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Tomato curly stunt virus (ToCSV) is a monopartite begomovirus infecting tomatoes in South Africa, with sequence similarity to tomato yellow leaf curl virus (TYLCV). While there are numerous reports on the mechanism of TYLCV resistance in tomato, the underlying mechanisms in the tomato-ToCSV pathosystem is still relatively unknown. The main aim of this study was to investigate and compare the global methylation profile of ToCSV in two near-isogenic tomato lines, one with a tolerant phenotype (T, NIL396) and one with a susceptible phenotype (S, NIL395). Bisulfite conversion and PCR amplification, coupled with a next-generation sequencing approach, were used to elucidate the global pattern of methylation of ToCSV cytosine residues in T and S leave tissue at 35 days post-infection (dpi). The extent of methylation was more pronounced in tolerant plants compared to susceptible plants in all sequence (CG, CHG and CHH) contexts, however, the overall methylation levels were relatively low (<3%). Notably, a significant interaction (p < 0.05) was observed between the viral genomic region and susceptible vs. tolerant status for CG methylated regions where it was observed that the 3’IR CG methylation was significantly (p < 0.05) higher than CG methylation of other genomic regions in tolerant and susceptible plants. Additionally, statistically significant (EdgeR p < 0.05) differentially methylated cytosines were located primarily in the genomic regions V2/V1 and C4/C1 of ToCSV. The relative expression, using RT-qPCR, was also employed in order to quantify the expression of various key methylation-related genes, MET1, CMT2, KYP4/SUVH4, DML2, RDM1, AGO4 and AGO6 in T vs. S plants at 35dpi. The differential expression between T and S was significant for MET1, KYP4/SUVH4 and RDM1 at p<0.05 which further supports more pronounced methylation observed in ToCSV from T plants vs. S plants. While this study provides new insights into the differences in methylation profiles of ToCSV in S vs. T tomato plants, further research is required to link tolerance and susceptibility to ToCSV.

Tomato Curly Stunt Virus (ToCSV) is one of the most catastrophic tomato diseases witnessed in South African fields since 1997. It is one of the utmost economically important begomoviruses reported in South Africa with infected crops portraying symptoms such as yellowing, leaf curling, stunting and fruit set reduction. The molecular mechanisms employed by the ToCSV-tomato pathosystem has not been fully elucidated yet, thus this study aims to contribute to the current body of knowledge using this pathosystem. In this study, an infectivity study of mock and ToCSV-inoculated resistant (R, NIL 396) and susceptible (S, NIL395) near isogenic tomato lines was investigated at 8-, 15-, and 35-dpi. Thereafter, to investigate if epigenetic defence takes place, Chromatin immunoprecipitation (ChIP) coupled to quantitative real-time PCR (qPCR) was used to identify presence of activation mark H3K9Ac and H3K9me2 at 15- and 35-dpi. This was conducted as a pilot study since this is the first report of using ChIP to identify host induced histone modification as a TGS defence mechanism in ToCSV. The infectivity study portrayed results revealing that the S line accumulates larger viral load compared to the R line at all time points throughout viral progression with a fold change of 1.06 between 8 and 15 dpi, and a fold change of 2.43 between 15 and 35 dpi. In addition, mild symptoms were observed for ToCSV-inoculated R plants along with the viral load, tolerant like behaviour is observed in R plants rather than that of a resistant phenotype, thus the R line will be referred to as the tolerant (T) line from hereon. Bokhale et al., (2020) reported elevated levels of RNA dependent RNA polymerase 1 (RDR1) was observed in R plants as compared to S plants in addition to lower viral load when compared to the S line. Therefore, NIL396 line may be linked to tolerance, however, this hypothesis requires further characterization and investigation.ChIP is one of the most commonly used methods with high sensitivity that is used when detecting the presence of epigenetic modifications, especially histone post translational modifictions such as acetylation and methylation. Histone 3 lysine 9 modifications are common in geminivirus infected crops with H3K9Ac and H3K9me2 one of the most common marks identified during epigenetic defence using transcriptional gene silencing (TGS). Unlike TYLCV that has been extensively studied globally characterizing and understanding the various molecular processes governing epidemiology, ToCSV