes thousands of deaths annually. HMGB1 is highly conserved, ubiquitously expressed, non-histone nuclear protein which plays important role in diseases like metabolic disorders, cancer, and viral infections. However, the importance of HMGB1 in DENV infection is understudied. In this study, we observed that DENV-2 induces cytoplasmic translocation and secretion of HMGB1. Interestingly, inhibition of HMGB1 secretion by ethyl pyruvate (EP) enhanced viral propagation while silencing of HMGB1 resulted in abrogated viral replication in DENV-2 infected A549 cells. RNA-Electrophoretic mobility shift assay and immunoprecipitation showed that HMGB1 interacts with 5'-3' UTRs of DENV-2 genome. This interaction further stimulates production of proinflammatory cytokines like TNF-α, IL-6 and IL-1ß which have been implicated in pathogenesis of severe DENV disease. Together, our finding suggests that DENV-2 modulates HMGB1 translocation and HMGB1-DENV-2 UTRs RNA interaction further induces proinflammatory cytokines production in A549 cells. This study discloses HMGB1 as an important host factor contributing to disease pathogenesis and hence can be targeted as an alternative approach for antiviral development against DENV virus infection.

Structural analysis of extrafloral nectaries of Senna occidentalis L.: insights on diversity and evolution.

MAIN CONCLUSION: The extrafloral nectaries of S. occidentalis were studied structurally and anatomically (at secretory and post-secretory developmental stages). Role of extrafloral nectaries as a common plant-adoptive characteristic in context to diversity and phylogenetic pattern was also speculated while exploring other collaborative evolutionary implications of this plant. Extrafloral nectaries (EFNs) are widespread and evolutionarily labile traits that have repeatedly and remarkably evolved in vascular plants. Morphological descriptions of the EFNs of certain plant species are common in the literature, but they rarely relate morphology with histology, gland distribution and secretory characteristics. Studies relating EFNs features, i.e., morphology and distribution with their differential visitation by insects, viz. ants and the cost of maintenance to the plants are important to understand the evolution of these glands. Therefore, in this study a morphological, anatomical (structure and ultrastructure) and secretory characterization of EFNs occurring on Senna occidentalis L. is made with the implications of gland attributes discussed from a functional perspective. S. occidentalis L. (Caesalpiniaceae) is an economically important species from industrial, medicinal and agricultural perspective. Observations from the result showed that shape of the EFNs (size 1-2 mm) ranged to globular, ovoid-conical, dome-shaped, fusiform or cylindrical with conical tip. The EFNs were sessile, positioned interpetiolar or seated at the base of petiole. Light and transmission electron microscopic studies showed the specific internal structures of the extrafloral nectary. Two developmental stages of the EFNs (secretory and post-secretory) were recognized. Our current understanding of the phylogenetic patterns of EFNs makes them powerful candidates for future work exploring the drivers of their evolutionary origins, shifts, and losses.

Experiences From an Implementation Model of ARI Diagnostic Device in Pneumonia Case Management Among Under-5 Children in Peripheral Healthcare Centers in India.

OBJECTIVES: To address pneumonia, a major killer of under-5 children in India, a multimodal pulse oximeter was implemented in Health and Wellness Centers. Given the evidence of pulse oximetry in effective pneumonia management and taking into account the inadequate skills of front-line healthcare workers in case management, the device was introduced to help them readily diagnose and treat a child and to examine usability of the device. DESIGN: The implementation was integrated with the routine OPD of primary health centers for 15 months after healthcare workers were provided with an abridged IMNCI training. Monthly facility data was collected to examine case management with the diagnostic device. Feedback on usefulness of the device was obtained. SETTING: Health and Wellness Centers (19) of 7 states were selected in consultation with state National Health Mission based on patient footfall. PARTICIPANTS: Under-5 children presenting with ARI symptoms at the OPD. RESULTS: Of 4846 children, 0.1% were diagnosed with severe pneumonia and 23% were diagnosed with pneumonia. As per device readings, correct referrals were made of 77.6% of cases of severe pneumonia, and 81% of pneumonia cases were correctly given antibiotics. The Pulse oximeter was highly acceptable among health workers as it helped in timely classification and treatment of pneumonia. It had no maintenance issue and battery was long-lasting. CONCLUSION: Pulse oximeter implementation was doable and acceptable among health workers. Together with IMNCI training, PO in primary care settings is a feasible approach to provide equitable care to under-5 children.

Signal transducer and activator of transcription 3 (STAT3) acts as a proviral factor for dengue virus propagation.

