A novel RIPK1 inhibitor reduces GVHD in mice via a nonimmunosuppressive mechanism that restores intestinal homeostasis.

Intestinal epithelial cells (IECs) are implicated in the propagation of T-cell-mediated inflammatory diseases, including graft-versus-host disease (GVHD), but the underlying mechanism remains poorly defined. Here, we report that IECs require receptor-interacting protein kinase-3 (RIPK3) to drive both gastrointestinal (GI) tract and systemic GVHD after allogeneic hematopoietic stem cell transplantation. Selectively inhibiting RIPK3 in IECs markedly reduces GVHD in murine intestine and liver. IEC RIPK3 cooperates with RIPK1 to trigger mixed lineage kinase domain-like protein-independent production of T-cell-recruiting chemokines and major histocompatibility complex (MHC) class II molecules, which amplify and sustain alloreactive T-cell responses. Alloreactive T-cell-produced interferon gamma enhances this RIPK1/RIPK3 action in IECs through a JAK/STAT1-dependent mechanism, creating a feed-forward inflammatory cascade. RIPK1/RIPK3 forms a complex with JAK1 to promote STAT1 activation in IECs. The RIPK1/RIPK3-mediated inflammatory cascade of alloreactive T-cell responses results in intestinal tissue damage, converting the local inflammation into a systemic syndrome. Human patients with severe GVHD showed highly activated RIPK1 in the colon epithelium. Finally, we discover a selective and potent RIPK1 inhibitor (Zharp1-211) that significantly reduces JAK/STAT1-mediated expression of chemokines and MHC class II molecules in IECs, restores intestinal homeostasis, and arrests GVHD without compromising the graft-versus-leukemia (GVL) effect. Thus, targeting RIPK1/RIPK3 in IECs represents an effective nonimmunosuppressive strategy for GVHD treatment and potentially for other diseases involving GI tract inflammation.

Inhibition of neuronal pentraxin 2 relieved epileptic seizure via reducing GluA1 phosphorylation.

Neuronal pentraxin 2 (Nptx2), a member of the synaptic protein family linked to excitatory synaptic formation, is found to be upregulated in epileptic mice, yet its role in epilepsy has been unclear. In vivo, we constructed a mouse model of epilepsy by using kainic acid induction. In vitro experiments, a Mg2+-free medium was used to induce epileptiform discharges in neurons. The results showed that the Nptx2 was upregulated in epileptic mice. Moreover, Nptx2 knockdown reduced the number of seizures and seizure duration. Knocking down Nptx2 not only reduced the number and duration of seizures but also showed a decrease in electroencephalogram amplitude. Behavioral tests indicated improvements in learning and memory abilities after Nptx2 knockdown. The Nissl staining and Timms staining revealed that Nptx2 silencing mitigated epilepsy-induced brain damage. The immunofluorescence staining revealed that Nptx2 absence resulted in a reduction of apoptosis. Nptx2 knockdown reduced Bax, cleaved caspase3, and cleaved caspase9 expression, while increased Bcl-2 expression. Notably, Nptx2 knockdown inhibited GluA1 phosphorylation at the S831 site and reduced the GluA1 membrane expression. The PSD95 expression declined in the epilepsy model, while the Nptx2 knockdown reversed it. Collectively, our study indicated that Nptx2 silencing not only alleviated brain damage and neuron apoptosis but also improved learning and memory ability in epileptic mice, suggesting Nptx2 as a promising target for epilepsy treatment.

Bacillus subtilis WB800N alleviates diabetic wounds in mice by regulating gut microbiota homeostasis and TLR2.

