Termite Fungus Comb Polysaccharides Alleviate Hyperglycemia and Hyperlipidemia in Type 2 Diabetic Mice by Regulating Hepatic Glucose/Lipid Metabolism and the Gut Microbiota.

Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by hyperglycemia and dyslipidemia. The termite fungus comb is an integral component of nests of termites, which are a global pest. Termite fungus comb polysaccharides (TFCPs) have been identified to possess antioxidant, anti-aging, and immune-enhancing properties. However, their physicochemical characteristics and their role in fighting diabetes have not been previously reported. In the current study, TFCPs were isolated and structurally characterized. The yield of TFCPs was determined to be 2.76%, and it was found to be composed of a diverse array of polysaccharides with varying molecular weights. The hypoglycemic and hypolipidemic effects of TFCPs, as well as their potential mechanisms of action, were investigated in a T2D mouse model. The results demonstrated that oral administration of TFCPs could alleviate fasting blood glucose levels, insulin resistance, hyperlipidemia, and the dysfunction of pancreatic islets in T2D mice. In terms of mechanisms, the TFCPs enhanced hepatic glycogenesis and glycolysis while inhibiting gluconeogenesis. Additionally, the TFCPs suppressed hepatic de novo lipogenesis and promoted fatty acid oxidation. Furthermore, the TFCPs altered the composition of the gut microbiota in the T2D mice, increasing the abundance of beneficial bacteria such as Allobaculum and Faecalibaculum, while reducing the levels of pathogens like Mailhella and Acetatifactor. Overall, these findings suggest that TFCPs may exert anti-diabetic effects by regulating hepatic glucose and lipid metabolism and the composition of the gut microbiota. These findings suggest that TFCPs can be used as a promising functional ingredient for the prevention and treatment of T2D.

Effects of Dietary Inclusion of ß-Hydroxy-ß-Methylbutyrate on Growth Performance, Fat Deposition, Bile Acid Metabolism, and Gut Microbiota Function in High-Fat and High-Cholesterol Diet-Challenged Layer Chickens.

Excessive lipid deposition in layer chickens due to inappropriate feeding adversely affects egg production; however, nutritional manipulation methods to deal with this issue are still limited. ß-hydroxy-ß-methylbutyrate (HMB), a metabolite of L-leucine, was recently reported as a lipid-lowering nutrient in mice and pigs, although its role in layers had not been investigated. Here, we employed high-fat and high-cholesterol diet (HFHCD)-challenged growing layers as an obese model to explore HMB function in the regulation of lipid metabolism and the potential mechanisms involved. We found that dietary supplementation with (0.05% or 0.10%) HMB significantly reduced HFHCD-induced bodyweight growth in layers, mainly due to reduction in abdominal fat deposition. Mechanistically, HMB supplementation enhanced hepatic bile acid synthesis from cholesterol through elevating expression of Cyp7a1, a gene coding a key enzyme in bile acid synthesis. Furthermore, 16S rRNA gene sequencing revealed that HMB supplementation remodeled the diversity and composition of the layers' cecal microbiota, and the abundance of Bacteroidetes at the phylum level were especially affected. Correlation analysis further indicated a strong negative association between Bacteroidetes abundance and lipid metabolism-related parameters. Taken together, these data suggest that dietary HMB supplementation could improve abdominal fat deposition in layers, probably through modulating hepatic bile acid synthesis and gut microbiota function.

Discovery of New Inhibitors of eEF2K from Traditional Chinese Medicine Based on In Silico Screening and In Vitro Experimental Validation.

Eukaryotic elongation factor 2 kinase (eEF2K) is a highly conserved α kinase and is increasingly considered as an attractive therapeutic target for cancer as well as other diseases. However, so far, no selective and potent inhibitors of eEF2K have been identified. In this study, pharmacophore screening, homology modeling, and molecular docking methods were adopted to screen novel inhibitor hits of eEF2K from the traditional Chinese medicine database (TCMD), and then cytotoxicity assay and western blotting were performed to verify the validity of the screen. Resultantly, after two steps of screening, a total of 1077 chemicals were obtained as inhibitor hits for eEF2K from all 23,034 compounds in TCMD. Then, to verify the validity, the top 10 purchasable chemicals were further analyzed. Afterward, Oleuropein and Rhoifolin, two reported antitumor chemicals, were found to have low cytotoxicity but potent inhibitory effects on eEF2K activity. Finally, molecular dynamics simulation, pharmacokinetic and toxicological analyses were conducted to evaluate the property and potential of Oleuropein and Rhoifolin to be drugs. Together, by integrating in silico screening and in vitro biochemical studies, Oleuropein and Rhoifolin were revealed as novel eEF2K inhibitors, which will shed new lights for eEF2K-targeting drug development and anticancer therapy.

