Biohybrid CO2 electrolysis for the direct synthesis of polyesters from CO2.

Converting anthropogenic CO2 to value-added products using renewable energy has received much attention to achieve a sustainable carbon cycle. CO2 electrolysis has been extensively investigated, but the products have been limited to some C1-3 products. Here, we report the integration of CO2 electrolysis with microbial fermentation to directly produce poly-3-hydroxybutyrate (PHB), a microbial polyester, from gaseous CO2 on a gram scale. This biohybrid system comprises electrochemical conversion of CO2 to formate on Sn catalysts deposited on a gas diffusion electrode (GDE) and subsequent conversion of formate to PHB by Cupriavidus necator cells in a fermenter. The electrolyzer and the electrolyte solution were optimized for this biohybrid system. In particular, the electrolyte solution containing formate was continuously circulated through both the CO2 electrolyzer and the fermenter, resulting in the efficient accumulation of PHB in C. necator cells, reaching a PHB content of 83% of dry cell weight and producing 1.38 g PHB using 4 cm2 Sn GDE. This biohybrid system was further modified to enable continuous PHB production operated at a steady state by adding fresh cells and removing PHB. The strategies employed for developing this biohybrid system will be useful for establishing other biohybrid systems producing chemicals and materials directly from gaseous CO2.

DeepFold: enhancing protein structure prediction through optimized loss functions, improved template features, and re-optimized energy function.

MOTIVATION: Predicting protein structures with high accuracy is a critical challenge for the broad community of life sciences and industry. Despite progress made by deep neural networks like AlphaFold2, there is a need for further improvements in the quality of detailed structures, such as side-chains, along with protein backbone structures. RESULTS: Building upon the successes of AlphaFold2, the modifications we made include changing the losses of side-chain torsion angles and frame aligned point error, adding loss functions for side chain confidence and secondary structure prediction, and replacing template feature generation with a new alignment method based on conditional random fields. We also performed re-optimization by conformational space annealing using a molecular mechanics energy function which integrates the potential energies obtained from distogram and side-chain prediction. In the CASP15 blind test for single protein and domain modeling (109 domains), DeepFold ranked fourth among 132 groups with improvements in the details of the structure in terms of backbone, side-chain, and Molprobity. In terms of protein backbone accuracy, DeepFold achieved a median GDT-TS score of 88.64 compared with 85.88 of AlphaFold2. For TBM-easy/hard targets, DeepFold ranked at the top based on Z-scores for GDT-TS. This shows its practical value to the structural biology community, which demands highly accurate structures. In addition, a thorough analysis of 55 domains from 39 targets with publicly available structures indicates that DeepFold shows superior side-chain accuracy and Molprobity scores among the top-performing groups. AVAILABILITY AND IMPLEMENTATION: DeepFold tools are open-source software available at https://github.com/newtonjoo/deepfold.

Developing Catalysts Integrated in Gas-Diffusion Electrodes for CO2 Electrolyzers.

ConspectusAs the demand for a carbon-neutral society grows rapidly, research on CO2 electrolysis has become very active. Many catalysts are reported for converting CO2 into value-added products by electrochemical reactions, which have to perform at high Faradaic and energy efficiency to become commercially viable. Various types of CO2 electrolyzers have been used in this effort, such as the H-cell, flow cell, and zero-gap membrane-electrode assembly (MEA) cell. H-cell studies are conducted with electrodes immersed in CO2-saturated electrolyte and have been used to elucidate reaction pathways and kinetic parameters of electrochemical CO2 reduction on many types of catalytic surfaces. From a transport phenomenological perspective, the low solubility and diffusion of CO2 to the electrode surface severely limit the maximum attainable current density, and this metric has been shown to have significant influence on the product spectrum. Flow and MEA cells provide a solution in the form of gas-diffusion electrodes (GDEs) that enable gaseous CO2 to closely reach the catalyst layer and yield record-high current densities. Because GDEs involve a complicated interface consisting of the catalyst surface, gaseous CO2, polymer overlayers, and liquid electrolyte, catalysts with high intrinsic activity might not show high performance in these GDE-based electrolyzers. Catalysts showing low overpotentials at low current densities may suffer from poor electron conductivity and mass transfer limitations at high current densities. Furthermore, the stability of the GDE-based catalysts is often compromised as CO2 electrolysis is pursued with high activity, most notoriously by electrolyte flooding.In this Account, we introduce recent examples where the electrocatalysts were integrated in GDEs, achieving high production rates. The performance of such systems is contingent on both GDE and cell design, and various parameters that affect the cell performance are discussed. Gaseous products, such as carbon monoxide, methane, and ethylene, and liquid products, such as formate and ethanol, have been mainly reported with high partial current density using the flow or MEA cells. Different strategies to this end are described, such as controlling microenvironments by the use of polymers mixed within the catalyst layer or the functionalization of catalyst surfaces with ligands to increase local concentrations of intermediates. Unique CO2 electrolyzer designs are also treated, including the incorporation of light-responsive plasmonic catalysts in the GDE, and combining the electrolyzer with a fermenter utilizing a microbial biocatalyst to synthesize complex multicarbon products. Basic conditions which the catalyst should satisfy to be adapted in the GDEs are listed, and our perspective is provided.

