Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen.

Objective: This study aimed to identify and characterize a novel endo-ß-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. Methods: A novel endo-ß-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley ß-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. Results: The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40 °C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley ß-glucan (109.59 ± 3.61 µmol·mg-1·min-1) and Icelandic moss lichenan (35.35 ± 1.55 µmol·mg-1·min-1). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. Conclusion: The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.

Gut microbiota-derived LCA mediates the protective effect of PEDV infection in piglets.

BACKGROUND: The gut microbiota is a critical factor in the regulation of host health, but the relationship between the differential resistance of hosts to pathogens and the interaction of gut microbes is not yet clear. Herein, we investigated the potential correlation between the gut microbiota of piglets and their disease resistance using single-cell transcriptomics, 16S amplicon sequencing, metagenomics, and untargeted metabolomics. RESULTS: Porcine epidemic diarrhea virus (PEDV) infection leads to significant changes in the gut microbiota of piglets. Notably, Landrace pigs lose their resistance quickly after being infected with PEDV, but transplanting the fecal microbiota of Min pigs to Landrace pigs alleviated the infection status. Macrogenomic and animal protection models identified Lactobacillus reuteri and Lactobacillus amylovorus in the gut microbiota as playing an anti-infective role. Moreover, metabolomic screening of the secondary bile acids' deoxycholic acid (DCA) and lithocholic acid (LCA) correlated significantly with Lactobacillus reuteri and Lactobacillus amylovorus, but only LCA exerted a protective function in the animal model. In addition, LCA supplementation altered the distribution of intestinal T-cell populations and resulted in significantly enriched CD8+ CTLs, and in vivo and in vitro experiments showed that LCA increased SLA-I expression in porcine intestinal epithelial cells via FXR receptors, thereby recruiting CD8+ CTLs to exert antiviral effects. CONCLUSIONS: Overall, our findings indicate that the diversity of gut microbiota influences the development of the disease, and manipulating Lactobacillus reuteri and Lactobacillus amylovorus, as well as LCA, represents a promising strategy to improve PEDV infection in piglets. Video Abstract.

"Mini-community" simulation revealed the differences of endophytic fungal communities between the above- and below-ground tissues of Ephedra sinica Stapf.

The microecology of endophytic fungi in special habitats, such as the interior of different tissues from a medicinal plant, and its effects on the formation of metabolites with different biological activities are of great importance. However, the factors affecting fungal community formation are unclear. This study is the first to utilize "mini-community" remodeling to understand the above phenomena. First, high-throughput sequencing technology was applied to explore the community composition and diversity of endophytic fungi in the above-ground tissues (Ea) and below-ground tissues (Eb) of Ephedra sinica. Second, fungi were obtained through culture-dependent technology and used for "mini-community" remodeling in vitro. Then, the effects of environmental factors, partner fungi, and plant tissue fluid (internal environment) on endophytic fungal community formation were discussed. Results showed that environmental factors played a decisive role in the selection of endophytic fungi, that is, in Ea and Eb, 93.8% and 25.3% of endophytic fungi were halophilic, respectively, and 10.6% and 60.2% fungi were sensitive to high temperature (33 °C), respectively. Meanwhile, pH had little effect on fungal communities. The internal environment of the plant host further promoted the formation of endophytic fungal communities.

Soluble CD4 effectively prevents excessive TLR activation of resident macrophages in the onset of sepsis.