Type VI secretion system contributes to Enterohemorrhagic Escherichia coli virulence by secreting catalase against host reactive oxygen species (ROS).

Enterohemorrhagic Escherichia coli (EHEC) is one major type of contagious and foodborne pathogens. The type VI secretion system (T6SS) has been shown to be involved in the bacterial pathogenicity and bacteria-bacteria competition. Here, we show that EHEC could secrete a novel effector KatN, a Mn-containing catalase, in a T6SS-dependent manner. Expression of katN is promoted by RpoS and OxyR and repressed by H-NS, and katN contributes to bacterial growth under oxidative stress in vitro. KatN could be secreted into host cell cytosol after EHEC is phagocytized by macrophage, which leads to decreased level of intracellular reactive oxygen species (ROS) and facilitates the intramacrophage survival of EHEC. Finally, animal model results show that the deletion mutant of T6SS was attenuated in virulence compared with the wild type strain, while the deletion mutant of katN had comparable virulence to the wild type strain. Taken together, our findings suggest that EHEC could sense oxidative stress in phagosome and decrease the host cell ROS by secreting catalase KatN to facilitate its survival in the host cells.

Activation of skeletal carbohydrate-response element binding protein (ChREBP)-mediated de novo lipogenesis increases intramuscular fat content in chickens.

The intracellular lipids in muscle cells of farm animals play a crucial role in determining the overall intramuscular fat (IMF) content, which has a positive impact on meat quality. However, the mechanisms underlying the deposition of lipids in muscle cells of farm animals are not yet fully understood. The purpose of this study was to determine the roles of carbohydrate-response element binding protein (ChREBP) and fructose in IMF deposition of chickens. For virus-mediated ChREBP overexpression in tibialis anterior (TA) muscle of chickens, seven 5-d-old male yellow-feather chickens were used. At 10 d after virus injection, the chickens were slaughtered to obtain TA muscles for analysis. For fructose administration trial, sixty 9-wk-old male yellow-feather chickens were randomly divided into 2 groups, with 6 replicates per group and 5 chickens per replicate. The chickens were fed either a basal diet or a basal diet supplemented with 10% fructose (purity ≥ 99%). At 4 wk later, the chickens were slaughtered, and breast and thigh muscles were collected for analysis. The results showed that the skeletal ChREBP mRNA levels were positively associated with IMF content in multiple species, including the chickens, pigs, and mice (P < 0.05). ChREBP overexpression increased lipid accumulation in both muscle cells in vitro and the TA muscles of mice and chickens in vivo (P < 0.05), by activation of the de novo lipogenesis (DNL) pathway. Moreover, activation of ChREBP by dietary fructose administration also resulted in increased IMF content in mice and notably chickens (P < 0.05). Furthermore, the lipidomics analysis revealed that ChREBP activation altered the lipid composition of chicken IMF and tented to improve the flavor profile of the meat. In conclusion, this study found that ChREBP plays a pivotal role in mediating the deposition of fat in chicken muscles in response to fructose-rich diets, which provides a novel strategy for improving meat quality in the livestock industry.

Effects of dietary supplementation of Anoectochilus roxburghii extract (ARE) on growth performance, abdominal fat deposition, meat quality, and gut microbiota in broilers.

The broiler industry frequently encounters 2 common problems: excessive deposition of abdominal fat and poor quality of meat. However, there are limited nutritional manipulation strategies to address these issues. While Anoectochilus roxburghii (Wall.) Lindl., a traditional Chinese herb, has been shown to have multiple beneficial effects in humans, its potential roles in broiler chickens remain unexplored. In this study, the effects of dietary supplementation with Anoectochilus roxburghii extract (ARE) on growth performance, abdominal fat deposition, meat quality, blood indices, and gut microbiota were investigated in yellow-feather broiler chickens. A total of 90 twenty-one-day-old yellow-feather broilers were randomly divided into 3 treatments, and each treatment included 5 replicates with 6 birds per replicate. Birds were fed a basal diet supplemented with 0, 0.15, or 0.30% ARE for 6 wk. The results showed that the inclusion of ARE in the diet did not have any significant effect on meat yield (P > 0.05). However, it did lead to a reduction in abdominal fat deposition and an improvement in meat quality (P < 0.05). Mechanistically, the addition of ARE inhibited lipid biosynthesis and enhanced lipid breakdown in both the liver and adipose tissue of the broilers. Furthermore, ARE supplementation increased the antioxidase activities in the muscle and serum of the broilers (P < 0.05). In addition, the supplementation of ARE optimized the diversity and composition of the cecal microbiota, particularly by lowering the ratio of Firmicutes to Bacteroidetes (P < 0.05). Moreover, the abundance of some bacteria that were positively correlated with abdominal fat deposition was reduced by ARE, and vice versa (P < 0.05). Collectively, the results suggest that ARE is a promising candidate as a feed additive for reducing abdominal fat deposition and improving meat quality in the broiler industry.

