Predicting valuable missense variants with AlphaMissense in a multiple pulmonary infection patient.

AlphaMissense is proficient in predicting the clinical classification of missense variants. we utilized AlphaMissense to find disease-relevant variants within a polymicrobial pulmonary infection case. Exome sequencing was performed in this patient, and AlphaMissense and Phenolyzer were combined to investigate disease-relevant variants screening from exome sequencing results.

Effective-compound combination of Bufei Yishen formula III combined with ER suppress airway mucus hypersecretion in COPD rats: via EGFR/MAPK signaling.

BACKGROUND: The aim of this study was to explore the combined efficacy ofeffective-component compatibility of Bufei Yishen formula III (ECC-BYF III) and exercise rehabilitation (ER) in inhibiting airway mucus hypersecretion in a chronic obstructive pulmonary disease (COPD) rat model. METHODS: A total of 48 SD rats were divided into control, model, acetylcysteine (NAC), ECC-BYF III, ER, and ECC-BYF III + ER groups (n=8). COPD rats were exposed to cigarette smoke and bacteria for 8 weeks and administered various treatments over the next eight weeks. Rats were euthanized at week 17 after pulmonary function testing. Pathological examination of lung tissues was performed. IL-6 and IL-10 levels were measured in bronchoalveolar lavage fluid (BALF) and protein levels of MUC5AC, MUC5B, AQP-5, EGFR, ERK, JNK, and p38 were measured in lung tissues. RESULTS: Improved pulmonary function and pathological changes were observed in ECC-BYF III, ECC-BYF III + ER, and NAC groups. ECC-BYF III and ECC-BYF III + ER had greater mean alveolar number (MAN) compared with NAC. Lung inflammation and goblet cell generation were reduced and MUC5AC, MUC5B and AQP-5 expressions were lower in all treatment groups. ECC-BYF III has more significant effect on MUC5AC than ER and NAC. ECC-BYFIII + ER had a greater effect on suppressing IL-6 in BALF compared with other treatments. ECC-BYFIII, ER, and ECC-BYF III + ER reduced EGFR, ERK, JNK, and p38 phosphorylated protein levels. ECC-BYFIII+ER had a greater effect on p-JNK and p-p38 than ECC-BYFIII and NAC. CONCLUSION: ECC-BYF III, ER, and ECC-BYF III + ER have efficacy in inhibiting airway mucus hypersecretion with improved pulmonary function and pathological changes. ECC-BYF III had a greater effect in improving MAN and MUC5AC in lung tissue. ECC-BYF III+ER had a greater effect in alleviating pulmonary pathology and inflammation. These effects may be mediated by inhibition of the EGFR/MAPK pathway.

Flavorubredoxin, a Candidate Trigger Related to Thrombotic Thrombocytopenic Purpura: Screening of the Complete Genome of a Salmonella enterica Serovar Typhimurium Isolate From an AIDS Case.

Thrombotic thrombocytopenic purpura (TTP) is one of the two classic thrombotic microangiopathy (TMA) diseases which could be induced by infections. To the best of our knowledge, this is the first report of an acquired immunodeficiency syndrome (AIDS) patient with acquired TTP induced by infection with Salmonella enterica serovar Typhimurium (hereafter, S. Typhimurium) isolate, S. Typhimurium_zhang, which was confirmed by serology and genetic taxonomy. The literature review identified 17 TMA-related genes encoding the candidate triggers, which were searched in the annotated genome sequence of S. Typhimurium_zhang. Anaerobic nitric oxide reductase flavorubredoxin (FlRd), encoded by norV which is related to another TMA, haemolytic uraemic syndrome (HUS), was found in S. Typhimurium_zhang. Basic local alignment search tool (BLAST) analysis revealed that norV and FlRd in S. Typhimurium_zhang, as well as eight S. Typhimurium type strains, have high identity with HUS-related Escherichia coli O157:H7 strain TW14359. Similar results were obtained from the BLAST analysis of 73 S. enterica isolates for congenital TTP which was also previously reported to be triggered by S. enterica. Phylogenetic analysis and amino acid sequence alignment revealed that FlRd was functional and highly conservative on 69 Enterobacteriaceae, including S. Typimurium_zhang and TW14359. In brief, we found norV in the genome of a S. Typhimurium clinical isolate that induced TTP in an AIDS patient. FlRd, the protein encoded by norV, probably triggered the TTP and was highly conservative, functional, and widespread in S. enterica and Enterobacteriaceae. More in vitro and in vivo studies are required to confirm our findings and determine the underlying mechanism.

Effective Component Compatibility of Bufei Yishen Formula III Which Regulates the Mucus Hypersecretion of COPD Rats via the miR-146a-5p/EGFR/MEK/ERK Pathway.