T lymphopenia, occurring in the early phase of sepsis in response to systemic inflammation, is commonly associated with morbidity and mortality of septic infections. We have previously shown that a sufficient number of T cells is required to constrain Toll-like receptors (TLRs) mediated hyperinflammation. However, the underlying mechanisms remains unsolved. Herein, we unveil that CD4+ T cells engage with MHC II of macrophages to downregulate TLR pro-inflammatory signaling. We show further that the direct contact between CD4 molecule of CD4+ T cells or the ectodomain of CD4 (soluble CD4, sCD4), and MHC II of resident macrophages is necessary and sufficient to prevent TLR4 overactivation in LPS and cecal ligation puncture (CLP) sepsis. sCD4 serum concentrations increase after the onset of LPS sepsis, suggesting its compensatory inhibitive effects on hyperinflammation. sCD4 engagement enables the cytoplasmic domain of MHC II to recruit and activate STING and SHP2, which inhibits IRAK1/Erk and TRAF6/NF-κB activation required for TLR4 inflammation. Furthermore, sCD4 subverts pro-inflammatory plasma membrane anchorage of TLR4 by disruption of MHC II-TLR4 raft domains that promotes MHC II endocytosis. Finally, sCD4/MHCII reversal signaling specifically interferes with TLR4 but not TNFR hyperinflammation, and independent of the inhibitive signaling of CD40 ligand of CD4+ cells on macrophages. Therefore, a sufficient amount of soluble CD4 protein can prevent excessive inflammatory activation of macrophages via alternation of MHC II-TLR signaling complex, that might benefit for a new paradigm of preventive treatment of sepsis.

AGEs-RAGE-KCa3.1 pathway mediates palmitic acid-induced migration of PBMCs from patients with type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is characterized by chronic low-grade systemic inflammation. Tissue infiltration by monocyte migration contributes to the pathogenesis of vascular complications in T2DM. We studied the role of intermediate-conductance Ca2+-activated K+ (KCa3.1) channels in the palmitic acid (PA)-induced migration of peripheral blood mononuclear cells (PBMCs) from T2DM patients and the influence of advanced glycation endproducts (AGEs). A total of 49 T2DM patients and 33 healthy subjects was recruited into this study. Using flow cytometry and Western blotting analysis as well as cell migration assay, we found that there was a significant decrease in frequency of T lymphocytes and monocytes in CD45+ leukocyte population. PA at 100 µM stimulated migration of PBMCs from T2DM individuals, which was inhibited by the specific KCa3.1 channel blocker TRAM-34 (1 µM). The PBMC migration was positively correlated with glycosylated hemoglobin A1 chain (HbA1c) level of T2DM patients, an indicator of AGEs, and PBMCs with higher level of HbA1c showed upregulated expression of toll-like receptor (TLR) 2/4 and KCa3.1 channels. In THP-1 cells, AGEs at 200 µg/ml increased protein expression of TLR 2/4 and KCa3.1 channels, and were synergistically involved in PA-induced migration through receptors of AGEs (RAGE)-mediated KCa3.1 upregulation. In conclusion, in PBMCs of T2DM patients, AGEs promotes PA-induced migration via upregulation of TLR2/4 and KCa3.1 channels.

Treatment of pyogenic liver abscess by surgical incision and drainage combined with platelet-rich plasma: A case report.

BACKGROUND: Pyogenic liver abscesses are insidious in the early stage. Some cases progress rapidly, and the patient's condition can worsen and even become life-threatening if timely treatment is not provided. Surgery and prolonged antibiotic treatment are often required if the abscess is large and liquefied and becomes separated within the lumen. CASE SUMMARY: We report a case of bacterial liver abscess with a poor outcome following pharmacological treatment, review the literature related to the use of platelet-rich plasma (PRP) in the treatment of hepatic impairment and partial hepatectomy in animals, and discuss the prognostic features of surgical incision and drainage combined with PRP in the treatment of bacterial liver abscesses. This is the first case describing the use of PRP in the treatment of a bacterial liver abscess in humans, providing new ideas for the treatment of this condition. CONCLUSION: This case highlights the importance of surgical treatment for bacterial liver abscesses that are well liquefied and poorly managed medically. PRP may produce antimicrobial effects and promote the regeneration and repair of liver tissue.

Relationship between the mean of 24-h venous blood glucose and in-hospital mortality among patients with subarachnoid hemorrhage: A matched cohort study.