SHMT2 promotes cell viability and inhibits ROS-dependent, mitochondrial-mediated apoptosis via the intrinsic signaling pathway in bladder cancer cells.

Mitochondrial serine hydroxymethyltransferase (SHMT2) catalyzes the conversion of serine to glycine and concomitantly produces one-carbon units to support cell growth and is upregulated in various cancer cells. SHMT2 knockdown triggers cell apoptosis; however, the detailed mechanism of apoptosis induced by SHMT2 inactivation remains unknown. Here, we demonstrate that SHMT2 supports the proliferation of bladder cancer (BC) cells by maintaining redox homeostasis. SHMT2 knockout decreased the pools of purine and one-carbon units and delayed cell cycle progression in a manner that was rescued by formate, demonstrating that SHMT2-mediated one-carbon units are essential for BC cell proliferation. SHMT2 deficiency promoted the accumulation of intracellular reactive oxygen species (ROS) by decreasing the NADH/NAD+, NADPH/NADP+, and GSH/GSSG ratios, leading to a loss in mitochondrial membrane potential, release of cytochrome c, translocation of Bcl-2 family protein and activation of caspase-3. Notably, blocking ROS production with the one-carbon donor formate and the ROS scavenger N-acetyl-cysteine (NAC) effectively rescued SHMT2 deficiency-induced cell apoptosis via the intrinsic signaling pathway. Treatment with the SHMT inhibitor SHIN1 resulted in a significant inhibitory effect on cell proliferation and induced cell apoptosis. Formate and NAC rescued SHIN1-induced cell apoptosis. Our findings reveal an important mechanism by which the loss of SHMT2 triggers ROS-dependent, mitochondrial-mediated apoptosis, which gives insight into the link between serine metabolism and cell apoptosis and provides a promising target for BC treatment and drug discovery.

High-throughput sequencing unravels the cell heterogeneity of cerebrospinal fluid in the bacterial meningitis of children.

Bacterial meningitis (BM) is a common life-threatening infection in children that occurs in the central nervous system (CNS). The cytologic examination of cerebrospinal fluid (CSF) is a key parameter in the diagnosis of BM, but the heterogeneity of cells in the CSF has not been elucidated, which limits the current understanding of BM neuroinflammation. In this study, CSF samples were collected from a number of BM patients who were in different stages of disease progression. Single-cell RNA-sequencing (scRNA-seq), with additional bulk transcriptome sequencing, was conducted to decipher the characteristics of CSF cells in BM progression. A total of 18 immune cell clusters in CSF were identified, including two neutrophils, two monocytes, one macrophage, four myeloid dendritic cells, five T cells, one natural killer cell, one B cell, one plasmacytoid dendritic cell, and one plasma cell subtype. Their population profiles and dynamics in the initial onset, remission, and recovery stages during BM progression were also characterized, which showed decreased proportions of myeloid cells and increased proportions of lymphoid cells with disease progression. One novel neutrophil subtype, FFAR2+TNFAIP6+ neutrophils, and one novel monocyte subtype, THBS1+IL1B+ monocytes, were discovered, and their quantity changes positively correlated with the intensity of the inflammatory response in the CSF during BM. In addition, the CSF of BM patients with unsatisfactory therapeutic responses presented with different cell heterogeneity compared to the CSF of BM patients with satisfactory therapeutic responses, and their CSF featured altered intercellular communications and increased proportions of type II myeloid dendritic cells and plasmacytoid dendritic cells. Moreover, the bulk transcriptome profiles of autologous CSF cells and peripheral blood leukocytes of BM patients showed that the immune cells in these two physiological compartments exhibited distinct immune responses under different onset conditions. In particular, the CSF cells showed a high expression of macrophage characteristic genes and a low expression of platelet characteristic genes compared with peripheral blood leukocytes. Our study conducted an in-depth exploration of the characteristics of CSF cells in BM progression, which provided novel insights into immune cell engagement in acute CNS infection.

Scavenger receptor A1 participates in uptake of Leptospira interrogans serovar Autumnalis strain 56606v and inflammation in mouse macrophages.

Leptospirosis, caused by pathogenic Leptospira species, has emerged as a widespread zoonotic disease worldwide. Macrophages mediate the elimination of pathogens through phagocytosis and cytokine production. Scavenger receptor A1 (SR-A1), one of the critical receptors mediating this process, plays a complicated role in innate immunity. However, the role of SR-A1 in the immune response against pathogenic Leptospira invasion is unknown. In the present study, we found that SR-A1 is an important nonopsonic phagocytic receptor on murine macrophages for Leptospira. However, intraperitoneal injection of leptospires into WT mice presented with more apparent jaundice, subcutaneous hemorrhaging, and higher bacteria burdens in blood and tissues than that of SR-A1-/- mice. Exacerbated cytokine and inflammatory mediator levels were also observed in WT mice and higher recruited macrophages in the liver than those of SR-A1-/- mice. Our findings collectively reveal that although beneficial in the uptake of Leptospira by macrophage, SR-A1 might be exploited by Leptospira to modulate inflammatory activation and increase the susceptibility of infection in the host. These results provide our new insights into the innate immune response during early infection by L. interrogans.