Background: The effective-component compatibility of Bufei Yishen formula III (ECC-BYF III) with 5 ingredients (ginsenoside Rh1, astragaloside, icariin, nobiletin, and paeonol) has been shown to protect against chronic obstructive pulmonary disease (COPD). The present study aimed to observe the effects of ECC-BYF III in a COPD rat model and dissect its potential mechanisms in regulating mucus hypersecretion via the miR-146a-5p/epidermal growth factor receptor (EGFR)/MEK/ERK pathway. Methods: COPD model rats were treated with normal saline, ECC-BYF III, or N-acetylcysteine (NAC). Pulmonary function, lung tissue histology with H & E and AB-PAS staining, expression levels of interleukin (IL)-4, IL-6, IL-1ß, MUC5AC, MUC5B, and FOXA2 in lung tissues and the mRNA and proteins involved in the miR-146a-5p/EGFR/MEK/ERK pathway were evaluated. Results: The COPD rats showed a significant decrease in the pulmonary function and serious pathological damage to the lung tissue. ECC-BYF III and NAC significantly improved the ventilation function and small airway pathological damage in the COPD rats. The goblet cells and the expression levels of IL-1ß, IL-6, MUC5AC, and MUC5B were increased in the COPD rats and were significantly decreased after ECC-BYF III or NAC intervention. The expression levels of IL-4 and FOXA2 in the COPD rats were markedly decreased and were improved in the ECC-BYF III and NAC groups. ECC-BYF III appeared to have a potent effect in restoring the reduced expression of miR-146a-5p. The increased phosphorylation levels of EGFR, MEK, and ERK1/2 and the protein expression levels of SPDEF in the lungs of COPD rats could be significantly reduced by ECC-BYF III. Conclusions: ECC-BYF III has a significant effect in improving the airway mucus hypersecretion in COPD model rats, as well as a protective effect against limited pulmonary function and injured lung histopathology. The protective effect of ECC-BYF III in reducing airway mucus hypersecretion in COPD may involve the miR-146a-5p/EGFR/MEK/ERK pathway.

Effective-constituent compatibility-based analysis of Bufei Yishen formula, a traditional herbal compound as an effective treatment for chronic obstructive pulmonary disease.

OBJECTIVE: Critical effective constituents were identified from Bufei Yishen formula (BYF), a traditional herbal compound and combined as effective-constituent compatibility (ECC) of BYF I, which may have potential bioactive equivalence to BYF. METHODS: The active constituents of BYF were identified using four cellular models and categorised into Groups 1 (Bufeiqi), 2 (Bushen), 3 (Huatan) and 4 (Huoxue) according to Chinese medicinal theory. An orthogonal design and a combination method were used to determine the optimal ratios of effective constituents in each group and the ratios of "Groups 1 to 4" according to their pharmacological activity. We also comprehensively assessed bioactive equivalence between the BYF and the ECC of BYF I in a rat model of chronic obstructive pulmonary disease (COPD). RESULTS: We identified 12 active constituents in BYF. The numbers of constituents in Groups 1 to 4 were 3, 2, 5 and 2, respectively. We identified the optimal ratios of effective constituents within each group. In Group 1, total ginsenosides:Astragalus polysaccharide:astragaloside IV ratio was 9:5:2. In Group 2, icariin:schisandrin B ratio was 100:12.5. In Group 3, nobiletin:hesperidin:peimine:peiminine:kaempferol ratio was 4:30:6.25:0:0. In Group 4, paeoniflorin:paeonol ratio was 4:1. An orthogonal design was then used to establish the optimal ratios of Group 1, Group 2, Group 3 and Group 4 in ECC of BYF I. The ratio for total ginsenosides:Astragalus polysaccharide:astragaloside IV:icariin:schisandrin B:nobiletin:hesperidin:peimine:paeoniflorin:paeonol was determined to be 22.5:12.5:5:100:12.5:4:30:6.25:25:6.25. A comprehensive evaluation confirmed that ECC of BYF I presented with bioactive equivalence to the original BYF. CONCLUSION: Based on the ECC of traditional Chinese medicine formula method, the effective constituents of BYF were identified and combined in a fixed ratio as ECC of BYF I that was as effective as BYF itself in treating rats with COPD.