Dengue fever is a significant mosquito-borne viral disease that affects millions of people every year. As a co-existing mechanism, DENV has evolved to evade elimination by the host antiviral immune system. DENV is reported to modulate host interferon response either by attenuating the factors that mediate interferon response like STAT1 and STAT2 or inhibiting the activation of STAT1 or by STAT2 degradation. Through this study we aim to understand how DENV modulates STAT3 mediated interferon response to its own advantage. We employed various techniques like Western blot, Confocal microscopy, RT-PCR to show that STAT3 acts as a pro-viral factor for DV-2 propagation. As per result of the present study STAT3 is upregulated as well as activated by phosphorylation in DV-2 infected A549 cells. Additionally, STAT3 knockdown led to a significant decrease in expression of viral proteins as well as viral replication. We show that DV-2 strategically tweaks STAT3 which is a negative regulator of Type I IFN signaling, in order to evade host Type I and Type III interferon response by upregulating its expression and activation. Our results demonstrate the proviral role of STAT3 for DV-2 propagation which is correlated to activation by tyrosine phosphorylation. Furthermore, since STAT3 is critical factor for DV-2 propagation, its modulation can facilitate targeted development of antivirals against Dengue.

Neem (Azadirachta Indica A. Juss) Capsules for Prophylaxis of COVID-19 Infection: A Pilot, Double-Blind, Randomized Controlled Trial.

CONTEXT: SARS-CoV-2 is a global public-health concern. Interventions to prevent infection are urgently needed. The anti-inflammatory and antiviral effects of neem make it a potential agent for COVID-19 prophylaxis. OBJECTIVE: The study intended to evaluate the prophylactic effects of neem capsules for persons at high risk of COVID-19 infection due to contact with COVID-19 positive patients. DESIGN: The research team designed a prospective, randomized, double-blind, placebo-controlled, parallel-design study. SETTING: The study was conducted at a single center in India. PARTICIPANTS: Participants were 190 healthcare workers at the hospital or relatives of patients with COVID-19 infection. INTERVENTION: Of the 190 participants, 95 were in the intervention group and 95 in the control group. Participants received 50 mg of a proprietary, patent-pending, neem-leaf extract or a placebo orally in capsules, twice a day for 28 days. OUTCOME MEASURES: The number of individuals positive for COVID-19 between baseline and follow-up on day 56 was the primary outcome measure. Secondary measures included an evaluation of neem's safety and its effects on quality of life (QOL) and changes in biomarkers. RESULTS: The mean age of participants was 36.97 years, and 68.42% were male. Total 13 subjects tested positive during the study. All were asymptomatic. Of the 154 participants who completed the study per-protocol, 11 tested positive, 3 in the intervention group and 8 in the control group. The probability of COVID-19 infection in participants receiving the intervention was 0.45 times that of participants receiving the placebo, a relative risk of 0.45, with the effectiveness of the intervention being around 55%. Treatment-emergent adverse events (TEAEs) in both groups were minimal and were of grade 1 or 2 in severity. Biomarkers and QOL remained stable in both groups. CONCLUSIONS: The study found a reduced risk of COVID-19 infection in participants receiving neem capsules, which demonstrates its potential as a prophylactic treatment for the prevention of COVID-19 infection. The findings warrant further investigation in clinical trials.

Structural Analysis of the Ash2L/Dpy-30 Complex Reveals a Heterogeneity in H3K4 Methylation.

Dpy-30 is a regulatory subunit controlling the histone methyltransferase activity of the KMT2 enzymes in vivo. Paradoxically, in vitro methyltransferase assays revealed that Dpy-30 only modestly participates in the positive heterotypic allosteric regulation of these methyltransferases. Detailed genome-wide, molecular and structural studies reveal that an extensive network of interactions taking place at the interface between Dpy-30 and Ash2L are critical for the correct placement, genome-wide, of H3K4me2 and H3K4me3 but marginally contribute to the methyltransferase activity of KMT2 enzymes in vitro. Moreover, we show that H3K4me2 peaks persisting following the loss of Dpy-30 are found in regions of highly transcribed genes, highlighting an interplay between Complex of Proteins Associated with SET1 (COMPASS) kinetics and the cycling of RNA polymerase to control H3K4 methylation. Overall, our data suggest that Dpy-30 couples its modest positive heterotypic allosteric regulation of KMT2 methyltransferase activity with its ability to help the positioning of SET1/COMPASS to control epigenetic signaling.

Comparative Evaluation of Efficiency of Different File Systems in Terms of Remaining Dentin Thickness Using Cone-Beam Computed Tomography: An In vitro Study.

