Innexin hemichannel activation by Microplitis bicoloratus ecSOD monopolymer reduces ROS.

The extracellular superoxide dismutases (ecSODs) secreted by Microplitis bicoloratus reduce the reactive oxygen species (ROS) stimulated by the Microplitis bicoloratus bracovirus. Here, we demonstrate that the bacterial transferase hexapeptide (hexapep) motif and bacterial-immunoglobulin-like (BIg-like) domain of ecSODs bind to the cell membrane and transiently open hemichannels, facilitating ROS reductions. RNAi-mediated ecSOD silencing in vivo elevated ROS in host hemocytes, impairing parasitoid larva development. In vitro, the ecSOD-monopolymer needed to be membrane bound to open hemichannels. Furthermore, the hexapep motif in the beta-sandwich of ecSOD49 and ecSOD58, and BIg-like domain in the signal peptides of ecSOD67 were required for cell membrane binding. Hexapep motif and BIg-like domain deletions induced ecSODs loss of adhesion and ROS reduction failure. The hexapep motif and BIg-like domain mediated ecSOD binding via upregulating innexins and stabilizing the opened hemichannels. Our findings reveal a mechanism through which ecSOD reduces ROS, which may aid in developing anti-redox therapy.

Metagenome-assembled genome reveals species and functional composition of Jianghan chicken gut microbiota and isolation of Pediococcus acidilactic with probiotic properties.

BACKGROUND: Chickens are one of the most widely farmed animals worldwide and play a crucial role in meat and egg production. Gut microbiota is essential for chickens' health, disease, growth, and egg production. However, native chickens such as Jianghan chickens have better meat and egg production quality than centralized chickens, their intestinal microbial diversity is richer, and the potential gut microbial resources may bring health benefits to the host. RESULTS: The bacterial species composition in the gut microbiota of Jianghan chickens is similar to that of other chicken breeds, with Phocaeicola and Bacteroides being the most abundant bacterial genera. The LEfSe analysis revealed significant differences in species composition and functional profiles between samples from Jingzhou and the other three groups. Functional annotation indicated that the gut microbiota of Jianghan chickens were dominated by metabolic genes, with the highest number of genes related to carbohydrate metabolism. Several antibiotic resistance genes (ARGs) were found, and the composition of ARGs was similar to that of factory-farmed chickens, suggesting that antibiotics were widely present in the gut microbiota of Jianghan chickens. The resistance genes of Jianghan chickens are mainly carried by microorganisms of the Bacteroidota and Bacillota phylum. In addition, more than 829 isolates were selected from the microbiota of Jianghan chickens. Following three rounds of acid and bile tolerance experiments performed on all the isolated strains, it was determined that six strains of Pediococcus acidilactici exhibited consistent tolerance. Further experiments confirmed that three of these strains (A4, B9, and C2) held substantial probiotic potential, with P. acidilactici B9 displaying the highest probiotic potential. CONCLUSIONS: This study elucidates the composition of the intestinal microbiota and functional gene repertoire in Jianghan chickens. Despite the absence of antibiotic supplementation, the intestinal microbial community of Jianghan chickens still demonstrates a profile of antibiotic resistance genes similar to that of intensively reared chickens, suggesting resistance genes are prevalent in free-ranging poultry. Moreover, Jianghan and intensively reared chickens host major resistance genes differently, an aspect seldom explored between free-range and pastured chickens. Furthermore, among the 829 isolates, three strains of P. acidilatici exhibited strong probiotic potential. These findings provide insights into the unique gut microbiota of Jianghan chickens and highlight potential probiotic strains offering benefits to the host. Video Abstract.

Platelet-rich plasma-derived exosomes enhance mesenchymal stem cell paracrine function and nerve regeneration potential.

