Enhancement of Biocontrol Activities and Cyclic Lipopeptides Production by Chemical Mutagenesis of Bacillus subtilis XF-1, a Biocontrol Agent of Plasmodiophora brassicae and Fusarium solani.

Bacillus subtilis XF-1 has been used as a biocontrol agent of clubroot disease of crucifers infected by Plasmodiophora brassicae, an obligate pathogen. In order to maximize the growth inhibition of the pathogen, random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine was applied to strain XF-1. The efficacy of 226 selected mutants was assessed against the growth of an indicator fungal pathogen: Fusarium solani using agar plate assay and the disruptive effects on the resting spores of P. brassicae. Four mutants exhibited inhibition activity significantly higher than the wild type. The cell extracts of these mutants and the XF-1 were subjected to matrix-assisted laser desorption ionization-time of flight mass spectra analysis, and three families of cyclic lipopeptides (CLPs) fengycin, surfactin and iturin were identified from the parental strain and the screened mutants. However, the relative contents and compound diversity changed after mutagenesis, and there was slight variation in the surfactin and fengycin. Notably, only 5 iturin components were discovered from the wild strain XF-1, but 13 were obtained from the mutant strains, and the relative CLPs contents of all mutant strains increased substantially. The results suggested that CLPs might be one of main biocontrol mechanisms of the clubroot disease by XF-1. The 4 mutants are far more effective than the parental strain, and they would be promising biocontrol candidates not only against P. brassicae but probably other plant diseases caused by fungi.

[Association of vitamin D receptor gene haplotypes and genotype combinations with susceptibility to occupational elevated blood lead].

OBJECTIVE: To investigate the association of the haplotypes and genotype combinations of vitamin D receptor (VDR) BsmI (rs1544410), Tru9I (rs757343), ApaI (rs7975232), and TaqI (rs731236) with the susceptibility to elevated blood lead in Chinese Han population. METHODS: According to Diagnostic Criteria of Occupational Chronic Lead Poisoning (GBZ 37-2002) and Occupational Exposure Limits for Hazardous Agents in the Workplace Part 1: Chemical Hazardous Agents (GBZ 2.1-2007), the workers were divided into high-exposure group (lead dust ≥ 0.05 mg/m(3), lead fume ≥ 0.03 mg/m(3)) and low-exposure group based on the concentrations of lead fume and lead dust in the workplace. The high-exposure group was further divided into normal-blood lead subgroup and high-blood lead subgroup. Fasting peripheral venous blood (5 ml) was collected using a heparin tube; genomic DNA was extracted from the peripheral blood cells with a Qiagen kit; single nucleotide polymorphisms were detected by allelic discrimination assay using TaqMan probes (carrying fluorescent dyes); haplotypes were analyzed and compared by Haploview. RESULTS: VDR BsmI, Tru9I, ApaI, and TaqI were in Hardy-Weinberg equilibrium between the normal-blood lead subgroup and high-blood lead subgroup (P > 0.05). Compared with haplotype CCCA which had the highest distribution frequency, haplotypes CCAA and CTCA were the high-risk factors for elevated blood lead (OR = 1.814, 95%CI = 1.055 ∼ 3.119; OR = 1.919, 95%CI = 1.040 ∼ 3.540). Compared with genotype combination CC + CC + CC + AA which had the highest distribution frequency, genotype combination CC + CC + AC + AA was the high-risk factor for elevated blood lead (OR = 2.800, 95%CI = 1.282 ∼ 6.116). CONCLUSION: As for VDR BsmI, Tru9I, ApaI, and TaqI, haplotypes CCAA and CTCA and genotype combination CC + CC + AC + AA are associated with the susceptibility to elevated blood lead.

ROS induced the Rab26 promoter hypermethylation to promote cigarette smoking-induced airway epithelial inflammation of COPD through activation of MAPK signaling.