OBJECTIVE: This study aims to investigate the effect of Bacillus subtilis WB800N on diabetic wounds. METHODS: Haematoxylin & eosin (H&E) staining was used to observe the healing of skin wounds. Collagen deposition was assessed by Masson staining. Western blotting and qRT-PCR were used to detect vascular endothelial-related factors (VWF), CD31, TLR2, NLRP3, ASC and Caspase-1 expression. 16S rDNA sequencing detected microbiota distribution. The concentrations of IL-1ß and IL-37 were measured by ELISA. Apoptosis was measured by the TUNEL assay. RESULTS: Compared with the control group, wound healing was delayed in diabetic mice. The wound area in the Bacillus subtilis group decreased more significantly than the diabetic wound group. H&E staining showed that Bacillus subtilis WB800N promoted wound healing and increased re-epithelialization. Masson staining showed that Bacillus subtilis WB800N increased collagen deposition in mice with diabetic wounds. Bacillus subtilis WB800N upregulated VWF and CD31 protein expression in diabetic wounds mice. The 16S rDNA results showed that Bacillus subtilis WB800N reduced the diversity of the gut microbiota of diabetic wounds mice and regulated the microbial composition. At the genus level, Bacillus subtilis WB800N reduced the relative abundance of Muribaculaceae and CG - 005 in diabetic wounds mice, whilst increasing the relative abundance of Lactobacillus. Bacillus subtilis WB800N increased the expression of TLR2, NLRP3, ASC and Caspase-1. Bacillus subtilis WB800N increased the concentrations of IL-1ß and IL-37 in serum. Bacillus subtilis WB800N upregulated cell apoptosis. The TLR2 antagonist Sparstolonin B (SsnB) reduced the expression of TLR2, NLRP3, ASC, Caspase-1, IL-1ß and IL-37 and the apoptosis in diabetic wounds mice, whilst the combined intervention of Bacillus subtilis and SsnB reversed the effect of SsnB treatment alone. CONCLUSION: Bacillus subtilis WB800N alleviated diabetic wound healing by regulating gut microbiota homeostasis and TLR2. SIGNIFICANCE AND IMPACT OF RESEARCH: Our findings might provide potential therapeutic targets for diabetic wounds.

Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas.

Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity.

[Research progress in pathogenesis of hemolytic anemia and its treatment with traditional Chinese medicine].

Hemolytic anemia is a common clinical disease with diverse pathogenesis. In recent years, the incidence of hemolytic anemia is increasing dramatically. The present clinical treatment of immunosuppressive agents or splenectomy is effective to some extent; however, the accompanied clinical adverse reactions are also significant. Traditional Chinese medicine (TCM) has beneficial therapeutic effect on hemolytic anemia, with the obvious advantages including curative effect, less adverse reactions, and low price. The pathogenesis of hemolytic anemia as well as the pharmacological effects and mechanisms of the compound, single herb, and monomer composition of TCM in the treatment of hemolytic anemia were reviewed, aiming to provide the basis for the clinical treatment of hemolytic anemia and the modern research on the mechanism of TCM.

Mechanisms and effective control of physiological browning phenomena in plant cell cultures.

Browning phenomena are ubiquitous in plant cell cultures that severely hamper scientific research and widespread application of plant cell cultures. Up to now, this problem still has not been well controlled due to the unclear browning mechanisms in plant cell cultures. In this paper, the mechanisms were investigated using two typical materials with severe browning phenomena, Taxus chinensis and Glycyrrhiza inflata cells. Our results illustrated that the browning is attributed to a physiological enzymatic reaction, and phenolic biosynthesis regulated by sugar plays a decisive role in the browning. Furthermore, to confirm the specific compounds which participate in the enzymatic browning reaction, transcriptional profile and metabolites of T. chinensis cells, and UV scanning and high-performance liquid chromatography-mass spectrometry (HPLC-MS) profile of the browning compounds extracted from the brown-turned medium were analyzed, flavonoids derived from phenylpropanoid pathway were found to be the main compounds, and myricetin and quercetin were deduced to be the main substrates of the browning reaction. Inhibition of flavonoid biosynthesis can prevent the browning occurrence, and the browning is effectively controlled via blocking flavonoid biosynthesis by gibberellic acid (GA3 ) as an inhibitor, which further confirms that flavonoids mainly contribute to the browning. On the basis above, a model elucidating enzymatic browning mechanisms in plant cell cultures was put forward, and effective control approaches were presented.