BACKGROUND: Peripheral nerve injury (PNI) presents a significant clinical challenge, leading to enduring sensory-motor impairments. While mesenchymal stem cell (MSC)-based therapy holds promise for PNI treatment, enhancing its neurotrophic effects remains crucial. Platelet-rich plasma-derived exosomes (PRP-Exo), rich in bioactive molecules for intercellular communication, offer potential for modulating cellular biological activity. METHODS: PRP-Exo was isolated, and its impact on MSC viability was evaluated. The effects of PRP-Exo-treated MSCs (MSCPExo) on Schwann cells (SCs) from injured sciatic nerves and human umbilical vein endothelial cells (HUVECs) were assessed. Furthermore, the conditioned medium from MSCPExo (MSCPExo-CM) was analyzed using a cytokine array and validated through ELISA and Western blot. RESULTS: PRP-Exo enhanced MSC viability. Coculturing MSCPExo with SCs ameliorated apoptosis and promoted SC proliferation following PNI. Similarly, MSCPExo-CM exhibited pro-proliferative, migratory, and angiogenic effects. Cytokine array analysis identified 440 proteins in the MSCPExo secretome, with 155 showing upregulation and 6 showing downregulation, many demonstrating potent pro-regenerative properties. ELISA confirmed the enrichment of several angiotrophic and neurotrophic factors. Additionally, Western blot analysis revealed the activation of the PI3K/Akt signaling pathway in MSCPExo. CONCLUSION: Preconditioning MSCs with PRP-Exo enhanced the paracrine function, particularly augmenting neurotrophic and pro-angiogenic secretions, demonstrating an improved potential for neural repair.

Platelet-rich plasma-derived exosomes boost mesenchymal stem cells to promote peripheral nerve regeneration.

Peripheral nerve injury (PNI) remains a severe clinical problem with debilitating consequences. Mesenchymal stem cell (MSC)-based therapy is promising, but the problems of poor engraftment and insufficient neurotrophic effects need to be overcome. Herein, we isolated platelet-rich plasma-derived exosomes (PRP-Exos), which contain abundant bioactive molecules, and investigated their potential to increase the regenerative capacity of MSCs. We observed that PRP-Exos significantly increased MSC proliferation, viability, and mobility, decreased MSC apoptosis under stress, maintained MSC stemness, and attenuated MSC senescence. In vivo, PRP-Exo-treated MSCs (pExo-MSCs) exhibited an increased retention rate and heightened therapeutic efficacy, as indicated by increased axonal regeneration, remyelination, and recovery of neurological function in a PNI model. In vitro, pExo-MSCs coculture promoted Schwann cell proliferation and dorsal root ganglion axon growth. Moreover, the increased neurotrophic behaviour of pExo-MSCs was mediated by trophic factors, particularly glia-derived neurotrophic factor (GDNF), and PRP-Exos activated the PI3K/Akt signalling pathway in MSCs, leading to the observed phenotypes. These findings demonstrate that PRP-Exos may be novel agents for increasing the ability of MSCs to promote neural repair and regeneration in patients with PNI.

Effect of simplified inoculum agent on performance and microbiome during cow manure-composting at industrial-scale.

A simplified inoculum agent, only comprising Bacillus subtilis and Aspergillus niger, was utilized for industrial-scale cow-manure composting to investigate its impact on composting performance and microbiome. Inoculants elevated the average and peak temperatures by up to 7 and 10 °C, respectively, during the thermophilic stage, reduced organic matter content, and raised germination index. Inoculation also extended the period of composting above 50 °C from 12 to 26 days. Sequencing unveiled significant shifts in microbial diversity, composition, and function. Aspergillus thrived during the mesophilic phase, potentially initiating composting, whereas Bacillus, Lysinibacillus, and Clostridium were enriched during the thermophilic stage. Metagenomic sequencing revealed an increased abundance of carbohydrate-active enzymes and glycometabolism-related genes responsible for lignocellulose degradation and heat generation after inoculation. These enriched microbes and functional genes contributed to organic matter degradation and temperature maintenance during thermophilic stage, expediting composting. This suggests the effectiveness of this simplified inoculum in industrial-level cow-manure composting.

Simulating immunosuppressive mechanism of Microplitis bicoloratus bracovirus coordinately fights Spodoptera frugiperda.