Cigarette smoking (CS) exposure-induced airway inflammatory responses drive the occurrence and development of emphysema and chronic obstructive pulmonary disease (COPD). However, its precise mechanisms have not been fully elucidated. In this study, we explore the role of Rab26 in CS exposure modulating the inflammatory response of airway epithelium and the novel mechanism of CS exposure regulation Rab26. These data showed that CS exposure and H2O2 (a type of ROS) suppressed the expression of Rab26 and increased the expression of DNMT3b in vivo and in vitro. GEO data analysis found the level of Rab26 was decreased in the lung tissue of COPD patients. CSE-induced ROS promoted DNA methylation of the Rab26 promoter and inhibited its promoter activity by elevating the DNMT3b level. Antioxidants N-Acetyl-l-cysteine (NAC), 5-Aza-2'-deoxycytidine (5-AZA) (DNA methylation inhibitor) and DNMT3B siRNA alleviated CSE's inhibitory effect on Rab26 expression in vitro. Importantly, NAC alleviated the improved expression of Rab26 and reduced DNMT3B expression, in the airway of smoking exposure as well as attenuated the inflammatory response in vivo. Overexpression of Rab26 attenuated CSE-induced production of inflammatory mediators through part inactivation of p38 and JNK MAPK. On the contrary, silencing Rab26 enhanced p38 and JNK activation and aggravated inflammatory response. These findings suggest that ROS-mediated Rab26 promoter hypermethylation is a critical step in cigarette smoking-induced airway epithelial inflammatory response. Restoring Rab26 in the airway epithelium might be a potential strategy for treating airway inflammation and COPD.

Genetic evidence on the causality between gut microbiota and various asthma phenotypes: a two-sample Mendelian randomization study.

Introduction: Asthma is a multifarious disease that manifests in various phenotypes. Among the various factors that contribute to the development of asthma, the gut microbiota has recently emerged as a compelling area of investigation. This study aims to investigate the causal relationships between gut microbiota and distinct asthma phenotypes. Methods: The genome-wide association study (GWAS) summary statistics for 211 gut microbial taxa were used as study exposure. Five traits pertaining to various asthma phenotypes (asthma, allergic asthma, childhood asthma, suggestive for eosinophilic asthma and obesity-related asthma) were included as study outcome. We conducted Mendelian randomization (MR) analysis and sensitivity analysis for each bacterial taxa and asthma phenotypes. Result: We discovered a total of 58 associations that exhibited evidence of causality. Out of these, 4 associations remained significant even after applying multiple correction. An increased risk of asthma was causally associated with higher abundance of genus Holdemanella (OR = 1.11; CI: 1.05-1.17; p = 0.027), genus Oxalobacter (OR = 1.09; CI: 1.04-1.15; p = 0.025) and genus Butyricimonas (OR = 1.14; CI: 1.06-1.22; p = 0.027). Order NB1n was causally linked with an increased risk of obesity-related asthma (OR = 1.17; CI: 1.07-1.29; p = 0.015). There was limited overlap among the taxa that exhibited potential causal relationships with distinct asthma phenotypes. Conclusion: Our research has provided genetic evidence that establishes multiple causal relationships between the gut microbiota and distinct asthma phenotypes, supporting the role of the gut microbiota in various asthma phenotypes. It is possible that different taxa play a role in the development of distinct asthma phenotypes. The causal relationships identified in this study require further investigation.

Prevention of exacerbation in patients with moderate-to-very severe COPD with the intent to modulate respiratory microbiome: a pilot prospective, multi-center, randomized controlled trial.

Introduction: Considering the role of bacteria in the onset of acute exacerbation of COPD (AECOPD), we hypothesized that the use of influenza-Streptococcus pneumoniae vaccination, oral probiotics or inhaled amikacin could prevent AECOPD. Methods: In this pilot prospective, muti-central, randomized trial, moderate-to-very severe COPD subjects with a history of moderate-to-severe exacerbations in the previous year were enrolled and assigned in a ratio of 1:1:1:1 into 4 groups. All participants were managed based on the conventional treatment recommended by GOLD 2019 report for 3 months, with three groups receiving additional treatment of inhaled amikacin (0.4 g twice daily, 5-7 days monthly for 3 months), oral probiotic Lactobacillus rhamnosus GG (1 tablet daily for 3 months), or influenza-S. pneumoniae vaccination. The primary endpoint was time to the next onset of moderate-to-severe AECOPD from enrollment. Secondary endpoints included CAT score, mMRC score, adverse events, and survival in 12 months. Results: Among all 112 analyzed subjects (101 males, 96 smokers or ex-smokers, mean ± SD age 67.19 ± 7.39 years, FEV1 41.06 ± 16.09% predicted), those who were given dual vaccination (239.7 vs. 198.2 days, p = 0.044, 95%CI [0.85, 82.13]) and oral probiotics (248.8 vs. 198.2 days, p = 0.017, 95%CI [7.49, 93.59]) had significantly delayed onset of next moderate-to-severe AECOPD than those received conventional treatment only. For subjects with high symptom burden, the exacerbations were significantly delayed in inhaled amikacin group as compared to the conventional treatment group (237.3 vs. 179.1 days, p = 0.009, 95%CI [12.40,104.04]). The three interventions seemed to be safe and well tolerated for patient with stable COPD. Conclusion: The influenza-S. pneumoniae vaccine and long-term oral probiotic LGG can significantly delay the next moderate-to-severe AECOPD. Periodically amikacin inhalation seems to work in symptomatic patients. The findings in the current study warrants validation in future studies with microbiome investigation.Clinical trial registration:https://clinicaltrials.gov/, identifier NCT03449459.