Objective: The aim of this study was to explore the correlation between the mean of 24-h venous blood glucose (BG) and in-hospital mortality and all-cause mortality (ACM) in patients with subarachnoid hemorrhage (SAH). Methods: Detailed clinical information was acquired from the Medical Information Mart for Intensive IV (MIMIC-IV) database. The best cutoff value of mean BG was calculated using the X-tile program. Univariate and multivariate logistic regressive analyses were utilized to analyze the prognosis significance of mean BG, and survival curves were drawn using the Kaplan-Meier (K-M) approach. To improve the reliability of results and balance the impact of underlying confounders, the 1:1 propensity score matching (PSM) approach was utilized. Results: An overall of 1,230 subjects were selected herein. The optimal cutoff value of the mean BG for in-hospital mortality was 152.25. In addition, 367 pairs of score-matched subjects were acquired after PSM analysis, and nearly all variables' differences were balanced. K-M analysis showed that patients with mean BG ≥ 152.25 mg/dl had significantly higher in-hospital, 3-month, and 6-month mortalities compared with patients with mean BG < 152.25 mg/dl (p < 0.001). The multivariable logistic regressive analyses revealed that patients with mean BG ≥ 152.25 mg/dl had significantly increased in-hospital mortality compared with patients with mean BG < 152.25 mg/dl after the adjustment for possible confounders (OR = 1.994, 95% CI: 1.321-3.012, p = 0.001). Similar outcomes were discovered in the PSM cohort. Conclusion: Our data suggested that mean BG was related to ACM of patients with SAH. More studies are needed to further analyze the role of the mean of 24-h venous BG in patients with SAH.

Transcriptional Regulatory Mechanism of Differential Metabolite Formation in Root and Stem of Ephedra sinica.

Ephedra sinica, a well-known Chinese medicinal plant, is characterized as having the opposite medicinal effect among its root and stem. However, there is a lack of understanding to differentiate the active components present in the root and stem of E. sinica, as well as the molecular mechanisms underlying the formation of the differential compounds, which has significantly hampered the further development and utilization of E. sinica resource. In this study, forty-five differential metabolic markers are affiliated to alkaloids, flavonoids, terpenoids, and organic acids between root and stem of E. sinica, and sixty genes of key enzymes are involved in their biosynthesis distributed in metabolic pathway branches such as phenylalanine metabolism, flavonoid biosynthesis and phenylpropane biosynthesis, based on combination non-targeted metabolome with transcriptome technologies. The finding revealed that the expression activity changes of these enzyme genes had a direct impact on the distinction of differential metabolic markers in the root and stem of E. sinica. This study will help to understand the molecular mechanism of the differentiation and biosynthesis of the primary active metabolites in the root and stem of E. sinica, providing a theoretical foundation for its quality control and promotion in cultivation.

Endophytic fungi and their bioactive secondary metabolites in medicinal leguminosae plants: nearly untapped medical resources.

There are many species of Chinese traditional leguminosae family plants that are well known for their medicinal applications, such as Astragalus membranaceus, Catsia tora, Glycyrrhiza uralensis, Sophora flavescens and Albacia acacia. Their unique bioactive composition and internal phenological environment contribute to the formation of specific and unique endophytic fungal communities, which are important resources for new compounds used in a variety of pharmacological activities. Nonetheless, they have not been systematically studied. In the last decade, nearly 64 genera and thousands of species of endophytic fungi have been discovered from leguminosae plants, as well as 138 secondary metabolites (with 34 new compounds) including flavonoid, alkaloids, phenol, anthraquinone, macrolide, terpenoid, phytohormone and many more. These were shown to have diverse applications and benefits, such as antibacterial, antitumor, antioxidative, immunoregulatory and neuroprotective properties. Here, we provide a summarized overview with the aim of raising awareness of endophytic fungi from medicinal leguminosae plants and providing a comprehensive review of the discoveries of new natural products that may be of medicinal and pharmaceutical importance.

Heterologous expression and characterization of two novel glucanases derived from sheep rumen microbiota.

