Identification and bioinformatic analysis of CircRNAs in the plasma of patients with very severe chronic obstructive pulmonary disease.

BACKGROUND: The differential expression of circular RNAs (circRNAs) in individuals with very severe chronic obstructive pulmonary disease (COPD) and healthy individuals was screened using microarray technology. The related functions and mechanisms were analyzed using bioinformatic methods to explore the potential of target circRNAs as biomarkers of COPD and provide insights for future pathogenesis. PATIENTS AND METHODS: Thirty patients with very severe COPD and thirty healthy controls were diagnosed at The Second People's Hospital of Hefei from September 2021 to September 2022. The differential expression of circRNAs was compared and analyzed using a gene microarray and verified using quantitative real-time polymerase chain reaction (qRT-PCR) technology. RESULTS: A total of 90 upregulated and 29 downregulated circRNAs were screened in patients with very severe COPD and compared with those in healthy controls. qRT-PCR analysis showed that hsa_circ_0062683 of patients with very severe COPD was significantly upregulated, and hsa_circ_0089763 and hsa_circ_0008882 were significantly downregulated. By constructing the circRNA-miRNA interaction network, it was found that hsa-miR-612, hsa-miR-593-5p, hsa-miR-765, and hsa-miR-103a-2-5p are the miRNAs regulated by more differentially expressed circRNAs (DEcircRNAs). DEcircRNAs may participate in the development of COPD through hypoxia or regulation of various immune cells. CONCLUSION: Plasma circRNAs may play a helpful role in the diagnosis and assessment of COPD and be valuable disease biomarkers.

Tumor necrosis factor-α requires Ezrin to regulate the cytoskeleton and cause pulmonary microvascular endothelial barrier damage.

Acute respiratory distress syndrome (ARDS) is a rapidly progressive disease with unknown pathogenesis. Damage of pulmonary microvascular endothelial cells (PMVECs) caused by inflammatory storm caused by cytokines such as TNF-α is the potential pathogenesis of ARDS. In this study, we examined the role of ezrin and Rac1 in TNF-α-related pathways, which regulates the permeability of PMVECs. Primary rat pulmonary microvascular endothelial cells (RPMVECs) were isolated and cultured. RPMVECs were treated with rat TNF-α (0, 1, 10, 100 ng/ml), and the cell activity of each group was measured using a CCK8 kit. The integrity of endothelial barrier was measured by transendothelial resistance (TEER) and FITC-BSA flux across RPMVECs membranes. Pulldown assay and Western blot was used to detect the activity of RAS-associated C3 botulinum toxin substrate 1 (Rac1) and Ezrin phosphorylation. Short hairpin RNA (shRNA) targeting ezrin and Rac1 was utilized to evaluate the effect of RPMVECs permeability and related pathway. The effects of ezrin and Rac1 on cytoskeleton were confirmed by immunofluorescence. Our results revealed that active Rac1 was essential for protecting the RPMVEC barrier stimulated by TNF-α, while active ezrin could partially destroy the PMVEC barrier by reducing Rac1 activity and regulating the subcellular structure of the cytoskeleton. These findings may be used to create new therapeutic strategies for targeting Rac1 in the treatment of ARDS.

Izhakiella australiensis sp. nov. isolated from an Australian desert soil.

A Gram-stain-negative, rod-shaped, non-motile bacterium, designated D4N98T, was isolated from a desert soil near Glendambo, Australia. The taxonomic position of strain D4N98T was investigated using a polyphasic approach. Strain D4N98T shared 97 % 16S rRNA gene sequence similarity with the only reference strain of the genus Izhakiella (Izhakiella capsodis N6PO6T), and less than 96 % similarity with other species of genera in the family Enterobacteriaceae with validly published names. Phylogenetic analysis, based on 16S rRNA, rpoB, atpD, gyrB genes and a concatenated sequence comprising 37 single copy marker genes, as well as chemotaxonomic data (major polar lipids: phosphatidyl ethanolamine, phosphatidylglycerol, diphosphatidylglycerol, a neutral lipid, and a glycolipid. Major fatty acids (>10 %): C16 : 0, C17 : 0 cyclo, summed feature 2 (C14 : 0 3-OH and/or iso-C16 : 1 I), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) support the affiliation of this strain to the genus Izhakiella. The results of in silico DNA-DNA hybridisation plus physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain D4N98T from the other species of the genus Izhakiellawith validly published names. Therefore, strain D4N98T represents a novel species, for which the name Izhakiella australiensis sp. nov. is proposed. The type strain is D4N98T (LMG 30066T=DSM 105030T).

