A genetic association study of circulating coagulation factor VIII and von Willebrand factor levels.

ABSTRACT: Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

CRISPR/Cas9 targeted mutations of OsDSG1 gene enhanced salt tolerance in rice.

Salinization is one of the leading causes of arable land shrinkage and rice yield decline, recently. Therefore, developing and utilizing salt-tolerant rice varieties have been seen as a crucial and urgent strategy to reduce the effects of saline intrusion and protect food security worldwide. In the current study, the CRISPR/Cas9 system was utilized to induce targeted mutations in the coding sequence of the OsDSG1, a gene involved in the ubiquitination pathway and the regulation of biochemical reactions in rice. The CRISPR/Cas9-induced mutations of the OsDSG1 were generated in a local rice cultivar and the mutant inheritance was validated at different generations. The OsDSG1 mutant lines showed an enhancement in salt tolerance compared to wild type plants at both germination and seedling stages indicated by increases in plant height, root length, and total fresh weight as well as the total chlorophyll and relative water contents under the salt stress condition. In addition, lower proline and MDA contents were observed in mutant rice as compared to wild type plants in the presence of salt stress. Importantly, no effect on seed germination and plant growth parameters was recorded in the CRISRP/Cas9-induced mutant rice under the normal condition. This study again indicates the involvement of the OsDSG1 gene in the salt resistant mechanism in rice and provides a potential strategy to enhance the tolerance of local rice varieties to the salt stress.

Synergistic Polymer Blending Informs Efficient Terpolymer Design and Machine Learning Discerns Performance Trends for pDNA Delivery.

Cationic polymers offer an alternative to viral vectors in nucleic acid delivery. However, the development of polymer vehicles capable of high transfection efficiency and minimal toxicity has remained elusive, and continued exploration of the vast design space is required. Traditional single polymer syntheses with large monomer bases are very time-intensive, limiting the speed at which new formulations are identified. In this work, we present an experimental method for the quick probing of the design space, utilizing a combinatorial set of 90 polymer blends, derived from 6 statistical copolymers, to deliver pDNA. This workflow facilitated rapid screening of polyplex compositions, successfully tailoring polyplex hydrophobicity, particle size, and payload binding affinity. This workflow identified blended polyplexes with high levels of transfection efficiency and cell viability relative to single copolymer controls and commercial JetPEI, indicating synergistic benefits from copolymer blending. Polyplex composition was coupled with biological outputs to guide the synthesis of single terpolymer vehicles, with high-performing polymers P10 and M20, providing superior transfection of HEK293T cells in serum-free and serum-containing media, respectively. Machine learning coupled with SHapley Additive exPlanations (SHAP) was used to identify polymer/polyplex attributes that most impact transfection efficiency, viability, and overall effective efficiency. Subsequent transfections on ARPE-19 and HDFn cells found that P10 and M20 were surpassed in performance by M10, contrasting with results in HEK293T cells. This cell type dependency reinforced the need to evaluate transfection conditions with multiple cell models to potentially identify moieties more beneficial to delivery in certain tissues. Overall, the workflow employed can be used to expedite the exploration of the polymer design space, bypassing extensive synthesis, and to develop improved polymer delivery vehicles more readily for nucleic acid therapies.

Super Bound States in the Continuum on a Photonic Flatband: Concept, Experimental Realization, and Optical Trapping Demonstration.

In this Letter, we theoretically propose and experimentally demonstrate the formation of a super bound state in a continuum (BIC) on a photonic crystal flat band. This unique state simultaneously exhibits an enhanced quality factor and near-zero group velocity across an extended region of the Brillouin zone. It is achieved at the topological transition when a symmetry-protected BIC pinned at k=0 merges with two Friedrich-Wintgen quasi-BICs, which arise from the destructive interference between lossy photonic modes of opposite symmetries. As a proof of concept, we employ the ultraflat super BIC to demonstrate three-dimensional optical trapping of individual particles. Our findings present a novel approach to engineering both the real and imaginary components of photonic states on a subwavelength scale for innovative optoelectronic devices.

Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification.