Selective Interbundle Cross-Linking for Lightweight and Superstrong Carbon Nanotube Yarns.

In this study, a range of carbon nanotube yarn (CNTY) architectures was examined and controlled by chemical modification to gain a deeper understanding of CNTY load-bearing systems and produce lightweight and superstrong CNTYs. The architecture of CNTY, which has polymer layers surrounding a compact bundle without hampering the original state of the CNTs in the bundle, is a favorable design for further chemical cross-linking and for enhancing the load-transfer efficiency, as confirmed by in situ Raman spectroscopy under a stress load. The resulting CNTY exhibited excellent mechanical performance that exceeded the specific strength of the benchmark, high-performance fibers. This exceptional strength of the CNTY makes it a promising candidate for the cable of a space elevator traveling from the Earth to the International Space Station given its strength of 4.35 GPa/(g cm-3), which can withstand the self-weight of a 440 km cable.

Early Surgery or Conservative Care for Asymptomatic Aortic Stenosis.

BACKGROUND: The timing and indications for surgical intervention in asymptomatic patients with severe aortic stenosis remain controversial. METHODS: In a multicenter trial, we randomly assigned 145 asymptomatic patients with very severe aortic stenosis (defined as an aortic-valve area of ≤0.75 cm2 with either an aortic jet velocity of ≥4.5 m per second or a mean transaortic gradient of ≥50 mm Hg) to early surgery or to conservative care according to the recommendations of current guidelines. The primary end point was a composite of death during or within 30 days after surgery (often called operative mortality) or death from cardiovascular causes during the entire follow-up period. The major secondary end point was death from any cause during follow-up. RESULTS: In the early-surgery group, 69 of 73 patients (95%) underwent surgery within 2 months after randomization, and there was no operative mortality. In an intention-to-treat analysis, a primary end-point event occurred in 1 patient in the early-surgery group (1%) and in 11 of 72 patients in the conservative-care group (15%) (hazard ratio, 0.09; 95% confidence interval [CI], 0.01 to 0.67; P = 0.003). Death from any cause occurred in 5 patients in the early-surgery group (7%) and in 15 patients in the conservative-care group (21%) (hazard ratio, 0.33; 95% CI, 0.12 to 0.90). In the conservative-care group, the cumulative incidence of sudden death was 4% at 4 years and 14% at 8 years. CONCLUSIONS: Among asymptomatic patients with very severe aortic stenosis, the incidence of the composite of operative mortality or death from cardiovascular causes during the follow-up period was significantly lower among those who underwent early aortic-valve replacement surgery than among those who received conservative care. (Funded by the Korean Institute of Medicine; RECOVERY ClinicalTrials.gov number, NCT01161732.).

Developing Hemodynamic Valve Deterioration and Mortality in Aortic Valve Replacement.

