Evaluating the diagnostic value of using metagenomic next-generation sequencing on bronchoalveolar lavage fluid and tissue in infectious pathogens located in the peripheral lung field.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) exerts a vital part in accurately diagnosing pulmonary infection. However, the diagnostic value of different samples obtained by virtual bronchoscopic navigation (VBN) combined with mNGS for pathogen detection in infections located in the peripheral lung field (PLF) is still unclear. METHODS: Patients infected from July 2018 to February of the following year were carefully analyzed and divided into two parts, namely, non-infectious disease group and the infectious disease group. Then bronchial expansion tests were performed for each subject, collected liquid specimens and tissue standards, and conducted regular mNGS and microbiological detection and analysis. The value of mNGS and culture in pathogen detection was compared, at the same time, the performance of tissue mNGS and bronchoalveolar lavage fluid (BALF) mNGS in the diagnosis process was compared. When discrete variables were processed, Pearson χ2 and Fisher's exact test could be used to perform categorical variables analysis. Continuous variables were analyzed and compared by Mann-Whitney U test. RESULTS: After mNGS diagnosis, Acinetobacter baumannii, Pseudomonas aeruginosa and Rothia mucilaginosa were the bacterial species showing the highest abundances. In addition, mNGS achieved the sensitivities in the detection of pathogens in tissues and BALF of 72.9% and 81.4%, respectively, and it is higher than conventional culture. Bacterial diagnostic sensitivity was significantly different between BALF and tissue using mNGS (95.0% vs. 62.5%, P=0.03). The sensitivity and specificity of BALF in detecting fungal infections were not significantly different from those of mNGS. A consistency test showed that these two methods had some degree of consistency (k=0.673, P=0.000). CONCLUSIONS: This study showed that the mNGS in BALF samples and the mNGS in tissue samples which could be used to test for pathogens in the lungs. The sensitivity will increase when mNGS is combined with culture. Also, mNGS of BALF and tissues had some degree of consistency to detect fungal infections, whereas mNGS of BALF had better sensitivity to detect bacterial infection than mNGS of tissues.

Long Non-coding RNA LINC00628 Interacts Epigenetically with the LAMA3 Promoter and Contributes to Lung Adenocarcinoma.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular progress in lung adenocarcinoma. In this study, to identify cancer-related lncRNAs and genes, we screened for those lncRNAs that were differentially expressed in lung adenocarcinoma, which revealed LINC00628 overexpression and low expression of laminin subunit alpha 3 (LAMA3). This was further validated in the cancerous tissues from patients diagnosed with lung adenocarcinoma. Thereafter, we explored the functional relevance of LINC00628 and LAMA3 in lung adenocarcinoma by analyzing the recruitment of DNA methyltransferase (DNMT) and the cellular processes of lung adenocarcinoma cells following treatments that induced LINC00628 overexpression or LINC00628 silencing or with 5-azacytidine (5-Aza, a DNMT inhibitor). The results showed that LINC00628 silencing decreased cell proliferation, migration, and invasion as well as the drug resistance of lung adenocarcinoma cells to vincristine (VCR). The results were opposite in the cells with LAMA3 demethylation induced by 5-Aza treatment. Further research indicated that LINC00628 recruited DNMT1, DNMT3A, and DNMT3B to promote the methylation of LAMA3 promoter, thereby decreasing its expression. Moreover, an in vivo experiment was performed in nude mice to assess the tumor growth ability and drug resistance of human lung adenocarcinoma cells. It was observed that LINC00628 silencing or 5-Aza treatment inhibited the in vivo tumor growth ability of the human lung adenocarcinoma cells and reduced their resistance to VCR. Altogether, our results provide evidence of a mechanism by which LINC00628 silencing exerts an inhibitory role in lung adenocarcinoma by modulating the DNA methylation of LAMA3, indicative of a novel molecular target for treatment of lung adenocarcinoma patients showing resistance to VCR.

Long non-coding RNA H19 is responsible for the progression of lung adenocarcinoma by mediating methylation-dependent repression of CDH1 promoter.

Lung adenocarcinoma is a common histologic type of lung cancer with a high death rate globally. Increasing evidence shows that long non-coding RNA H19 (lncRNA H19) and CDH1 methylation are involved in multiple tumours. Here, we tried to investigate whether lncRNA H19 or CDH1 methylation could affect the development of lung adenocarcinoma. First, lung adenocarcinoma tissues were collected to detect CDH1 methylation. Then, the regulatory mechanisms of lncRNA H19 were detected mainly in concert with the treatment of overexpression of lncRNA H19, siRNA against lncRNA H19, overexpression of CDH1 and demethylating agent A-5az in lung adenocarcinoma A549 cell. The expression of lncRNA H19 and epithelial-mesenchymal transition (EMT)-related factors as well as cell proliferation, sphere-forming ability, apoptosis, migration and invasion were detected. Finally, we observed xenograft tumour in nude mice so as to ascertain tumorigenicity of lung adenocarcinoma cells. LncRNA H19 and methylation of CDH1 were highly expressed in lung adenocarcinoma tissues. A549 cells with silencing of lncRNA H19, overexpression of CDH1 or reduced CDH1 methylation by demethylating agent 5-Az had suppressed cell proliferation, sphere-forming ability, apoptosis, migration and invasion, in addition to inhibited EMT process. Silencing lncRNA H19 could reduce methylation level of CDH1. In vivo, A549 cells with silencing lncRNA H19, overexpression of CDH1 or reduced CDH1 methylation exhibited low tumorigenicity, reflected by the smaller tumour size and lighter tumour weight. Taken together, this study demonstrates that silencing of lncRNA H19 inhibits EMT and proliferation while promoting apoptosis of lung adenocarcinoma cells by inhibiting methylation of CDH1 promoter.