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads. Our examination involved FTIR, EDS, XRD, SDS-PAGE, and Western blotting methods. FTIR spectra revealed notable changes in the stretching patterns and intensities of hydroxyl, amine, carbonyl, imine and amide chemical groups, when Mpro protein was present in the Ni-M/A sample. XRD spectra demonstrated the occurrence of two Nickel peaks at 35-40 deg and 40-45 deg in Ni-M/A, but only one nickel peak at 35-40 deg in Ni-M/A-Mpro, indicating the binding of Mpro on the Nickel ions. EDS analysis reported a decrease in the concentration of Nickel on the surface of Ni-M/A from 16% to 7% when Mpro protein was loaded into the stationary phase. Importantly, our data indicated that the purity of the His-tagged protein Mpro after purification reached 97% after just one-step purification using the Ni-M/A stationary phase. Moreover, the binding capacity of Ni-M/A for Mpro was approximately 5.2 mg/g with recovery efficiency of 40%. Our results suggested Ni-M/A as a highly potential solid phase for affinity chromatography in the purification of His-tagged protein.

Antithrombin, Protein C, and Protein S: Genome and Transcriptome-Wide Association Studies Identify 7 Novel Loci Regulating Plasma Levels.

BACKGROUND: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total. METHODS: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. RESULTS: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (GCKR, BAZ1B, and HP-TXNL4B) and 1 with PS levels (ORM1-ORM2). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (FCGRT), 1 with PC (GOLM2), and 1 with PS (MYL7). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17, and HP genes in antithrombin regulation. CONCLUSIONS: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.

Understanding the biases to sepsis surveillance and quality assurance caused by inaccurate coding in administrative health data.

PURPOSE: Timely and accurate data on the epidemiology of sepsis are essential to inform policy decisions and research priorities. We aimed to investigate the validity of inpatient administrative health data (IAHD) for surveillance and quality assurance of sepsis care. METHODS: We conducted a retrospective validation study in a disproportional stratified random sample of 10,334 inpatient cases of age ≥ 15 years treated in 2015-2017 in ten German hospitals. The accuracy of coding of sepsis and risk factors for mortality in IAHD was assessed compared to reference standard diagnoses obtained by a chart review. Hospital-level risk-adjusted mortality of sepsis as calculated from IAHD information was compared to mortality calculated from chart review information. RESULTS: ICD-coding of sepsis in IAHD showed high positive predictive value (76.9-85.7% depending on sepsis definition), but low sensitivity (26.8-38%), which led to an underestimation of sepsis incidence (1.4% vs. 3.3% for severe sepsis-1). Not naming sepsis in the chart was strongly associated with under-coding of sepsis. The frequency of correctly naming sepsis and ICD-coding of sepsis varied strongly between hospitals (range of sensitivity of naming: 29-71.7%, of ICD-diagnosis: 10.7-58.5%). Risk-adjusted mortality of sepsis per hospital calculated from coding in IAHD showed no substantial correlation to reference standard risk-adjusted mortality (r = 0.09). CONCLUSION: Due to the under-coding of sepsis in IAHD, previous epidemiological studies underestimated the burden of sepsis in Germany. There is a large variability between hospitals in accuracy of diagnosing and coding of sepsis. Therefore, IAHD alone is not suited to assess quality of sepsis care.

Expression of virus-like particles (VLPs) of foot-and-mouth disease virus (FMDV) using Saccharomyces cerevisiae.

We engineered Saccharomyces cerevisiae to express structural proteins of foot-and-mouth disease virus (FMDV) and produce virus-like particles (VLPs). The gene, which encodes four structural capsid proteins (VP0 (VP4 and VP2), VP3, and VP1), followed by a translational "ribosomal skipping" sequence consisting of 2A and protease 3C, was codon-optimized and chemically synthesized. The cloned gene was used to transform S. cerevisiae 2805 strain. Western blot analysis revealed that the polyprotein consisting of VP0, VP3, and VP1 was processed into the discrete capsid proteins. Western blot analysis of 3C confirmed the presence of discrete 3C protein, suggesting that the 2A sequence functioned as a "ribosomal skipping" signal in the yeast for an internal re-initiation of 3C translation from a monocistronic transcript, thereby indicating polyprotein processing by the discrete 3C protease. Moreover, a band corresponding to only VP2, which was known to be non-enzymatically processed from VP0 to both VP4 and VP2 during viral assembly, further validated the assembly of processed capsid proteins into VLPs. Electron microscopy showed the presence of the characteristic icosahedral VLPs. Our results clearly demonstrate that S. cerevisiae processes the viral structural polyprotein using a viral 3C protease and the resulting viral capsid subunits are assembled into virion particles. KEY POINTS: • Ribosomal skipping by self-cleaving FMDV peptide in S. cerevisiae. • Proteolytic processing of a structural polyprotein from a monocistronic transcript. • Assembly of the processed viral capsid proteins into a virus-like particle.