is a relatively new virus identified in South Africa with little research been completed using this pathosystem, therefore, this study was conducted as a pilot study aiming to improve research quality on the overall study. Hence, this study is aimed to identify the presence of epigenetic activation and repressive marks H3K9Ac and H3k9me2, respectively. This is the first time ChIP has been used in the ToCSV-tomato pathosystem to detect the presence of epigenetic defence ii at the TGS level. Post crosslinking, sonication, antibody assay and DNA purification, immunoprecipitated DNA was used to detect presence of ToCSV using conventional PCR. Gene expression of DNA and histone methyltransferases CMT2, MET1, KYP/SUVH4 and AGO4 were investigated using qPCR. It was observed that ToCSV-inoculated immunoprecipitated DNA associated with marks of activation and repression was present in both T and S lines at 15 and 35 dpi; however, the ChIP negative control, Normal Mouse IgG, produced a viral amplicon when compared to the mock ChIP negative IgG control. To determine if this observation was due to plant DNA, 18S rRNA was amplified in mock and ToCSV-inoculated T and S plants suggesting plant DNA co-precipitates along with viral DNA. This data highlights the presence of activation and repressive epigenetic marks of acetylation and methylation in the ToCSV-tomato pathosystem suggesting the role TGS has as a defence mechanism against ToCSV; however, the data remains inconclusive to report if epigenetic defence does occur during infection. In addition, it was observed that T plants displayed upregulation of all DNA and histone methyltransferase genes whereas S plants show downregulation except for AGO4. Hence, the gene expression data portrays methylation occurring in both T and S plants between 15 and 35 dpi, thereby substantiating the H3K9me2 repressive mark observed in ChIP. Thus, several aspects relating to the experiment should be considered such as the downstream application, choice of IgG used, and method of viral DNA isolation, as well as additional epigenetic methods such as DNA methylation used in conjunction with ChIP allowing further accurate results of ChIP related experiments.

Background Anemia is a worldwide problem with iron deficiency being the most common cause. When anemia occurs in pregnancy, it increases the risk of adverse maternal, fetal, and postnatal outcomes. It induces preterm births and low birth weight (LBW) deliveries, long-term neurodevelopmental sequelae, and an increased risk of earlier onset of postnatal iron deficiency. Anemia rates are among the highest in South Asia, and India’s National Family Health Survey (NFHS-5) for 2019–2021 indicated that over half of pregnant women, and more than 65% of children, in the country are classified as anemic (Sciences IIfP, National Family Health Survey-5, 2019–21, India Fact Sheet). In 2021, the parent RAPIDIRON Trial (Derman et al., Trials 22:649, 2021) was initiated in two states in India, with the goal of assessing whether a dose of intravenous (IV) iron given to anemic women during early pregnancy results in a greater proportion of participants with normal hemoglobin concentrations in the third trimester and a lower proportion of participants with LBW deliveries compared to oral iron. As a follow-up to the RAPIDIRON Trial, the RAPIDIRON-KIDS Study will follow the offspring of previously randomized mothers to assess, neurobehavioral, hematological, and health outcomes. Methods This prospective observational cohort study will follow a subset of participants previously randomized as part of the RAPIDIRON Trial and their newborns. Study visits occur at birth, 6 weeks, 4 months, 12 months, 24 months, and 36 months and include blood sample collection with both maternal and infant participants and specific neurobehavioral assessments conducted with the infants (depending on the study visit). The primary outcomes of interest are (1) infant iron status as indicated by both hemoglobin and ferritin (a) at birth and (b) at 4 months of age and (2) the developmental quotient (DQ) for the cognitive domain of the Bayley Scales of Infant Development Version IV (BSID-IV) at 24 months of age. Discussion This RAPIDIRON-KIDS Study builds upon its parent RAPIDIRON Trial by following a subset of the previously randomized participants and their offspring through the first 3 years of life to assess neurodevelopmental and neurobehavioral (infants, children), hematological, and health outcomes. Trial registration ClinicalTrials.gov NCT05504863 , Registered on 17 August 2022. Clinical Trials Registry – India CTRI/2022/05/042933 . Registered on 31 May 2022.

