Metagenomics of gut microbiome for migratory seagulls in Kunming city revealed the potential public risk to human health.

BACKGROUND: Seagull as a migratory wild bird has become most popular species in southwest China since 1980s. Previously, we analyzed the gut microbiota and intestinal pathogenic bacteria configuration for this species by using 16S rRNA sequencing and culture methods. To continue in-depth research on the gut microbiome of migratory seagulls, the metagenomics, DNA virome and RNA virome were both investigated for their gut microbial communities of abundance and diversity in this study. RESULTS: The metagenomics results showed 99.72% of total species was bacteria, followed by viruses, fungi, archaea and eukaryota. In particular, Shigella sonnei, Escherichia albertii, Klebsiella pneumonia, Salmonella enterica and Shigella flexneri were the top distributed taxa at species level. PCoA, NMDS, and statistics indicated some drug resistant genes, such as adeL, evgS, tetA, PmrF, and evgA accumulated as time went by from November to January of the next year, and most of these genes were antibiotic efflux. DNA virome composition demonstrated that Caudovirales was the most abundance virus, followed by Cirlivirales, Geplafuvirales, Petitvirales and Piccovirales. Most of these phages corresponded to Enterobacteriaceae and Campylobacteriaceae bacterial hosts respectively. Caliciviridae, Coronaviridae and Picornaviridae were the top distributed RNA virome at family level of this migratory animal. Phylogenetic analysis indicated the sequences of contigs of Gammacoronavirus and Deltacoronavirus had highly similarity with some coronavirus references. CONCLUSIONS: In general, the characteristics of gut microbiome of migratory seagulls were closely related to human activities, and multiomics still revealed the potential public risk to human health.

Identification of Novel Vascular Genes Downstream of Islet2 and Nr2f1b Transcription Factors.

The genetic regulation of vascular development is not elucidated completely. We previously characterized the transcription factors Islet2 (Isl2) and Nr2f1b as being critical for vascular growth. In this study, we further performed combinatorial microarrays to identify genes that are potentially regulated by these factors. We verified the changed expression of several targets in isl2/nr2f1b morphants. Those genes expressed in vessels during embryogenesis suggested their functions in vascular development. We selectively assayed a potential target follistatin a (fsta). Follistatin is known to inhibit BMP, and BMP signaling has been shown to be important for angiogenesis. However, the fsta's role in vascular development has not been well studied. Here, we showed the vascular defects in ISV growth and CVP patterning while overexpressing fsta in the embryo, which mimics the phenotype of isl2/nr2f1b morphants. The vascular abnormalities are likely caused by defects in migration and proliferation. We further observed the altered expression of vessel markers consistent with the vascular defects in (fli:fsta) embryos. We showed that the knockdown of fsta can rescue the vascular defects in (fli:fsta) fish, suggesting the functional specificity of fsta. Moreover, the decreased expression of fsta rescues abnormal vessel growth in isl2 and nr2f1b morphants, indicating that fsta functions downstream of isl2/nr2f1b. Lastly, we showed that Isl2/Nr2f1b control vascular development, via Fsta-BMP signaling in part. Collectively, our microarray data identify many interesting genes regulated by isl2/nr2f1b, which likely function in the vasculature. Our research provides useful information on the genetic control of vascular development.

Metabolomics studies on cachexia in patients with cancer: a scoping review protocol.

INTRODUCTION: Cancer seriously threatens human health worldwide. Cancer cachexia is one of the life-threatening consequences that occurs commonly in patients with cancer, and severely worsens patient survival, prognosis and quality of life. Previous studies have demonstrated that cancer cachexia is closely related to differential metabolites and metabolic pathways based on metabolomics analysis. This scoping review protocol, therefore, aims to provide the strategy for a formal scoping review that will summarise the differential metabolites and related metabolic pathways of cachexia in patients with cancer. METHODS AND ANALYSIS: The proposed scoping review will follow the Arksey and O'Malley's methodological framework, Levac et al's recommendations for applying this framework, and Peters' enhancements of the framework. The key information from the selected studies will be extracted, including author, year of publication, cachexia definition, country/origin, study design, setting, population and sample size, biological specimens, independent variables, independent variables' measure and statistical analysis. A summary of metabolites will be divided into several sections depending on the biological specimen. Differential metabolites will be compared between paired groups, and the number and names of related metabolic pathways will be counted and described. The reporting of this scoping review will be in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews checklist. This is a scoping review protocol and describes the planned review process and provides data examples extracted from a pilot study to confirm the feasibility of further investigation of the subject. ETHICS AND DISSEMINATION: An ethical approval is not required for this scoping review protocol, nor for the scoping review. The results of this scoping review will be disseminated through publication in a peer-reviewed journal, or presentation at a national or international conference.

