Genetic control of root architectural traits under drought stress in spring barley (Hordeum vulgare L.).

Root architectural traits play pivotal roles in plant adaptation to drought stress, and hence they are considered promising targets in breeding programs. Here, we phenotyped eight root architecture traits in response to well-watered and drought stress conditions in 200 spring barley (Hordeum vulgare L.) inbred lines over two consecutive field seasons. Root architecture traits were less developed under drought in both seasons when compared with control treatments. Genetic variation in root architectural traits was dissected employing a genome-wide association study (GWAS) coupled with linkage disequilibrium mapping. GWAS uncovered a total of 186 significant single nucleotide polymorphism-trait associations for eight root traits under control, drought, and drought-related indices. Of these, a few loci for root traits were detected on chromosomes 3 and 5, which co-located with QTL identified in previous studies. Interestingly, 13 loci showed simultaneou associations with multiple root traits under drought and drought-related indices. These loci harbored candidate genes, which included a wide range of drought-responsive components such as transcription factors, binding proteins, protein kinases, nutrient and ion transporters, and stress signaling factors. For instance, two candidate genes, HORVU7Hr3G0713160 and HORVU6H r3G0626550, are orthologous to AtACX3 and AtVAMPs, which have reported functions in root length-mediated drought tolerance and as a key protein in abiotic stress tolerance, respectively. Interestingly, one of these loci underlying a high-confidence candidate gene NEW ENHANCER OF ROOT DWARFISM1 (NERD1) showed involvement with root development. An allelic variation of this locus in non-coding region was significantly associated with increased root length under drought. Collectively, these results offer promising multi-trait affecting loci and candidate genes underlying root phenotypic responses to drought stress, which may provide valuable resources for genetic improvement of drought tolerance in barley.

Cholera toxin and O-specific polysaccharide immune responses after oral cholera vaccination with Dukoral in different age groups of Bangladeshi participants.

Vaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Bakuchiol, a natural constituent and its pharmacological benefits.

Background and aims: Natural compounds extracted from medicinal plants have recently gained attention in therapeutics as they are considered to have lower Toxicity and higher tolerability relative to chemically synthesized compounds. Bakuchiol from Psoralea corylifolia L. is one such compound; it is a type of meroterpene derived from the leaves and seeds of Psoralea corylifolia plants. Natural sources of bakuchiol have been used in traditional Chinese and Indian medicine for centuries due to its preventive benefits against tumors and inflammation. It plays a strong potential role as an antioxidant with impressive abilities to remove Reactive Oxygen Species (ROS). This review has focused on bakuchiol's extraction, therapeutic applications, and pharmacological benefits. Methods: A search strategy has been followed to retrieve the relevant newly published literature on the pharmacological benefits of bakuchiol. After an extensive study of the retrieved articles and maintaining the inclusion and exclusion criteria, 110 articles were finally selected for this review. Results: Strong support of primary research on the protective effects via antitumorigenic, anti-inflammatory, antioxidative, antimicrobial, and antiviral activities are delineated. Conclusions: From ancient to modern life, medicinal plants have always been drawing the attention of human beings to alleviate ailments for a healthy and balanced lifestyle. This review is a comprehensive approach to highlighting bona fide essential pharmacological benefits and mechanisms underlying their therapeutic applications.

Vaccination of Rabbits with a Cholera Conjugate Vaccine Comprising O-Specific Polysaccharide and a Recombinant Fragment of Tetanus Toxin Heavy Chain Induces Protective Immune Responses against Vibrio cholerae O1.