Two jasmonate-responsive factors, TcERF12 and TcERF15, respectively act as repressor and activator of tasy gene of taxol biosynthesis in Taxus chinensis.

Methyl jasmonate (MeJA) is one of the most effective inducers of taxol biosynthetic genes, particularly the tasy gene. However, the mechanism underlying the regulation of tasy by MeJA is still unknown. In this study, a 550-bp 5'-flanking sequence was obtained and confirmed as the promoter of the tasy gene. Deletion analysis revealed that the fragment containing a GCC-box from -150 to -131 was the crucial jasmonate (JA)-responsive element, designated as JRE. Using JRE as bait, two binding proteins, namely TcERF12 and TcERF15, were discovered. Sequence alignment and phylogenetic analysis showed that TcERF12 was related to the repressor AtERF3, while TcERF15 was more related to the activator ORA59; these are typical GCC-box-binding ethylene-responsive factors. Both could significantly respond to MeJA for 10 and 4.5 times, respectively, in 0.5 h. When the two TcERFs were overexpressed in Taxus cells, tasy gene expression decreased by 2.1 times in TcERF12-overexpressing cells, but increased by 2.5 times in TcERF15-overexpressing cells. Results indicated that TcERF12 and TcERF15 were negative and positive regulators, respectively, in the JA signal transduction to the tasy gene by binding the GCC-box in the JRE of the tasy promoter. Our results promote further research on regulatory mechanisms of taxol biosynthesis.

Pre-exposure of Triclosan compromise tetracycline-derived antibiotic resistance in methanogenic digestion microbiome.

Triclosan (TCS) and tetracycline (TC) are commonly detected antibacterial agents in sewage and environment matrices. Nonetheless, the impact of sequential exposure to TCS and TC on the methanogenic digestion microbiome remains unknown. In this study, TCS was shown to reduce COD removal efficiency to 69.8%, but alleviated the inhibitive effect of consequent TC-amendment on the digestion microbiome. Interestingly, TCS pre-exposure resulted in abundance increase of acetotrophic Methanosaeta to 2.68%, being 2.91 folds higher than that without TCS amendment. Microbial network analyses showed that TCS pre-exposure caused microorganisms to establish a co-ecological relationship against TC disturbance. Further analyses of total antibiotic resistance genes (ARGs) showed the TCS-derived compromise of TC-induced ARGs enrichment in digestion microbiomes, e.g., 238.2% and 152.1% ARGs increase upon TC addition in digestion microbiomes without and with TCS pre-exposure, respectively. This study provides new insights into the impact of antibacterial agents on the methanogenic digestion microbiome.

Glycoprotein Non-metastatic Melanoma Protein B (GPNMB) Protects Against Neuroinflammation and Neuronal Loss in Pilocarpine-induced Epilepsy via the Regulation of Microglial Polarization.

Epilepsy is a progressive neurodegenerative disease highlighted by recurrent seizures, neuroinflammation, and the loss of neurons. Microglial dysfunction is commonly found in epileptic foci and contributes to neuroinflammation in the initiation and progression of epilepsy. Glycoprotein non-metastatic melanoma protein B (GPNMB), a transmembrane glycoprotein, has been involved in the microglial activation and neuroinflammation response. The present study investigated the functional significance of GPNMB in epilepsy. A proven model of epilepsy was established by intraperitoneal injection of pilocarpine to male Sprague Dawley rats. Lentivirus vectors carrying GPNMB or GPNMB short hairpin RNA (shGPNMB) were injected into the hippocampus to induce overexpression or knockdown of GPNMB. GPNMB expression was significantly upregulated and overexpression of GPNMB in the hippocampus reduced seizure activity and neuronal loss after status epilepticus (SE). We here focused on the effects of GPNMB deficiency on neuronal injury and microglia polarization 28 days after SE. GPNMB knockdown accelerated neuronal damage in the hippocampus, evidenced by increased neuron loss and neuronal cell apoptosis. Following GPNMB knockdown, M1 polarization (iNOS) and secretion of pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α were increased, and M2 polarization (Arg1) and secretion of anti-inflammatory cytokines IL-4, IL-10, and TGF-ß were decreased. BV2 cells were used to further confirm the regulatory role of GPNMB in modulating phenotypic transformations and inflammatory cytokine expressions in microglia. In conclusion, these results indicated that GPNMB suppressed epilepsy through repression of hippocampal neuroinflammation, suggesting that GPNMB might be considered the potential neurotherapeutic target for epilepsy management and play a protective role against epilepsy by modulating the polarization of microglia.