Preparation of progenin III from total steroidal saponins of Dioscorea nipponica Makino using a crude enzyme from Aspergillus oryzae strain.

Progenin III, one of the most active spirostanol saponins, is a potential candidate for anti-cancer therapy due to its strong antitumor activity and low hemolytic activity. However, the concentration of progenin III is extremely low in natural Dioscorea plants. In this paper, the progenin III production from total steroidal saponins of Dioscorea nipponica Makino was studied using the crude enzyme from Aspergillus oryzae DLFCC-38. The crude enzyme converting total steroidal saponins into progenin III was obtained from the A. oryzae DLFCC-38 culture. For enzyme production, the strain was cultured for 72 h at 30 °C with shaking at 150 rpm in 5 % (w/v) malt extract medium containing 2 % (v/v) extract of D. nipponica as the enzyme inducer. The crude enzyme converted total steroidal saponins into major progenin III with a high yield when the reaction was carried out for 9 h at 50 °C and pH 5.0 with the 20 mg/ml of substrate. In the preparation of progenin III, 117 g of crude progenin III was obtained from 160 g of substrate, and the crude product was purified with silica gel column to obtain 60.3 g progenin III of 93.4 % purity.

Plasma miR-150-5p as a Biomarker for Chronic Obstructive Pulmonary Disease.

Purpose: To investigate the potential of plasma microRNA-150-5p (miR-150-5p) as a biomarker for chronic obstructive pulmonary disease (COPD) and its relationship with clinical indicators such as pulmonary function. Patients and Methods: Fifty-nine patients with COPD and twenty-six healthy control individuals were recruited in the Second People's Hospital of Hefei from September 2021 to September 2022. The plasma expression level of miR-150-5p was measured by quantitative real-time polymerase chain reaction. Results: The miR-150-5p level in the COPD group was significantly lower than that in the control group, and the relative expression was lower in patients with severe airflow limitation than those with mild limitation. Plasma miR-150-5p levels were positively correlated with pulmonary function indicators and negatively correlated with the white blood cell count and C-reactive protein level. The receiver operating characteristic curve suggested that plasma miR-150-5p had predictive value for COPD (area under curve = 0.819, sensitivity 64.4%, specificity 92.3%). Conclusion: MiR-150-5p can be useful for the diagnosis and disease assessment of COPD, and has value as a biomarker for COPD.

let-7b-5p suppresses the proliferation and migration of pulmonary artery smooth muscle cells via down-regulating IGF1.

OBJECTIVES: Some previous studies indicated that the excessive proliferation and migration of Pulmonary Artery Smooth Muscle Cells (PASMCs) could be observed in pulmonary artery intima after Pulmonary Embolism (PE) occurred. In addition, recent studies identified some miRNAs that are differentially expressed in the blood of PE patients, which might be used as a diagnostic biomarker for PE, including let-7a-5p, let-7b-5p, and miR-150-5p. Hence, the authors sought to explore the effects of let-7b-5p in PASMC proliferation and migration and the corresponding regulatory mechanism. METHODS: Platelet-Derived Growth Factor (PDGF) was utilized to induce the hyper-proliferation model in PASMCs. The mRNA and protein expression levels were detected by RT-qPCR and western blot, respectively. The proliferation of PASMCs was evaluated by the detection of PCNA expression, as well as CCK-8 and Edu assays. Wound healing and Transwell assays were exploited to assess the migration ability of PASMCs. The targets of let-7b-5p were predicted based on two bioinformatics online tools. Dual-luciferase and Ago2 pull-down assays were applied to confirm the interaction between let-7b-5p and IGF1. RESULTS: 40 ng/mL PDGF was selected as the optimal concentration to induce PASMCs. let-7b-5p mimics suppressed the proliferation and migration of PDGF-induced PASMCs, while let-7b-5p inhibitor led to the opposite result. In further mechanism exploration, IGF1 was predicted and confirmed as the direct target gene of let-7b-5p. The promotion role of IGF1 overexpression on the proliferation and migration of PDGF-induced PASMCs was dramatically countered by let-7b-5p mimics. CONCLUSION: let-7b-5p prohibits the proliferation and migration of PDGF-induced PASMCs by modulating IGF1.

LPS induces pulmonary microvascular endothelial cell barrier dysfunction by upregulating ceramide production.

