Host-derived CEACAM-laden vesicles engage enterotoxigenic E. coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections.

The Impact of Non-Dysentery Shigella Infection on the Growth and Health of Children over Time (INSIGHT)-A Prospective Case-Control Study Protocol.

(1) Shigella spp. (Shigella) is known for causing dysentery with blood in stool, but most children infected with Shigella have non-dysentery Shigella-associated diarrhea (NDSD). The World Health Organization recommends the use of antibiotics when diarrhea is bloody, leaving most NDSD cases untreated. The absence of dysentery may not indicate a low risk of death and severe morbidity. Rapid diagnosis and treatment of shigellosis in vulnerable, young children may be lifesaving. INSIGHT aims to determine the potential benefit of identifying NDSD cases (n = 296) and their outcomes compared to cases of Shigella dysentery [DS (n = 148)] and non-Shigella watery diarrhea [WD (n = 148)]. (2) Children seeking care at hospitals in Bangladesh will be enrolled and followed for 1 year (NDSD and DS) or 90 days (WD). We will determine the impact of NDSD on morbidity, mortality, gut health, nutritional status, and cognitive development compared to DS and WD in children and whether the simple "Rapid LAMP-based Diagnostic Test (RLDT)" can accelerate the detection and treatment of shigellosis in the clinical settings of rural Bangladesh. (3) INSIGHT will determine the impact of NDSD in children and determine if the treatment guidelines of shigellosis need to be revisited to improve clinical outcomes and the development of these children.

Comprehensive multi-layered analyses of genotype-dependent proteo-metabolic networks reveal organellar crosstalk and biochemical pathways regulating aroma formation in rice.

Molecular basis of rice aroma formation is sparsely known and developmental programs driving biochemical pathways towards aroma is in infancy. Here, discovery and targeted proteo-metabolome of non-aromatic and aromatic rice seeds across developmental stages identified a total of 442 aroma-responsive proteins (ARPs) and 824 aroma-responsive metabolites (ARMs) involved in metabolism, calcium and G-protein signaling. Biochemical examination revealed ARM/Ps were linked to 2-acetylpyrrolidine, γ-aminobutyrate, anthocyanin, tannins, flavonoids and related enzymes. Pairwise correlation and clustering showed positive correlation among ARM/Ps. Consistent with aroma-related QTLs, ARPs were mapped on chromosomes 3,4,5,8 and were mainly compartmentalized in cytoplasm and mitochondria. ARM/P-correlation network identified associations related to metabolism and signaling. Multiple reaction monitoring (MRM) confirmed role of catechins, quinic acid and quercetin in aroma formation. Pathway enrichment, multivariate analysis and qRT-PCR validated that calcium and G-protein signaling, aromatic/branched-chain aminoacid, 2-acetylpyrrolidine, oxylipin, melvonate and prenylpyrophosphate pathways, indole, phenylacetate, flavonoid, cinnamoic ester govern aroma formation in rice.

Wheat TaNACα18 functions as a positive regulator of high-temperature adaptive responses and improves cell defense machinery.

Global wheat production amounted to >780 MMT during 2022-2023 whose market size are valued at >$128 billion. Wheat is highly susceptible to high-temperature stress (HTS) throughout the life cycle and its yield declines 5-7% with the rise in each degree of temperature. Previously, we reported an array of HTS-response markers from a resilient wheat cv. Unnat Halna and described their putative role in heat acclimation. To complement our previous results and identify the key determinants of thermotolerance, here we examined the cytoplasmic proteome of a sensitive cv. PBW343. The HTS-triggered metabolite reprograming highlighted how proteostasis defects influence the formation of an integrated stress-adaptive response. The proteomic analysis identified several promising HTS-responsive proteins, including a NACα18 protein, designated TaNACα18, whose role in thermotolerance remains unknown. Dual localization of TaNACα18 suggests its crucial functions in the cytoplasm and nucleus. The homodimerization of TaNACα18 anticipated its function as a transcriptional coactivator. The complementation of TaNACα18 in yeast and overexpression in wheat demonstrated its role in thermotolerance across the kingdom. Altogether, our results suggest that TaNACα18 imparts tolerance through tight regulation of gene expression, cell wall remodeling and activation of cell defense responses.

Proteomic signatures uncover phenotypic plasticity of susceptible and resistant genotypes by wall remodelers in rice blast.