BACKGROUND: As life span increases, in patients having a bioprosthetic valve, the development of hemodynamic valve deterioration (HVD) is an important concern. We evaluated the association of developing HVD to survival in patients undergoing surgical aortic valve replacement (SAVR). METHODS: The individuals undergoing isolated SAVR and serial echocardiography exams (interval >30 d) were included in this study. HVD was defined as mean pressure gradient ≥ 20 mmHg, mean pressure gradient ≥10 mmHg higher than in the baseline exam, or more than moderate regurgitation on Doppler echocardiography (moderate and severe grade). A time-dependent Cox proportional hazard model was used for this study. RESULTS: A total of 631 patients were included. The mean age was 71.8 ± 6.1 y old (female: 53.6%). HVD was found in 259 patients (41%) during echocardiographic follow-up (mean 3.3 ± 3.0 y). Patient-prosthetic mismatch was found in 174 patients. One hundred and twenty-six patients died during follow-up (median 62.1 mo, interquartile range 31.1-96.8). The development of HVD was an independent risk factor for death during follow-up (P = 0.038, hazard ratio 1.46, 95% confidential interval: 1.02-2.08). CONCLUSIONS: HVD was common after bioprosthetic SAVR during mid-term follow-up. Developing HVD, including moderate and severe grades, was associated with a poor survival rate compared with patients without HVD.

Plausibility of Daphnia magna as an alternative experimental model to evaluate effects on eicosanoid synthesis.

Eicosanoids play important roles in inflammation, allergy, fever, and immune responses. In the eicosanoid pathway, cyclooxygenase (COX) catalyzes the conversion of arachidonic acid to prostaglandins and is a crucial target of nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, toxicological studies on the eicosanoid pathway are important for drug discovery and the evaluation of adverse health outcomes due to environmental contaminants. However, experimental models are limited owing to concerns regarding ethical standards. Thus, new alternative models for evaluating toxic effects on the eicosanoid pathway must be developed. To this end, we adopted an invertebrate species, Daphnia magna, as an alternative model. D. magna was exposed to ibuprofen, a major NSAID, for 6 and 24 h. Transcription of eicosanoid-related genes (pla2, cox, pgd synthase, pgd2r2, ltb4dh, and lox) was analyzed by qPCR, eicosanoids (arachidonic acid, prostaglandin F2, dihydroxy prostaglandin F2, and 5-hydroxyeicosatetraenoate) were quantified by multiple reaction monitoring, and enzyme-linked immunosorbent assay was used to determine protein levels of arachidonic acid and prostaglandin E2 (PGE2). After 6 h of exposure, transcription of the pla2 and cox genes was downregulated. In addition, the whole-body level of arachidonic acid, an upstream of COX pathway, increased by over 1.5-fold. The levels of PGE2, a downstream of COX pathway, decreased after 24 h of exposure. According to our results, it is expected that the eicosanoid pathway might be conserved in D. magna, at least partially. This indicates the plausibility of D. magna as an alternative model for the screening of new drugs or chemical toxicity.

The Neuroprotective Effects of Dendropanax morbifera Water Extract on Scopolamine-Induced Memory Impairment in Mice.

This study investigated the neuroprotective effects of Dendropanax morbifera leaves and stems (DMLS) water extract on scopolamine (SCO)-induced memory impairment in mice. First, we conducted experiments to determine the protective effect of DMLS on neuronal cells. Treatment with DMLS showed a significant protective effect against neurotoxicity induced by Aß(25-35) or H2O2. After confirming the neuroprotective effects of DMLS, we conducted animal studies. We administered DMLS orally at concentrations of 125, 250, and 375 mg/kg for 3 weeks. In the Y-maze test, SCO decreased spontaneous alternation, but treatment with DMLS or donepezil increased spontaneous alternation. In the Morris water-maze test, the SCO-treated group showed increased platform reach time and decreased swim time on the target platform. The passive avoidance task found that DMLS ingestion increased the recognition index in short-term memory. Furthermore, memory impairment induced by SCO reduced the ability to recognize novel objects. In the Novel Object Recognition test, recognition improved with DMLS or donepezil treatment. In the mouse brain, except for the cerebellum, acetylcholinesterase activity increased in the SCO group and decreased in the DMLS and donepezil groups. We measured catalase and malondialdehyde, which are indicators of antioxidant effectiveness, and found that oxidative stress increased with SCO but was mitigated by DMLS or donepezil treatment. Thus, our findings suggest that ingestion of DMLS restored memory impairment by protecting neuronal cells from Aß(25-35) or H2O2-induced neurotoxicity, and by reducing oxidative stress.