Parasitoid wasps control pests via a precise attack leading to the death of the pest. However, parasitoid larvae exhibit self-protection strategies against bracovirus-induced reactive oxygen species impairment. This has a detrimental effect on pest control. Here, we report a strategy for simulating Microplitis bicoloratus bracovirus using Mix-T dsRNA targeting 14 genes associated with transcription, translation, cell-cell communication, and humoral signaling pathways in the host, and from wasp extracellular superoxide dismutases. We implemented either one-time feeding to the younger instar larvae or spraying once on the corn leaves, to effectively control the invading pest Spodoptera frugiperda. This highlights the conserved principle of "biological pest control," as elucidated by the triple interaction of parasitoid-bracovirus-host in a cooperation strategy of bracovirus against its pest host.

Intervention of muscle-building and antifrailty exercise combined with Baduanjin for frailty of different functional levels: study protocol for a randomised controlled trial.

INTRODUCTION: Frailty has been currently considered as a multidimensional concept, including physical, cognitive and social frailty. Frailty has also been associated with a range of adverse events, which might increase the risks of disability, falls, fractures, delirium and death. Increasing evidence has shown that multicomponent exercise training can improve physical and cognitive function, delay or reverse frailty. However, there is still a lack of exercise intervention programmes for the frail older adults in China. This trial aims to investigate the effects of the muscle-building and antifrailty exercise combined with Baduanjin on the physical function of frail older adults, as well as the effectiveness and safety of the intervention. METHODS AND ANALYSIS: This study is a prospective randomised controlled trial. A total of 192 patients, aged 70 years or older, who are diagnosed as prefrailty or frailty based on the Fried criteria will be included. Prior written and informed consent will be obtained from every subject. These subjects will be randomly assigned to the exercise intervention group (n=96) and the control group (n=96). The exercise intervention group will undergo different exercise programmes for different levels of physical function. They will perform the muscle-building and antifrailty exercise three times per week for 30-60 min for 24 weeks. The control group will implement health education on frailty and maintain the old lifestyle without any intervention.The primary outcomes include the change in frailty and functional capacity, assessed according to the Fried Scale and the Short Physical Performance Battery. Secondary outcomes include the changes in body composition, Activities of daily living, Mini-Mental State Examination, The Geriatric Depression Scale-15 and the haematological indicators. ETHICS STATEMENT: The study has been approved by the Medical Ethics Committee of the PLA General Hospital (approval no.: S2022-600-02). TRIAL REGISTRATION NUMBER: ChiCTR2300070535.

Hospitalization Costs for Patients with Acute Appendicitis: An Update Using Real-World Data from a Large Province in China.

Purpose: The aim of this study is to investigate the factors influencing hospitalization costs for patients diagnosed with acute appendicitis in China. Methods: We conducted a cross-sectional study using data from Provincial Health Statistics Support System Database from S Province in China. This dataset contained all hospital's electronic medical records from January 1, 2015 to December 31, 2018 including both public and private hospitals. The target population was identified based on the principal diagnosis of appendicitis (ICD-10: K35). To examine the impact of various factors on hospitalization costs, we conducted a multivariate linear regression analysis. Furthermore, we employed the Shapley value decomposition method to gain a more comprehensive understanding of the factors that influenced hospitalization costs and their respective levels of importance. Results: Our study comprised 317,200 cases. During the period from 2015 to 2018, the average hospitalization expenses for patients with acute appendicitis were estimated at approximately 7014 RMB (1061 USD), which accounts for a considerable 12% of China's per capita GDP. The results of this study demonstrate a significant correlation between various factors, such as the patient's age, gender, marital status, occupation, payment method, number of complications, treatment method, hospital tier, and ownership, and the total hospitalization costs and subcomponents of hospitalization costs. Notably, the treatment method employed had the most substantial impact on hospitalization costs. Conclusion: To the best of knowledge, this is one of the first studies to investigate the hospitalization costs of acute appendicitis incorporating both patient-level and hospital-level covariates, using a large sample size. To reduce the costs associated with acute appendicitis in China, it is recommended to consider suitable treatment options and explore the option of receiving medical care at lower-tier and privately-owned healthcare facilities.

Research on the Operational Efficiency of Basic Medical Insurance for Urban Employees in China-Based on Three-Stage DEA Model.