Molecular communication between macromolecules dictates extracellular matrix (ECM) dynamics during pathogen recognition and disease development. Extensive research has shed light on how plant immune components are activated, regulated and function in response to pathogen attack. However, two key questions remain largely unresolved: (i) how does ECM dynamics govern susceptibility and disease resistance, (ii) what are the components that underpin these phenomena? Rice blast, caused by Magnaporthe oryzae adversely affects rice productivity. To understand ECM regulated genotype-phenotype plasticity in blast disease, we temporally profiled two contrasting rice genotypes in disease and immune state. Morpho-histological, biochemical and electron microscopy analyses revealed that increased necrotic lesions accompanied by electrolyte leakage governs disease state. Wall carbohydrate quantification showed changes in pectin level was more significant in blast susceptible compared to blast resistant cultivar. Temporally resolved quantitative disease- and immune-responsive ECM proteomes identified 308 and 334 proteins, respectively involved in wall remodelling and integrity, signalling and disease/immune response. Pairwise comparisons between time and treatment, messenger ribonucleic acid expression, diseasome and immunome networks revealed novel blast-related functional modules. Data demonstrated accumulation of α-galactosidase and phosphatase were associated with disease state, while reactive oxygen species, induction of Lysin motif proteins, CAZymes and extracellular Ca-receptor protein govern immune state.

Proteo-metabolomic Dissection of Extracellular Matrix Reveals Alterations in Cell Wall Integrity and Calcium Signaling Governs Wall-Associated Susceptibility during Stem Rot Disease in Jute.

The plant surveillance system confers specificity to disease and immune states by activating distinct molecular pathways linked to cellular functionality. The extracellular matrix (ECM), a preformed passive barrier, is dynamically remodeled at sites of interaction with pathogenic microbes. Stem rot, caused by Macrophomina phaseolina, adversely affects fiber production in jute. However, how wall related susceptibility affects the ECM proteome and metabolome remains undetermined in bast fiber crops. Here, stem rot responsive quantitative temporal ECM proteome and metabolome were developed in jute upon M. phaseolina infection. Morpho-histological examination revealed that leaf shredding was accompanied by reactive oxygen species production in patho-stressed jute. Electron microscopy showed disease progression and ECM architecture remodeling due to necrosis in the later phase of fungal attack. Using isobaric tags for relative and absolute quantitative proteomics and liquid chromatography-tandem mass spectrometry, we identified 415 disease-responsive proteins involved in wall integrity, acidification, proteostasis, hydration, and redox homeostasis. The disease-related correlation network identified functional hubs centered on α-galactosidase, pectinesterase, and thaumatin. Gas chromatography-mass spectrometry analysis pointed toward enrichment of disease-responsive metabolites associated with the glutathione pathway, TCA cycle, and cutin, suberin, and wax metabolism. Data demonstrated that wall-degrading enzymes, structural carbohydrates, and calcium signaling govern rot responsive wall-susceptibility. Proteomics data were deposited in Pride (PXD046937; PXD046939).

Validation of a Human Challenge Model Using an LT-Expressing Enterotoxigenic E. coli Strain (LSN03-016011) and Characterization of Potential Amelioration of Disease by an Investigational Oral Vaccine Candidate (VLA1701).

Controlled human infection models are important tools for the evaluation of vaccines against diseases where an appropriate correlate of protection has not been identified. Enterotoxigenic Escherichia coli (ETEC) strain LSN03-016011/A (LSN03) is an LT enterotoxin and CS17-expressing ETEC strain useful for evaluating vaccine candidates targeting LT-expressing strains. We sought to confirm the ability of the LSN03 strain to induce moderate-to-severe diarrhea in a healthy American adult population, as well as the impact of immunization with an investigational cholera/ETEC vaccine (VLA-1701) on disease outcomes. A randomized, double-blinded pilot study was conducted in which participants received two doses of VLA1701 or placebo orally, one week apart; eight days after the second vaccination, 30 participants (15 vaccinees and 15 placebo recipients) were challenged with approximately 5 × 109 colony-forming units of LSN03. The vaccine was well tolerated, with no significant adverse events. The vaccine also induced serum IgA and IgG responses to LT. After challenge, 11 of the placebo recipients (73.3%; 95%CI: 48.0-89.1) and 7 of the VLA1701 recipients (46.7%; 95%CI: 24.8-68.8) had moderate-to-severe diarrhea (p = 0.26), while 14 placebo recipients (93%) and 8 vaccine recipients (53.3%) experienced diarrhea of any severity, resulting in a protective efficacy of 42.9% (p = 0.035). In addition, the vaccine also appeared to provide protection against more severe diarrhea (p = 0.054). Vaccinees also tended to shed lower levels of the LSN03 challenge strain compared to placebo recipients (p = 0.056). In addition, the disease severity score was lower for the vaccinees than for the placebo recipients (p = 0.046). In summary, the LSN03 ETEC challenge strain induced moderate-to-severe diarrhea in 73.3% of placebo recipients. VLA1701 vaccination ameliorated disease severity, as observed by several parameters, including the percentage of participants experiencing diarrhea, as well as stool frequency and ETEC severity scores. These data highlight the potential value of LSN03 as a suitable ETEC challenge strain to evaluate LT-based vaccine targets (NCT03576183).

