Determining Conformational Change within the Escherichia coli Outer Membrane Protein FepA

Bacteria have been able to circumvent antibiotic treatment in several ways, e.g., reorganization of the membrane and its permeability, decrease porin content, over expression of efflux pumps, and genetic changes in target sites. Most aerobic and facultative anaerobic microorganisms synthesize at least one siderophore, which chelates iron, making iron transport systems an appealing target in determining new methods of pathogenic preventions.Ferric enterobactin (FeEnt) is the native siderophore of Escherichia coli (E. coli). The mechanism of FeEnt transport through the outer membrane (OM) receptor protein, FepA, is still unknown. FepA is a ligand-gated porin with a globular N-domain occluding the 22-stranded β-barrel C-domain. The occlusion of the ligand-gated porin makes it necessary for conformational change within the N-domain of FepA during FeEnt transport. Two models have been proposed: the ball-and-chain model and the transient pore model; in the former the N-domain is completely expelled into the periplasmic space and the latter requires a structural rearrangement within the β-barrel of FepA.TonB is essential for transporting all metal complexes through the OM, including FeEnt. The role of TonB is unknown and the concentration of TonB proteins and TonB-dependent receptor proteins is disproportionate. This suggests that there are two populations of TonB-dependent receptor proteins: active transporters associated with TonB and inactive transporters unassociated with TonB. This discrepancy in protein concentration between the OM transport protein and TonB may explain the slow turnover rate of FeEnt through FepA. The slow turnover rate may also be a result of an intrinsically slow transport mechanism across the OM. Existing radioisotopic assays measure the transport of FeEnt through the passage of the inner membrane (IM), as the accumulation of the iron complex in the cytoplasm. We devised a new assay to observe FeEnt transport through the OM.The novel post-uptake binding (PUB) determinations provided information on the mechanism of FeEnt transport through FepA. It revealed that all of the FepA proteins were functionally active and could transport FeEnt through the OM. It is possible that the interaction between FepA and TonB is the rate-limiting step, which may explain the low turnover number. PUB determinations from strains lacking FepB or a complete FepCDG inner membrane complex suggested that these proteins were necessary for FeEnt transport through the OM. After determining the FepA proteins were actively transporting FeEnt in the strain lacking FepB, it was determined that FeEnt was not retained in the cell. Accumulation of the ligand in the cytoplasm was impaired in strains lacking the periplasmic binding protein or a complete inner membrane FepCDG complex. Retention experiments and PUB determinations revealed TolC exported FeEnt out of the cell.Kinetic data suggested that ligand uptake through FepA is triphasic with the initial rate being the most rapid, the second rate has an intermediate rate, and the last rate is the slowest. These results have not been previously observed and it may reflect the mechanistic connections to TonB-ExbBD, FepB and FepCDG-Fes. We determined the activation energy of FeEnt internalization through the OM was approximately 35 kcal/mol. The results indicated that the transport of FeEnt through FepA may involve significant conformational changes. These studies have resulted in a publication, Newton, S. M., Trinh, V., Pi, H., and Klebba, P. E. (2010) J Biol Chem 285, 17488-17497.After determining that all FepA proteins bound to FeEnt actively transport FeEnt, we tried to determine the mechanism of FeEnt transport through FepA using FRET analyses. We engineered several double Cys substituted mutants in FepA. These FepA derivatives were labeled with the donor dye, fluorescein maleimide (FM), and the acceptor dye, Alexa Fluor 546 maleimide (A546M). The fluorescence intensities from an emission scan with an excitation at 488 nm was used to determine if energy transfer was occurring between the two dyes. Using the energy transfer efficiency, the distance between the two dyes could be calculated. Comparing the distance between the dyes in the absence and presence of FeEnt transport through FepA would help determine the mechanism of ligand transport through FepA.After determining the double Cys mutant derivatives of FepA transported FeEnt similar to wild-type FepA, we optimized fluorescent labeling conditions with FM and A546M. Then, we ran excitation and emission scans of the single and double Cys substituted mutants in FepA. We were unable to show reproducible results that indicated energy transfer between the dyes in the absence or presence of FeEnt transport through FepA.It was possible that the dipole orientation of the dyes are more static than anticipated, this would result in poor energy transfer between the fluors. By engineering new double Cys substituted mutants in FepA, it should be possible to remedy this problem. The labeling efficiency of A546M may also contribute to the results that indicated energy transfer did not occur between the dyes. If fractional labeling of A546M occurred, it was possible that FepA could be labeled with two FM dyes rather than just one. If the fractional labeling of A546M was determined, it would be possible to adjust the values obtained in the emission scan and determine the distance between the fluors. If we determined the energy transfer between the dyes in the absence and presence of FeEnt transport through FepA, we would determine the mechanism of FeEnt transport through FepA: ball-and-chain or transient pore model.After determining FRET analysis could not be used for studies involving the conformational change of FepA during FeEnt transport, disulfide bond formation studies were conducted to determine the conformational change of the N-domain of FepA during FeEnt transport through FepA. We constructed two classes of double Cys mutants in FepA: N-terminus to N-terminus mutants in FepA and N-terminus to C-terminus mutants in FepA.Siderophore nutrition tests, FeEnt accumulation determinations, and mobility shift assays were conducted under oxidizing and reducing conditions. The first two assays were used to determine if the formation of a disulfide bond hindered the transport of FeEnt through FepA. The N-terminus to N-terminus Cys substituted mutants in FepA were designed so the Cys residues were within cross-linking distance. With the exception of L125C/V141C, all N-terminus to N-terminus mutants in FepA indicated a formation of a disulfide bond within the globular N-domain of FepA. This cross-link hindered FeEnt accumulation through FepA. These results suggested that the N-domain of FepA needed to undergo conformational changes during FeEnt uptake through the OM.N-terminus to C-terminus mutants were engineered where the Cys residues could not form a disulfide bond in the native FepA or in the absence of FeEnt transport. If the N-domain of FepA was displaced into the periplasmic space, like the ball-and-chain model, it was possible that the Cys residue in the N-domain could come in proximity of the Cys residue in the C-domain and form a disulfide bond. Based on the FeEnt accumulation assays and the siderophore nutrition tests a cross-link did not form between the two Cys substituted residues in FepA. If a disulfide bond formed, it did not hinder FeEnt transport through FepA. The electrophoretic mobility results indicated that all of the double Cys mutants in FepA formed a disulfide cross-link. This result was just qualitative and there were no quantitative results to determine the effects of the disulfide bond during FeEnt binding or transport