Purpose: To conduct a three-stage data envelopment analysis (DEA) of the operational efficiency of urban employee basic medical insurance (UEBMI) across 31 provinces in China from 2012 to 2021. Methods: Utilized a three-stage DEA model, this study measured and evaluated the operational efficiency of urban employee basic medical insurance within China's 31 provinces. Results: The operational efficiency of urban employee basic medical insurance in China was notably low, displaying significant disparities across different regions and periods. Substantial room for improvement exists. Environmental factors, including urbanization level and unemployment rate, wielded a pronounced influence on the operational efficiency of China's employee medical insurance. Conversely, the fiscal revenue and expenditure ratio hindered the enhancement of employee medical insurance efficiency. Conclusion: This study held valuable insights for enhancing the operational efficiency of China's urban employee basic medical insurance. To effectively improve insurance efficiency, the following recommendations are put forth: firstly, relevant departments should escalate resource investments and optimize resource utilization based on rational allocation; secondly, enhance legislation and regulations, fortify fund oversight, and ensure equitable and judicious utilization of the medical insurance fund; finally, leveraging modern high-tech advancements can comprehensively elevate the operational efficiency of the employee medical insurance fund. Ultimately, with the advent of cutting-edge technology, the operational efficiency of employee medical insurance fund can be comprehensively elevated.

Effect of enzyme-assisted fermentation on quality, safety, and microbial community of black soldier fly larvae (Hermetia illucens L.) as a novel protein source.

Considering the significance and scarcity of quality protein, this study aims to obtain a novel safe protein source through fermenting the black soldier fly larvae (BSFL). Lactobacillus crispatus M1027 and Pichia kudriavzevii DHX19 were added as starters together with neutral protease for enzymolysis during fermentation. The results showed that the low pH value (from 6.60 to 3.99), generated by lactic acid accumulation, created an environment where the pathogen could hardly grow. During fermentation, the flavor compound ethyl acetate content reached up to 406.55 mg/L, and the melanization was effectively inhibited by the starters. The increase of trichloroacetic acid-soluble protein content (from 8.73 % to 17.96 %) contributed to improving the absorbability of product by animals after feeding. Notably, the contents of detrimental substances, including total volatile basic nitrogen and histamine, were both below specified limits after fermentation. Simultaneously, the malonic dialdehyde content remained stable during fermentation. Relative abundance of Lactobacillus and Pichia gradually increased and finally dominated in the culture during fermentation, accompanied by pathogens decline below detection limit (1.0 Log cfu/g). Moreover, there was a close relationship between the dynamics of physicochemical indices and microbial succession. Overall, our studies explored a new process to ferment the BSFL paste which would improve the quality and safety of fermented BSFL paste. This research provided theoretical support for fermented insect as a novel protein source.

Developing the Limosilactobacillus reuteri Chassis through an Endogenous Programmable Endonuclease-Based Genome Editing Tool.

Using genetically tractable probiotics to engineer live biotherapeutic products (LBPs) for disease treatment is urgently needed. Limosilactobacillus reuteri is an important vertebrate gut symbiont, which has great potential for developing LBPs. However, in L. reuteri, synthetic biology work is largely limited by the long editing cycle. In this study, we identified a subtype II-A CRISPR-Cas9 system in L. reuteri 03 and found the endogenous Cas9 (LrCas9) recognizing a broad protospacer-adjacent motif (PAM) sequence (3'-NDR; N = A, G, T, C; D = A, G, T; R = A, G). We reprogrammed the LrCas9 for efficient gene deletion (95.46%), point mutation (86.36%), large fragment deletion (40 kb), and gene integration (1743 bp, 73.9%), which uncovered the function of the repeated conserved domains in mucus-binding protein. Moreover, we analyzed the distribution of endogenous endonucleases in 304 strains of L. reuteri and found the existence of programmable endonucleases in 98.36% of L. reuteri strains suggesting the potential to reprogram endogenous endonucleases for genetic manipulation in the majority of L. reuteri strains. In conclusion, this study highlights the development of a new probiotic chassis based on endogenous endonucleases in L. reuteri 03, which paves the way for the development of genome editing tools for functional genetic studies in other L. reuteri. We believe that the development of an endogenous endonuclease-based genetic tool will greatly facilitate the construction of LBPs.