Verproside, the Most Active Ingredient in YPL-001 Isolated from Pseudolysimachion rotundum var. subintegrum, Decreases Inflammatory Response by Inhibiting PKCδ Activation in Human Lung Epithelial Cells.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease which causes breathing problems. YPL-001, consisting of six iridoids, has potent inhibitory efficacy against COPD. Although YPL-001 has completed clinical trial phase 2a as a natural drug for COPD treatment, the most effective iridoid in YPL-001 and its mechanism for reducing airway inflammation remain unclear. To find an iridoid most effectively reducing airway inflammation, we examined the inhibitory effects of the six iridoids in YPL-001 on TNF or PMA-stimulated inflammation (IL-6, IL-8, or MUC5AC) in NCI-H292 cells. Here, we show that verproside among the six iridoids most strongly suppresses inflammation. Both TNF/NF-κB-induced MUC5AC expression and PMA/PKCδ/EGR-1-induced IL-6/-8 expression are successfully reduced by verproside. Verproside also shows anti-inflammatory effects on a broad range of airway stimulants in NCI-H292 cells. The inhibitory effect of verproside on the phosphorylation of PKC enzymes is specific to PKCδ. Finally, in vivo assay using the COPD-mouse model shows that verproside effectively reduces lung inflammation by suppressing PKCδ activation and mucus overproduction. Altogether, we propose YPL-001 and verproside as candidate drugs for treating inflammatory lung diseases that act by inhibiting PKCδ activation and its downstream pathways.

Black Ginseng Extract Exerts Potentially Anti-Asthmatic Activity by Inhibiting the Protein Kinase Cθ-Mediated IL-4/STAT6 Signaling Pathway.

Asthma is a chronic inflammatory lung disease that causes respiratory difficulties. Black ginseng extract (BGE) has preventative effects on respiratory inflammatory diseases such as asthma. However, the pharmacological mechanisms behind the anti-asthmatic activity of BGE remain unknown. To investigate the anti-asthmatic mechanism of BGE, phorbol 12-myristate 13-acetate plus ionomycin (PMA/Iono)-stimulated mouse EL4 cells and ovalbumin (OVA)-induced mice with allergic airway inflammation were used. Immune cells (eosinophils/macrophages), interleukin (IL)-4, -5, -13, and serum immunoglobulin E (IgE) levels were measured using an enzyme-linked immunosorbent assay. Inflammatory cell recruitment and mucus secretion in the lung tissue were estimated. Protein expression was analyzed via Western blotting, including that of inducible nitric oxide synthase (iNOS) and the activation of protein kinase C theta (PKCθ) and its downstream signaling molecules. BGE decreased T helper (Th)2 cytokines, serum IgE, mucus secretion, and iNOS expression in mice with allergic airway inflammation, thereby providing a protective effect. Moreover, BGE and its major ginsenosides inhibited the production of Th2 cytokines in PMA/Iono-stimulated EL4 cells. In EL4 cells, these outcomes were accompanied by the inactivation of PKCθ and its downstream transcription factors, such as nuclear factor of activated T cells (NFAT), nuclear factor kappa B (NF-κB), activator of transcription 6 (STAT6), and GATA binding protein 3 (GATA3), which are involved in allergic airway inflammation. BGE also inhibited the activation of PKCθ and the abovementioned transcriptional factors in the lung tissue of mice with allergic airway inflammation. These results highlight the potential of BGE as a useful therapeutic and preventative agent for allergic airway inflammatory diseases such as allergic asthma.

Genomic diversity and comprehensive taxonomical classification of 61 Bacillus subtilis group member infecting bacteriophages, and the identification of ortholog taxonomic signature genes.