There is a need for next-generation cholera vaccines that provide high-level and durable protection in young children in cholera-endemic areas. A cholera conjugate vaccine (CCV) is in development to address this need. This vaccine contains the O-specific polysaccharide (OSP) of Vibrio cholerae O1 conjugated via squaric acid chemistry to a recombinant fragment of the tetanus toxin heavy chain (OSP:rTTHc). This vaccine has been shown previously to be immunogenic and protective in mice and found to be safe in a recent preclinical toxicological analysis in rabbits. We took advantage of excess serum samples collected as part of the toxicological study and assessed the immunogenicity of CCV OSP:rTTHc in rabbits. We found that vaccination with CCV induced OSP-, lipopolysaccharide (LPS)-, and rTTHc-specific immune responses in rabbits, that immune responses were functional as assessed by vibriocidal activity, and that immune responses were protective against death in an established virulent challenge assay. CCV OSP:rTTHc immunogenicity in two animal model systems (mice and rabbits) is encouraging and supports further development of this vaccine for evaluation in humans.

Acetic acid positively modulates proline metabolism for mitigating PEG-mediated drought stress in Maize and Arabidopsis.

Introduction: Osmotic imbalance is one of the major consequences of drought stress, negatively affecting plant growth and productivity. Acetic acid has modulatory roles in osmotic balance in plants; however, the mechanistic insights into acetic acid-mediated osmotic adjustment under drought stress remains largely unknown. Methods: Here, we investigated how seed priming and seedling root treatment with acetic acid enabled maize plants overcoming polyethylene glycol (PEG)-induced drought effects. Results: Maize seeds primed with acetic acid showed better growth performance when compared with unprimed seeds under PEG application. This growth performance was mainly attributed to improved growth traits, such as fresh weight, dry weight, length of shoots and roots, and several leaf spectral indices, including normalized difference vegetation index (NDVI) and chlorophyll absorption in reflectance index (MCARI). The levels of oxidative stress indicators hydrogen peroxide (H2O2) and malondialdehyde (MDA) did not alter significantly among the treatments, but proline content as well as the expression of proline biosynthetic gene, Δ1-PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 (P5CS1) was significantly elevated in plants receiving acetic acid under PEG-treatments. On the other hand, treating the seedlings root with acetic acid led to a significant recovery of maize plants from drought-induced wilting. Although growth traits remained unchanged among the treatments, the enhancement of leaf water content, photosynthetic rate, proline level, expression of P5CS1, and antioxidant enzyme activities along with reduced level of H2O2 and MDA in acetic acid-supplemented drought plants indicated a positive regulatory role of acetic acid in maize tolerance to drought. Moreover, the high expression of P5CS1 and the subsequent elevation of proline level upon acetic acid application were further validated using wild type and proline biosynthetic mutant p5cs1 of Arabidopsis. Results showed that acetic acid application enabled wild type plants to maintain better phenotypic appearance and recovery from drought stress than p5cs1 plants, suggesting a crosstalk between acetic acid and proline metabolism in plants under drought stress. Discussion: Our results highlight the molecular and intrinsic mechanisms of acetic acid conferring plant tolerance to drought stress.

Comparison of O-specific polysaccharide responses in patients following infection with Vibrio cholerae O139 versus vaccination with a bivalent (O1/O139) oral killed cholera vaccine in Bangladesh.

