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.

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.

Genetic dissection of bread wheat diversity and identification of adaptive loci in response to elevated tropospheric ozone.

Rising tropospheric ozone affects the performance of important cereal crops thus threatening global food security. In this study, genetic variation of wheat regarding its physiological and yield responses to ozone was explored by exposing a diversity panel of 150 wheat genotypes to elevated ozone and control conditions throughout the growing season. Differential responses to ozone were observed for foliar symptom formation quantified as leaf bronzing score (LBS), vegetation indices and yield components. Vegetation indices representing the carotenoid to chlorophyll pigment ratio (such as Lic2) were particularly ozone-responsive and were thus considered suitable for the non-invasive diagnosing of ozone stress. Genetic variation in ozone-responsive traits was dissected by a genome-wide association study (GWAS). Significant marker-trait associations were identified for LBS on chromosome 5A and for vegetation indices (NDVI and Lic2) on chromosomes 6B and 6D. Analysis of linkage disequilibrium (LD) in these chromosomal regions revealed distinct LD blocks containing genes with a putative function in plant redox biology such as cytochrome P450 proteins and peroxidases. This study gives novel insight into the natural genetic variation in wheat ozone response, and lays the foundation for the molecular breeding of tolerant wheat varieties.

Impact of elemental uptake in the root chemistry of wetland plants.

Plants play a key role in the accumulation of metals in contaminated environment. Ephemeral plants, such as cyperus vaginatus, from the family Cyperaceae have been used in constructed wetlands to alter the biogeochemistry of waterlogged soils. High elemental content in wetlands often induces chemical changes in the root, stem and leaf of wetland plants. Elemental uptake and possible chemical changes in the roots of Cyperus vaginatus was investigated and compared with plants grown away from the wetland. Among the 9 heavy metals (Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, and Pb) and metalloid (As) measured, with the exception of Mn, all metals had higher content in the plant roots grown within the wetland. This was followed by plants grown near to the wetland that receive stormwater occasionally and then plants grown far from the wetland. The 3-D fluorescence spectra record showed notable differences in the chemical composition of roots grown in the three locations. The spectra combined with parallel factor analysis showed three dominant fluorescence components. Comparison of the fluorescence signatures showed a continuum of spectral properties constrained by the degree of metal contamination.

Spectroscopic study of the interaction between adenosine disodium triphosphate and gatifloxacin-Al3+ complex and its analytical application.

A new and sensitive spectrofluorimetric method has been proposed to determine trace amount of adenosine disodium triphosphate (ATP). The method is based on the fluorimetric interaction between gatifloxacin (GFLX)-aluminium (III) (Al(3+) ) complex and ATP and studied using UV-visible and fluorescence spectroscopy. Weak luminescence spectra of Al(3+) were enhanced after complexation with GFLX at 423 nm upon excitation at 272 nm due to energy transfer from the ligand to the Al(3+) ion. It was observed that the FL emission spectrum of GFLX-Al(3+) was enhanced significantly by the addition of ATP. Under the optimal conditions, the enhancement of FL intensity at 423 nm was responded linearly with the concentration of ATP in the range 1.3 × 10(-10) - 1.0 × 10(-8) mol L(-1) with correlation coefficient (r) of 0.9981. The limit of detection (LOD) was found to be 1.1 × 10(-11) mol L(-1) for ATP with the standard deviation (RSD) of 1.21% for five repeated measurement of 2.3 × 10(-8) mol L(-1) ATP. The presented method is simple, sensitive, free from coexisting interferents and can be applied successfully to determine ATP in the real samples.

Chemiluminescence determination of moxifloxacin in pharmaceutical and biological samples based on its enhancing effect of the luminol-ferricyanide system using a microfluidic chip.

A sensitive determination of a synthetic fluoroquinolone antibacterial agent, moxifloxacin (MOX), by an enhanced chemiluminescence (CL) method using a microfluidic chip is described. The microfluidic chip was fabricated by a soft-lithographic procedure using polydimethyl siloxane (PDMS). The fabricated PDMS microfluidic chip had three-inlet microchannels for introducing the sample, chemiluminescent reagent and oxidant, and a 500 µm wide, 250 µm deep and 82 mm long microchannel. An enhanced CL system, luminol-ferricyanide, was adopted to analyze the MOX concentration in a sample solution. CL light was emitted continuously after mixing luminol and ferricyanide in the presence of MOX on the PDMS microfluidic chip. The amount of MOX in the luminol-ferricyanide system influenced the intensity of the CL light. The linear range of MOX concentration was 0.14-55.0 ng/mL with a correlation coefficient of 0.9992. The limit of detection (LOD) and limit of quantification (LOQ) were 0.06 and 0.2 ng/mL respectively. The presented method afforded good reproducibility, with a relative standard deviation (RSD) of 1.05% for 10 ng/mL of MOX, and has been successfully applied for the determination of MOX in pharmaceutical and biological samples.