Tumor-suppressive effects of microRNA-181d-5p on non-small-cell lung cancer through the CDKN3-mediated Akt signaling pathway in vivo and in vitro.

The involvement of several microRNAs (miRs) in the initiation and development of tumors through the suppression of the target gene expression has been highlighted. The aberrant expression of miR-181d-5p and cyclin-dependent kinase inhibitor 3 (CDKN3) in non-small-cell lung cancer (NSCLC) was then screened by microarray analysis. In the present study, we performed a series of in vivo and in vitro experiments for the purpose of investigating their roles in NSCLC and the underlying mechanism. There was a high expression of CDKN3, whereas miR-181d-5p was downregulated in NSCLC. Quantitative RT-PCR, Western blot analysis, and dual-luciferase reporter gene assay further identified that CDKN3 could be negatively regulated by miR-181d-5p. Moreover, the upregulation of miR-181d-5p or silencing of CDKN3 could inactivate the Akt signaling pathway. A549 with the lowest miR-181d-5p and H1975 with the highest CDKN3 among the five NSCLC cell lines (H1299, A549, H1975, NCI-H157, and GLC-82) were adopted for in vitro experiments, in which expression of miR-181d-5p and CDKN3 was altered by transfection of miR-181d-5p mimic/inhibitor or siRNA-targeting CDKN3. Afterwards, cell proliferation, apoptosis, invasion, migration, and angiogenesis, as well as epithelial-mesenchymal transition (EMT), were evaluated, and tumorigenicity was assessed. In addition, an elevation in miR-181d-5p or depletion in CDKN3 led to significant reductions in proliferation, invasion, migration, angiogenesis, EMT, and tumorigenicity of NSCLC cells, coupling with increased cell apoptosis. In conclusion, this study highlights the tumor-suppressive effects of miR-181d-5p on NSCLC via Akt signaling pathway inactivation by suppressing CDKN3, thus providing a promising therapeutic strategy for the treatment of NSCLC.

Insights into biomethane production and microbial community succession during semi-continuous anaerobic digestion of waste cooking oil under different organic loading rates.

High content of lipids in food waste could restrict digestion rate and give rise to the accumulation of long chain fatty acids in anaerobic digester. In the present study, using waste cooking oil skimmed from food waste as the sole carbon source, the effect of organic loading rate (OLR) on the methane production and microbial community dynamics were well investigated. Results showed that stable biomethane production was obtained at an organic loading rate of 0.5-1.5 g VS L-1 days-1. The specific biogas/methane yield values at OLR of 1.0 were 1.44 ± 0.15 and 0.98 ± 0.11 L g VS-1, respectively. The amplicon pyrosequencing revealed the distinct microbial succession in waste cooking oil AD reactors. Acetoclastic methanogens belonging to the genus Methanosaeta were the most dominant archaea, while the genera Syntrophomona, Anaerovibrio and Synergistaceae were the most common bacteria during AD process. Furthermore, redundancy analysis indicated that OLR showed more significant effect on the bacterial communities than that of archaeal communities. Additionally, whether the OLR of lipids increased had slight influence on the acetate fermentation pathway.

Adaption of microbial community during the start-up stage of a thermophilic anaerobic digester treating food waste.

A successful start-up enables acceleration of anaerobic digestion (AD) into steady state. The microbial community influences the AD performance during the start-up. To investigate how microbial communities changed during the start-up, microbial dynamics was analyzed via high-throughput sequencing in this study. The results confirmed that the AD was started up within 25 d. Thermophilic methanogens and bacterial members functioning in hydrolysis, acidogenesis, and syntrophic oxidation became predominant during the start-up stage, reflecting a quick adaption of microorganisms to operating conditions. Such predominance also indicated the great contribution of these members to the fast start-up of AD. Redundancy analysis confirmed that the bacterial abundance significantly correlated with AD conditions. The stable ratio of hydrogenotrophic methanogens to aceticlastic methanogens is also important to maintain the stability of the AD process. This work will be helpful to understand the contribution of microbial community to the start-up of AD.

Isolation and characterization of Methanoculleus receptaculi sp. nov. from Shengli oil field, China.

