Identification of signaling pathways induced by porcine parvovirus in porcine kidney-15 cells based on weighted correlation network analysis.

Porcine parvovirus (PPV) is an important trigger of reproductive issues in pigs. Infection of the porcine kidney-15 (PK-15) cells with PPV induces cell death and inflammation. To explore the impact of PPV infection on gene expression in PK-15 cells and to identify the associated signaling pathways, we performed weighted correlation network analysis (WGCNA) on both PPV-infected and uninfected cells. We identified the blue and brown modules, with the blue module demonstrating decreased gene expression and the brown module showing increased gene expression at 48 hr post-PPV induction. Gene Ontology (GO) analysis revealed that genes in the blue module were predominantly associated with cellular components, while those in the brown module were enriched in biological processes, including the immunological response to PPV infection. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the modules indicated that the "Cytokine-cytokine receptor interaction" was linked to PPV. Furthermore, we screened 124 target genes involved in more than 100 pathways that interact with C-X-C motif chemokine ligand 8 (CXCL8), which plays an essential role in regulating numerous biological processes. The signaling pathways we identified facilitate a comprehensive examination of the mechanisms of interactions initiated by PPV infection.

Hyperuricemia Facilitates Uric Acid-Mediated Vascular Endothelial Cell Damage by Inhibiting Mitophagy.

Hyperuricemia remains an elusive factor in the pathogenesis of vascular endothelial injury. This study elucidates the role of hydroxychloroquine (HCQ) in the context of uric acid (UA)-induced vascular endothelial cell damage. Human umbilical vein endothelial cells (HUVECs) were exposed to varying UA concentrations (6 mg/dL to 50 mg/dL) for 48 h, or to 50 mg/dL UA for different time points (6 to 72 h). We observed a concentration- and time-dependent inhibition of cell proliferation, particularly at 40 mg/dL and 50 mg/dL UA. The autophagy marker LC3 exhibited reduced fluorescence intensity post-UA treatment, along with decreased expression of LC3-II/LC3I, beclin1, and p62, indicating impaired autophagy. The mechanistic exploration revealed that HCQ, in conjunction with the mitochondrial autophagy inhibitor Cyclosporine A (CsA), exacerbated the inhibitory effects of UA on HUVEC autophagy. This was evidenced by a further reduction in mitochondrial autophagy-related proteins and diminished fluorescence of LC3-II/LC3-I and Parkin, culminating in suppressed cell proliferation and accelerated cell senescence and apoptosis. Conversely, the co-treatment with the mitochondrial autophagy inducer carbonyl cyanide m-chlorophenyl hydrazine (CCCP) and HCQ mitigated the detrimental effects of UA on HUVEC autophagy. This intervention led to increased expression of PINK1, Parkin, Bnip3, and Nix, along with enhanced fluorescence of LC3-II/LC3-I and Parkin, effectively inhibiting cell senescence and apoptosis while promoting cell proliferation. In conclusion, our findings underscore the pivotal role of HCQ in modulating UA-mediated vascular endothelial cell damage through the inhibition of mitophagy, providing novel insights into the therapeutic potential of targeting HCQ in the management of hyperuricemia-associated vascular complications.

Unraveling the molecular landscape of breast muscle development in domestic Yuzhong pigeons and European meat pigeon: Insights from Iso-seq and RNA-seq analysis.

The mechanisms governing gene regulation in domestic Yuzhong pigeon breast muscle development remain largely elusive. Here, we conducted a comparative analysis using Iso-seq and RNA-seq data from domestic Yuzhong pigeons and European meat pigeons to uncover signaling pathways and genes involved in breast muscle development. The Iso-seq data from domestic Yuzhong pigeons yielded 131,377,075 subreads, resulting in 16,587 non-redundant high-quality full-length transcripts post-correction. Furthermore, utilizing pfam, CPC, PLEK, and CPAT, we predicted 5575, 4973, 2333, and 4336 lncRNAs, respectively. Notably, several genes potentially implicated in breast muscle development were identified, including tropomyosin beta chain, myosin regulatory light chain 2, and myosin binding protein C. KEGG enrichment analysis revealed critical signaling pathways in breast muscle development, spanning carbon metabolism, biosynthesis of amino acids, glycolysis/gluconeogenesis, estrogen signaling, PI3K-AKT signaling, protein processing in the endoplasmic reticulum, oxidative phosphorylation, pentose phosphate pathway, fructose and mannose metabolism, and tight junctions. These findings offer insights into the biological processes driving breast muscle development in domestic Yuzhong pigeon, contributing to our understanding of this complex phenomenon.