The specific role of ceramides in pulmonary microvascular endothelial cell (PMVEC) barrier dysfunction remains unclear. In the present study, pretreatment with pan-caspase inhibitors significantly reduced LPS-induced PMVEC apoptosis and helped to stimulate PMVEC barrier reconstruction after 12 h but had no effect on PMVEC barrier dysfunction in the first 8 h. Further studies showed that imipramine, an acid sphingomyelinase (ASMase) inhibitor, significantly inhibited LPS-induced barrier dysfunction, while an siRNA targeting serine palmityl transferase subunit 1 (SPTLC1) and the pharmacological inhibitor myriocin did not inhibit early acute barrier dysfunction but significantly inhibited PMVEC apoptosis and apoptosis-dependent delayed barrier dysfunction. In addition, LPS was shown to activate RhoA by inducing transient receptor potential channel 6 (TRPC6) overexpression and calcium influx through the ASMase/ceramide pathway, and activation of RhoA further induced the cytoskeletal rearrangement of PMVECs and destruction of intercellular junctions, ultimately leading to early acute PMVEC barrier dysfunction. However, regarding apoptosis-dependent delayed barrier dysfunction, the ceramide-induced de novo synthesis pathway in paracellular cells induced the apoptosis of PMVECs, in which Txnip overexpression inhibited Trx activity and subsequently activated ASK1 in the context of LPS-induced PMVEC apoptosis, acting upstream of the ceramide-induced activation of p38 MAPK and JNK. At the same time, in rats with LPS- or exogenous C8 ceramide-induced ALI, ceramide was demonstrated to play an important role in lung injury by inducing the Txnip/TRX/ASK1/P38 and JNK pathways. Thus, the Txnip/TRX/ASK1/p38 and JNK pathways might be involved in ceramide-mediated PMVEC apoptosis in LPS-induced ALI.

The dipeptidyl peptidase-4 inhibitor linagliptin ameliorates LPS-induced acute lung injury by maintenance of pulmonary microvascular barrier via activating the Epac1/AKT pathway.

Pulmonary microvascular endothelial cells (PMVECs) barrier dysfunction is a main pathophysiological feature of sepsis-related acute lung injury (ALI). This study aimed to investigate whether the dipeptidyl peptidase (DPP)-4 inhibitor linagliptin could protect against LPS-induced PMVECs barrier disruption and its underlying molecular mechanisms. A classical ALI animal model and LPS-treated PMVECs were applied and all were treated with or without linagliptin. Cellular experiments demonstrated that linagliptin could mitigate LPS-induced PMVECs hyperpermeability and intercellular junction (VE-cadherin, ß-catenin, and ZO-1) disruption in a dose-dependent manner. Correspondingly, it was observed that linagliptin pretreatment distinctly relieved LPS-induced lung injury, oxidative stress, and pulmonary edema in vivo. Furthermore, we found that the inhibition of oxidative stress by linagliptin may be achieved by reversing impaired mitochondrial function. Mechanistically, linagliptin administration promoted the activation of the Epac1 pathway and its downstream AKT pathway, while inhibition of the Epac1/Akt signaling pathway significantly alleviated the above-mentioned protective effect of linagliptin on the PMVECs barrier. Taken together, these data suggest that linagliptin can effectively reserve PMVECs barrier dysfunction and inhibit oxidative stress to protect against ALI via activating the Epac1/AKT signaling pathway, and thus may become a potential clinical therapeutic strategy for ALI.

Predictive value of neutrophil-lymphocyte ratio and platelet-lymphocyte ratio in non-small cell lung cancer patients treated with immune checkpoint inhibitors: A meta-analysis.