Epidemiology of enterotoxigenic Escherichia coli and impact on the growth of children in the first two years of life in Lima, Peru.

Background: Enterotoxigenic E. coli (ETEC) is a leading cause of diarrheal morbidity and mortality in children, although the data on disease burden, epidemiology, and impact on health at the community level are limited. Methods: In a longitudinal birth cohort study of 345 children followed until 24 months of age in Lima, Peru, we measured ETEC burden in diarrheal and non-diarrheal samples using quantitative PCR (LT, STh, and STp toxin genes), studied epidemiology and measured anthropometry in children. Results: About 70% of children suffered from one or more ETEC diarrhea episodes. Overall, the ETEC incidence rate (IR) was 73 per 100 child-years. ETEC infections began early after birth causing 10% (8.9-11.1) ETEC-attributable diarrheal burden at the population level (PAF) in neonates and most of the infections (58%) were attributed to ST-ETEC [PAF 7.9% (1.9-13.5)] and LT + ST-ETEC (29%) of which all the episodes were associated with diarrhea. ETEC infections increased with age, peaking at 17% PAF (4.6-27.7%; p = 0.026) at 21 to 24 months. ST-ETEC was the most prevalent type (IR 32.1) with frequent serial infections in a child. The common colonization factors in ETEC diarrhea cases were CFA/I, CS12, CS21, CS3, and CS6, while in asymptomatic ETEC cases were CS12, CS6 and CS21. Only few (5.7%) children had repeated infections with the same combination of ETEC toxin(s) and CFs, suggested genotype-specific immunity from each infection. For an average ETEC diarrhea episode of 5 days, reductions of 0.060 weight-for-length z-score (0.007 to 0.114; p = 0.027) and 0.061 weight-for-age z-score (0.015 to 0.108; p = 0.009) were noted in the following 30 days. Conclusion: This study showed that ETEC is a significant pathogen in Peruvian children who experience serial infections with multiple age-specific pathotypes, resulting in transitory growth impairment.

Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children.

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected additional 12% Shigella and 13% ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

Combining extracellular matrix proteome and phosphoproteome of chickpea and meta-analysis reveal novel proteoforms and evolutionary significance of clade-specific wall-associated events in plant.

Extracellular matrix (ECM) plays central roles in cell architecture, innate defense and cell wall integrity (CWI) signaling. During transition to multicellularity, modular domain structures of ECM proteins and proteoforms have evolved due to continuous adaptation across taxonomic clades under different ecological niche. Although this incredible diversity has to some extent been investigated at protein level, extracellular phosphorylation events and molecular evolution of ECM proteoform families remains unexplored. We developed matrisome proteoform atlas in a grain legume, chickpea and performed meta-analyses of 74 plant matrisomes. MS/MS analysis identified 1,424 proteins and 315 phosphoproteins involved in diverse functions. Cross-species ECM protein network identified proteoforms associated with CWI maintenance system. Phylogenetic characterization of eighteen matrix protein families highlighted the role of taxon-specific paralogs and orthologs. Novel information was acquired on gene expansion and loss, co-divergence, sub functionalization and neofunctionalization during evolution. Modular networks of matrix protein families and hub proteins showed higher diversity across taxonomic clades than among organs. Furthermore, protein families differ in nonsynonymous to synonymous substitution rates. Our study pointed towards the matrix proteoform functionality, sequence divergence variation, interactions between wall remodelers and molecular evolution using a phylogenetic framework. This is the first report on comprehensive matrisome proteoform network illustrating presence of CWI signaling proteins in land plants.

Effects of breastfeeding on children's gut colonization with multidrug-resistant Enterobacterales in peri-urban Lima, Peru.