Synapses do not facilitate prion-like transfer of alpha-synuclein: a quantitative study in reconstructed unidirectional neural networks.

Alpha-synuclein (aSyn) aggregation spreads between cells and underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson's diseases. The mechanisms of cell-to-cell propagation of aggregates, which dictate how aggregation progresses at the network level, remain poorly understood. Notably, while prion and prion-like spreading is often simplistically envisioned as a "domino-like" spreading scenario where connected neurons sequentially propagate protein aggregation to each other, the reality is likely to be more nuanced. Here, we demonstrate that the spreading of preformed aSyn aggregates is a limited process that occurs through molecular sieving of large aSyn seeds. We further show that this process is not facilitated by synaptic connections. This was achieved through the development and characterization of a new microfluidic platform that allows reconstruction of binary fully oriented neuronal networks in vitro with no unwanted backward connections, and through the careful quantification of fluorescent aSyn aggregates spreading between neurons. While this allowed us for the first time to extract quantitative data of protein seeds dissemination along neural pathways, our data suggest that prion-like dissemination of proteinopathic seeding aggregates occurs very progressively and leads to highly compartmentalized pattern of protein seeding in neural networks.

Barriers to and facilitators of user engagement with web-based mental health interventions in young people: a systematic review.

Many young people (YP) are diagnosed with mental illnesses and require support. Web-based mental health interventions (W-MHIs) have been increasingly utilized by YP, healthcare providers, and parents due to reasons including convenience and anonymity. W-MHIs are effective in improving mental health in YP. However, real-world engagement with W-MHIs remains low. Therefore, understanding barriers/facilitators of user engagement with W-MHIs is necessary to promote W-MHIs and help users gain optimal benefits through higher engagement. This review aims to identify barriers/facilitators of user engagement with W-MHIs in YP aged 10-24 years. A systematic search of five databases for English language, peer-reviewed publications was conducted between January 2010 and February 2023. Studies examining factors influencing user engagement with W-MHIs, described as barriers or facilitators, were included. Study quality was assessed using the Mixed Methods Appraisal Tool. A narrative synthesis was performed. Of 4088 articles identified, 69 studies were included. Barriers/facilitators were reported by young people (63 studies), providers (17 studies), and parents/caregivers (8 studies). YP perceived that usefulness and connectedness were the most common facilitators, whereas low-perceived need was the most reported barrier. Both providers and parents reported that perceived usefulness for YP was the most common facilitator, whereas concerns about program effectiveness and privacy were noted as barriers. This review found that program- and individual-related factors were important determinants of engagement with W-MHIs. This review provides guidance on the future design and development of new interventions, narrowing the gap between existing W-MHIs and unmet needs of users.

Expanded vaccination practice in 2-year-old children and maternal knowledge on expanded vaccination in Tien Giang, Vietnam.

Background: Vaccination plays a central role in protecting children against severe diseases and preventing child mortality. Objectives: This study aimed to determine the rate and factors associated with complete and timely vaccination in 2-year-old children, as well as maternal knowledge on expanded vaccination in Go Cong Tay district, Tien Giang province, Vietnam. Methods: A cross-sectional descriptive study was conducted on 558 2-year-old children and their mothers residing in Go Cong Tay district, Tien Giang province, Vietnam. The study employed a systematic random sampling method from June to September 2021. Results: The rate of complete vaccination in children was 74.7%. Factors associated with the rate of complete vaccination were occupation (OR=0.3; 95%CI: 0.1-0.7; p=0.006), economic status (OR=3.8; 95%CI: 1.7-8.6; p=0.001), and maternal general knowledge on expanded vaccination (OR=1.7; 95%CI: 1.1-2.6; p=0.01). The rate of timely vaccination was 47.8%. Factors associated with the rate of timely vaccination were maternal age group (OR=3.1; 95%CI: 1.6-6.0; p=0.001; OR=3.0; 95%CI: 1.3-6.6; p=0.006) and economic status (OR=0.4; 95%CI: 0.2-0.9; p=0.04). The rate of both complete and timely vaccination was 22.6%. Factors associated with the rate of complete and timely vaccination were maternal age group (OR=3.1; 95%CI: 1.3-7.2; p=0.009; OR=3.3; 95%CI: 1.2-9.1; p=0.02) and maternal general knowledge on expanded vaccination (OR=1.5; 95%CI: 1.0-2.4; p=0.03). The rate of maternal general knowledge on expanded vaccination was 57.5%. Conclusion: The rates of complete and timely vaccination are still low, and various factors influence expanded vaccination. The-refore, it is crucial to continue health education campaigns to improve knowledge on expanded vaccination, remind mothers of vaccination schedules, strengthen confidence in vaccination programs and vaccine safety, attract customers to vaccination services, provide adequate healthcare for children, and ensure vaccination activities during disease outbreaks.