Symptom Clusters and Quality of Life in Gastric Cancer Patients Receiving Chemotherapy.

CONTEXT: Although gastric cancer is one of the most common tumors worldwide, there is little knowledge about symptom clusters and quality of life (QoL) in this population. OBJECTIVES: The objectives were to identify the symptom clusters in gastric cancer patients receiving chemotherapy, and explore their effects on QoL. METHODS: Gastric cancer patients receiving chemotherapy were recruited. Data were collected using the Memorial Symptom Assessment Scale Short Form, the Functional Assessment of Cancer Therapy-Gastric and the self-designed General Information Evaluation Form. The symptom clusters were extracted through the exploratory factor analysis. The influencing factors of symptom clusters and their effects on QoL were identified using multiple linear regression analysis. RESULTS: A total of 322 participants were enrolled from three medical centers. Five factors were identified in this exploratory factor analysis based on symptom prevalence, namely fatigue related symptom cluster, epithelial symptom cluster, neurologic symptom cluster, malnutrition related symptom cluster and psychological symptom cluster (χ2 = 31.470, P < 0.05). The affecting factors across symptom clusters and QoL subscales were relatively stable, but also different. Generally, fatigue related symptom cluster, malnutrition related symptom cluster and psychological symptom cluster demonstrated significantly negative effects on all aspects of QoL except social well being. CONCLUSION: Five symptom clusters were identified in gastric cancer patients receiving chemotherapy in mainland China. The symptom clusters negatively contributed to the variance in all aspects of QoL except social well being. Further studies should examine interventions for symptom clusters, their influencing factors, and their effects on improving QoL.

Pulmonary AngII promotes LPS-induced lung inflammation by regulating microRNA-143.

Angiotensin converting enzyme 2 (ACE2) is a terminal carboxypeptidase, which cleaves single terminal residues from several bioactive peptides such as Angiotensin II (AngII). Many investigations indicated that ACE2 functions in angiotensin system and plays a crucial role in inflammatory lung diseases. However, the mechanism behind the involvement of ACE2 in inflammatory lung disease has not been fully elucidated. In this study, BEAS-2B cells were treated with gradient concentration of AngII and lipopolysaccharide (LPS) to induce inflammatory condition. Quantitative RT-PCR was performed to detect the level of ACE2 and miR-143-3p. Western blotting and immunofluorescence assays were performed to measure the expression of related proteins. The levels of inflammatory cytokines and cell viability were respectively measured by ELISA and CCK-8 kits. And ACE2 activity was detected by corresponding commercial kits. Bioinformatics methods were employed to predict the potential microRNA targeting ACE2, which was then confirmed by dual luciferase reporter assay. The results showed that ACE2 expression and activity were time-dependently decreased in LPS group for the first 12 h, after which this tendency was reversed. AngII addition enhanced these effects, compared with LPS group. Additionally, the lowest ACE2 activity level was found in both LPS and AngII + LPS groups at 6 h. The number of nuclei and the ACE2 expression were decreased in LPS groups at 6 h and further reduced by addition of AngII, detected by immunofluorescence. Moreover, ACE2 was validated to be a direct target of miR-143-3p. Pretreatment of AngII and LPS regulated the activity of ACE2, increased the expression of proinflammatory cytokines and cell apoptosis and regulated the expression of Bax, Bcl-2 and cleaved caspase-3 in BEAS-2B cells, which could be reversed by transfecting miR-143-3p inhibitor. The results collectively suggest that AngII promotes LPS-induced inflammation by regulating miR-143-3p in BEAS-2B cells. Therefore, miR-143-3p is considered a potential molecular target for the treatment of lung inflammation.