BACKGROUND: Despite the applications of Bacillus subtilis group species in various sectors, limited information is available regarding their phages. Here, 61 B. subtilis group species-infecting phages (BSPs) were studied for their taxonomic classification considering the genome-size, genomic diversity, and the host, followed by the identification of orthologs taxonomic signature genes. RESULTS: BSPs have widely ranging genome sizes that can be bunched into groups to demonstrate correlations to family and subfamily classifications. Comparative analysis re-confirmed the existing, BSPs-containing 14 genera and 21 species and displayed inter-genera similarities within existing subfamilies. Importantly, it also revealed the need for the creation of new taxonomic classifications, including 28 species, nine genera, and two subfamilies (New subfamily1 and New subfamily2) to accommodate inter-genera relatedness. Following pangenome analysis, no ortholog shared by all BSPs was identified, while orthologs, namely, the tail fibers/spike proteins and poly-gamma-glutamate hydrolase, that are shared by more than two-thirds of the BSPs were identified. More importantly, major capsid protein (MCP) type I, MCP type II, MCP type III and peptidoglycan binding proteins that are distinctive orthologs for Herelleviridae, Salasmaviridae, New subfamily1, and New subfamily2, respectively, were identified and analyzed which could serve as signatures to distinguish BSP members of the respective taxon. CONCLUSIONS: In this study, we show the genomic diversity and propose a comprehensive classification of 61 BSPs, including the proposition for the creation of two new subfamilies, followed by the identification of orthologs taxonomic signature genes, potentially contributing to phage taxonomy.

Flicker-free dual-volume augmented reality display using a pixelated interwoven integral floating technique with a geometric phase lens.

A geometric phase (GP) integral floating display can provide multifocal three-dimensional (3D) augmented reality (AR) images with enhanced depth expression by switching the focal modes of the GP lens via polarization control. However, using temporal multiplexing to switch between the focal modes of GP optics causes flickering as each 3D AR image is fully presented in different frames and their temporal luminance profile becomes easily recognizable, particularly as the number of available focal modes increases. Here, we propose a novel integral floating technique to generate pixelated interwoven 3D AR images; a half of each image is spatially mixed with another and presented in both focal modes simultaneously to resolve the flickering issue. The principle was verified via experimental demonstration and optically measured data.

Genome-wide transcriptional regulation in Saccharomyces cerevisiae in response to carbon dioxide.

Sugar metabolism by Saccharomyces cerevisiae produces ample amounts of CO2 under both aerobic and anaerobic conditions. High solubility of CO2 in fermentation media, contributing to enjoyable sensory properties of sparkling wine and beers by S. cerevisiae, might affect yeast metabolism. To elucidate the overlooked effects of CO2 on yeast metabolism, we examined glucose fermentation by S. cerevisiae under CO2 as compared to N2 and O2 limited conditions. While both CO2 and N2 conditions are considered anaerobic, less glycerol and acetate but more ethanol were produced under CO2 condition. Transcriptomic analysis revealed that significantly decreased mRNA levels of GPP1 coding for glycerol-3-phosphate phosphatase in glycerol synthesis explained the reduced glycerol production under CO2 condition. Besides, transcriptional regulations in signal transduction, carbohydrate synthesis, heme synthesis, membrane and cell wall metabolism, and respiration were detected in response to CO2. Interestingly, signal transduction was uniquely regulated under CO2 condition, where upregulated genes (STE3, MSB2, WSC3, STE12, and TEC1) in the signal sensors and transcriptional factors suggested that MAPK signaling pathway plays a critical role in CO2 sensing and CO2-induced metabolisms in yeast. Our study identifies CO2 as an external stimulus for modulating metabolic activities in yeast and a transcriptional effector for diverse applications.

Toxicological profile of bisphenol F via comprehensive and extensive toxicity evaluations following dermal exposure.

Bisphenol F (BPF) is classified as a harmful substance by the U.S. Environmental Protection Agency. Although previous studies focused on human exposure to BPF via direct consumption or inhalation, few investigators assessed potential toxicological effects following skin contact. The aim of this study was to examine (1) the degree and pattern by which BPF is absorbed onto the skin in vivo, and (2) determination of toxicity and safety using the following tests: acute dermal; a 28-day repeat dermal; a skin irritation; an eye irritation; and a skin sensitization. As indicated by the amount of BPF remaining in the epidermis or dermis, data demonstrated that BPF was absorbed through the skin at a 26.5% rate. BPF penetrated the subcutaneous layer at a "fast rate" (Kp: 2.2E-02). Although no toxicological changes or local irritation were observed following skin exposure, BPF induced potent sensitization. In summary, the findings of this study showed that BPF penetrated and was absorbed into the skin at a high rate which was associated with enhanced chemical-induced skin sensitization and this may have significant implications following exposure of skin to BPF.

Methyl P-Coumarate Ameliorates the Inflammatory Response in Activated-Airway Epithelial Cells and Mice with Allergic Asthma.