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.

Identification of Differentially Expressed Genes and Protein-Protein Interaction in Patients With COVID-19 and Diabetes Peripheral Neuropathy: A Bioinformatics and System Biology Approach.

The coronavirus disease 2019 (COVID-19) pandemic has had a significant impact globally, resulting in a higher death toll and persistent health issues for survivors, particularly those with pre-existing medical conditions. Numerous studies have demonstrated a strong correlation between catastrophic COVID-19 results and diabetes. To gain deeper insights, we analysed the transcriptome dataset from COVID-19 and diabetic peripheral neuropathic patients. Using the R programming language, differentially expressed genes (DEGs) were identified and classified based on up and down regulations. The overlaps of DEGs were then explored between these groups. Functional annotation of those common DEGs was performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Bio-Planet, Reactome, and Wiki pathways. A protein-protein interaction (PPI) network was created with bioinformatics tools to understand molecular interactions. Through topological analysis of the PPI network, we determined hub gene modules and explored gene regulatory networks (GRN). Furthermore, the study extended to suggesting potential drug molecules for the identified mutual DEG based on the comprehensive analysis. These approaches may contribute to understanding the molecular intricacies of COVID-19 in diabetic peripheral neuropathy patients through insights into potential therapeutic interventions.

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.

Chemiluminescence microfluidic system of gold nanoparticles enhanced luminol-silver nitrate for the determination of vitamin B12.

A rapid and sensitive chemiluminescence (CL) system coupled with a microfluidic chip has been presented to determine vitamin B12 (VB12) based on the reaction of luminol and silver nitrate (AgNO(3)) in the presence of gold nanoparticles (AuNPs). A microfluidic chip was fabricated by a soft-lithographic procedure using polydimethyl siloxane (PDMS) having four inlets and one outlet with a 200 µm wide, 250 µm deep, and 100 mm long microchannel. Ag(+) was used as a chemiluminogenic oxidant in this CL reaction which oxidized luminol to produce strong CL signal in the presence of AuNPs. Luminol reacted with AgNO(3) under the catalysis of AuNPs to produce luminol radicals which reacted with dissolved oxygen and emitted CL light. The proposed CL system was applied to determine the amount of VB12 in VB12 tablets and multivitamin. Under the optimum conditions, the CL intensity of the system was increased with the concentration of VB12 in the range of 0.25-100 ng mL(-1) with the correlation coefficient of 0.9982. The limit of detection was found to be 0.04 ng mL(-1) with the relative standard deviation of 1.56 % for five replicate determinations of 25 ng mL(-1) of VB12. The CL reaction mechanism was demonstrated by UV-visible spectra and CL emission spectra.

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.

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.

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.

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.

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.

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.

Silver nanoparticle-enhanced chemiluminescence method for determining naproxen based on europium(III)-sensitized Ce(IV)-Na2S2O4 reaction.

A simple and sensitive chemiluminescence (CL) method coupled with flow-injection technique is proposed to determine naproxen (NAP). The method is based upon the enhancement of the weak CL signal arising from the reaction of Ce(IV) and Na(2)S(2)O(4) with Eu(3+) to form the Eu(3+)-Ce(IV)-Na(2)S(2)O(4) system. The CL intensity was significantly increased by the introduction of NAP into this system in the presence of silver nanoparticles (Ag NPs). Examination of the recorded UV-vis spectra and fluorescence spectra indicated that the energy of the intermediate SO(2)*, which originated from the redox reaction of Ce(IV) and Na(2)S(2)O(4), was transferred to Eu(3+) via NAP and that the process was accelerated by Ag NPs due to their catalytic activity. Under the optimum conditions, the CL intensity was increased with increasing NAP concentration and the correlation was linear (r = 0.9992) over the NAP concentration range of 1-420 ng mL(-1). The limit of detection (LOD) was 0.11 ng mL(-1) with a relative standard deviation (RSD) of 1.15% for 5 replicate determinations of 200 ng mL(-1) NAP. The method was successfully applied to determine NAP in pharmaceutical and biological samples.