HyperComm: Hypergraph-based communication in multi-agent reinforcement learning.

In the realm of fully cooperative multi-agent reinforcement learning (MARL), effective communication can induce implicit cooperation among agents and improve overall performance. In current communication strategies, agents are allowed to exchange local observations or latent embeddings, which can augment individual local policy inputs and mitigate uncertainty in local decision-making processes. Unfortunately, in previous communication schemes, agents may potentially receive irrelevant information, which increases training difficulty and leads to poor performance in complex settings. Furthermore, most existing works lack the consideration of the impact of small coalitions formed by agents in the multi-agent system. To address these challenges, we propose HyperComm, a novel framework that uses the hypergraph to model the multi-agent system, improving the accuracy and specificity of communication among agents. Our approach brings the concept of hypergraph for the first time in multi-agent communication for MARL. Within this framework, each agent can communicate more effectively with other agents within the same hyperedge, leading to better cooperation in environments with multiple agents. Compared to those state-of-the-art communication-based approaches, HyperComm demonstrates remarkable performance in scenarios involving a large number of agents.

CXCR6 defines therapeutic subtypes of CD4+ cytotoxic T cell lineage for adoptive cell transfer therapy in pediatric B cell acute lymphoblastic leukemia.

The potential of cytotoxic CD4+ T cells and tissue resident memory T cells (Trm) in achieving adult leukemia remission have been highlighted [1,2]. We hypothesized that CXCR6 could serve as a marker for cytotoxic CD4+ Trm cells in the bone marrow (BM) of pediatric B-ALL patients. Flow cytometry (FCM) and published single cell RNA sequencing (scRNA-seq) datasets were employed to characterize CXCR6+CD4+ T cells in the BM and peripheral blood (PB) of pediatric B-ALL patients and healthy donors. FCM, scRNA-seq and co-culture were utilized to explore the cytotoxicity of CXCR6+CD4+ T cells in vitro based on in vitro induction of CXCR6+CD4+ T cells using tumor antigens and peripheral blood mononuclear cells (PBMCs). The ssGSEA based on the cell markers identified according to the in vivo scRNA-seq data, the TARGET-ALL-P2 datasets, and integrated machine learning algorithm were employed to figure out the key cells with prognostic values, followed by simulation of adoptive cell transfer therapy (ACT). Integrated machine learning identified the high-risk cells for disease free survival, and overall survival, while simulation of ACT therapy using CXCR6+CD4+T cells indicated that CXCR6+CD4+ T cells could remodel the bone marrow microenvironments towards anti-tumor. Based on the expression of genes involved in formation of resident memory T cells, CXCR6 is not a marker of resident memory CD4+T cells but defines therapeutic subtypes of CD4+ cytotoxic T cell lineage for pediatric B-ALL.

Elevated enteric putrescine suppresses differentiation of intestinal germinal center B cells.