Methyl p-coumarate (methyl p-hydroxycinnamate) (MH) is a natural compound found in a variety of plants. In the present study, we evaluated the ameliorative effects of MH on airway inflammation in an experimental model of allergic asthma (AA). In this in vitro study, MH was found to exert anti-inflammatory activity on PMA-stimulated A549 airway epithelial cells by suppressing the secretion of IL-6, IL-8, MCP-1, and ICAM-1. In addition, MH exerted an inhibitory effect not only on NF-κB (p-NF-κB and p-IκB) and AP-1 (p-c-Fos and p-c-Jun) activation but also on A549 cell and EOL-1 cell (eosinophil cell lines) adhesion. In LPS-stimulated RAW264.7 macrophages, MH had an inhibitory effect on TNF-α, IL-1ß, IL-6, and MCP-1. The results from in vivo study revealed that the increases in eosinophils/Th2 cytokines/MCP-1 in the bronchoalveolar lavage fluid (BALF) and IgE in the serum of OVA-induced mice with AA were effectively inhibited by MH administration. MH also exerted a reductive effect on the immune cell influx, mucus secretion, and iNOS/COX-2 expression in the lungs of mice with AA. The effects of MH were accompanied by the inactivation of NF-κB. Collectively, the findings of the present study indicated that MH attenuates airway inflammation in mice with AA, suggesting its potential as an adjuvant in asthma therapy.

Lipid-Oriented Live-Cell Distinction of B and T Lymphocytes.

The identification of each cell type is essential for understanding multicellular communities. Antibodies set as biomarkers have been the main toolbox for cell-type recognition, and chemical probes are emerging surrogates. Herein we report the first small-molecule probe, CDgB, to discriminate B lymphocytes from T lymphocytes, which was previously impossible without the help of antibodies. Through the study of the origin of cell specificity, we discovered an unexpected novel mechanism of membrane-oriented live-cell distinction. B cells maintain higher flexibility in their cell membrane than T cells and accumulate the lipid-like probe CDgB more preferably. Because B and T cells share common ancestors, we tracked the cell membrane changes of the progenitor cells and disclosed the dynamic reorganization of the membrane properties over the lymphocyte differentiation progress. This study casts an orthogonal strategy for the small-molecule cell identifier and enriches the toolbox for live-cell distinction from complex cell communities.

Assessment of rapidly advancing bone age during puberty on elbow radiographs using a deep neural network model.

OBJECTIVES: Bone age is considered an indicator for the diagnosis of precocious or delayed puberty and a predictor of adult height. We aimed to evaluate the performance of a deep neural network model in assessing rapidly advancing bone age during puberty using elbow radiographs. METHODS: In all, 4437 anteroposterior and lateral pairs of elbow radiographs were obtained from pubertal individuals from two institutions to implement and validate a deep neural network model. The reference standard bone age was established by five trained researchers using the Sauvegrain method, a scoring system based on the shapes of the lateral condyle, trochlea, olecranon apophysis, and proximal radial epiphysis. A test set (n = 141) was obtained from an external institution. The differences between the assessment of the model and that of reviewers were compared. RESULTS: The mean absolute difference (MAD) in bone age estimation between the model and reviewers was 0.15 years on internal validation. In the test set, the MAD between the model and the five experts ranged from 0.19 to 0.30 years. Compared with the reference standard, the MAD was 0.22 years. Interobserver agreement was excellent among reviewers (ICC: 0.99) and between the model and the reviewers (ICC: 0.98). In the subpart analysis, the olecranon apophysis exhibited the highest accuracy (74.5%), followed by the trochlea (73.7%), lateral condyle (73.7%), and radial epiphysis (63.1%). CONCLUSIONS: Assessment of rapidly advancing bone age during puberty on elbow radiographs using our deep neural network model was similar to that of experts. KEY POINTS: • Bone age during puberty is particularly important for patients with scoliosis or limb-length discrepancy to determine the phase of the disease, which influences the timing and method of surgery. • The commonly used hand radiographs-based methods have limitations in assessing bone age during puberty due to the less prominent morphological changes of the hand and wrist bones in this period. • A deep neural network model trained with elbow radiographs exhibited similar performance to human experts on estimating rapidly advancing bone age during puberty.

Production of neoagarooligosaccharides by probiotic yeast Saccharomyces cerevisiae var. boulardii engineered as a microbial cell factory.