ß-Glucanases are a suite of glycoside hydrolases that depolymerize ß-glucan into cellooligosaccharides and/or monosaccharides and have been widely used as feed additives in livestock. In this study, two novel glucanase genes, IDSGluc5-26 and IDSGluc5-37, derived from sheep rumen microbiota, were expressed and functionally characterized. The optimal temperatures/pH of recombinant IDSGLUC5-26 and IDSGLUC5-37 were 50 °C/5.0 and 40 °C/6.0, respectively. Notably, IDSGLUC5-26 showed considerable stability under acidic conditions. Both IDSGLUC5-26 and IDSGLUC5-37 showed the highest activities toward barley ß-glucan, with Vmax values of 89.96 ± 9.19 µmol/min/mg and 459.50 ± 25.02 µmol/min/mg, respectively. Additionally, these two glucanases demonstrated hydrolysis of Icelandic moss lichenan and konjac gum, IDSGLUC5-26 releasing cellobiose (G2; occupying 17.37% of total reducing sugars), cellotriose (G3; 23.97%), and cellotetraose (G4; 30.93%) from barley ß-glucan and Icelandic moss lichenan after 10 min and suggestive of a typical endo-ß-1,4-glucanase (EC.3.2.1.4). In contrast, IDSGLUC5-37 was capable of liberating dominant G3 (64.11% or 67.55%) from barley ß-glucan or Icelandic moss lichenan, suggesting that the enzyme was likely an endo-ß-1,3 - 1,4-glucanases/lichenase (EC3.2.1.73). These findings describe the expression and characterization of two novel glucanase genes from sheep rumen microbiota. The two recombinant enzymes, particularly the acid-stable IDSGLUC5-26, will be of interest for potential application in food-/feed-additive development.

Biotransformation ability of endophytic fungi: from species evolution to industrial applications.

Increased understanding of the interactions between endophytic fungi and plants has led to the discovery of a new generation of chemical compounds and processes between endophytic fungi and plants. Due to the long-term co-evolution between fungal endophytes and host plants, endophytes have evolved special biotransformation abilities, which can have critical consequences on plant metabolic processes and their composition. Biotransformation or bioconversion can impact the synthesis and decomposition of hormones, sugars, amino acids, vitamins, lipids, proteins, and various secondary metabolites, including flavonoids, polysaccharides, and terpenes. Endophytic fungi produce enzymes and various bioactive secondary metabolites with industrial value and can degrade or sequester inorganic and organic small molecules and macromolecules (e.g., toxins, pollutants, heavy metals). These fungi also have the ability to cause highly selective catalytic conversion of high-value compounds in an environmentally friendly manner, which can be important for the production/innovation of bioactive molecules, food and nutrition, agriculture, and environment. This work mainly summarized recent research progress in this field, providing a reference for further research and application of fungal endophytes. KEY POINTS: •The industrial value of degradation of endophytes was summarized. • The commercial value for the pharmaceutical industry is reviewed.

Lactobacillus plantarum surface-displayed ASFV (p54) with porcine IL-21 generally stimulates protective immune responses in mice.

African classical swine fever virus (ASFV) has spread seriously around the world and has dealt with a heavy blow to the pig breeding industry due to the lack of vaccines. In this study, we produced recombinant Lactobacillus plantarum (L. plantarum) expressing an ASFV p54 and porcine IL-21 (pIL-21) fusion protein and evaluated the immune effect of NC8-pSIP409-pgsA'-p54-pIL-21 in a mouse model. First, we verified that the ASFV p54 protein and p54-pIL-21 fusion protein were anchored on the surface of L. plantarum NC8 by flow cytometry, immunofluorescence and Western blotting. Then, the results were verified by flow cytometry, ELISA and MTT assays. Mouse-specific humoral immunity and mucosal and T cell-mediated immune responses were induced by recombinant L. plantarum. The results of feeding mice recombinant L. plantarum showed that the levels of serum IgG and mucosal secreted IgA (SIgA), the number of CD4 and CD8 T cells, and the expression of IFN-γ in CD4 and CD8 T cells increased significantly, and lymphocyte proliferation occurred under stimulation with the ASFV p54 protein. Our data lay a foundation for the development of oral vaccines against ASFV in the future.