Children living in low-resource settings are frequently gut-colonized with multidrug-resistant bacteria. We explored whether breastfeeding may protect against children's incident gut colonization with extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-Ec) and Klebsiella, Enterobacter, or Citrobacter spp. (ESBL-KEC). We screened 937 monthly stool samples collected from 112 children aged 1-16 months during a 2016-19 prospective cohort study of enteric infections in peri-urban Lima. We used 52,816 daily surveys to examine how exposures to breastfeeding in the 30 days prior to a stool sample were associated with children's risks of incident gut-colonization, controlling for antibiotic use and other covariates. We sequenced 78 ESBL-Ec from 47 children to explore their diversity. Gut-colonization with ESBL-Ec was increasingly prevalent as children aged, approaching 75% by 16 months, while ESBL-KEC prevalence fluctuated between 18% and 36%. Through 6 months of age, exclusively providing human milk in the 30 days prior to a stool sample did not reduce children's risk of incident gut-colonization with ESBL-Ec or ESBL-KEC. From 6 to 16 months of age, every 3 additional days of breastfeeding in the prior 30 days was associated with 6% lower risk of incident ESBL-Ec gut-colonization (95% CI: 0.90, 0.98, p = .003). No effects were observed on incident ESBL-KEC colonization. We detected highly diverse ESBL-Ec among children and few differences between children who were predominantly breastfed (mean age: 4.1 months) versus older children (10.8 months). Continued breastfeeding after 6 months conferred protection against children's incident gut colonization with ESBL-Ec in this setting. Policies supporting continued breastfeeding should be considered in efforts to combat antibiotic resistance.

Impact of Rotaviral Diarrhea on Child Growth in Sub-Saharan Africa and South Asia in the Global Enteric Multicenter Study.

Rotavirus is the leading cause of dehydrating diarrhea among children in developing countries. The impact of rotaviral diarrhea on nutritional status is not well understood. We aimed to determine the association between rotavirus-positive moderate-to-severe diarrhea and nutrition in children under 5 years of age. We analyzed data from the Global Enteric Multicenter Study on children 0-59 months old from South Asia and sub-Saharan Africa. The relationships between explanatory variables and outcome variables were assessed using multiple linear regression; the explanatory variable was the presence of rotavirus in the stool sample, and the outcome variables were z scores [length/height-for-age (LAZ/HAZ), weight-for-age (WAZ), and weight-for-length/height (WLZ/WHZ)] at follow-up (∼60 days). The prevalence of rotaviral diarrhea was 17.3% (905/5,219) in South Asia and 19.95% (842/4,220) in sub-Saharan Africa. Rotavirus was associated with higher LAZ/HAZ (ß: 0.19; 95% CI: 0.12, 0.26; P <0.001) and WAZ (ß: 0.15; 95% CI: 0.79, 0.22; P <0.001) in sub-Saharan Africa and with lower WLZ/WHZ (ß coefficient: -0.08; 95% CI: -0.15, -0.009; P = 0.027) in South Asia. Our study indicates that rotaviral diarrhea is positively associated with nutritional status in sub-Saharan Africa and is negatively associated with nutritional status in South Asia. An expedited implementation policy of ongoing preventive and control strategies, including vaccination against rotavirus, is necessary to reduce the burden of rotaviral diarrhea, which may further help to reduce the potential nutritional ramifications.

The Antimicrobial Resistance of Enterotoxigenic Escherichia coli from Diarrheal Patients and the Environment in Two Geographically Distinct Rural Areas in Bangladesh over the Years.

Antimicrobial resistance (AMR) is an unprecedented global health challenge, involving the transfer of bacteria and genes between humans and the environment. We simultaneously and longitudinally determined the AMR of enterotoxigenic Escherichia coli (ETEC) strains isolated from diarrheal patients and an aquatic environment over two years from two geographically distinct locations, Coastal Mathbaria and Northern Chhatak in Bangladesh. A total of 60% and 72% of ETEC strains from the patients in Mathbaria and Chhatak, respectively, were multi-drug resistant (MDR) with a high proportion of ETEC resistant to nalidixic acid (80.7%), macrolides (49.1-89.7%), ampicillin (57.9-69%), and trimethoprim/sulfamethoxazole (55.2%). From the surface water, 68.8% and 30% of ETEC were MDR in Mathbaria and Chhatak, respectively, with a high proportion of ETEC strains resistant to macrolides (87.5-100%), ampicillin (50-75%), ceftriaxone (62.5%), and nalidixic acid (40%). Notably, 80-100% of the ETEC strains were susceptible to tetracycline and quinolones (ciprofloxacin and norfloxacin), both in clinical and aquatic ETEC. The AMR varied by the ETEC toxin types. The patterns of excessive or limited consumption of drugs to treat diarrhea over time in Bangladesh were reflected in the ETEC AMR from the patients and the environment. The high prevalence of MDR-ETEC strains in humans and the environment is of concern, which calls for vaccines and other preventative measures against ETEC.