Evaluation of Bruton tyrosine kinase inhibitors monotherapy and combination therapy in lymphocytic leukemia.

BTKi is an effective treatment in chronic lymphocytic leukemia. However, head-to-head clinical trials between BTKi are rare. To explore evidence-based treatment decisions, we conducted this network meta-analysis. We searched in PubMed, Cochrane Library and Embase and selected articles of BTKi treatment in CLL patients, with English restrictions. Objective response rate (ORR), progression-free survival (PFS) and safety were outcomes. Combination therapy and acalabrutinib monotherapy achieved great ORR (greater than 80%). Combination therapy (AO and IR) also performed terrific PFS (> 80%). Compared with ibrutinib monotherapy, zanubrutinib, acalabrutinib and IR showed no significance in overall survival. Diarrhea, hypertension, cardiac events, neutropenia were common adverse events of BTKi therapy. IR had higher incidence of hypertension (0.38, 95% CI 0.28-0.48), and IU was more likely occurred cardiac events. Zanubrutinib monotherapy had lower incidence of total serious adverse reaction (0.42, 95% confidence interval (95% CI): 0.36-0.47),while ibrutinib monotherapy occurred higher adverse reactions of grade ≥ 3 (0.77, 95% CI 0.72-0.82). Although both BTKi monotherapy and combination therapy showed great efficacy, combination therapy did not display priority. Meanwhile, safety of BTKi combination therapy needs to be fully and comprehensively considered.Registration number: CRD42022378732.

CRISPR-Cas-Based Engineering of Probiotics.

Probiotics are the treasure of the microbiology fields. They have been widely used in the food industry, clinical treatment, and other fields. The equivocal health-promoting effects and the unknown action mechanism were the largest obstacles for further probiotic's developed applications. In recent years, various genome editing techniques have been developed and applied to explore the mechanisms and functional modifications of probiotics. As important genome editing tools, CRISPR-Cas systems that have opened new improvements in genome editing dedicated to probiotics. The high efficiency, flexibility, and specificity are the advantages of using CRISPR-Cas systems. Here, we summarize the classification and distribution of CRISPR-Cas systems in probiotics, as well as the editing tools developed on the basis of them. Then, we discuss the genome editing of probiotics based on CRISPR-Cas systems and the applications of the engineered probiotics through CRISPR-Cas systems. Finally, we proposed a design route for CRISPR systems that related to the genetically engineered probiotics.

An archaeal virus-encoded anti-CRISPR protein inhibits type III-B immunity by inhibiting Cas RNP complex turnover.

CRISPR-Cas systems are widespread in prokaryotes and provide adaptive immune against viral infection. Viruses encode a type of proteins called anti-CRISPR to evade the immunity. Here, we identify an archaeal virus-encoded anti-CRISPR protein, AcrIIIB2, that inhibits Type III-B immunity. We find that AcrIIIB2 inhibits Type III-B CRISPR-Cas immunity in vivo regardless of viral early or middle-/late-expressed genes to be targeted. We also demonstrate that AcrIIIB2 interacts with Cmr4α subunit, forming a complex with target RNA and Cmr-α ribonucleoprotein complex (RNP). Furtherly, we discover that AcrIIIB2 inhibits the RNase activity, ssDNase activity and cOA synthesis activity of Cmr-α RNP in vitro under a higher target RNA-to-Cmr-α RNP ratio and has no effect on Cmr-α activities at the target RNA-to-Cmr-α RNP ratio of 1. Our results suggest that once the target RNA is cleaved by Cmr-α RNP, AcrIIIB2 probably inhibits the disassociation of cleaved target RNA, therefore blocking the access of other target RNA substrates. Together, our findings highlight the multiple functions of a novel anti-CRISPR protein on inhibition of the most complicated CRISPR-Cas system targeting the genes involved in the whole life cycle of viruses.

GTF2E2 downregulated by miR-340-5p inhibits the malignant progression of glioblastoma.