Effective-component compatibility of Bufei Yishen formula II inhibits mucus hypersecretion of chronic obstructive pulmonary disease rats by regulating EGFR/PI3K/mTOR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: The effective-component compatibility of Bufei Yishen formula I (ECC-BYF I), a combination of 10 compounds, including total ginsenosides, astragaloside IV, icariin, and paeonol, etc., is derived from Bufei Yishen formula (BYF). The efficacy and safety of ECC-BYF I is equal to BYF. However, the composition of ECC-BYF I needs to be further optimized. Based on the beneficial effects of BYF and ECC-BYF I on chronic obstructive pulmonary disease (COPD), this study aimed to optimize the composition of ECC-BYF I and to explore the effects and mechanisms of optimized ECC-BYF I (ECC-BYF II) on mucus hypersecretion in COPD rats. MATERIALS AND METHODS: ECC-BYF I was initially optimized to six groups: optimized ECC-BYF I (OECC-BYF I)-A~F. Based on a COPD rat model, the effects of OECC-BYF I-A~F on COPD rats were evaluated. R-value comprehensive evaluation was used to evaluate the optimal formula, which was named ECC-BYF II. The changes in goblet cells and expression of mucins and the mRNA and proteins involved in the epidermal growth factor receptor/phosphoinositide-3-kinase/mammalian target of rapamycin (EGFR/PI3K/mTOR) pathway were evaluated to explore the effects and mechanisms of ECC-BYF II on mucus hypersecretion. RESULTS: ECC-BYF I and its six optimized groups, OECC-BYF I-A~F, had beneficial effects on COPD rats in improving pulmonary function and lung tissue pathology, reducing inflammation and oxidative stress, and improving the protease/anti-protease imbalance and collagen deposition. R-value comprehensive evaluation found that OECC-BYF I-E (paeonol, icariin, nobiletin, total ginsenoside, astragaloside IV) was the optimal formula for improving the comprehensive effects (lung function: VT, MV, PEF, EF50, FVC, FEV 0.1, FEV 0.1/FVC; histological changes: MLI, MAN; IL-1ß, IL-6, TNF-α, MMP-9, TIMP-1, T-AOC, LPO, MUC5AC, Collagen I and Collagen III). OECC-BYF I-E was named ECC-BYF II. Importantly, the effect of ECC-BYF II showed no significant difference from BYF and ECC-BYF I. ECC-BYF II inhibited mucus hypersecretion in COPD rats, which manifested as reducing the expression of MUC5AC and MUC5B and the hyperplasia rate of goblet cells. The mRNA and protein expression levels of EGFR, PI3K, Akt, and mTOR were increased in COPD rats and were obviously downregulated after ECC-BYF II administration. CONCLUSION: ECC-BYF II, which consists of paeonol, icariin, nobiletin, total ginsenoside and astragaloside IV, has beneficial effects equivalent to BYF and ECC-BYF I on COPD rats. ECC-BYF II significantly inhibited mucus hypersecretion, which may be related to the regulation of the EGFR/PI3K/mTOR pathway.

Effect of Bufei Yishen Granules Combined with Electroacupuncture in Rats with Chronic Obstructive Pulmonary Disease via the Regulation of TLR-4/NF-κB Signaling.

BACKGROUND: The combined therapy of Bufei Yishen granules (BY) and electroacupuncture (EA) has shown good effects clinically in treating chronic obstructive pulmonary disease (COPD). The present study aimed to observe the effects of the BY + EA combination in a COPD rat model and dissect the potential mechanisms via Toll-like receptor (TLR) 4/nuclear factor kappa B (NF-κB) signaling. METHODS: The COPD rats were treated with normal saline, aminophylline, Bufei Yishen granules, electroacupuncture, or Bufei Yishen granules combined with electroacupuncture. The pulmonary function; lung tissue histology; levels of inflammatory factors; expression levels of TLR-4, inhibitor of nuclear factor kappa B (IκB), and NF-κB; and activation of NF-κB in the lung tissues were evaluated. RESULTS: Pulmonary function was markedly decreased in the COPD rats, and the lung tissue histology of the COPD rats showed severe pathological changes. The pulmonary function and lung tissue morphology in the treatment groups (APL, BY, EA, and BY + EA) were improved. The increased levels of the inflammatory cytokines interleukin (IL)-1ß and IL-6 indicated a chronic inflammatory state in the COPD rats. In the BY, EA, and BY + EA groups, the levels of IL-1ß and IL-6 were decreased, especially in the BY + EA group. In addition, the mRNA and protein expression levels of TLR-4, IκB, and NF-κB were obviously downregulated in the BY and BY + EA groups; and the NF-κB p65 activation was significantly decreased in the BY, EA, and BY + EA groups. CONCLUSIONS: Bufei Yishen granules and electroacupuncture have curative effects in COPD rats, and the combination therapy of Bufei Yishen granules and electroacupuncture is superior. The TLR-4/NF-κB pathway may be involved in the potential mechanisms by which Bufei Yishen granules and electroacupuncture reduce inflammation.

Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,ß-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Šresolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/ß)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

Structure of yeast sulfhydryl oxidase erv1 reveals electron transfer of the disulfide relay system in the mitochondrial intermembrane space.

The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX(9)C or twin CX(3)C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 Å resolution of the CTD and at 3.0 Å resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.

Structural insights into the cofactor-assisted substrate recognition of yeast quinone oxidoreductase Zta1.

Quinone oxidoreductase (QOR EC1.6.5.5) catalyzes the reduction of quinone to hydroxyquinone using NADPH as a cofactor. Here we present the crystal structure of the ζ-crystallin-like QOR Zta1 from Saccharomycescerevisiae in apo-form at 2.00 Šand complexed with NADPH at 1.59 Šresolution. Zta1 forms a homodimer, with each subunit containing a catalytic and a cofactor-binding domain. Upon NADPH binding to the interdomain cleft, the two domains shift towards each other, producing a better fit for NADPH, and tightening substrate binding. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis defined a potential quinone-binding site that determines the stringent substrate specificity. Moreover, multiple-sequence alignment and kinetics assays implied that a single-residue change from Arg in lower organisms to Gly in vertebrates possibly resulted in elevation of enzymatic activity of ζ-crystallin-like QORs throughout evolution.