Mesenchymal stem cell therapy in pulmonary fibrosis: a meta-analysis of preclinical studies.

BACKGROUND: Pulmonary fibrosis (PF) is a devastating disease characterized by remodeling of lung architecture and abnormal deposition of fibroblasts in parenchymal tissue and ultimately results in respiratory failure and death. Preclinical studies suggest that mesenchymal stem cell (MSC) administration may be a safe and promising option in treating PF. The objective of our meta-analysis is to assess the efficacy of MSC therapy in preclinical models of PF. METHODS: We performed a comprehensive literature search in PubMed, EMBASE, Web of Science, and Cochrane Library databases from inception to March 17, 2021. Studies that assessed the efficacy of MSC therapy to animals with PF were included. The SYRCLE bias risk tool was employed to evaluate the bias of included studies. The primary outcomes included survival rate and pulmonary fibrosis scores. Meta-analysis was conducted via Cochrane Collaboration Review Manager (version 5.4) and Stata 14.0 statistical software. RESULTS: A total of 1120 articles were reviewed, of which 24 articles met inclusion criteria. Of these, 12 studies evaluated the survival rate and 20 studies evaluated pulmonary fibrosis scores. Compared to the control group, MSC therapy was associated with an improvement in survival rate (odds ratios (OR) 3.10, 95% confidence interval (CI) 2.06 to 4.67, P < 0.001, I2 = 0%) and a significant reduction in pulmonary fibrosis scores (weighted mean difference (WMD) 2.05, 95% CI -2.58 to -1.51, P < 0.001, I2 = 90%). CONCLUSIONS: MSC therapy is a safe and effective method that can significantly improve the survival and pulmonary fibrosis of PF animals. These results provide an important basis for future translational clinical studies.

Comprehensive analysis of the circRNA expression profile and circRNA-miRNA-mRNA network in the pathogenesis of EV-A71 infection.

Enterovirus A71 (EV-A71) is an important emerging virus posing a threat to children under five years old. Circular RNAs (circRNAs), a novel class of endogenous RNAs, have been recognized to play important roles in the onset and development of viral diseases. However, it has not been determined which specific circRNAs are involved in the pathological mechanisms of EV-A71 infection. In this study, RNA-sequencing (RNA-seq) was conducted to characterize differentially expressed circRNAs during the process of EV-A71 infection. Overall, 8726, 10405 and 4710 circRNAs were detected in the control, EV-A71-12 h and EV-A71-24 h groups, respectively, of which 1851 and 951 circRNAs were differentially expressed in the EV-A71-12 h and EV-A71-24 h groups versus the control group. The overlapping circRNAs in the EV-A71-infected groups were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, which further demonstrated that the host genes of these circRNAs were principally implicated in activities associated with the progression of viral infection, such as immune system process, Wnt signaling pathway, etc. Additionally, qRT-PCR detection showed that six selected circRNAs were identical to the sequencing data. To excavate the key circRNAs in EV-A71 infection, we comprehensively evaluated and integrated the relationship of circRNA/miRNA/mRNA, and eventually screened 2 key circRNA regulatory axes, namely hsa_circ_0017115/hsa-miR-150-5p/EGR1 and hsa_circ_0005060/hsa-miR-4685-5p/MMP2. In summary, our findings not only provide the first comprehensive expression and functional profile of circRNAs in response to EV-A71 infection, but also offer a novel direction to elucidate the molecular mechanism underlying viral pathogenesis and the cellular immune response in host-EV-A71 interactions.

Ginsenoside Rb3 alleviates smoke-induced lung injury via the H19/miR-29b-3p/HGMB1/TLR4 signalling pathway.