The Clinical Impact of Metagenomic Next-Generation Sequencing (mNGS) Test in Hospitalized Patients with Suspected Sepsis: A Multicenter Prospective Study.

Background: Metagenomic Next Generation Sequencing (mNGS) has the potential to detect pathogens rapidly. We aimed to assess the diagnostic performance of mNGS in hospitalized patients with suspected sepsis and evaluate its role in guiding antimicrobial therapy. Methods: A multicenter, prospective cohort study was performed. We enrolled patients with suspected sepsis, collected clinical characteristics and blood samples, and recorded the 30-day survival. Diagnostic efficacy of mNGS test and blood culture was compared, and the clinical impact of mNGS on antibiotic regimen modification was analyzed. Results: A total of 277 patients were enrolled, and 162 were diagnosed with sepsis. The mortality was 44.8% (121/270). The mNGS test exhibited shorter turn-out time (27.0 (26.0, 29.0) vs. 96.0 (72.0, 140.3) hours, p < 0.001) and higher sensitivity (90.5% vs. 36.0%, p < 0.001) compared with blood culture, especially for fungal infections. The mNGS test showed better performance for patients with mild symptoms, prior antibiotic use, and early stage of infection than blood culture, and was capable of guiding antibiotic regimen modification and improving prognosis. Higher reads of pathogens detected by mNGS were related to 30-day mortality (p = 0.002). Conclusions: Blood mNGS testing might be helpful for early etiological diagnosis of patients with suspected sepsis, guiding the antibiotic regimen modification and improving prognosis.

Respiratory Aspergillus Colonization Was Associated With Relapse of Acute Exacerbation in Patients With Chronic Obstructive Pulmonary Disease: Analysis of Data From A Retrospective Cohort Study.

Background: Patients with chronic obstructive pulmonary disease (COPD) are more susceptible to Aspergillus colonization or infection. Several studies have demonstrated that invasive pulmonary Aspergillosis (IPA) and Aspergillus hypersensitivity (AH) have a detrimental effect on COPD. However, it remains to be clarified whether Aspergillus colonization is associated with acute exacerbation of COPD (AECOPD). This study aimed to explore the impact of Aspergillus colonization in the lower respiratory tract on AECOPD. Method: Patients with Aspergillus colonization were identified from a retrospective cohort of hospitalized AECOPD from 2011 to 2016 in eight centers in Shanghai, China. The demographic information, conditions of the stable stage, clinical characteristics during hospitalization, and 1-year follow-up information after discharge were collected and compared to participants without fungi colonization. Result: Twenty-six hospitalized AECOPD patients with Aspergillus colonization and 72 controls were included in the final analysis after excluding patients with other fungi isolation and matching. The rates of recurrence of acute exacerbation within 90 days and 180 days after discharge in the patients with Aspergillus colonization were both significantly higher than that in the fungi negative patients (90 days: 19.2 vs. 4.2%, p = 0.029; 180 days: 23.1 vs. 4.2%, p = 0.010), and the all-cause mortality within 1 year was also higher (11.5 vs. 0.0%, p = 0.017). Multivariate logistic regression analysis showed that Aspergillus colonization was an independent risk factor for the recurrence of acute exacerbation within 90 days and 180 days (90 days: OR = 8.661, 95% CI: 1.496-50.159, p = 0.016; 180 days: OR =10.723, 95% CI: 1.936-59.394, p = 0.007). Conclusion: Aspergillus colonization may predict poor prognosis of AECOPD while leading to an increased risk of recurrent AECOPD in a short period.