The dysregulation of B cell maturation and putrescine metabolism has been implicated in various diseases. However, the causal relationship between them and the underlying mechanisms remain unclear. In this study, we investigated the impact of exogenous putrescine on B cell differentiation in the intestinal microenvironment. Our results demonstrated that administration of exogenous putrescine significantly impaired the proportion of germinal center B (GC B) cells in Peyer's patches (PPs) and lamina propria. Through integration of bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq), we identified putrescine-mediated changes in gene drivers, including those involved in the B cell receptor (BCR) signaling pathway and fatty acid oxidation. Furthermore, putrescine drinking disrupted T-B cell interactions and increased reactive oxygen species (ROS) production in B cells. In vitro activation of B cells confirmed the direct suppression of putrescine on GC B cells differentiation and ROS production. Additionally, we explored the Pearson correlations between putrescine biosynthesis activity and B cell infiltration in pan-cancers, revealing negative correlations in colon adenocarcinoma, stomach adenocarcinoma, and lung adenocarcinoma, but positive correlations in liver hepatocellular carcinoma, and breast invasive carcinoma. Our findings provided novel insights into the suppressive effects of elevated enteric putrescine on intestinal B cells differentiation and highlighted the complex and distinctive immunoregulatory role of putrescine in different microenvironments. These findings expand our understanding of the role of polyamines in B cell immunometabolism and related diseases.

Finite-Time Convergent Primal-Dual Gradient Dynamics With Applications to Distributed Optimization.

This article studies the finite-time (FT) convergence of a fast primal-dual gradient dynamics (PDGD), called FT-PDGD, for solving constrained optimization with general constraints and cost functions. Based on the nonsmooth analysis and augmented Lagrangian function, sufficient conditions are established for FT-PDGD to enable the realization of primal-dual optimization in FT. A specific class of nonsmooth sign-preserving functions is defined and analyzed for ensuring FT stability. Particularly, the matrix of linear equations is not required to have a full-row rank and the cost function is not necessary to be strictly convex. By introducing auxiliary variables for general linear inequality constraints, reduced sufficient conditions are further derived for the optimization with linear equality and inequality constraints after transformation. In addition, by the nonsmooth analysis, the switching dynamics evolved in both primal and dual variables are carefully investigated and the upper bound on the convergence time is explicitly provided. Moreover, as applications of FT-PDGD, several FT convergent distributed algorithms are designed to solve distributed optimization with separated and coupled linear equations, respectively. Finally, two case studies are conducted to show the performance of the proposed algorithms.

An accelerated end-to-end method for solving routing problems.

The application of neural network models to solve combinatorial optimization has recently drawn much attention and shown promising results in dealing with similar problems, like Travelling Salesman Problem. The neural network allows to learn solutions based on given problem instances, using reinforcement learning or supervised learning. In this paper, we present a novel end-to-end method to solve routing problems. In specific, we propose a gated cosine-based attention model (GCAM) to train policies, which accelerates the training process and the convergence of policy. Extensive experiments on different scale of routing problems show that the proposed method can achieve faster convergence of the training process than the state-of-the-art deep learning models while achieving solutions of the same quality.

Cumulative Capacitated Colored Traveling Salesman Problem.

A colored traveling salesman problem (CTSP) is a generalization of the well-known multiple traveling salesman problem, which introduces colors to distinguish the accessibility of its cities to salesmen. This work proposes a city/customer-centric model called cumulative capacitated CTSP ( C2 -CTSP) to tackle some practical problems with fast response requirements. Its hypergraph and mathematical programming formulations are developed for the first time. A general variable neighborhood search (GVNS) metaheuristic is designed to solve it. Specifically, greedy backtracking is proposed to initialize a solution taking into account the cumulative cost and two constraints including colors and capacities. Next, 2-swap, reinsertion, and double-bridge operations are randomly selected and carried out to execute the perturbation. Moreover, neighborhood-list-2-opt, relocation move, and generalized partition crossover are organized as variable neighborhood descent to constitute the local search for better solutions. Extensive experiments are conducted to compare the proposed GVNS with four genetic algorithms, two hybrid ant colony systems, two variable neighborhood search methods, and a perturb-based local search in 20 regular and random cases. The statistical results demonstrate that GVNS is superior to all competitors tuned by irace package in terms of both search ability and convergence rate. In addition, the study of six GVNS variants lacking different operators validates the significant role of each corresponding operator in GVNS's outstanding performance.

Metal(loid)s in organic-matter-polluted urban rivers in China: Spatial pattern, ecological risk and reciprocal interactions with aquatic microbiome.