Cholera caused by Vibrio cholerae O139 emerged in the early 1990s and spread rapidly to 11 Asian countries before receding for unclear reasons. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). V. cholerae O139 also expresses the OSP-capsule. We, therefore, assessed antibody responses targeting V. cholerae O139 OSP, LPS, capsule, and vibriocidal responses in patients in Bangladesh with cholera caused by V. cholerae O139. We compared these responses to those of age-gender-blood group-matched recipients of the bivalent oral cholera vaccine (OCV O1/O139). We found prominent OSP, LPS, and vibriocidal responses in patients, with a high correlation between these responses. OSP responses primarily targeted the terminal tetrasaccharide of OSP. Vaccinees developed OSP, LPS, and vibriocidal antibody responses, but of significantly lower magnitude and responder frequency (RF) than matched patients. We separately analyzed responses in pediatric vaccinees born after V. cholerae O139 had receded in Bangladesh. We found that OSP responses were boosted in children who had previously received a single dose of bivalent OCV 3 yr previously but not in vaccinated immunologically naïve children. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 despite the presence of capsules, that vaccination with bivalent OCV is poorly immunogenic in the short term in immunologically naïve individuals, but that OSP-specific immune responses can be primed by previous exposure, although whether such responses can protect against O139 cholera is uncertain. IMPORTANCE Cholera is a severe dehydrating illness in humans caused by Vibrio cholerae serogroups O1 or O139. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) of V. cholerae LPS. Yet, little is known about immunity to O139 OSP. In this study, we assessed immune responses targeting OSP in patients from an endemic region with cholera caused by V. cholerae O139. We compared these responses to those of the age-gender-blood group-matched recipients of the bivalent oral cholera vaccine. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 and that the OSP responses primarily target the terminal tetrasaccharide of OSP. Our results further suggest that vaccination with the bivalent vaccine is poorly immunogenic in the short term for inducing O139-specific OSP responses in immunologically naïve individuals, but OSP-specific immune responses can be primed by previous exposure or vaccination.

Development of Shigella conjugate vaccines targeting Shigella flexneri 2a and S. flexneri 3a using a simple platform-approach conjugation by squaric acid chemistry.

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.

Genetic dissection of root architectural plasticity and identification of candidate loci in response to drought stress in bread wheat.

BACKGROUND: The frequency of droughts has dramatically increased over the last 50 years, causing yield declines in cereals, including wheat. Crop varieties with efficient root systems show great potential for plant adaptation to drought stress, however; genetic control of root systems in wheat under field conditions is not yet well understood. RESULTS: Natural variation in root architecture plasticity (phenotypic alteration due to changing environments) was dissected under field-based control (well-irrigated) and drought (rain-out shelter) conditions by a genome-wide association study using 200 diverse wheat cultivars. Our results revealed root architecture and plasticity traits were differentially responded to drought stress. A total of 25 marker-trait associations (MTAs) underlying natural variations in root architectural plasticity were identified in response to drought stress. They were abundantly distributed on chromosomes 1 A, 1B, 2 A, 2B, 3 A, 3B, 4B, 5 A, 5D, 7 A and 7B of the wheat genome. Gene ontology annotation showed that many candidate genes associated with plasticity were involved in water-transport and water channel activity, cellular response to water deprivation, scavenging reactive oxygen species, root growth and development and hormone-activated signaling pathway-transmembrane transport, indicating their response to drought stress. Further, in silico transcript abundance analysis demonstrated that root plasticity-associated candidate genes were highly expressed in roots across different root growth stages and under drought treatments. CONCLUSION: Our results suggest that root phenotypic plasticity is highly quantitative, and the corresponding loci are associated with drought stress that may provide novel ways to enable root trait breeding.

Predicted changes in future precipitation and air temperature across Bangladesh using CMIP6 GCMs.

Understanding spatiotemporal variability in precipitation and temperature and their future projections is critical for assessing environmental hazards and planning long-term mitigation and adaptation. In this study, 18 Global Climate Models (GCMs) from the most recent Coupled Model Intercomparison Project phase 6 (CMIP6) were employed to project the mean annual, seasonal, and monthly precipitation, maximum air temperature (Tmax), and minimum air temperature (Tmin) in Bangladesh. The GCM projections were bias-corrected using the Simple Quantile Mapping (SQM) technique. Using the Multi-Model Ensemble (MME) mean of the bias-corrected dataset, the expected changes for the four Shared Socioeconomic Pathways (SSP1-2.6, SSP2-4.5, SSP3-7.0, and SSP5-8.5) were evaluated for the near (2015-2044), mid (2045-2074), and far (2075-2100) futures in comparison to the historical period (1985-2014). In the far future, the anticipated average annual precipitation increased by 9.48%, 13.63%, 21.07%, and 30.90%, while the average Tmax (Tmin) rose by 1.09 (1.17), 1.60 (1.91), 2.12 (2.80), and 2.99 (3.69) °C for SSP1-2.6, SSP2-4.5, SSP3-7.0, and SSP5-8.5, respectively. According to predictions for the SSP5-8.5 scenario in the distant future, there is expected to be a substantial rise in precipitation (41.98%) during the post-monsoon season. In contrast, winter precipitation was predicted to decrease most (11.12%) in the mid-future for SSP3-7.0, while to increase most (15.62%) in the far-future for SSP1-2.6. Tmax (Tmin) was predicted to rise most in the winter and least in the monsoon for all periods and scenarios. Tmin increased more rapidly than Tmax in all seasons for all SSPs. The projected changes could lead to more frequent and severe flooding, landslides, and negative impacts on human health, agriculture, and ecosystems. The study highlights the need for localized and context-specific adaptation strategies as different regions of Bangladesh will be affected differently by these changes.