Three strictly anaerobic, thermophilic methanogens (ZC-2T, ZC-3 and ZC-6) were isolated from Shengli oil field, China. The 16S rRNA gene sequences of the three strains were nearly identical, possessing > 99.8% sequence similarity. They also possessed high sequence similarity, 97.4%, to Methanoculleus palmolei strain INSLUZ(T) (97.4% and 97.5%, respectively), indicating that they represented a novel species within the genus Methanoculleus. Cells of strain ZC-2T were nonmotile cocci, 0.8-1.7 microm in diameter, and always occurred singly or in pairs. The three strains used H2/CO2 or sodium formate as substrates for methanogenesis but not sodium acetate, trimethylamine, monomethylamine, ethanol, dimethyl sulfide, isopropanol, isobutanol, butan-2-ol or H2/CO. Optimum growth of strain ZC-2T occurred in the presence of 0.2 M NaCl, pH 7.5-7.8 and temperature 50-55 degrees C with a specific growth rate of 0.084 h(-1). The mol% G+C content of the genomic DNA was 55.2 mol%. Based on these phenotypic and phylogenetic characteristics, strains ZC-2T, ZC-3 and ZC-6 are proposed to represent a novel species in the genus Methanoculleus and named Methanoculleus receptaculi sp. nov. The type strain is ZC-2T (CGMCC 1.5087T=DSM 18860T).

Methermicoccus shengliensis gen. nov., sp. nov., a thermophilic, methylotrophic methanogen isolated from oil-production water, and proposal of Methermicoccaceae fam. nov.

A thermophilic, methylotrophic methanogen, strain ZC-1(T), was isolated from the Shengli oilfield, China. Cells of strain ZC-1(T) were motile cocci, 0.7-1.0 microm in diameter and always occurred in clusters of two to four cells. Lysis-susceptibility experiments and analysis of transmission electron micrographs of strain ZC-1(T) suggested the presence of a proteinaceous cell wall. Strain ZC-1(T) used methanol, methylamine and trimethylamine as substrates for methanogenesis. Optimal growth, with a doubling time of around 5 h, occurred at pH 6.0-6.5, 65 degrees C, 0.3-0.5 M NaCl and 0.05-0.20 M MgCl(2). The DNA G+C content of this organism was 56 mol%. Analysis of 16S rRNA gene sequence and the inferred amino acid sequence of the mcrA gene of strain ZC-1(T) indicated that it is related specifically to members of the family Methanosaetaceae (90.6 and 76.6 % sequence similarity, respectively). However, strain ZC-1(T) failed to grow with acetate as substrate for methanogenesis, which is a special characteristic of the family Methanosaetaceae. Based on these phenotypic and phylogenic characteristics, strain ZC-1(T) is proposed to represent a novel genus and species, for which the name Methermicoccus shengliensis gen. nov., sp. nov. is proposed. The type strain is ZC-1(T) (=CGMCC 1.5056(T)=DSM 18856(T)). Methermicoccaceae fam. nov. is also proposed.

[Changes of scavenger receptor class B type I and peroxisome proliferator-activated receptor gamma expression in atherosclerotic mini swine].

AIM: To study the expressions of scavenger receptor class B type I(SR-BI) and peroxisome proliferator-activated receptor gamma (PPARgamma) in atherosclerotic mini swine and provide a new mechanism for investigating the pathogenesis of atherosclerosis. METHODS: Chinese mini swine were fed by a normal control diet or a high fat/high cholesterol diet for 12 months after common carotid artery injury induced by balloon denudation. Plasma total cholesterol(TC), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were determined by commercially enzymatic methods every two months. The sections, which were taken from liver and abdominal aorta, were stained with hematoxylin eosin. The expressions of SR-BI and PPARgamma mRNA and protein in liver and aorta tissue were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry respectively. RESULTS: At the end of 12 months, plasma TC, HDL-C and TG in HFHC mini swine were increased. There were fatty liver and atherosclerotic plaque in mini swine live and aorta respectively. The expression of SR-BI was upregulated in HFHC mini swine liver and aorta tissue. CONCLUSION: HFHC may induce atherosclerosis and the expression of SR-BI and PPARgamma. Upregulating SR-BI expression may inhibit atherosclerosis. Increasing SR-BI expression in liver and aorta may accelerate SR-BI-mediated reverse cholesterol transport and develop a new anti-atherogenic strategy.

Formation of nanostructured copper filaments in electrochemical deposition.

In this paper, we report in detail the studies of a different self-organized copper electrodeposition carried out in an ultrathin layer of CuSO4 electrolyte. On a macroscopic scale, the morphology of the electrodeposit is fingerlike. Microscopically, each fingering branch consists of long, straight copper filaments with periodic corrugated nanostructures. Branching rate of the electrodeposit is significantly decreased, compared with the patterns grown in conventional systems. Detailed information of the growth environment in the ultrathin electrodeposition system is provided, the formation mechanism of the periodic nanostructures on the deposit filaments is explored, and the origin of the significant descent of branching rate of the electrodeposit is discussed.