Black-odorous urban rivers can serve as reservoirs for heavy metals and other pollutants, in which sewage-derived labile organic matter triggering the water blackening and odorization largely determine the fate and ecological impact of the heavy metals. Nonetheless, information on the pollution and ecological risk of heavy metals and their reciprocal impact on microbiome in organic matter-polluted urban rivers remain unknown. In this study, sediment samples were collected and analyzed from 173 typical black-odorous urban rivers in 74 cities across China, providing a comprehensive nationwide assessment of heavy metal contamination. The results revealed substantial contamination levels of 6 heavy metals (i.e., Cu, Zn, Pb, Cr, Cd, and Li), with average concentrations ranging from 1.85 to 6.90 times higher than their respective background values in soil. Notably, the southern, eastern, and central regions of China exhibited particularly elevated contamination levels. In comparison to oligotrophic and eutrophic waters, the black-odorous urban rivers triggered by organic matter exhibited significantly higher proportions of the unstable form of these heavy metals, indicating elevated ecological risks. Further analyses suggested the critical roles of organic matter in shaping the form and bioavailability of heavy metals through fueling microbial processes. In addition, most heavy metals had significantly higher but varied impact on the prokaryotic populations relative to eukaryotes.

Precedence-Constrained Colored Traveling Salesman Problem: An Augmented Variable Neighborhood Search Approach.

A colored traveling salesman problem (CTSP) as a generalization of the well-known multiple traveling salesman problem utilizes colors to distinguish the accessibility of individual cities to salesmen. This work formulates a precedence-constrained CTSP (PCTSP) over hypergraphs with asymmetric city distances. It is capable of modeling the problems with operations or activities constrained to precedence relationships in many applications. Two types of precedence constraints are taken into account, i.e., 1) among individual cities and 2) among city clusters. An augmented variable neighborhood search (VNS) called POPMUSIC-based VNS (PVNS) is proposed as a main framework for solving PCTSP. It harnesses a partial optimization metaheuristic under special intensification conditions to prepare candidate sets. Moreover, a topological sort-based greedy algorithm is developed to obtain a feasible solution at the initialization phase. Next, mutation and multi-insertion of constraint-preserving exchanges are combined to produce different neighborhoods of the current solution. Two kinds of constraint-preserving k -exchange are adopted to serve as a strong local search means. Extensive experiments are conducted on 34 cases. For the sake of comparison, Lin-Kernighan heuristic, two genetic algorithms and three VNS methods are adapted to PCTSP and fine-tuned by using an automatic algorithm configurator-irace package. The experimental results show that PVNS outperforms them in terms of both search ability and convergence rate. In addition, the study of four PVNS variants each lacking an important operator reveals that all operators play significant roles in PVNS.

Rosmarinic acid inhibits cell proliferation, migration, and invasion and induces apoptosis in human glioma cells.

There is a growing evidence that Fyn kinase is upregulated in glioblastoma multiforme (GBM), where it plays a key role in tumor proliferation and invasion. In the present study, the antitumor effects of rosmarinic acid (RA), a Fyn inhibitor, were explored in human­derived U251 and U343 glioma cell lines. These cells were treated with various concentrations of RA to determine its effects on proliferation, migration, invasion, apoptosis, and gene and protein expression levels. The CCK­8 assay revealed that RA significantly suppressed cell viability of U251 and U343 cells. Furthermore, RA significantly reduced proliferation rates, inhibited migration and invasion, and decreased the expression levels of invasion­related factors, such as matrix metalloproteinase (MMP)­2 and MMP­9. TUNEL staining revealed that RA resulted in a dose­dependent increase of U251 and U343 cell apoptosis. In line with this finding, the expression of apoptosis suppressor protein Bcl­2 was downregulated and that of the pro­apoptotic proteins Bax and cleaved caspase­3 was increased. In addition, it was revealed that the phosphatidylinositol 3­kinase (PI3K)/Akt/nuclear factor­κB (NF­κB) signaling pathway was involved in RA­induced cytotoxicity in U251 and U343 cells. Collectively, the present study suggested RA as a drug candidate for the treatment of GBM.