Shigella O-specific polysaccharide functional IgA responses mediate protection against shigella infection in an endemic high-burden setting.

Shigella is the second leading cause of diarrheal disease-related death in young children in low and middle income countries. The mechanism of protection against shigella infection and disease in endemic areas is uncertain. While historically LPS-specific IgG titers have been associated with protection in endemic settings, emerging deeper immune approaches have recently elucidated a protective role for IpaB-specific antibody responses in a controlled human challenge model in North American volunteers. To deeply interrogate potential correlates of immunity in areas endemic for shigellosis, here we applied a systems approach to analyze the serological response to shigella across endemic and non-endemic populations. Additionally, we analyzed shigella-specific antibody responses over time in the context of endemic resistance or breakthrough infections in a high shigella burden location. Individuals with endemic exposure to shigella possessed broad and functional antibody responses across both glycolipid and protein antigens compared to individuals from non-endemic regions. In high shigella burden settings, elevated levels of OSP-specific FcαR binding antibodies were associated with resistance to shigellosis. OSP-specific FcαR binding IgA found in resistant individuals activated bactericidal neutrophil functions including phagocytosis, degranulation and reactive oxygen species production. Moreover, IgA depletion from resistant serum significantly reduced binding of OSP-specific antibodies to FcαR and antibody mediated activation of neutrophils and monocytes. Overall, our findings suggest that OSP-specific functional IgA responses contribute to protective immunity against shigella infection in high-burden settings. These findings will assist in the development and evaluation of shigella vaccines.

Longevity of memory B cells and antibodies, as well as the polarization of effector memory helper T cells, are associated with disease severity in patients with COVID-19 in Bangladesh.

The longevity of immune responses induced by different degrees of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection provides information important to understanding protection against coronavirus disease 2019 (COVID-19). Here, we report the persistence of SARS-CoV-2 spike receptor-binding domain (RBD) specific antibodies and memory B cells recognizing this antigen in sequential samples from patients in Bangladesh with asymptomatic, mild, moderate and severe COVID-19 out to six months following infection. Since the development of long-lived memory B cells, as well as antibody production, is likely to be dependent on T helper (Th) cells, we also investigated the phenotypic changes of Th cells in COVID-19 patients over time following infection. Our results show that patients with moderate to severe COVID-19 mounted significant levels of IgG antibodies out to six months following infection, while patients with asymptomatic or mild disease had significant levels of IgG antibodies out to 3 months following infection, but these then fell more rapidly at 6 months than in patients with higher disease severity. Patients from all severity groups developed circulating memory B cells (MBCs) specific to SARS-CoV-2 spike RBD by 3 months following infection, and these persisted until the last timepoint measured at 6 months. A T helper cell response with an effector memory phenotype was observed following infection in all symptomatic patients, while patients with asymptomatic infection had no significant increases in effector Th1, Th2 and Th17 effector memory cell responses. Our results suggest that the strength and magnitude of antibody and memory B cells induced following SARS-CoV-2 infection depend on the severity of the disease. Polarization of the Th cell response, with an increase in Th effector memory cells, occurs in symptomatic patients by day 7 following infection, with increases seen in Th1, Th2, Th17 and follicular helper T cell subsets.