A seamless and iterative DNA assembly method named PS-Brick and its assisted metabolic engineering for threonine and 1-propanol production.

BACKGROUND: DNA assembly is an essential technique enabling metabolic engineering and synthetic biology. Combining novel DNA assembly technologies with rational metabolic engineering can facilitate the construction of microbial cell factories. Amino acids and derived biochemicals are important products in industrial biotechnology with wide application and huge markets. DNA assembly scenarios encountered in metabolic engineering for the construction of amino acid and related compound producers, such as design-build-test-learn cycles, construction of precise genetic circuits and repetitive DNA molecules, usually require for iterative, scarless and repetitive sequence assembly methods, respectively. RESULTS: Restriction endonuclease (RE)-assisted strategies constitute one of the major categories of DNA assembly. Here, we developed a Type IIP and IIS RE-assisted method named PS-Brick that comprehensively takes advantage of the properties of PCR fragments and REs for iterative, seamless and repetitive sequence assembly. One round of PS-Brick reaction using purified plasmids and PCR fragments was accomplished within several hours, and transformation of the resultant reaction product from this PS-Brick assembly reaction exhibited high efficiency (104-105 CFUs/µg DNA) and high accuracy (~ 90%). An application of metabolic engineering to threonine production, including the release of feedback regulation, elimination of metabolic bottlenecks, intensification of threonine export and inactivation of threonine catabolism, was stepwise resolved in E. coli by rounds of "design-build-test-learn" cycles through the iterative PS-Brick paradigm, and 45.71 g/L threonine was obtained through fed-batch fermentation. In addition to the value of the iterative character of PS-Brick for sequential strain engineering, seamless cloning enabled precise in-frame fusion for codon saturation mutagenesis and bicistronic design, and the repetitive sequence cloning ability of PS-Brick enabled construction of tandem CRISPR sgRNA arrays for genome editing. Moreover, the heterologous pathway deriving 1-propanol pathway from threonine, composed of Lactococcus lactis kivD and Saccharomyces cerevisiae ADH2, was assembled by one cycle of PS-Brick, resulting in 1.35 g/L 1-propanol in fed-batch fermentation. CONCLUSIONS: To the best of our knowledge, the PS-Brick framework is the first RE-assisted DNA assembly method using the strengths of both Type IIP and IIS REs. In this study, PS-Brick was demonstrated to be an efficient DNA assembly method for pathway construction and genome editing and was successfully applied in design-build-test-learn (DBTL) cycles of metabolic engineering for the production of threonine and threonine-derived 1-propanol. The PS-Brick presents a valuable addition to the current toolbox of synthetic biology and metabolic engineering.

A new model of self-resolving leptospirosis in mice infected with a strain of Leptospira interrogans serovar Autumnalis harboring LPS signaling only through TLR4.

Leptospirosis is an emerging worldwide zoonosis caused by pathogenic Leptospira spp. Our understanding of leptospirosis pathogenesis and host immune response remains limited, while mechanistic studies are hindered by a lack of proper animal models and immunological reagents. Here we established a murine model of acute and self-resolving leptospirosis by infecting 10-week-old C57BL/6 mice with Leptospira interrogans serovar Autumnalis strain 56606v, with characteristic manifestations including jaundice as well as subcutaneous and pulmonary bleeding, but no kidney lesions. We also verified that the lipopolysaccharide (LPS) of strain 56606v signaled through a TLR4-dependent pathway in murine bone marrow-derived macrophages (BMDMs), rather than the previously reported TLR2. In addition, upon infection with Leptospira strain 56606v, TLR4-/- C57BL/6 mice presented more severe jaundice and liver injury as well as higher bacterial loads than WT mice but milder pulmonary hemorrhaging. Molecular studies showed that leptospirosis-related bleeding coincides with the temporal kinetics of iNOS production, while jaundice and liver injury are probably due to insufficiently controlled bacterial loads in the liver. These results suggested that TLR4 is essential in mediating host leptospiral clearance and, to some extent, is associated with pulmonary and subcutaneous hemorrhage, probably through downstream inflammatory mediators, iNOS in particular. Overall, our murine model using immunocompetent mice might facilitate future studies into the pathogenesis of jaundice and bleeding in leptospirosis. Meanwhile, our study suggests the prospect of combining antibiotics and immunosuppressants in the treatment of severe leptospirosis presenting with pulmonary hemorrhage.