The over-activation of inflammation is involved in the pathogenesis of smoke-induced lung injury (SILI), while Rb3 treatment may alleviate smoke-induced lung injury by down-regulating the expression of H19, a regulator of miR-29b expression. Moreover, HMGB1 is an important mediator of inflammation. Therefore, in this study, we set up an animal model of SILI and treated it with Rb3 to study the effect of Rb3 on the treatment of SILI and the involvement of H19/miR-29b/HMGB1/TLR4 signalling. SILI mice treated with Rb3 before H&E staining and TUNEL assay were conducted to observe the pathological damages and status of apoptosis in each group. Real-time PCR, Western blot, computational analysis and luciferase assays were utilized to establish the signalling pathway involved in the pathogenesis of SILI and the action of Rb3 treatment. Rb3 treatment alleviated pathological changes in the lungs while decreasing the levels of W/D ratio and cell apoptotic index. H19 was validated to sponge miR-29b-3p, while HMGB1 mRNA was validated to be a target gene of miR-29b-3. As a result, a signalling pathway of H19/miR-29b-3p/HMGB1 was established. Cell viability was evidently reduced after 72 hours of treatment with CSE, but the treatment of Rb3 elevated the expression of H19 and HMBG1 in the presence of CSE. Also, CSE-induced inhibition of miR-29b-3p expression was restored by Rb3. The findings of this study collectively demonstrated that Rb3 exhibited its therapeutic effect during the treatment of SILI via modulating the H19/miR-29b-3p/HMBG1 signalling pathway.

Group 2 innate lymphoid cells contribute to IL-33-mediated alleviation of cardiac fibrosis.

Rationale: The major cause of heart failure is myocardium death consequent to detrimental cardiac remodeling and fibrosis following myocardial infarction. The cardiac protective cytokine interleukin (IL)-33, which signals by ST2 receptor binding, is associated with group 2 innate lymphoid cell (ILC2) activation and regulates tissue homeostasis and repair following tissue injury in various tissues. However, the distribution and role of IL-33-responsive ILC2s in cardiac fibrosis remain unclear. In this study, we elucidated the roles of IL-33-responsive cardiac-resident ILC2s and IL-33-mediated immunomodulatory functions in cardiac fibrosis. Methods: We examined the distribution of cardiac ILC2s by using flow cytometry. The roles of IL-33-mediated ILC2 expansion in cardiac fibrosis was evaluated in the mouse model of catecholamine-induced cardiac fibrosis. ILC-deficient Rag2‒/‒IL2Rγc‒/‒ mice were implemented to determine the contribution of endogenous ILC in the progression of cardiac fibrosis. Histopathological assessments, speckle tracking echocardiography, and transcriptome profile analysis were performed to determine the effects of IL-33-mediated cardiac protective functions. Results: We identified the resident cardiac ILC2s, which share similar cell surface marker and transcriptional factor expression characteristics as peripheral blood and lung tissue ILC2s. IL-33 treatment induced ILC2 expansion via ST2. In vivo, ILC-deficient Rag2‒/‒IL2Rγc‒/‒ mice developed exacerbated cardiac fibrosis following catecholamine-induced stress cardiac injury. IL-33 treatment expanded cardiac ILC2s and revealed protective effects against cardiac tissue damage with reduced cardiomyocyte death, immune cell infiltration, tissue fibrosis, and improved myocardial function. Transcriptome analysis revealed that IL-33 attenuated extracellular matrix synthesis- and fibroblast activation-associated gene expressions. IL13-knockout or epidermal growth factor receptor (EGFR) inhibition abolished IL-33-mediated cardiac protective function, confirming IL-13 and EGFR signaling as crucial for IL-33-mediated cardioprotective responses. Moreover, ILC2-produced BMP-7 served as a novel anti-fibrotic factor to inhibit TGF-ß1-induced cardiac fibroblast activation. Conclusion: Our findings indicate the presence of IL-33-responsive ILC2s in cardiac tissue and that IL-33-mediated ILC2 expansion affords optimal cardioprotective function via ILC2-derived factors. IL-33-mediated immunomodulation is thus a promising strategy to promote tissue repair and alleviate cardiac fibrosis following acute cardiac injury.

Dihydroartemisinin Inhibits the Proliferation, Colony Formation and Induces Ferroptosis of Lung Cancer Cells by Inhibiting PRIM2/SLC7A11 Axis.