Genomic landscape of prominent XDR Acinetobacter clonal complexes from Dhaka, Bangladesh.

BACKGROUND: Acinetobacter calcoaceticus-A. baumannii (ACB) complex pathogens are known for their prevalence in nosocomial infections and extensive antimicrobial resistance (AMR) capabilities. While genomic studies worldwide have elucidated the genetic context of antibiotic resistance in major international clones (ICs) of clinical Acinetobacter spp., not much information is available from Bangladesh. In this study, we analysed the AMR profiles of 63 ACB complex strains collected from Dhaka, Bangladesh. Following this, we generated draft genomes of 15 of these strains to understand the prevalence and genomic environments of AMR, virulence and mobilization associated genes in different Acinetobacter clones. RESULTS: Around 84% (n = 53) of the strains were extensively drug resistant (XDR) with two showing pan-drug resistance. Draft genomes generated for 15 strains confirmed 14 to be A. baumannii while one was A. nosocomialis. Most A. baumannii genomes fell under three clonal complexes (CCs): the globally dominant CC1 and CC2, and CC10; one strain had a novel sequence type (ST). AMR phenotype-genotype agreement was observed and the genomes contained various beta-lactamase genes including blaOXA-23 (n = 12), blaOXA-66 (n = 6), and blaNDM-1 (n = 3). All genomes displayed roughly similar virulomes, however some virulence genes such as the Acinetobactin bauA and the type IV pilus gene pilA displayed high genetic variability. CC2 strains carried highest levels of plasmidic gene content and possessed conjugative elements carrying AMR genes, virulence factors and insertion sequences. CONCLUSION: This study presents the first comparative genomic analysis of XDR clinical Acinetobacter spp. from Bangladesh. It highlights the prevalence of different classes of beta-lactamases, mobilome-derived heterogeneity in genetic architecture and virulence gene variability in prominent Acinetobacter clonal complexes in the country. The findings of this study would be valuable in understanding the genomic epidemiology of A. baumannii clones and their association with closely related pathogenic species like A. nosocomialis in Bangladesh.

Pinpointing genomic loci for drought-induced proline and hydrogen peroxide accumulation in bread wheat under field conditions.

BACKGROUND: Proline (Pro) and hydrogen peroxide (H2O2) play a critical role in plants during drought adaptation. Genetic mapping for drought-induced Pro and H2O2 production under field conditions is very limited in crop plants since their phenotyping with large populations is labor-intensive. A genome-wide association study (GWAS) of a diversity panel comprised of 184 bread wheat cultivars grown in natural field (control) and rain-out shelter (drought) environments was performed to identify candidate loci and genes regulating Pro and H2O2 accumulation induced by drought. RESULTS: The GWAS identified top significant marker-trait associations (MTAs) on 1A and 2A chromosomes, respectively for Pro and H2O2 in response to drought. Similarly, MTAs for stress tolerance index (STI) of Pro and H2O2 were identified on 5B and 1B chromosomes, respectively. Total 143 significant MTAs were identified including 36 and 71 were linked to drought and 2 and 34 were linked to STI for Pro and H2O2, respectively. Next, linkage disequilibrium analysis revealed minor alleles of significant single-markers and haplotypes were associated with higher Pro and H2O2 accumulation under drought. Several putative candidate genes for Pro and H2O2 content encode proteins with kinase, transporter or protein-binding activities. CONCLUSIONS: The identified genetic factors associated with Pro and H2O2 biosynthesis underlying drought adaptation lay a fundamental basis for functional studies and future marker-assisted breeding programs.

Spatiotemporal drought analysis in Bangladesh using the standardized precipitation index (SPI) and standardized precipitation evapotranspiration index (SPEI).