How Does Diabetes Impair Penile Tissues during Erectile Dysfunction?

BACKGROUND: Erectile dysfunction (ED) is a significant but underestimated complication during diabetes mellitus (DM). Currently, few special treatments are available clinically due to the lack of specific therapeutic targets. Genomic analysis can be helpful to find potential targets. In this study, the gene expression under diabetic ED condition was analyzed using a gene array, and the significance of the outcomes was evaluated through clinical data. METHODS: The expressions of 15923 genes were analyzed using R software. Differential expression genes (DEGs) were identified through the constructed volcano plot. The function enrichment of Gene Ontology (GO) and KEGG was screened with the DAVID online tool. The interaction between these DEGs was revealed through constructing a protein-protein interaction network and the hub genes were uncovered using the STRING and Cytoscape tool. Lastly, the data of diabetic ED patients were applied to verify the bioinformatics findings. RESULTS: The study showed that 75 genes in the rat penile tissues were upregulated, while 97 genes were downregulated on the diabetic ED condition. These genes were mainly involved in extracellular matrix composition, collagen fibril organization, as well as protein digestion & absorption. Additionally, insulin-related signaling pathways were affected. The clinical analysis indicated that insulin resistance was associated with the diabetic ED severity. Notably, the bioinformatics analysis also suggested that ferroptosis pathway was probably activated under the diabetic ED condition. CONCLUSION: The impaired protein synthesis induced by deficient insulin signaling is an important cause of the diabetic ED. The improvement of protein synthesis through restoring insulin function may be potentially useful for diabetic ED therapy.

Hydrogen sulfide primes diabetic wound to close through inhibition of NETosis.

Diabetes-induced neutrophil NETosis impairs wound healing through neutrophil extracellular traps (NETs). Reactive oxygen species (ROS)-triggered activation of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 is involved in NETosis. Hydrogen sulfide (H2S), an endogenous signaling molecule, accelerates diabetic wound healing (DWH), and inhibits ROS production, ERK1/2 and p38 activation, while its level is decreased in diabetes. However, it remains unknown whether H2S could accelerate DWH through inhibition of NETosis, and whether this inhibitory effect was associated with blockage of ROS-induced ERK1/2 and p38 activation. In order to solve these problems, serum NETs content was measured in diabetic foot patients and healthy individuals. Wound was created in dorsal skin of LepRdb/db and control mice and NETs content in wound tissues was tested. An in vitro NETosis model was induced by phorbol 12-myristate 13-acetate (PMA) in isolated neutrophils. Effects of H2S in form of Na2S on skin wound healing and NETosis were investigated both in vivo and in vitro. It was found that NETs level was highly increased in diabetic foot patients. Comparing with LepRm+/db mice, DWH was delayed in LepRdb/db mice, accompanied with high NETs level. In PMA-induced NETosis model, peptidylarginine deiminase (PAD)-4 and citrullinated histone H3, as well as NETs components dsDNA framework, myeloperoxidase and neutrophil elastase, were significantly increased. PMA-induced neutrophil NETosis and NETs formation were abolished by treatment with H2S. The delayed DWH of diabetic mice was partially restored by intraperitoneal injection of H2S, meanwhile, the highly expressed NETosis and NETs release were also down-regulated. The treatment with H2S not only attenuated ROS production but also abolished MAPK ERK1/2 and p38 activation. Like the effects of H2S, inhibition of MAPK ERK1/2 or p38 could decrease NETs release. These findings suggests that H2S attenuates NETosis and primes diabetic wound to heal through blockage of ROS-mediated MAPK ERK1/2 and p38 activation.

[Clinical manifestation and influential factors in patients with acute arsenic poisoning].