OBJECTIVE: Lung cancer is the first leading cause of cancer-related deaths both worldwide and in China and threatens human health and quality of life. New drugs and therapeutic methods are urgently needed. Our study evaluated the roles of dihydroartemisinin (DHA) in lung cancer and further explored its underlying mechanisms. METHODS: CCK-8, colony formation and trypan blue exclusion assays were used to detect the cell viability, colony formation ability and cell death. qRT-PCR and Western blotting assays were applied to analyze the expressions of key molecules. RESULTS: DHA inhibited the proliferation and colony formation abilities and enhanced the cell death and induced ferroptosis of lung NCI-H23 and XWLC-05 cancer cells. DHA reduced PRIM2 expression and silencing PRIM2 mimicked the inhibitory roles on proliferation and colony formation and promotive roles on cell death and ferroptosis of DHA in lung NCI-H23 and XWLC-05 cancer cells. We further found that DHA treatment and loss of PRIM2 reduced the GSH level and increased the cellular lipid ROS and mitochondrial MDA levels, and further downregulated the expressions of SLC7A11 and ß-catenin in lung cancer cells, respectively. Exogenetic overexpression of PRIM2 recovered the inhibitory effects of DHA on proliferation and colony formation in lung NCI-H23 cancer cells, meanwhile loss of PRIM2 sensitizes NCI-H23 cells to DHA therapy. In vivo experiment further showed that DHA treatment significantly suppressed the tumor growth and downregulated PRIM2 and SLC7A11. CONCLUSION: Our study suggested that DHA inhibited the proliferation, colony formation and enhanced cell death and induced ferroptosis of lung cancer cells by inactivating PRIM2/SLC7A11 axis. Loss of PRIM2 induced ferroptosis might developed to be a novel therapeutic method in lung cancer therapy.

IL-33/ST2 axis mediates hyperplasia of intrarenal urothelium in obstructive renal injury.

The monolayered intrarenal urothelium covers the renal papilla and ureteropelvic junction (UPJ). In response to increased renal pressure during obstruction or ischemic injuries, intrarenal urothelial cells begin to proliferate and form a multilayered urothelium. Little is known regarding the mechanism and pathophysiological role of urothelium hyperplasia during renal obstruction. In this study, we investigated the expression of interleukin (IL)-33, an IL-1 family cytokine, in kidneys with unilateral ureteral obstruction (UUO)-induced obstructive injury. IL-33 levels in hydronephrotic urine and serum were upregulated 2 days after UUO. The number of ST2-expressing immune cells was increased in the UUO kidney. We found that IL-33 was upregulated in vimentin-positive cells in the cortical and medullar layers and the UPJ stroma. Moreover, IL-33 expression was predominantly induced in multilayered keratin 5-positive urothelial cells in the UPJ. IL-33 was not detected in terminally differentiated superficial umbrella cells expressing uroplakin 3a. In vivo, we confirmed that deficiency of IL33 or its receptor ST2 attenuated UUO-induced hyperplasia of the UPJ urothelium. Deficiency of IL33 attenuated the expression of UUO-induced type 2 inflammatory cytokines and upregulated uroplakins and urothelial differentiation signaling in UPJ tissues. Our results collectively suggest that the IL-33/ST2 axis mediates the activation of innate immune responses and contributes to urothelial hyperplasia by regulating urothelial differentiation in obstructive kidney injury.

Molecular characterization, expression, and regulation of Gynostemma pentaphyllum squalene epoxidase gene 1.