Enriching Biomedical Knowledge for Vietnamese Low-resource Language Through Large-Scale Translation

Biomedical data and benchmarks are highly valuable yet very limited in low-resource languages other than English such as Vietnamese. In this paper, we make use of a state-of-the-art translation model in English-Vietnamese to translate and produce both pretrained as well as supervised data in the biomedical domains. Thanks to such large-scale translation, we introduce ViPubmedT5, a pretrained Encoder-Decoder Transformer model trained on 20 million translated abstracts from the high-quality public PubMed corpus. ViPubMedT5 demonstrates state-of-the-art results on two different biomedical benchmarks in summarization and acronym disambiguation. Further, we release ViMedNLI - a new NLP task in Vietnamese translated from MedNLI using the recently public En-vi translation model and carefully refined by human experts, with evaluations of existing methods against ViPubmedT5

Selective deployment of virulence effectors correlates with host specificity in a fungal plant pathogen

なぜ病原菌は特定の作物にのみ感染するのか --植物病原菌の宿主特異性の鍵因子--. 京都大学プレスリリース. 2023-03-23.Fungus has a host of issues: KyotoU discovers how toxic fungi target specific host plants. 京都大学プレスリリース. 2023-06-07.The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C. orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C. orbiculare ‘core’ effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C. orbiculare to cucurbit hosts, but not to the Solanaceae host N. benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C. orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts

The immunogenicity of plant-based COE-GCN4pII protein in pigs against the highly virulent porcine epidemic diarrhea virus strain from genotype 2

Porcine epidemic diarrhea virus (PEDV) is a serious infectious causative agent in swine, especially in neonatal piglets. PEDV genotype 2 (G2) strains, particularly G2a, were the primary causes of porcine epidemic diarrhea (PED) outbreaks in Vietnam. Here, we produced a plant-based CO-26K-equivalent epitope (COE) variant from a Vietnamese highly virulent PEDV strain belonging to genotype 2a (COE/G2a) and evaluated the protective efficacy of COE/G2a-GCN4pII protein (COE/G2a-pII) in piglets against the highly virulent PEDV G2a strain following passive immunity. The 5-day-old piglets had high levels of PEDV-specific IgG antibodies, COE-IgA specific antibodies, neutralizing antibodies, and IFN-γ responses. After virulent challenge experiments, all of these piglets survived and had normal clinical symptoms, no watery diarrhea in feces, and an increase in their body weight, while all of the negative control piglets died. These results suggest that the COE/G2a-pII protein produced in plants can be developed as a promising vaccine candidate to protect piglets against PEDV G2a infection in Vietnam