BACKGROUND: Endodontic therapy treats inside of the tooth and its success is based upon the triad of thorough canal debridement, effective disinfection, and obturation of the canal space. Thus, one of the most important steps is biomechanical preparation, which is the key stage of endodontic treatment with a predictive success factor if performed properly. AIM: The aim of the study is to evaluate and compare the cleaning efficiency of different file systems in terms of remaining dentin thickness. MATERIALS AND METHODS: A total of thirty permanent extracted anterior teeth were taken for the study and was divided into three groups - Group I - Manual Protapers, Group II - Rotary Mtwo, and Group III - Reciprocating WaveOne file systems. Pre-cone-beam computed tomography (CBCT) scan was taken followed by biomechanical preparation by the respective file groups. Post-CBCT scan was taken and the pre- and post-CBCT scans were compared for remaining dentin thickness. Data were analyzed with ANOVA (P = 0.001). STATISTICAL ANALYSIS USED: ANOVA test was used in this study. RESULTS: Protaper showed minimum reduction in dentin thickness followed by Mtwo and WaveOne showed maximum reduction in dentin thickness, but the intergroup comparison was found to be highly insignificant. CONCLUSION: WaveOne is a highly recommended rotary endodontic instrument which does not cause significant lowering in remaining dentin thickness after biomechanical preparation.

Improving Adolescent Health Services across High Priority Districts in 6 States of India: Learnings from an Integrated Reproductive Maternal Newborn Child and Adolescent Health Project.

BACKGROUND: India has been at the forefront of designing adolescent health (AH) policies. The National Adolescent Reproductive and Sexual Health policy (2006), the Reproductive, Maternal, Newborn Child, and AH strategy (2013), and the "Rashtriya Kishor Swasthya Karyakram (RKSK)" (2014) have been the critical milestones in this direction. However, despite policies being available, the AH outcomes need improvement through operationalization of focused and need-based AH interventions. OBJECTIVES: The objectives of this study were to improve services for RKSK interventions across select geographies of India. MATERIALS AND METHODS: USAID's VRIDDHI Project has been providing technical support at the national level and in six focus states to improve uptake of evidence-based high-impact reproductive, maternal, newborn, child, and AH interventions. To improve AH services and outcomes, two approaches were implemented, namely (a) strengthen the functioning of adolescent-friendly health clinics in 95 high caseload health facilities in 26 high priority districts across six states and (b) demonstrate other operational strategies outlined in RKSK program including strengthening of district committees on AH, undertaking formative research for developing adolescent-focused communication strategy, and operationalizing weekly iron and folic acid supplementation program. RESULTS: As a result of ongoing technical support over 2-year period (January 2016-December 2017), improvements were noted across multiple AH indicators. In addition, evidence-based learnings were also generated from the demonstration models for potential scale up to other geographies. CONCLUSION: The project was successful in improving AH services in the intervention facilities through an integrated approach which is replicable, sustainable, and scalable for driving the AH program in India.

Development and characterization of 2-dimensional culture for buffalo intestinal cells.

Small intestinal epithelial cells (IEC) play a major role in the absorption of nutrients and toxins. Due to the similarity of genome-wide single copy protein orthologues between cattle and human, establishment of ruminant's primary small IEC culture could be a valuable tool for toxicity studies. Therefore, the current study focused on the development and characterization of buffalo IEC culture, as cattle slaughter is banned in India. The buffalo jejunum fragments were washed consecutively several times in saline, warm phosphate buffered saline (PBS), PBS with 5 mM dithiothreitol, digesting solution and 2% sorbitol in PBS. The cells were cultured on 17 µg/cm2 collagen coated plates and transwell plates with serum (2% Fetal bovine serum (FBS) and 10% FBS) and serum-free culture conditions. The cells were differentiated into typical epithelial cobblestone morphology from day 5 onwards in 50% successful cultures. The cultured IEC were characterized by gene expression of epithelial cell markers, cytokeratin and vimentin, and enterocyte markers like villin, zonula occluden (ZO1), fatty acid binding protein 2 (FABP2) and small intestinal peptidase (IP). Based on the morphology and gene expression profile, 10% FBS has been recommended for culturing primary buffalo IEC on collagen coated plates for 10 days. However, 50% of the successful cultures could not show epithelial phenotype on 10% FBS culture conditions even on collagen coated plates. Interestingly, undifferentiated IEC showed an increasing expression of FABP2, IP and ZO1 transcripts compared to differentiated intestinal cells with 10% FBS on collagen plates. Therefore, future studies are needed to understand the role of FABP2, IP and ZO1 in differentiation of buffalo IEC.