BACKGROUND: High neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) are associated with poor prognosis in cancer patients treated with Immune checkpoint inhibitors (ICIs). However, whether this relationship exists in non-small cell lung cancer (NSCLC) patients remains unclear. Thus, this meta-analysis was conducted to investigate the prognostic role of NLR and PLR in NSCLC treated with ICIs. METHODS: Eligible studies that evaluated the value of pre-treatment or post-treatment NLR/PLR in NSCLC patients received ICIs were obtained by searching PubMed, Web of Science, Cochrane Library, and EMBASE. The pooled hazard ratio (HR) and 95% confidence interval (CI) were used to assess the relationship between NLR/PLR and overall survival (OS) and progression-free survival (PFS). Subgroup analysis and publication bias were conducted to investigate heterogeneity. RESULTS: 1845 NSCLC patients from 21 studies were included and three ICIs(nivolumab, pembrolizumab, and atezolizumab) were used. Overall, high NLR was associated with poor OS (HR: 2.50, 95% CI:1.79-3.51, P < 0.001) and PFS (HR: 1.77, 95% CI:1.51-2.01, P < 0.001). Subgroup analyses were consistent with the pooled results. Similarly, the pooled results for PLR showed that elevated PLR was related to inferior OS (HR: 1.93, 95% CI: 1.51-2.01, P < 0.001) and PFS (HR: 1.57, 95%CI: 1.30-1.90, P < 0.001). However, the subgroup analysis based on test time indicated that there was no significant correlation between post-treatment PLR and survival outcomes. CONCLUSION: NLR and pre-treatment PLR could serve as prognostic biomarkers in NSCLC patients treated with ICIs. However, the value of post-treatment PLR needs further to be evaluated.

Head-to-head comparison of the diagnostic accuracy of Xpert MTB/RIF and Xpert MTB/RIF Ultra for tuberculosis: a meta-analysis.

BACKGROUND: Tuberculosis (TB) diagnosis has significantly improved since the introduction of the automated molecular test Xpert MTB/RIF (Xpert) and the new version Xpert MTB/RIF Ultra (Ultra) that detect Mycobacterium tuberculosis. Due to the rapidly widespread use of Xpert and Ultra, we conducted a meta-analysis to compare the performances of Xpert and Ultra in diagnosing TB and discuss the advantages and limitations of these two tests. METHODS: Web of Science, Medline (via PubMed), Embase (via OvidSP), the Cochrane Central Register of Controlled Trials and Google Scholar (up to April 2020) were searched for relevant studies. The diagnostic performance of Xpert and Ultra for TB was determined using a bivariate random-effects regression model. The sources of heterogeneity were explored via meta-regression and subgroup analyses. RESULTS: Of 19 studies that examined a total of 5855 samples, the pooled sensitivity and specificity of Xpert in TB diagnosis were 0.69 (95% CI: 0.57-0.78) and 0.99 (95% CI: 0.98-0.99), respectively. However, the pooled sensitivity and specificity of Ultra in TB diagnosis were 0.84 (95% CI: 0.76-0.90) and 0.97 (95% CI: 0.96-0.98), respectively. Regardless of whether the comparisons were indirect or direct, Ultra was consistently found to be more sensitive, but with slightly lower specificity than Xpert in diagnosing TB. CONCLUSIONS: Compared with Xpert, Ultra had higher sensitivity but slightly lower specificity for the diagnosis of TB disease. The excellent upgrade in sensitivity of the Ultra test was particularly relevant in subjects with paucibacillary TB including tuberculous pleurisy, tuberculous meningitis and paediatric TB.

let-7b-5p suppresses the proliferation and migration of pulmonary artery smooth muscle cells via down-regulating IGF1

Abstract Objectives Some previous studies indicated that the excessive proliferation and migration of Pulmonary Artery Smooth Muscle Cells (PASMCs) could be observed in pulmonary artery intima after Pulmonary Embolism (PE) occurred. In addition, recent studies identified some miRNAs that are differentially expressed in the blood of PE patients, which might be used as a diagnostic biomarker for PE, including let-7a-5p, let-7b-5p, and miR-150-5p. Hence, the authors sought to explore the effects of let-7b-5p in PASMC proliferation and migration and the corresponding regulatory mechanism. Methods Platelet-Derived Growth Factor (PDGF) was utilized to induce the hyper-proliferation model in PASMCs. The mRNA and protein expression levels were detected by RT-qPCR and western blot, respectively. The proliferation of PASMCs was evaluated by the detection of PCNA expression, as well as CCK-8 and Edu assays. Wound healing and Transwell assays were exploited to assess the migration ability of PASMCs. The targets of let-7b-5p were predicted based on two bioinformatics online tools. Dual-luciferase and Ago2 pull-down assays were applied to confirm the interaction between let-7b-5p and IGF1. Results 40 ng/mL PDGF was selected as the optimal concentration to induce PASMCs. let-7b-5p mimics suppressed the proliferation and migration of PDGF-induced PASMCs, while let-7b-5p inhibitor led to the opposite result. In further mechanism exploration, IGF1 was predicted and confirmed as the direct target gene of let-7b-5p. The promotion role of IGF1 overexpression on the proliferation and migration of PDGF-induced PASMCs was dramatically countered by let-7b-5p mimics. Conclusion let-7b-5p prohibits the proliferation and migration of PDGF-induced PASMCs by modulating IGF1.