Countries depending on small-scale agriculture, such as Bangladesh, are susceptible to climate change and variability. Changes in the frequency and intensity of drought are a crucial aspect of this issue and the focus of this research. The goal of this work is to use SPI (standardized precipitation index) and SPEI (standardized precipitation evapotranspiration index) to investigate the differences in drought characteristics across different physiognomy types in Bangladesh and to highlight how drought characteristics change over time and spatial scales when considering different geomorphologies. This study used monthly precipitation and temperature data from 29 metrological stations for 39 years (1980-2018) for calculating SPI and SPEI values. To determine the significance of drought characteristic trends over different temporal and spatial scales, the modified Mann-Kendall trend test and multivariable linear regression (MLR) techniques were used. The results are as follows: (1) Overall, decreasing dry trend was found in Eastern hill regions, whereas an increasing drought trends were found in the in the rest of the regions in all time scaless (range is from - 0.08 decade-1 to - 0.15 decade-1 for 3-month time scale). However, except for the one-month time scale, the statistically significant trend was identified mostly in the north-central and northeast regions, indicating that drought patterns migrate from the northwest to the center region. (2) SPEI is anticipated to be better at capturing dry/wet cycles in more complex regions than SPI. (3) According to the MLR, longitude and maximum temperature can both influence precipitation. (4) Drought intensity increased gradually from the southern to the northern regions (1.26-1.56), and drought events occurred predominantly in the northwestern regions (27-30 times), indicating that drought meteorological hotspots were primarily concentrated in the Barind Tract and Tista River basin over time. Findings can be used to improve drought evaluation, hazard management, and application policymaking in Bangladesh. This has implications for agricultural catastrophe prevention and mitigation.

Identifying Recent Cholera Infections Using a Multiplex Bead Serological Assay.

Estimates of incidence based on medically attended cholera can be severely biased. Vibrio cholerae O1 leaves a lasting antibody signal and recent advances showed that these can be used to estimate infection incidence rates from cross-sectional serologic data. Current laboratory methods are resource intensive and challenging to standardize across laboratories. A multiplex bead assay (MBA) could efficiently expand the breadth of measured antibody responses and improve seroincidence accuracy. We tested 305 serum samples from confirmed cholera cases (4 to 1083 d postinfection) and uninfected contacts in Bangladesh using an MBA (IgG/IgA/IgM for 7 Vibrio cholerae O1-specific antigens) as well as traditional vibriocidal and enzyme-linked immunosorbent assays (2 antigens, IgG, and IgA). While postinfection vibriocidal responses were larger than other markers, several MBA-measured antibodies demonstrated robust responses with similar half-lives. Random forest models combining all MBA antibody measures allowed for accurate identification of recent cholera infections (e.g., past 200 days) including a cross-validated area under the curve (cvAUC200) of 92%, with simpler 3 IgG antibody models having similar accuracy. Across infection windows between 45 and 300 days, the accuracy of models trained on MBA measurements was non-inferior to models based on traditional assays. Our results illustrated a scalable cholera serosurveillance tool that can be incorporated into multipathogen serosurveillance platforms. IMPORTANCE Reliable estimates of cholera incidence are challenged by poor clinical surveillance and health-seeking behavior biases. We showed that cross-sectional serologic profiles measured with a high-throughput multiplex bead assay can lead to accurate identification of those infected with pandemic Vibrio cholerae O1, thus allowing for estimates of seroincidence. This provides a new avenue for understanding the epidemiology of cholera, identifying priority areas for cholera prevention/control investments, and tracking progress in the global fight against this ancient disease.

Virus-like Particle Display of Vibrio choleraeO-Specific Polysaccharide as a Potential Vaccine against Cholera.