OBJECTIVE: To investigate the clinical characteristics of acute arsenic poisoning and its influential factors. METHODS: Clinical data of 47 cases of arsenic poisoning were collected and analyzed. Two cases of observation, 40 cases of mild acute poisoning, and 5 severe acute poisoning were investigated in this group. RESULTS: Myocardial enzyme activity was correlated with age and urine arsenic concentrations. Myocardial enzyme, the liver ALT, total bilirubin (TBil) and indirect bilirubin (IBil) were negatively correlated with vomiting frequency, with statistical significance (P < 0.05). Urine arsenic concentration was correlated with vomiting frequency and amount of soup drunk, with statistically significant difference (P < 0.05). Despite no statistical significance in age and amount of soup drunk, the patients with more vomiting or diarrhea had less urine arsenic concentrations, cardiac enzymes and liver enzyme concentration. CONCLUSION: Acute arsenic poisoning can lead to multiple organ damage. The damage is relevant with amount of arsenic intake, vomiting, diarrhea and urinary frequency arsenic concentration. So early use of gastric lavage, vomiting, poison discharges, and adequate application of effective antidote (Na-DMPS) as soon as possible, symptomatic treatment and the reinforced monitoring, are the rescue key for patients with acute arsenic poisoning.

[Clinical cure for acute methanol poisoning].

OBJECTIVE: To analyze the treatment of 42 patients with acute methanol poisoning because of drinking alcohol containing methanol. METHODS: Clinical data of 42 cases of methanol poisoning were collected and analyzed. Methanol concentration in drinking alcohol and blood was determined by gas chromatography (GC). National standard for occupational medicine (GBZ53-2002) was used to diagnose the cases. RESULTS: The methanol concentration in the alcohol was 16% approximately 46%. 42 Patients (40 males, 2 females), at age of 46.1 (22 approximately 80), took 588.1 ml (50 approximately 2,000 ml) of the alcohol. The average methanol concentration in blood was 1.61 mmol/L (0.03 approximately 23.60 mmol/L). According to clinical diagnosis, there were 17 observed cases, 9 mild acute toxication, and 16 severe acute toxication. Among them, 35 (83.3%) patients were recovered, 2 (4.8%) blind, 4 (9.5%) with neuropsychic sequela and 1 (2.4%) dead after adopting 8 cure measures. CONCLUSION: To start using emergency plan for public health events suddenly happened, designate a special treatment hospital, clear blood methanol as soon as possible, correct acidosis, adequately administer folacin and hormone, protect optic nerve and retina, and take comprehensive symptomatic treatment as well as strict monitoring are the keys of clinical cure.

Diversity and active mechanism of fengycin-type cyclopeptides from Bacillus subtilis XF-1 against Plasmodiophora brassicae.

Bacillus subtilis XF-1, a strain with demonstrated ability to control clubroot disease caused by Plasmodiophora brassicae, was studied to elucidate its mechanism of antifungal activity against P. brassicae. Fengycin-type cyclopeptides (FTCPs), a well-known class of compounds with strong fungitoxic activity, were purified by acid precipitation, methanol extraction, and chromatographic separation. Eight homologs of fengycin, seven homologs of dehydroxyfengycin, and six unknown FTCPs were characterized with LC/ESI-MS, LC/ESI-MS/MS, and NMR. FTCPs (250 microg/ml) were used to treat the resting spores of P. brassicae (10(7)/ml) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A(260)) and at 280 nm (A(280)) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be cleaved by the FTCPs of B. subtilis XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol.

ESI LC-MS and MS/MS characterization of antifungal cyclic lipopeptides produced by Bacillus subtilis XF-1.

Bacillus subtilis XF-1 (CGMCC No. 2357), a patent strain with good effects for controlling the clubroot of crucifer and many pathogenic fungi, was predicted to produce cyclic lipopeptide (CLP) antibiotics based on its genomic analysis. In this study, the CLPs were purified and determined with the following protocol: the supernatant of XF-1 cultivating mixture was firstly precipitated, then the precipitants were extracted with methanol and further separated by Sephadex LH-20 chromatography to test its antifungal activities. Fungi-inhibiting fractions were further characterized with LC/ESI-MS and LC/ESI-MS/MS. The results show that four molecular ion peaks [M+H]⁺ (m/z 1,464, 1,478, 1,492 and 1,506) from fungi suppression fraction were identified as fengycin A with fatty acid of C16-C19, fengycin B (C14-C17), fengycin C (C15-C18), fengycin D (C15-C18) and fengycin S (C15-C18). Fengycin C (C15 and C18), fengycin D (C15, C16 and C18) and fengycin S (C15, C16 and C18) were reported for the first time. The diversity of the fengycins that exist in this strain will help the elucidation of their biocontrol mechanisms.