MicroRNA and circRNA Expression Analysis in a Zbtb1 Gene Knockout Monoclonal EL4 Cell Line.

Zinc finger and BTB domain containing 1(Zbtb1) is a transcriptional suppressor protein, and a member of the mammalian Zbtb gene family. Previous studies have shown that Zbtb1 is essential for T-cell development. However, the role of Zbtb1 in T-cell lymphoma is undetermined. In this study, an EL4 cell line with Zbtb1 deletion was constructed using the CRISPR-Cas9 technique. The expression profiles of microRNA and circRNA produced by the control and gene deletion groups were determined by RNA-seq. In general, 24 differentially expressed microRNA and 16 differentially expressed circRNA were found between normal group and gene deletion group. Through further analysis of differentially expressed genes, GO term histogram and KEGG scatter plot were drawn, and three pairs of miRNA and circRNA regulatory relationships were found. This study describes the differentially expressed microRNA and circRNA in normal and Zbtb1-deficient EL4 cell lines, thus providing potential targets for drug development and clinical treatment of T-cell lymphoma.

Progressive liver injury and increased mortality risk in COVID-19 patients: A retrospective cohort study in China.

BACKGROUND: Liver injury is common and also can be fatal, particularly in severe or critical patients with coronavirus disease 2019 (COVID-19). AIM: To conduct an in-depth investigation into the risk factors for liver injury and into the effective measures to prevent subsequent mortality risk. METHODS: A retrospective cohort study was performed on 440 consecutive patients with relatively severe COVID-19 between January 28 and March 9, 2020 at Tongji Hospital, Wuhan, China. Data on clinical features, laboratory parameters, medications, and prognosis were collected. RESULTS: COVID-19-associated liver injury more frequently occurred in patients aged ≥ 65 years, female patients, or those with other comorbidities, decreased lymphocyte count, or elevated D-dimer or serum ferritin (P < 0.05). The disease severity of COVID-19 was an independent risk factor for liver injury (severe patients: Odds ratio [OR] = 2.86, 95% confidence interval [CI]: 1.78-4.59; critical patients: OR = 13.44, 95%CI: 7.21-25.97). The elevated levels of on-admission aspartate aminotransferase and total bilirubin indicated an increased mortality risk (P < 0.001). Using intravenous nutrition or antibiotics increased the risk of COVID-19-associated liver injury. Hepatoprotective drugs tended to be of assistance to treat the liver injury and improve the prognosis of patients with COVID-19-associated liver injury. CONCLUSION: More intensive monitoring of aspartate aminotransferase or total bilirubin is recommended for COVID-19 patients, especially patients aged ≥ 65 years, female patients, or those with other comorbidities. Drug hepatotoxicity of antibiotics and intravenous nutrition should be alert for COVID-19 patients.

Inflammation-associated factors for predicting in-hospital mortality in patients with COVID-19.

The aim is to explore the relation between inflammation-associated factors and in-hospital mortality and investigate which factor is an independent predictor of in-hospital death in patients with coronavirus disease-2019. This study included patients with coronavirus disease-2019, who were hospitalized between February 9, 2020, and March 30, 2020. Univariate Cox regression analysis and least absolute shrinkage and selection operator regression (LASSO) were used to select variables. Multivariate Cox regression analysis was applied to identify independent risk factors in coronavirus disease-2019. A total of 1135 patients were analyzed during the study period. A total of 35 variables were considered to be risk factors after the univariate regression analysis of the clinical characteristics and laboratory parameters (p < .05), and LASSO regression analysis screened out seven risk factors for further study. The six independent risk factors revealed by multivariate Cox regression were myoglobin (HR, 5.353; 95% CI, 2.633-10.882; p < .001), C-reactive protein (HR, 2.063; 95% CI, 1.036-4.109; p = .039), neutrophil count (HR, 2.015; 95% CI, 1.154-3.518; p = .014), interleukin 6 (Il-6; HR, 9.753; 95% CI, 2.952-32.218; p < .001), age (HR, 2.016; 95% CI, 1.077-3.773; p = .028), and international normalized ratio (HR, 2.595; 95% CI, 1.412-4.769; p = .002). Our results suggested that inflammation-associated factors were significantly associated with in-hospital mortality in coronavirus disease-2019 patients. C-reactive protein, neutrophil count, and interleukin 6 were independent factors for predicting in-hospital mortality and had a better independent predictive ability. We believe these findings may allow early identification of the patients at high risk for death, and can also assist in better management of these patients.