Vibrio cholerae, a noninvasive mucosal pathogen, is endemic in more than 50 countries. Oral cholera vaccines, based on killed whole-cell strains of Vibrio cholerae, can provide significant protection in adults and children for 2-5 years. However, they have relatively limited direct protection in young children. To overcome current challenges, in this study, a potential conjugate vaccine was developed by linking O-specific polysaccharide (OSP) antigen purified from V. cholerae O1 El Tor Inaba strain PIC018 with Qß virus-like particles efficiently via squarate chemistry. The Qß-OSP conjugate was characterized with mass photometry (MP) on the whole particle level. Pertinent immunologic display of OSP was confirmed by immunoreactivity of the conjugate with convalescent phase samples from humans with cholera. Mouse immunization with the Qß-OSP conjugate showed that the construct generated prominent and long-lasting IgG antibody responses against OSP, and the resulting antibodies could recognize the native lipopolysaccharide from Vibrio cholerae O1 Inaba. This was the first time that Qß was conjugated with a bacterial polysaccharide for vaccine development, broadening the scope of this powerful carrier.

Scalable production and immunogenicity of a cholera conjugate vaccine.

There is a need to develop cholera vaccines that are protective in young children under 5 years of age, which induce long-term immunity, and which can be incorporated into the Expanded Programme of Immunization (EPI) in cholera-endemic countries. The degree of protection afforded by currently available oral cholera vaccines (OCV) to young children is significantly lower than that induced by vaccination of older vaccine recipients. Immune responses that protect against cholera target the O-specific polysaccharide (OSP) of Vibrio cholerae, and young children have poor immunological responses to bacterial polysaccharides, which are T cell independent antigens. To overcome this, we have developed a cholera conjugate vaccine (CCV) containing the OSP of V. cholerae O1, the main cause of endemic and epidemic cholera. Here, we describe production of CCV through a scalable manufacturing process and preclinical evaluation of immunogenicity in the presence and absence of aluminum phosphate (alum) as an adjuvant. The vaccine displays V. cholerae O1 Inaba OSP in sun-burst display via single point attachment of core oligosaccharide to a recombinant tetanus toxoid heavy chain fragment (rTTHc). Two different pilot-scale production batches of non-GMP CCV were manufactured and characterized in terms of physico-chemical properties and immunogenicity. In preclinical testing, the vaccine induced OSP- and lipopolysaccharide (LPS)-specific IgG and IgM responses, vibriocidal responses, memory B cell responses, and protection in a V. cholerae O1 challenge model. The addition of alum to the administered vaccine increased OSP-specific immune responses. These results support evaluation of CCV in humans.

Lr21 diversity unveils footprints of wheat evolution and its new role in broad-spectrum leaf rust resistance.

Aegilops tauschii, the progenitor of the wheat D genome, contains extensive diversity for biotic and abiotic resistance. Lr21 is a leaf rust resistance gene, which did not enter the initial gene flow from Ae. tauschii into hexaploid wheat due to restrictive hybridization events. Here, we used population genetics and high-resolution comparative genomics to study evolutionary and functional divergence of Lr21 in diploid and hexaploid wheats. Population genetics identified the original Lr21, lr21-1 and lr21-2 alleles and their evolutionary history among Ae. tauschii accessions. Comparative genetics of Lr21 variants between Ae. tauschii and cultivated genotypes suggested at least two independent polyploidization events in bread wheat evolution. Further, a recent re-birth of a unique Lr21-tbk allele and its neofunctionalization was discovered in the hexaploid wheat cv. Tobak. Altogether, four independent alleles were investigated and validated for leaf rust resistance in diploid, synthetic hexaploid and cultivated wheat backgrounds. Besides seedling resistance, we uncover a new role of the Lr21 gene in conferring an adult plant field resistance. Seedling and adult plant resistance turned out to be correlated with developmentally dependent variation in Lr21 expression. Our results contribute to understand Lr21 evolution and its role in establishing a broad-spectrum leaf rust resistance in wheat.