BACKGROUND: Saccharomyces cerevisiae var. boulardii is a representative probiotic yeast that has been widely used in the food and pharmaceutical industries. However, S. boulardii has not been studied as a microbial cell factory for producing useful substances. Agarose, a major component of red macroalgae, can be depolymerized into neoagarooligosaccharides (NAOSs) by an endo-type ß-agarase. NAOSs, including neoagarotetraose (NeoDP4), are known to be health-benefiting substances owing to their prebiotic effect. Thus, NAOS production in the gut is required. In this study, the probiotic yeast S. boulardii was engineered to produce NAOSs by expressing an endo-type ß-agarase, BpGH16A, derived from a human gut bacterium Bacteroides plebeius. RESULTS: In total, four different signal peptides were compared in S. boulardii for protein (BpGH16A) secretion for the first time. The SED1 signal peptide derived from Saccharomyces cerevisiae was selected as optimal for extracellular production of NeoDP4 from agarose. Expression of BpGH16A was performed in two ways using the plasmid vector system and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. The production of NeoDP4 by engineered S. boulardii was verified and quantified. NeoDP4 was produced by S. boulardii engineered using the plasmid vector system and CRISPR-Cas9 at 1.86 and 0.80 g/L in a 72-h fermentation, respectively. CONCLUSIONS: This is the first report on NAOS production using the probiotic yeast S. boulardii. Our results suggest that S. boulardii can be considered a microbial cell factory to produce health-beneficial substances in the human gut.

Complete nucleotide sequence analysis and identification of 7-cyano-7-deazaguanine (PreQ0) biosynthesis-related genes in the novel Bacillus subtilis-infecting Siphoviridae family phage BSP7.

In this study, bacteriophage BSP7, a novel Bacillus subtilis-infecting member of the family Siphoviridae, was isolated from a Korean soybean-based fermented food, Deonjang, using B. subtilis ATCC 21336 as a host. The genome is 55,455 bp long with 39.92% G+C content. A total of 70 ORFs with no tRNA were detected in the genome. A distinct feature of the BSP7 genome among B. subtilis-infecting Siphoviridae family phages is the presence of putative ORFs related to biosynthesis of 7-cyano-7-deazaguanine (PreQ0), a precursor of queuosine and archaeosine biosynthesis. Bioinformatic analysis revealed that the genome of BSP7 does not exhibit any significant similarities to other phages with sequences in the NCBI database. A comparative genomic analysis also confirmed the uniqueness of BSP7 within the family Siphoviridae.

Primary Graft Dysfunction After Isolated Heart Transplantation　- Incidence, Risk Factors, and Clinical Implications Based on a Single-Center Experience.

BACKGROUND: Since the international consensus on primary graft dysfunction (PGD) following heart transplantation (HT) was reported in 2014, few clinical studies have been reported. We aimed to analyze the incidence, predictive factors, and clinical implications of PGD following the International Society of Heart and Lung Transplant criteria in a single center.Methods and Results:This study enrolled 570 consecutive adult patients undergoing isolated HT between November 1992 and December 2017. Under a new set of criteria, PGD-left ventricle (PGD-LV) occurred in 35 patients (6.1%; mild, n=1 [0.2%]; moderate, n=14 [2.5%]; severe, n=20 [3.5%]), whereas PGD-right ventricle (PGD-RV) occurred in 3 (0.5%). Multivariable analysis demonstrated that preoperative admission (odds ratio [OR] 4.20; 95% confidence interval [CI] 1.24-14.26; P=0.021), preoperative extracorporeal membrane oxygenation (OR 4.03; 95% CI 1.75-9.26; P=0.001), and prolonged total ischemic time (OR 1.09; 95% CI 1.02-1.15; P=0.006) were significant predictors of moderate to severe PGD-LV. Moderate to severe PGD-LV was an independent and significant risk factor for early death (OR 55.64; 95% CI 11.65-265.73; P<0.001), with its effects extending up to 3 months after HT. CONCLUSIONS: Moderate to severe PGD-LV, as defined by the new guidelines, is an important predictor of early mortality, with effects extending up to 3 months after HT. Efforts to reduce the occurrence of moderate to severe PGD-LV may lead to better outcomes.