Co-occurrence of genes for aerobic and anaerobic biodegradation of dichloroethane in organochlorine-contaminated groundwater.

1,2-Dichloroethane (DCA) is a problematic groundwater pollutant. Factors influencing the distribution and activities of DCA-degrading bacteria are not well understood, which has hampered their application for bioremediation. Here, we used quantitative PCR to investigate the distribution of putative DCA-dehalogenating bacteria at a DCA-impacted site in Sydney (Australia). The dehalogenase genes dhlA, tceA and bvcA were detected in all groundwater samples (n = 15), while vcrA was found in 11/15 samples. The 16S rRNA gene sequences specific to the dehalogenating genera Dehalobacter, Desulfitobacterium and Dehalogenimonas were detected in 15/15, 13/15 and 13/15 samples, respectively, while Dehalococcoides sequences were found in 9/15 samples. The tceA, bvcA and vcrA genes occurred in the same samples as Dehalococcoides and Dehalobacter. Microcosm experiments confirmed the presence of bacteria capable of dechlorination under anoxic conditions. The abundance of the dhlA gene, which is found in hydrolytic DCA degraders, was positively correlated to the DCA concentration, and was unexpectedly most abundant in samples with low oxygen conditions. A dhlA-containing bacterium isolated from the site (Xanthobacter EL8) was capable of anaerobic growth on DCA under denitrifying conditions. The presence of diverse DCA-dehalogenating bacteria at this site indicates that natural attenuation or biostimulation could be valid approaches for site cleanup.

[Role of Ezrin in the injury of rat pulmonary microvascular endothelial cells induced by tumor necrosis factor-α and the impact of Rac 1].

OBJECTIVE: To investigate the role of Ezrin and its phosphorylation (p-Ezrin) in the modulation of rat pulmonary microvascular endothelial cell (PMVEC) injury induced by tumor necrosis factor-α (TNF-α) and the impact of Rac 1. METHODS: Cultured PMVECs of Sprague-Dawley (SD) rats were randomly divided into time-dependent injury group induced by TNF-α and intervention group in which cells were pretreated with Rac 1 inhibitor (NSC 23766). (1) In the time-dependent injury group, Western Blot was used to detect the expression of Ezrin and p-Ezrin after 10 µg/L TNF-α stimulation for 0, 0.25, 0.5, 1, 3, 6, 12, 24 hours. (2) In the intervention group, after pre-treatment with 200 µmol/L NSC 23766 for 0.5 h, PMVECs were treated with 10 µg/L TNF-α, and the expression of p-Ezrin was detected by Western Blot after 3 hours. Besides these groups, there were control (1% fetal bovine serum simulation), single NSC 23766 or TNF-α simulation groups. RESULTS: (1) Few Ezrin expression was found in PMVEC, and TNF-α could not affect Ezrin expression. p-Ezrin protein expression (p-Ezrin/Ezrin, gray scale) of PMVECs at 0 hour after TNF-α stimulation was 0.21 ± 0.03, and elevated at 0.25 hour (0.53 ± 0.19), peaked at 3 hours (1.68 ± 0.30), then it was gradually lowered, but it remained at higher level at 24 hours(0.87 ± 0.18) with significant difference (F = 62.200, P=0.000). It demonstrated that TNF-α could increase Ezrin phosphorylation in a time-dependent manner. (2) Compared with blank control group, in single NSC 23766 or TNF-α simulation group, p-Ezrin expression was induced (TNF-α group: 0.92 ± 0.12 vs. 0.68 ± 0.16, t = -2.864, P=0.020; NSC 23766 group:1.33 ± 0.24 vs. 0.68 ± 0.16, t = -5.429,P=0.000. When NSC 23766 was pre-treated with PMVECs, the expression of p-Ezrin was significantly increased compared with that in single TNF-α simulation group (2.14 ± 0.18 vs. 0.92 ± 0.12, t = -14.670, P=0.000) with significant difference (F = 73.810, P=0.000). CONCLUSIONS: Ezrin proteins are phosphorylated by TNF-α. Rac 1 signaling pathway inhibition plays an important role in TNF-α-induced injury by up-regulation of p-Ezrin in PMVECs.