The association between food environment, diet quality and malnutrition in low‐ and middle‐income adult populations across the rural—Urban gradient in Vietnam

Background: Economic reforms and trade liberalisation in Vaietnm have transformed the food environment, influencing dietary patterns and malnutrition status. The present study focuses on the relationship between food environments (proximity and density of food outlets) and malnutrition (underweight, overweight, obesity) through diet quality in adult populations across urban, periurban and rural areas of Vietnam. Methods: We evaluated food environment by geospatial mapping of food outlets through a transect walk across the “food ecosystem” from rural to urban areas. Diet quality was assessed using the Diet Quality Index – Vietnamese (DQI‐V) comprising Variety, Adequacy, Moderation and Balance components. Malnutrition status was determined using body mass index. We performed a mediation analysis utilising mixed effect models to control for neighbourhood clustering effects. Confounders included age, education, income and nutrition knowledge score. Results: Analysis of data from 595 adult participants (mean ± SD age: 31.2 ± 6.4 years; 50% female) found that longer distance to the nearest food outlet was associated with higher overall DQI‐V (β = 2.0; 95% confidence interval = 0.2–3.8; p = 0.036) and the Moderation component (β = 2.6; 95% confidence interval = 1.2–4.0; p = 0.001). Outlet density shows a negative association with the odds of underweight among women (odds ratio = 0.62; 95% confidence interval = 0.37–0.96). However, we did not observe statistically significant relationships between diet quality and malnutrition. Education and nutrition knowledge scores were positively associated with diet diversity, while income was negatively associated with diet moderation. Conclusions: The findings of the present study have important implications for nutrition and dietetics practice in Vietnam and globally. It emphasises the need to consider various dimensions of sustainable diets, including economic, health and socio‐cultural/political factors. Longer distances to food outlets are associated with higher diet quality, whereas lower food outlet density increases the odds of underweight among women. This poses challenges in balancing modernisation and its adverse effects on sustainable food systems. Socio‐economic status consistently correlated with diet quality and malnutrition, necessitating further research to promote healthy diets across socio‐economic strata

Diet Quality Index and food choice motives in Vietnam: The roles of sensory appeal, mood, convenience, and familiarity

Food choices that shape human diets and health are influenced by various socio-economic factors. Vietnam struggles to meet many nutrition targets where links between food choice and diet have not been widely explored. This study assesses the food choice motives, based on a 28-item food choice questionnaire (FCQ), and the diet quality of 603 adults in three sites (urban, peri-urban, and rural) in northern Vietnam. We assess diet quality using the Diet Quality Index–Vietnam (DQI-V) which consists of variety, adequacy, moderation, and balance components. Using factor analysis, we grouped FCQ items into five factors: health focus, sensory appeal, mood ethics, convenience, and familiarity. The structural equation modeling indicates that food choice motives significantly impact the DQI-V and its components but in different directions. The results show that sensory appeal has a positive association with the overall DQI-V score, while having a negative impact on the variety component. Findings present a potential trade-off issue for interventions and policies related to food products. Nutrition knowledge is positively associated with all elements of diet quality across all three study sites. Vietnamese agrobiodiversity could be better utilized to increase dietary diversity. Differentiated policies are necessary to address the poor dietary diversity and adequacy in northern Vietnam

EVALUATION OF GENETIC DIVERSITY OF THE BLACK GLUTINOUS RICE BASED ON AGRO-MORPHOLOGICAL CHARACTERS

The study assessed the variations in nine agro-morphological characters among and within the black glutinous rice (Oryza sativa) population from Chau Thanh District, Tra Vinh Province. The nine quantitative agromorphological characters that were measured include culm length, leaf length, leaf width, number of panicles, panicle length, grain length, grain width, number of firm grain, and number of grain per panicle. The unweighted pair group method with arithmetic mean method and principal coordinate analysis by the NTSYS program were applied in this study to classify the nine agro-morphological characters. In addition, to compare the variations in quantitative characters between O. sativa populations, one-way analysis of variance (ANOVA) was used. The results showed significant differences between the black glutinous rice populations for all quantitative agro-morphological characters. Moreover, some agro-morphological characters showed positive correlations to each other. The dendrogram generated from the analysis process of the agromorphological data divided the O. sativa populations into two groups with unfamiliar features. However, the O. sativa populations assessed exhibited a wide range of variations in morphological characteristics, both within the same population and among other populations with the same strains