Enhancing powdery mildew resistance in soybean by targeted mutation of MLO genes using the CRISPR/Cas9 system.

BACKGROUND: Powdery mildew is a major disease that causes great losses in soybean yield and seed quality. Disease-resistant varieties, which are generated by reducing the impact of susceptibility genes through mutation in host plants, would be an effective approach to protect crops from this disease. The Mildew Locus O (MLO) genes are well-known susceptibility genes for powdery mildew in plant. In this study, we utilized the CRISPR/Cas9 system to induce targeted mutations in the soybean GmMLO genes to improve powdery mildew resistance. RESULTS: A dual-sgRNA CRISPR/Cas9 construct was designed and successfully transferred into the Vietnamese soybean cultivar DT26 through Agrobacterium tumefaciens-mediated transformation. Various mutant forms of the GmMLO genes including biallelic, chimeric and homozygous were found at the T0 generation. The inheritance and segregation of CRISPR/Cas9-induced mutations were confirmed and validated at the T1 and T2 generations. Out of six GmMLO genes in the soybean genome, we obtained the Gmmlo02/Gmmlo19/Gmmlo23 triple and Gmmlo02/Gmmlo19/Gmmlo20/Gmmlo23 quadruple knockout mutants at the T2 generation. When challenged with Erysiphe diffusa, a fungus that causes soybean powdery mildew, all mutant plants showed enhanced resistance to the pathogen, especially the quadruple mutant. The powdery mildew severity in the mutant soybeans was reduced by up to 36.4% compared to wild-type plants. In addition, no pleiotropic effect on soybean growth and development under net-house conditions was observed in the CRISPR/Cas9 mutants. CONCLUSIONS: Our results indicate the involvement of GmMLO02, GmMLO19, GmMLO20 and GmMLO23 genes in powdery mildew susceptibility in soybean. Further research should be conducted to investigate the roles of individual tested genes and the involvement of other GmMLO genes in this disease infection mechanism. Importantly, utilizing the CRISPR/Cas9 system successfully created the Gmmlo transgene-free homozygous mutant lines with enhanced resistance to powdery mildew, which could be potential materials for soybean breeding programs.

Disrupting Sc-uORFs of a transcription factor bZIP1 using CRISPR/Cas9 enhances sugar and amino acid contents in tomato (Solanum lycopersicum).

MAIN CONCLUSION: Induced mutations in the SC-uORF of the tomato transcription factor gene SlbZIP1 by the CRISPR/Cas9 system led to the high accumulation of sugar and amino acid contents in tomato fruits. Tomato (Solanum lycopersicum) is one of the most popular and consumed vegetable crops in the world. Among important traits for tomato improvement such as yield, biotic and abiotic resistances, appearance, post-harvest shelf life and fruit quality, the last one seems to face more challenges because of its genetic and biochemical complexities. In this study, a dual-gRNAs CRISPR/Cas9 system was developed to induce targeted mutations in uORF regions of the SlbZIP1, a gene involved in the sucrose-induced repression of translation (SIRT) mechanism. Different induced mutations in the SlbZIP1-uORF region were identified at the T0 generation, stably transferred to the offspring, and no mutation was found at potential off-target sites. The induced mutations in the SlbZIP1-uORF region affected the transcription of SlbZIP1 and related genes in sugar and amino acid biosynthesis. Fruit component analysis showed significant increases in soluble solid, sugar and total amino acid contents in all SlbZIP1-uORF mutant lines. The accumulation of sour-tasting amino acids, including aspartic and glutamic acids, raised from 77 to 144%, while the accumulation of sweet-tasting amino acids such as alanine, glycine, proline, serine, and threonine increased from 14 to 107% in the mutant plants. Importantly, the potential SlbZIP1-uORF mutant lines with desirable fruit traits and no impaired effect on plant phenotype, growth and development were identified under the growth chamber condition. Our result indicates the potential utility of the CRISPR/Cas9 system for fruit quality improvement in tomato and other important crops.