Background Despite tuberculous lymphadenitis (TBL) being the most prevalent type of extrapulmonary tuberculosis, there are limitations in laboratory diagnosis of TBL due to high cost, inadequate diagnostic efficacy and feasibility. Xpert MTB/RIF (Xpert) assay is a design-locked, molecular diagnostic technique that detects Mycobacterium tuberculosis (MTB) genome and rifampicin resistance by targeting rpoB gene and mutations within the gene. Currently, there is limited evidence validating Xpert assay usage for diagnosing TBL in Bangladesh. Therefore, in this study, we evaluated diagnostic efficacy of Xpert, considering composite reference standard (CRS), i.e. combination of acid-fast bacilli (AFB) microscopy, culture, Xpert assay and cytology, as gold standard, and compared it to cytology. Methods AFB microscopy, culture, cytology, and Xpert assay were conducted on fine needle aspirates collected from 523 presumptive TBL patients. Genomic DNA was extracted from bacterial colonies of culture-positive specimens. In order to confirm presence of MTB, PCR and gel electrophoresis were performed on extracted DNA to detect RD9 region of MTB DNA. Sensitivity, specificity, positive and negative predictive values, and Cohen’s kappa coefficient were determined, and McNemar’s test was performed for Xpert and cytology with respect to CRS. Additionally, latent class analysis was performed to estimate sensitivity and specificity of all four diagnostic modalities. Results Xpert showed sensitivity and specificity of 72.9% (261/358) and 100% (165/165) respectively against CRS, with 69.8% sensitivity and 97.1% specificity using Bayesian latent class modeling. In contrast, cytology demonstrated sensitivity and specificity of 84.1% (301/358) and 100% (165/165) against CRS, and 81.9% and 99.9% upon latent class analysis, respectively. Furthermore, Xpert showed moderate agreement with cytology (κ = 0.45, p  < 0.0001), fair agreement with culture (κ = 0.30, p  < 0.0001), and poor agreement with AFB microscopy (κ = 0.09, p  < 0.0001). Conclusion Our study findings validate routinely using Xpert assay in TBL diagnosis and enable detecting patients with low bacterial load. However, further assessment via cytology is recommended for confirmation in Xpert-negative patients having patent symptoms. Clinical trial Not applicable.

To investigate the prevalence of vitamin A deficiency (VAD) among pregnant women in rural Bangladesh, and examine the relationship between various factors and vitamin A status. Community Nutrition Promoter (CNP) centres in Kapasia sub-district of Gazipur district, Bangladesh. A cross-sectional study. Two hundred women, aged 18-39 years, in their second or third trimester of pregnancy were selected from seventeen CNP centres in four unions of Kapasia sub-district where they usually visit for antenatal care. Various socio-economic, personal and pregnancy-related information, dietary intake of vitamin A and mid-upper arm circumference (MUAC) data were collected. Serum retinol (vitamin A) concentration was determined. More than half (51 %) of the pregnant women had low vitamin A status (serum retinol <1.05 micromol/l) with 18.5 % having VAD (serum retinol <0.70 micromol/l). Fifty-three per cent of the women's vitamin A intake was less than the recommended dietary allowance. By multiple regression analysis, MUAC, per-capita expenditure on food and wealth index were found to have significant independent positive relationship with serum retinol concentration, while gestational age of the pregnant women had a negative relationship. The overall F-ratio (10.3) was highly significant (P = 0.0001), the adjusted R2 was 0.18 (multiple R = 0.45). VAD is highly prevalent among rural pregnant women in Bangladesh. Gestational age, nutritional status, per-capita expenditure on food and wealth index appear to be important in influencing the vitamin A status of these women. An appropriate intervention is warranted in order to improve the vitamin A status.