Epidemiology of Enterotoxigenic Escherichia coli among Children and Adults Seeking Care at Hospitals in Two Geographically Distinct Rural Areas in Bangladesh.

Enterotoxigenic Escherichia coli (ETEC) infections undeniably continue to have substantial morbidity and mortality in younger children; however, limited data are available on the disease burden of older children and adults and on ETEC epidemiology by geographical location at the subnational level. Facility-based surveillance over the years was established to identify patients with ETEC diarrhea in two geographically distinct areas in rural Bangladesh, Chhatak in the north and Mathbaria in the southern coastal area. ETEC was highly prevalent in both areas, while the proportions, toxin types and colonization factors varied by location, season and age groups. Children < 5 years old and adults between 20 and 60 years old were at the highest risk of ETEC diarrhea which required urgent care. This study underscores the importance of capturing subnational and seasonal variations in ETEC epidemiology. ETEC vaccine developers and public health stakeholders may need to target adults between 20 and 60 years of age in addition to young children as new vaccines currently under development become licensed and introduction begins.

Dehydration-responsive cytoskeleton proteome of rice reveals reprograming of key molecular pathways to mediate metabolic adaptation and cell survival.

The plant cytoskeletal proteins play a key role that control cytoskeleton dynamics, contributing to crucial biological processes such as cell wall morphogenesis, stomatal conductance and abscisic acid accumulation in repercussion to water-deficit stress or dehydration. Yet, it is still completely unknown which specific biochemical processes and regulatory mechanisms the cytoskeleton uses to drive dehydration tolerance. To better understand the role of cytoskeleton, we developed the dehydration-responsive cytoskeletal proteome map of a resilient rice cultivar. Initially, four-week-old rice plants were exposed to progressive dehydration, and the magnitude of dehydration-induced compensatory physiological responses was monitored in terms of physicochemical indices. The organelle fractionation in conjunction with label-free quantitative proteome analysis led to the identification of 955 dehydration-responsive cytoskeletal proteins (DRCPs). To our knowledge, this is the first report of a stress-responsive plant cytoskeletal proteome, representing the largest inventory of cytoskeleton and cytoskeleton-associated proteins. The DRCPs were apparently involved in a wide array of intra-cellular molecules transportation, organelles positioning, cytoskeleton organization followed by different metabolic processes including amino acid metabolism. These findings presented open a unique view on global regulation of plant cytoskeletal proteome is intimately linked to cellular metabolic rewiring of adaptive responses, and potentially confer dehydration tolerance, especially in rice, and other crop species, in general.

The 2022 Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference: Summary of abstract-based presentations.

The global nonprofit organization PATH hosted the third Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference in Washington, DC, on November 29 to December 1, 2022. With a combination of plenary sessions and posters, keynote presentations, and breakout workshops, the 2022 VASE Conference featured key updates on research related to the development of vaccines against neglected diarrheal pathogens including Shigella, enterotoxigenic Escherichia coli (ETEC), Campylobacter, and Salmonella. The presentations and discussions highlighted the significant impact of these diarrheal pathogens, particularly on the health of infants and young children in low- and middle-income countries, reflecting the urgent need for the development and licensure of new enteric vaccines. Oral and poster presentations at the VASE Conference explored a range of topics, including: the global burden and clinical presentation of disease, epidemiology, and the impact of interventions; the assessment of the value of vaccines against enteric pathogens; preclinical evaluations of vaccine candidates and models of enteric diseases; vaccine candidates in clinical trials and human challenge models; host parameters and genomics that predict responses to infection and disease; the application of new omics technologies for characterization of emerging pathogens and host responses; novel adjuvants, vaccine delivery platforms, and immunization strategies; and strategies for combination/co-administered vaccines. The conference agenda also featured ten breakout workshop sessions on topics of importance to the enteric vaccine field, which are summarized separately. This article reviews key points and highlighted research presented in each of the plenary conference sessions and poster presentations at the 2022 VASE Conference.