Automatic Detection of Meniscus Tears Using Backbone Convolutional Neural Networks on Knee MRI.

BACKGROUND: Timely diagnosis of meniscus injuries is key for preventing knee joint dysfunction and improving patient outcomes because it decreases morbidity and facilitates treatment planning. PURPOSE: To train and evaluate a deep learning model for automated detection of meniscus tears on knee magnetic resonance imaging (MRI). STUDY TYPE: Bicentric retrospective study. SUBJECTS: In total, 584 knee MRI studies, divided among training (n = 234), testing (n = 200), and external validation (n = 150) data sets, were used in this study. The public data set MRNet was used as a second external validation data set to evaluate the performance of the model. SEQUENCE: A 3 T, coronal, and sagittal images from T1-weighted proton density (PD) fast spin-echo (FSE) with fat saturation and T2-weighted FSE with fat saturation sequences. ASSESSMENT: The detection system for meniscus tear was based on the improved YOLOv4 model with Darknet-53 as the backbone. The performance of the model was also compared with that of three radiologists of varying levels of experience. The determination of the presence of a meniscus tear from surgery reports was used as the ground truth for the images. STATISTICAL TESTS: Sensitivity, specificity, prevalence, positive predictive value, negative predictive value, accuracy, and receiver operating characteristic curve were used to evaluate the performance of the detection model. Two-way analysis of variance, Wilcoxon signed-rank test, and Tukey's multiple tests were used to evaluate differences in performance between the model and radiologists. RESULTS: The overall accuracies for detecting meniscus tears using our model on the internal testing, internal validation, and external validation data sets were 95.4%, 95.8%, and 78.8%, respectively. One radiologist had significantly lower performance than our model in detecting meniscal tears (accuracy: 0.9025 ± 0.093 vs. 0.9580 ± 0.025). DATA CONCLUSION: The proposed model had high sensitivity, specificity, and accuracy for detecting meniscus tears on knee MRIs. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 2.

Evolution of hypoxia and hypoxia-inducible factor asparaginyl hydroxylase regulation in chronic kidney disease.

BACKGROUND: The roles of hypoxia and hypoxia inducible factor (HIF) during chronic kidney disease (CKD) are much debated. Interventional studies with HIF-α activation in rodents have yielded contradictory results. The HIF pathway is regulated by prolyl and asparaginyl hydroxylases. While prolyl hydroxylase inhibition is a well-known method to stabilize HIF-α, little is known about the effect asparaginyl hydroxylase factor inhibiting HIF (FIH). METHODS: We used a model of progressive proteinuric CKD and a model of obstructive nephropathy with unilateral fibrosis. In these models we assessed hypoxia with pimonidazole and vascularization with three-dimensional micro-computed tomography imaging. We analysed a database of 217 CKD biopsies from stage 1 to 5 and we randomly collected 15 CKD biopsies of various severity degrees to assess FIH expression. Finally, we modulated FIH activity in vitro and in vivo using a pharmacologic approach to assess its relevance in CKD. RESULTS: In our model of proteinuric CKD, we show that early CKD stages are not characterized by hypoxia or HIF activation. At late CKD stages, some areas of hypoxia are observed, but these are not colocalizing with fibrosis. In mice and in humans, we observed a downregulation of the HIF pathway, together with an increased FIH expression in CKD, according to its severity. Modulating FIH in vitro affects cellular metabolism, as described previously. In vivo, pharmacologic FIH inhibition increases the glomerular filtration rate of control and CKD animals and is associated with decreased development of fibrosis. CONCLUSIONS: The causative role of hypoxia and HIF activation in CKD progression is questioned. A pharmacological approach of FIH downregulation seems promising in proteinuric kidney disease.

Improving expression and assembly of difficult-to-express heterologous proteins in Saccharomyces cerevisiae by culturing at a sub-physiological temperature.