Identification and Comparative Analysis of Long Non-coding RNAs in High- and Low-Fecundity Goat Ovaries During Estrus.

The ovary is the most important reproductive organ in goats and directly affects the fecundity. Long non-coding RNAs (lncRNAs) are involved in the biological process of oocyte maturation. However, in the context of reproduction in goats, few studies have explored the regulation of lncRNAs. Therefore, we herein used the ovaries of high and low fecundity Leizhou black goats to identify differentially expressed lncRNAs (DElncRNAs) by high-throughput RNA sequencing; moreover, we analyzed the target genes of lncRNAs by functional annotation to explore the role of DElncRNAs in ovarian development. Twenty DElncRNAs were identified, of which six were significantly upregulated and 14 were significantly downregulated in high fecundity goats. Gene Ontology analyses suggested that MSTRG.3782 positively influences the expression of the corresponding gene API5, exerting regulative effects on the development of follicles, through which litter size might show variations. The target gene KRR1 of ENSCHIT00000001883 is significantly enriched in cell components, and ENSCHIT00000001883 may regulate cell growth and thus affect follicular development. Further, as per Kyoto Encyclopedia of Genes and Genomes pathway analyses, MSTRG.2938 was found to be significantly enriched, and we speculate that MSTRG.2938 could regulate ribosomal biogenesis in the pre-snoRNP complex as well as cell transformation in eukaryotes. Quantitative real-time PCR results were consistent with sequencing data. To conclude, our research results indicate that some lncRNAs play a key role in regulating follicle development and cell growth during goat' s ovarian development.

Branched-Chain Amino Acids Catabolism Pathway Regulation Plays a Critical Role in the Improvement of Leukopenia Induced by Cyclophosphamide in 4T1 Tumor-Bearing Mice Treated With Lvjiaobuxue Granule.

Background: Cyclophosphamide is a common tumor chemotherapy drug used to treat various cancers. However, the resulting immunosuppression leads to leukopenia, which is a serious limiting factor in clinical application. Therefore, the introduction of immunomodulators as adjuvant therapy may help to reduce the hematological side effects of cyclophosphamide. Lvjiaobuxue granule has been widely used in the clinical treatment of gynecological diseases such as anemia and irregular menstruation. Recently, it has been found to increase the function of white blood cells, but its mechanism of action is still unclear. We aimed to reveal the mechanisms of Lvjiaobuxue granule against acute leukopenia by an integrated strategy combining metabolomics with network pharmacology. Methods: Subcutaneously inoculated 4T1 breast cancer cells to prepare tumor-bearing mice, intraperitoneal injection of cyclophosphamide to establish a 4T1 tumor-bearing mice leukopenia animal model, using pharmacodynamic indicators, metabolomics, network pharmacology and molecular biology and other technical methods. To comprehensively and systematically elucidate the effect and mechanism of Lvjiaobuxue granule in improving cyclophosphamide-induced leukopenia in 4T1 tumor-bearing mice. Results: Lvjiaobuxue granule can improve the blood routine parameters and organ index levels of the leukopenia model of 4T1 tumor-bearing mice. Metabolomics studies revealed that 15 endogenous metabolites in the spleen of mice were considered as potential biomarkers of Lvjiaobuxue granule for their protective effect. Metabonomics and network pharmacology integrated analysis indicated that Lvjiaobuxue granule exerted the leukocyte elevation activity by inhibiting the branched-chain amino acids (BCAAs) degradation pathway and increasing the levels of valine, leucine and isoleucine. The results of molecular biology also showed that Lvjiaobuxue granule can significantly regulate the key enzymes in the catabolism of BCAAs, which further illustrates the importance of BCAAs in improving leukopenia. Conclusion: Lvjiaobuxue granule exerts obvious pharmacological effects on the leukopenia model of 4T1 tumor-bearing mice induced by cyclophosphamide, which could be mediated by regulating the branched-chain amino acid degradation pathway and the levels of valine, leucine and isoleucine.