Supplementation with beta-1,3-glucan improves productivity, immunity and antioxidative status in transition Holstein cows.

Dairy cows undergo dramatic physiological changes during the transition from late pregnancy to early lactation, which make them vulnerable to metabolic stress and immune dysfunction. The objective of this study was to evaluate the effects of a commercial beta-1,3-glucan product (Aleta™, containing 50% beta-1,3-glucan) on productivity, immunity and antioxidative status in transition cows. Fifty-four multiparous Holstein cows received a control diet or a diet supplemented with 5 or 10 g of beta-1,3-glucan per cow per day from 21 days before expected calving to 21 days after parturition. Blood samples were collected at day -21, 1, and 21 relative to calving. Colostrum and milk were collected at day 1 and 21 after calving, respectively. Data showed that supplementation with beta-1,3-glucan had no effect on milk composition, but increased milk production. Beta-1,3-glucan treatment also improved the milk quality, as shown by reduced milk somatic cell count and increased immunoglobulin levels in colostrum. Notably, beta-1,3-glucan markedly reduced serum levels of pro-inflammatory cytokines and C-reactive protein, while elevated serum immunoglobulin levels, indicating its immunity enhancement in transition cows. Moreover, beta-1,3-glucan addition reduced the serum malondialdehyde level and enhanced the activities of serum superoxide dismutase and catalase, which enhanced the antioxidative capacity in transition cows. In summary, supplementation with beta-1,3-glucan improves productivity, immunity and antioxidative status in transition dairy cows.

[Spatio-temporal Evolution of PM2.5 Concentration During 2000-2019 in China].

An ensemble estimation model of PM2.5 concentration was proposed on the basis of extreme gradient boosting, gradient boosting, random forest model, and stacking model fusion technology. Measured PM2.5 data, MERRA-2 AOD and PM2.5 reanalysis data, meteorological parameters, and night light data sets were used. On this basis, the spatiotemporal evolution features of PM2.5 concentration in China during 2000-2019 were analyzed at monthly, seasonal, and annual temporal scales. The results showed that:① Monthly PM2.5 concentration in China from 2000-2019 can be estimated reliably by the ensemble model. ② PM2.5 annual concentration changed from rapid increase to remaining stable and then changed to significant decline from 2000-2019, with turning points in 2007 and 2014. The monthly variation of PM2.5 concentration showed a U shape that first decreased then increased, with the minimum value in July and the maximum value in December. ③ Natural geographic conditions and human activities laid the foundation for the annual spatial pattern change of PM2.5 concentration in China, and the main trend of monthly spatial pattern change of PM2.5 concentration was determined by meteorological conditions. ④ At an annual scale, the national PM2.5 concentration average center of standard deviation ellipse moved eastward from 2000-2014 and westward from 2014-2018. At a monthly scale, the average center shifted to the west from January to March, moved northward then southward from April to September, and shifted to the east from September to December.

Benzenebistriazole-strapped calix[4]pyrrole: a neutral anion receptor with CH and NH donor groups that exhibits high sulfate binding affinity and selectivity in aqueous solutions.

A calix[4]pyrrole strapped by benzenebistriazole has been prepared as an artificial anion binding receptor. This neutral anion receptor shows high sulfate binding affinity and selectivity in an aqueous solution. In solid state, the receptor binds the sulfate anion in a chair-like 3D cavity via multiple N-H and C-H hydrogen bonds.