BACKGROUND: Escherichia coli heat labile toxin B subunit (LTB) is one of the most popular oral vaccine adjuvants and intestine adsorption enhancers. It is often expressed as a fusion partner with target antigens to enhance their immunogenicity as well as gut absorbability. However, high expression levels of a fusion protein are critical to the outcome of immunization experiments and the success of subsequent vaccine development efforts. In order to improve the expression and functional assembly of LTB-fusion proteins using Saccharomyces cerevisiae, we compared their expression under culture conditions at a sub-physiological temperature 20 °C with their expression under a standard 30 °C. RESULTS: The assembled expression of LTB-EDIII2 (LTB fused to the envelope domain III (EDIII) of Dengue virus serotype 2), which was expressed at the level of 20 µg/L in our previous study, was higher when the expression temperature was 20 °C as opposed to 30 °C. We also tested whether the expression and functional assembly of a difficult-to-express LTB fusion protein could be increased. The assembled expression of the difficult-to-express LTB-VP1 fusion protein (LTB fused to VP1 antigen of Foot-and-Mouth Disease Virus) dramatically increased, although the total amount of expressed protein was still lower than that of LTB-EDIII2. Slight but significant increase in the expression of well-known reporter protein eGFP, which has previously been shown to be increased by cultivation at 20 °C, was also observed in our expression system. As no significant changes in corresponding transcripts levels and cell growth were observed between 20 °C and 30 °C, we infer that translation and post-translational assembly are responsible for these enhancements. CONCLUSIONS: The effects of lowering the expression temperature from 30 °C to 20 °C on protein expression and folding levels in S. cerevisiae, using several proteins as models, are reported. When heterologous proteins are expressed at 20 °C, a greater amount of (specially, more assembled) functional proteins accumulated than at 30 °C. Although further studies are required to understand the molecular mechanisms, our results suggest that lowering the expression temperature is a convenient strategy for improving the expression of relatively complexly structured and difficult-to-express proteins in S. cerevisiae.

Clinical benefit of AI-assisted lung ultrasound in a resource-limited intensive care unit.

BACKGROUND: Interpreting point-of-care lung ultrasound (LUS) images from intensive care unit (ICU) patients can be challenging, especially in low- and middle- income countries (LMICs) where there is limited training available. Despite recent advances in the use of Artificial Intelligence (AI) to automate many ultrasound imaging analysis tasks, no AI-enabled LUS solutions have been proven to be clinically useful in ICUs, and specifically in LMICs. Therefore, we developed an AI solution that assists LUS practitioners and assessed its usefulness in  a low resource ICU. METHODS: This was a three-phase prospective study. In the first phase, the performance of four different clinical user groups in interpreting LUS clips was assessed. In the second phase, the performance of 57 non-expert clinicians with and without the aid of a bespoke AI tool for LUS interpretation was assessed in retrospective offline clips. In the third phase, we conducted a prospective study in the ICU where 14 clinicians were asked to carry out LUS examinations in 7 patients with and without our AI tool and we interviewed the clinicians regarding the usability of the AI tool. RESULTS: The average accuracy of beginners' LUS interpretation was 68.7% [95% CI 66.8-70.7%] compared to 72.2% [95% CI 70.0-75.6%] in intermediate, and 73.4% [95% CI 62.2-87.8%] in advanced users. Experts had an average accuracy of 95.0% [95% CI 88.2-100.0%], which was significantly better than beginners, intermediate and advanced users (p < 0.001). When supported by our AI tool for interpreting retrospectively acquired clips, the non-expert clinicians improved their performance from an average of 68.9% [95% CI 65.6-73.9%] to 82.9% [95% CI 79.1-86.7%], (p < 0.001). In prospective real-time testing, non-expert clinicians improved their baseline performance from 68.1% [95% CI 57.9-78.2%] to 93.4% [95% CI 89.0-97.8%], (p < 0.001) when using our AI tool. The time-to-interpret clips improved from a median of 12.1 s (IQR 8.5-20.6) to 5.0 s (IQR 3.5-8.8), (p < 0.001) and clinicians' median confidence level improved from 3 out of 4 to 4 out of 4 when using our AI tool. CONCLUSIONS: AI-assisted LUS can help non-expert clinicians in an LMIC ICU improve their performance in interpreting LUS features more accurately, more quickly and more confidently.

Updated estimation of the burden of fungal disease in Vietnam.