Contribution of the Rapid LAMP-Based Diagnostic Test (RLDT) to the Evaluation of Enterotoxigenic Escherichia coli (ETEC) and Shigella in Childhood Diarrhea in the Peri-Urban Area of Ouagadougou, Burkina Faso.

The estimates of enterotoxigenic Escherichia coli (ETEC) and Shigella burden in developing countries are limited by the lack of rapid, accessible, and sensitive diagnostics and surveillance tools. We used a "Rapid LAMP based Diagnostic Test (RLDT)" to detect ETEC and Shigella in diarrheal and non-diarrheal stool samples from a 12-month longitudinal cohort of children under five years of age in a peri-urban area of Ouagadougou in Burkina Faso (West Africa). To allow comparison with the RLDT-Shigella results, conventional culture methods were used to identify Shigella strains in the stool samples. As conventional culture alone cannot detect ETEC cases, a subset of E. coli-like colonies was tested using conventional PCR to detect ETEC toxins genes. Of the 165 stool samples analyzed for ETEC, 24.9% were positive when using RLDT against 4.2% when using culture followed by PCR. ETEC toxin distribution when using RLDT was STp 17.6% (29/165), LT 11.5% (19/165), and STh 8.5% (14/165). Of the 263 specimens tested for Shigella, 44.8% were positive when using RLDT against 23.2% when using culture. The sensitivity and specificity of the RLDT compared to culture (followed by PCR for ETEC) were 93.44% and 69.8% for Shigella and 83.7% and 77.9% for ETEC, respectively. This study indicates that both Shigella and ETEC are substantially underdiagnosed when using conventional culture and highlights the potential contribution of the new RLDT method to improve enteric disease burden estimation and to guide future efforts to prevent and control bacterial enteric infection and disease.

Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children.

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected 12% more Shigella and 13% more ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

Development of a simple, rapid, and sensitive molecular diagnostic assay for cholera.

Cholera continues to inflict high rates of morbidity and mortality. Prompt identification of cholera cases facilitates rapid outbreak responses in the short term while providing reliable surveillance data to guide long-term policies and interventions. Microbiological stool culture, the current recognized gold standard for diagnosing cholera, has significant limitations. Rapid diagnostic tests (RDTs) represent promising alternatives for diagnosing cholera in areas with limited laboratory infrastructure. However, studies conducted with the current cholera RDTs demonstrated wide variations in sensitivity and specificity. To address this gap in the diagnosis of cholera, we developed a simple, rapid, and sensitive diagnostic assay, "Rapid LAMP based Diagnostic Test (RLDT)." With a novel, simple sample preparation method directly from the fecal samples along with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, cholera toxin gene (ctxA) and O1 (O1rfb) gene could be detected in less than an hour. Cholera RLDT assay is cold chain and electricity-free. To avoid any end-user bias, a battery-operated, handheld reader was used to read the RLDT results. The performance specifications of the cholera RLDT assay, including analytical sensitivity and specificity, were evaluated using direct fecal samples, dried fecal samples on filter paper, and environmental water samples spiked with cholera strain. The limit of detection (LOD) was ~104 CFU/gm of stool for both ctxA and O1 genes, corresponding to about 1 CFU of Vibrio cholerae per reaction within 40 minutes. The LOD was 10 bacteria per ml of environmental water when tested with RLDT directly, without enrichment. Being simple, RLDT has the potential to be applied in resource-poor endemic settings for rapid, sensitive, and reliable diagnosis of cholera.

The plant cytoskeleton takes center stage in abiotic stress responses and resilience.

Stress resilience behaviours in plants are defensive mechanisms that develop under adverse environmental conditions to promote growth, development and yield. Over the past decades, improving stress resilience, especially in crop species, has been a focus of intense research for global food security and economic growth. Plants have evolved specific mechanisms to sense external stress and transmit information to the cell interior and generate appropriate responses. Plant cytoskeleton, comprising microtubules and actin filaments, takes a center stage in stress-induced signalling pathways, either as a direct target or as a signal transducer. In the past few years, it has become apparent that the function of the plant cytoskeleton and other associated proteins are not merely limited to elementary processes of cell growth and proliferation, but they also function in stress response and resilience. This review summarizes recent advances in the role of plant cytoskeleton and associated proteins in abiotic stress management. We provide a thorough overview of the mechanisms that plant cells employ to withstand different abiotic stimuli such as hypersalinity, dehydration, high temperature and cold, among others. We also discuss the crucial role of the plant cytoskeleton in organellar positioning under the influence of high light intensity.