Gynostemma pentaphyllum (Thunb.) Makino is a perennial medicinal herb widely distributed in China. This herb contains important medicinal components called gypenosides, which belong to dammarane-type triterpenoid saponins. Squalene epoxidase (SE, EC 1.14.99.7) catalyzes the epoxidation of squalene to form oxidosqualene and is a key regulatory enzyme in triterpenoid saponin biosynthesis. In this study, a SE gene designated as GpSE1 was isolated from G. pentaphyllum leaves. The deduced protein sequence of GpSE1 contained two conserved domains involved in the catalytic function of SE. GpSE1 was expressed as inclusion bodies in Escherichia coli cells, and the HIS-tagged recombinant protein was successfully purified and renatured in vitro. Immunofluorescence indicated that the polygonal reticular fluorescence signal of GpSE1 was significantly stronger in young leaves than in mature leaves and rhizomes. This finding is consistent with the tissue-specific expression pattern of GpSE1 and suggests that the young leaves of G. pentaphyllum mainly serve as the active site of gypenoside synthesis. Methyl jasmonate (MeJA) treatment upregulated GpSE1 expression in both the young and mature leaves of G. pentaphyllum, with greater upregulation in young leaves than in mature leaves. However, the expression of GpSE1 was not enhanced continually with the increase in MeJA concentration. Moreover, the GpSE1 expression was maximally regulated in response to 50 µM MeJA but not to 100 µM MeJA. This result indicates that MeJA exerts a concentration-dependent effect on GpSE1 expression.

Nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development.

BACKGROUND: The specification of vein and the patterning of intersegmental vessels (ISV) controlled by transcription factor is not fully characterized. The orphan nuclear receptor Chicken ovalbumin upstream promoter transcription factor II (CoupTFII, a.k.a NR2F2) positively regulates vein identity in mice. In this study, we show that nr2f1b is important for vein and tip cell identity during zebrafish development. RESULTS: Nr2f1b mRNA is expressed in ventral lateral mesoderm at 15S stage and in vessels at 24 hpf consistent with a role in early vascular specification. Morpholino knockdown of nr2f1b results in a decrease in both vein cell number and expression of the vein specific marker flt4 and mrc1, suggested its role in venous specification. We also show loss of nr2f1b reduced ISV cell number and impairs ISV growth, which is likely due to the impairment of angiogenic cells migration and/or proliferation by time-lapse imaging. Consequently, nr2f1b morphants showed pericardial edema and circulation defects. Overexpression of nr2f1b under the fli promoter increases the number of venous cells and ISV endothelial cells indicated the function of nr2f1b is required and necessary for vascular development. We further showed that nr2f1b likely interact with Notch signalling. nr2f1b expression is increased in rbpsuh morphants and DAPT-treatment embryos suggested nr2f1b is negatively regulated by Notch activity. CONCLUSIONS: We show nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development, which is mediated by Notch signalings.

Identification and expression of a stearoyl-ACP desaturase gene responsible for oleic acid accumulation in Xanthoceras sorbifolia seeds.

Xanthoceras sorbifolia Bunge is an oilseed tree that grows well on barren lands in dry climate. Its seeds contain a large amount of oil rich in oleic acid (18:1(Δ9)) and linoleic acid (18:2(Δ9, 12)). However, the molecular regulation of oil biosynthesis in X. sorbifolia seeds is poorly understood. Stearoyl-ACP desaturase (SAD, EC 1.14.99.6) is a plastid-localized soluble desaturase that catalyzes the conversion of stearic acid (18:0) to oleic acid, which plays a key role in determining the ratio of saturated to unsaturated fatty acids. In this study, a full-length cDNA of XsSAD was isolated from developing X. sorbifolia embryos. The XsSAD open reading frame had 1194-bp, encoding a polypeptide of 397 amino acids. XsSAD expression in Escherichia coli cells resulted in increased 18:1(Δ9) level, confirming the biological activity of the enzyme encoded by XsSAD. XsSAD expression in Arabidopsis ssi2 mutants partially restored the morphological phenotype and effectively increased the 18:1(Δ9) level. The levels of other unsaturated fatty acids synthesized with 18:1(Δ9) as the substrate also increased to some degree. XsSAD in X. sorbifolia had a much higher expression in embryos than in leaves and petals. XsSAD expression also correlated well with the oleic acid, unsaturated fatty acid, and total fatty acid levels in developing embryos. These data suggested that XsSAD determined the synthesis of oleic acid and contributed to the accumulation of unsaturated fatty acid and total oil in X. sorbifolia seeds. A preliminary tobacco rattle virus-based virus-induced gene silencing system established in X. sorbifolia can also be helpful for further analyzing the functions of XsSAD and other oil synthesis-related genes in woody plants.