Defining Polysaccharide-Specific Antibody Targets against Vibrio cholerae O139 in Humans following O139 Cholera and following Vaccination with a Commercial Bivalent Oral Cholera Vaccine, and Evaluation of Conjugate Vaccines Targeting O139.

Cholera caused by Vibrio cholerae O139 could reemerge, and proactive development of an effective O139 vaccine would be prudent. To define immunoreactive and potentially immunogenic carbohydrate targets of Vibrio cholerae O139, we assessed immunoreactivities of various O-specific polysaccharide (OSP)-related saccharides with plasma from humans hospitalized with cholera caused by O139, comparing responses to those induced in recipients of a commercial oral whole-cell killed bivalent (O1 and O139) cholera vaccine (WC-O1/O139). We also assessed conjugate vaccines containing selected subsets of these saccharides for their ability to induce protective immunity using a mouse model of cholera. We found that patients with wild-type O139 cholera develop IgM, IgA, and IgG immune responses against O139 OSP and many of its fragments, but we were able to detect only a moderate IgM response to purified O139 OSP-core, and none to its fragments, in immunologically naive recipients of WC-O1/O139. We found that immunoreactivity of O139-specific polysaccharides with antibodies elicited by wild-type infection markedly increase when saccharides contain colitose and phosphate residues, that a synthetic terminal tetrasaccharide fragment of OSP is more immunoreactive and protectively immunogenic than complete OSP, that native OSP-core is a better protective immunogen than the synthetic OSP lacking core, and that functional vibriocidal activity of antibodies predicts in vivo protection in our model but depends on capsule thickness. Our results suggest that O139 OSP-specific responses are not prominent following vaccination with a currently available oral cholera vaccine in immunologically naive humans and that vaccines targeting V. cholerae O139 should be based on native OSP-core or terminal tetrasaccharide. IMPORTANCE Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae serogroup O1 or O139. Protection against cholera is serogroup specific, and serogroup specificity is defined by O-specific polysaccharide (OSP). Little is known about immunity to O139 OSP. In this study, we used synthetic fragments of the O139 OSP to define immune responses to OSP in humans recovering from cholera caused by V. cholerae O139, compared these responses to those induced by the available O139 vaccine, and evaluated O139 fragments in next-generation conjugate vaccines. We found that the terminal tetrasaccharide of O139 is a primary immune target but that the currently available bivalent cholera vaccine poorly induces an anti-O139 OSP response in immunologically naive individuals.

Abscisic acid-responsive element binding transcription factors contribute to proline synthesis and stress adaptation in Arabidopsis.

Proline accumulation is one of the most common adaptive responses of higher plants against abiotic stresses like drought. It plays multiple roles in osmotic adjustment, cell homeostasis and stress recovery. Genetic regulation of proline accumulation under drought is complex, and transcriptional cascades modulating proline is poorly understood. Here, we employed quadruple mutant (abf1 abf2 abf3 abf4) to dissect the role of ABA-responsive elements (ABREs) binding transcription factors (ABFs) in modulating proline accumulation across varying stress scenarios. ABREs are present across the promoter of the P5CS1 gene, whose upregulation is considered a hallmark for drought inducible proline accumulation. Upon ABA treatment, P5CS1 mRNA expression and proline content in the shoot were significantly higher in Col-0 compared to the quadruple mutant. Similar results were found at 2 h and 3 h after acute dehydration. We quantified proline at different time points after drought stress treatment. The proline content was higher in wild type (Col-0) than the quadruple mutant at the early stage of drought. Notably, the proline accumulation in wild type increased at a slower rate than the quadruple mutant 7 d after drought stress. Besides, the quadruple mutant displayed significant oxidative damage, low tissue turgidity and higher membrane damage under terminal drought stress. Both terminal drought stress and long-term constant water stress revealed substantial differences in growth rate between wild type and quadruple mutant. The study provides evidence that ABFs are involved in drought stress response, such as proline biosynthesis in Arabidopsis.