Concerted loop motion triggers induced fit of FepA to ferric enterobactin

Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles

GRADIENT KIẾN TẠO HIỆN ĐẠI KHU VỰC NINH THUẬN VÀ LÂN CẬN

The estimation of the present day tectonic movement and tectonic gradient (strain rate) has an important practical signification in the assessment of active fault and seismic hazards for the selection of Ninh Thuan nuclear power plant. Based on the three campaigns of GPS measurement between 2012 - 2013, we used BERNESE 5.0 software to determine present day slip rates of 13 stations in ITRF08 frame. The GPS stations move eastwards at the slip rates of 22 - 25 mm/yr, southwards at the velocities of 5 - 10 mm/yr. The standard errors in latitudinal and longitudinal directions are 1.2 mm/yr and 0.9 mm/yr, respectively. Combined with GPS data from the project of the study on actual geodynamics in Tay Nguyen TN3/06, we determined the strain rate ranging from 50 to 100 nanostrains with the standard error of 50 nanostrains. The direction of  maximum compressive strain rate is from northwest - southeast to east - west.Đánh giá vận tốc chuyển động kiến tạo hiện đại và gradient kiến tạo hiện đại có ý nghĩa thực tiễn quan trọng trong việc đánh giá đứt gãy hoạt động nguy hiểm động đất phục vụ xây dụng nhà máy điện hạt nhân Ninh Thuận. Trên cơ sở đo 3 chu kỳ GPS vào các năm 2012 - 2013, sử dụng phần mềm BERNESE 5.0, chúng tôi đã xác lập được vận tốc chuyển động kiến tạo hiện đại tại 13 điểm đo GPS trong khu vực lân cận bao gồm kéo dài từ Nha Trang tới đảo Phú Quý. Vận tốc chuyển dịch kiến tạo hiện đại về phía đông thay đổi từ 22 - 25 mm/năm và chuyển dịch về phía nam với vận tốc giao động từ 5 - 10 mm/năm trên hệ tọa độ toàn cầu ITRF08. Sai số vận tốc chuyển dịch kiến tạo về phía đông giao động trong khoảng 1,2 - 1,5 mm/năm và về phía nam giao động trong khoảng 0,9 - 1,2 mm/năm. Liên kết với giá trị đo GPS từ đề tài nghiên cứu địa động lực hiện đại khu vực Tây Nguyên mã số TN3/T06, chúng tôi đã xác định được giá trị vận tốc biến dạng giao động từ 50 nano tới 100 nano biến dạng với sai số giao động trong khoảng 50 nano biến dạng. Trục biến dạng nén cực đại giao động theo phương thay đổi từ bắc nam sang đông bắc - tây nam. Trục biến dạng căng cực đại có phương thay đổi từ tây bắc - đông nam sang phương đông - tây

The anti-SARS-CoV-2 monoclonal antibody, bamlanivimab, minimally impacts the endogenous immune response to COVID-19 vaccination

As the coronavirus disease 2019 (COVID-19) pandemic evolves and vaccine rollout progresses, the availability and demand for monoclonal antibodies for the prevention and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are also accelerating. This longitudinal serological study evaluated the magnitude and potency of the endogenous antibody response to COVID-19 vaccination in participants who first received a COVID-19 monoclonal antibody in a prevention study. Over the course of six months, serum samples were collected from a population of nursing home residents and staff enrolled in a clinical trial who were randomized to either bamlanivimab treatment or placebo. In an unplanned component of this trial, a subset of these participants was subsequently fully vaccinated with two doses of either SpikeVax (Moderna) or Comirnaty (BioNTech/Pfizer) COVID-19 mRNA vaccines. This post-hoc analysis assessed the immune response to vaccination for 135 participants without prior SARS-CoV-2 infection. Antibody titers and potency were assessed using three assays against SARS-CoV-2 proteins that bamlanivimab does not efficiently bind to, thereby reflecting the endogenous antibody response. All bamlanivimab and placebo recipients mounted a robust immune response to full COVID-19 vaccination, irrespective of age, risk-category, and vaccine type with any observed differences of uncertain clinical importance. These findings are pertinent for informing public health policy with results that suggest that the benefit of receiving COVID-19 vaccination at the earliest opportunity outweighs the minimal effect on the endogenous immune response due to prior prophylactic COVID-19 monoclonal antibody infusion