Herbal combat against E. faecalis - An in vitro study.

BACKGROUND: Herbs have renewed importance in this modern era for their antimicrobial properties and fewer side effects. So a novel idea to use these herbs for pulpectomy; was implemented. There are chances of failure of pulpectomy due to inefficient removal of microbial load. Thus, for a successful endodontic treatment, obturating material with optimum antimicrobial properties is advocated in the present study. AIM: To compare and evaluate the antibacterial efficacy of zinc oxide eugenol, zinc oxide with tulsi extract and zinc oxide with aloe vera as obturating materials against E. faecalis bacteria. MATERIALS AND METHODS: Antimicrobial efficacy of zinc oxide eugenol as control, zinc oxide with tulsi extract and zinc oxide with aloe vera as experimental groups were assessed by using agar diffusion method. The statistical analysis was done. RESULTS: Intergroup comparison revealed significant difference amongst all the groups except between zinc oxide eugenol and zinc oxide with tulsi extract. Zinc oxide eugenol had significantly higher zone of inhibition among all the groups. CONCLUSION: According to results obtained from the present study can be summarized as follows: Zinc oxide eugenol > Zinc oxide with tulsi extract > Zinc oxide with aloe vera.

Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68.

Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK-MAPK pathway that regulates alternative splicing facilitates ERK-1/2-mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1-HDAC6-Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation.

Epigenetic manifestation of metabolic syndrome and dietary management.

SIGNIFICANCE: Metabolic syndrome constitutes a group of disorders such as insulin resistance, hypertension, and hypertriglyceridemia, predisposing an individual to risk factors such as cardiovascular disease, diabetes, obesity, and dyslipidemia. A majority of these diseases are influenced by the environmental factors, nutrient uptake, and genetic profile of an individual that together dysregulate gene function. These genetic and nongenetic factors are reported to introduce epigenetic cues that modulate the gene function which is inherited by the offspring. RECENT ADVANCES: Considering the epigenetic modulation of the metabolic disorders, nutrigenomics has been distinctly categorized as a branch that deals with modulatory effect of nutrients on metabolic disorders and disease progression by supplementing the individuals with key nutrient-enriched diets which are derived from plant and animal sources. CRITICAL ISSUES: Nutritional components of the diet regulate the metabolic health of an individual either by controlling the expression of some key genes related to metabolic pathways or by modulating the epigenetic events on such genes. The present article discusses various metabolic disorders in detail and the effect of nutrients on the specific genes causing those disorders. We also highlight the molecular mechanisms of some metabolic disorders through epigenetic modifications and possible therapeutic interventions. FUTURE DIRECTIONS: With the advent of high-throughput technologies and epigenetic modulation of the metabolic disorders, an altered epigenetic code that is programmed due to improper nutrients can be reverted back by supplementing the diet with various plant-derived compounds. The implication of small molecular drugs is also of utmost significance for challenging the metabolic disorders.

Integrated management of childhood illness (IMCI) follow-up of basic health workers.

OBJECTIVE: To assess the practice of skills learnt by basic health workers for 4 - 8 weeks and one year after IMCI training, and to identify the gaps in practices due to various constraints. METHODS: The Anganwadi Workers (AWWS) and the supervisory staff were given 5 days IMCI training using WHO package. The supervisors gave follow up visits to AWWs using standardized follow up forms adapted from WHO material. The supervisors gave follow up visit to the 1st batch of AWWs 1 year after training in IMCI and a second visit was given 4-8 weeks after the 1st visit. The 2nd batch of AWWs was followed up 4-8 weeks after training in IMCI. RESULTS: The performance on correct treatment of cases by AWWs weeks were trained 4-6 weeks prior to follow up was better than group followed up one year after the completion of training (81.8% and 47.9% respectively). At the same time, the performance on correct treatment showed significant improvement during the second follow up (47.9% and 83.8% respectively). Performance on counseling improved from 15.6% during 1st follow up to 52.1% during 2nd follow up visit. The average number of cases seen by AWWs increased from 6.6 in 1st follow up to 9.3 during second follow up of the same AWWs. CONCLUSION: The basic health workers (AWWs) are capable of correct case management of sick children using the IMCI guidelines. The first follow up visit should not be delayed as delay leads to loss of skills. The health workers benefit from frequent and regular follow up by supervisors. Provision of requisite supplies is essential for practice of skills after training in IMCI by basic health worker.