Glioblastoma is the most common malignant tumor in the central nervous system. The general transcription factor IIE subunit beta (GTF2E2) is crucial for physiological and pathological functions, but its roles in the malignant biological function of glioma remain ambiguous. CCK-8, colony formation assays, TUNEL assays, cell migration assays, wound-healing assays, and xenograft model were established to investigate the biological functions of GTF2E2 both in vitro and in vivo. GTF2E2 was overexpressed in glioma and was associated with poor prognosis of glioma patients. Biological functions of GTF2E2 were investigated both in vitro and in vi0vo by multiple experiments. Moreover, we explored the possible mechanisms of GTF2E2. In our results, we demonstrated that GTF2E2 could be regulated by miR-340-5p directly or indirectly. CCND1 was transcriptionally affected by GTF2E2 and glioma progression was then regulated. Our data presented the overexpression of GTF2E2 in glioma and indicated the association between GTF2E2 and glioma prognosis. GTF2E2 was found to be regulated by miR-340-5p and thus affect downstream gene expressions and glioma progression. Our results indicate that GTF2E2 might be a potential target in the diagnosis and treatments of glioblastoma.

The involvement of PDIA2 gene in the progression of renal cell carcinoma is potentially through regulation of JNK signaling pathway

Renal cell carcinoma (RCC) with poor prognosis and high incidence rate is a common malignant disease. Current therapies could bring little benefit for the patients with advanced-stage RCC. PDIA2 is an isomerase responsible for protein folding and its role in cancer including RCC is under investigation. In this study, we found that PDIA2 was expressed much higher in RCC tissues than the control but the methylation level of PDIA2 promoter was lower based on the TCGA data. Patients with higher PDIA2 expression exerted worse survival. In clinical specimen, PDIA2 expression was correlated to patients’ clinical factors such as TNM stage (I/II vs III/IV, p = 0.025) and tumor size (≤ 7 cm vs > 7 cm, p = 0.004). Moreover, K-M analysis showed that PDIA2 was associated with patients’ survival in RCC. PDIA2 was expressed much higher in cancer cells A498 than 786-O than that in 293 T cells. After PDIA2 was knocked down, cell proliferation, migration and invasion was potently inhibited. But cell apoptotic rate increased reversely. Furthermore, the efficacy of Sunitinib on RCC cells was strengthened after PDIA2 knockdown. In addition, knockdown of PDIA2 gene leaded to downregulation of levels of JNK1/2, phosphorylated JNK1/2, c-JUN, and Stat3. But this inhibition was partially released when JNK1/2 was overexpressed. In consistent, cell proliferation was also partially recovered. In summary, PDIA2 plays important role in progression of RCC and JNK signaling pathway might be regulated by PDIA2. This study suggests PDIA2 as a candidate target for therapy of RCC (AU)

Metagenome-assembled genomes reveal carbohydrate degradation and element metabolism of microorganisms inhabiting Tengchong hot springs, China.

A hot spring is a distinctive aquatic environment that provides an excellent system to investigate microorganisms and their function in elemental cycling processes. Previous studies of terrestrial hot springs have been mostly focused on the microbial community, one special phylum or category, or genes involved in a particular metabolic step, while little is known about the overall functional metabolic profiles of microorganisms inhabiting the terrestrial hot springs. Here, we analyzed the microbial community structure and their functional genes based on metagenomic sequencing of six selected hot springs with different temperature and pH conditions. We sequenced a total of 11 samples from six hot springs and constructed 162 metagenome-assembled genomes (MAGs) with completeness above 70% and contamination lower than 10%. Crenarchaeota, Euryarchaeota and Aquificae were found to be the dominant phyla. Functional annotation revealed that bacteria encode versatile carbohydrate-active enzymes (CAZYmes) for the degradation of complex polysaccharides, while archaea tend to assimilate C1 compounds through carbon fixation. Under nitrogen-deficient conditions, there were correspondingly fewer genes involved in nitrogen metabolism, while abundant and diverse set of genes participating in sulfur metabolism, particularly those associated with sulfide oxidation and thiosulfate disproportionation. In summary, archaea and bacteria residing in the hot springs display distinct carbon metabolism fate, while sharing the common energy preference through sulfur metabolism. Overall, this research contributes to a better comprehension of biogeochemistry of terrestrial hot springs.