BACKGROUND: Anecdotally, the burden of fungal diseases in Vietnam is rapidly rising, but there has been no updated estimate on this issue since a previous report in 2015. OBJECTIVES: In this study, we aimed at estimating the incidence and prevalence of serious fungal infections for the year 2020. METHODS: We made estimates with a previously described methodology, using reports on the incidence and prevalence of various established risk factors for fungal infections from local, regional or global sources. RESULTS: We estimated 2,389,661 cases of serious fungal infection occurred in Vietnam in 2020. The most common condition was recurrent vaginal candidiasis (4047/100,000 women annually). Among people living with HIV, we estimated 451 cases of cryptococcal meningitis, 1030 of pneumocystis pneumonia, 166 of histoplasmosis and 1612 of talaromycosis annually. Candidaemia incidence was estimated at 12/100,000 population each year. Owing to its high burden of tuberculosis and respiratory diseases, Vietnam had high rates of severe infections caused by Aspergillus species. Incidence of invasive aspergillosis is 24/100,000 population, allergic bronchopulmonary aspergillosis 78/100,000 and severe asthma with fungal sensitisation 102/100,000. Five-year period prevalence of chronic pulmonary aspergillosis is 120/100,000 population /5-year period. Mucormycosis, fungal keratitis and tinea capitis were estimated at 192, 14,431 and 201 episodes each year, respectively. CONCLUSIONS: The number of patients with mycoses in Vietnam is likely underestimated due to a lack of local data and limited diagnostic capacity, but at least 2.5% of the population might have some form of serious fungal disease.

Health-related quality of life in children, adolescents and young adults with self-harm or suicidality: A systematic review.

OBJECTIVE: Self-harm and suicidality are associated with substantial social and economic burden, especially among children, adolescents and young adults. The aim of this review was to systematically synthesize the literature on the association between health-related quality of life and self-harm/suicidality in children, adolescents and young adults. METHODS: Searches were conducted via MEDLINE, PsycINFO, CINAHL, EconLit and EMBASE. Search terms were the combination of the following blocks: (1) self-harm/suicidality, (2) health-related quality of life/well-being/life satisfaction and (3) children/adolescents/young adults. The quality of studies was assessed using the Effective Public Health Practice Project tool. RESULTS: We identified 23 relevant studies. Findings showed that participants who reported self-harm had lower well-being, life satisfaction or overall health-related quality of life compared to those without self-harm. There was also evidence supporting the association between health-related quality of life and suicidal attempt. However, the results for the association with suicidal ideation remained inconsistent. Additionally, mental health, emotional well-being, physical health, oral health, existential well-being and family quality of life were found to be significant domains associated with self-harm or suicidality. Regarding the quality of included studies, 35% (n = 8), 39% (n = 9) and 26% (n = 6) of studies were scored as 'Strong', 'Moderate' and 'Weak', respectively. CONCLUSION: Findings from the review showed that health-related quality of life varied according to the severity of suicidality (from ideation to attempt). There was also no evidence to infer the direction of causality between health-related quality of life and self-harm/suicidality. The findings suggest a need for further research, in particular longitudinal studies to fill identified gaps in the literature.

Transcriptional regulation of genes bearing intronic heterochromatin in the rice genome.

Intronic regions of eukaryotic genomes accumulate many Transposable Elements (TEs). Intronic TEs often trigger the formation of transcriptionally repressive heterochromatin, even within transcription-permissive chromatin environments. Although TE-bearing introns are widely observed in eukaryotic genomes, their epigenetic states, impacts on gene regulation and function, and their contributions to genetic diversity and evolution, remain poorly understood. In this study, we investigated the genome-wide distribution of intronic TEs and their epigenetic states in the Oryza sativa genome, where TEs comprise 35% of the genome. We found that over 10% of rice genes contain intronic heterochromatin, most of which are associated with TEs and repetitive sequences. These heterochromatic introns are longer and highly enriched in promoter-proximal positions. On the other hand, introns also accumulate hypomethylated short TEs. Genes with heterochromatic introns are implicated in various biological functions. Transcription of genes bearing intronic heterochromatin is regulated by an epigenetic mechanism involving the conserved factor OsIBM2, mutation of which results in severe developmental and reproductive defects. Furthermore, we found that heterochromatic introns evolve rapidly compared to non-heterochromatic introns. Our study demonstrates that heterochromatin is a common epigenetic feature associated with actively transcribed genes in the rice genome.