Isolation and genomic characterization of one novel goose astrovirus causing acute gosling gout in China.

Novel goose astrovirus (NGAstV) is a member of the genus Avain Avastrovirus (AAstV) and the family Astroviridae. NGAstV-associated gout disease has caused huge economic losses to the goose industry worldwide. Since early 2020, NGAstV infections characterized by articular and visceral gout emerged continuously in China. Herein, we isolated a GAstV strain from goslings with fatal gout disease and sequenced its complete genome nucleotide sequence. Then we conducted systematic genetic diversity and evolutionary analysis. The results demonstrated that two genotypic species of GAstV (GAstV-I and GAstV-II) were circulating in China, and GAstV-II sub-genotype IId had become the dominant one. Multiple alignments of amino acid sequences of GAstV capsid protein revealed that several characteristic mutations (E456D, A464N, and L540Q) in GAstV-II d strains, as well as additional residues in the newly identified isolate which varied over time. These findings enrich the understanding of the genetic diversity and evolution of GAstV and may facilitate the development of effective preventive strategies.

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 µm nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

IFN-γ-STAT1-mediated CD8+ T-cell-neural stem cell cross talk controls astrogliogenesis after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) causes nearly all patients to suffer from protracted disabilities. An emerging therapeutic strategy involving the recruitment of endogenous neural stem cells (NSCs) has been developed. However, endogenous NSCs in the adult spinal cord differentiate into mostly astrocytes after traumatic injury, forming glial scars, which is a major cause of regeneration failure in SCI. Thus, understanding which factors drive the activation and differentiation of endogenous NSCs after SCI is critical for developing therapeutic drugs. METHODS: The infiltration, state, and location of CD8+ T cells in spinal cord after traumatic injury were analyzed by flow cytometry and immunofluorescence (IF) staining. The Basso Mouse Scale (BMS) scores and rotarod testing were used for motor behavioral analysis. NSCs were co-cultured with CD8+ T cells. EdU assay was used to detect proliferative cells. Western blotting was used to analyze the expression levels of STAT1, p-STAT1, and p27. ChIP-seq and ChIP-qRT-PCR analyses were used to detect the downstream of STAT1. Nestin-CreERT2::Ai9 transgenic mice were used to genetic lineage tracing of Nestin+ NSCs after SCI in vivo. RESULTS: A prolonged increase of activated CD8+ T cells occurs in the injured spinal cords. The behavioral analysis demonstrated that the administration of an anti-CD8 antibody promotes the recovery of locomotor function. Then, we discovered that CD8+ T cells suppressed the proliferation of NSCs and promoted the differentiation of NSCs into astrocytes by the IFN-γ-STAT1 pathway in vitro. ChIP-seq and ChIP-qRT-PCR analysis revealed that STAT1 could directly bind to the promoters of astrocyte marker genes GFAP and Aldh1l1. Genetic lineage tracing of Nestin+ NSCs demonstrated that most NSCs differentiated into astrocytes following SCI. Depleting CD8+ T cells reduced the differentiation of NSCs into astrocytes and instead promoted the differentiation of NSCs into oligodendrocytes. CONCLUSION: In conclusion, CD8+ T cells suppressed the proliferation of NSCs and promoted the differentiation of NSCs into astrocytes by the IFN-γ-STAT1-GFAP/Aldhl1l axis. Our study identifies INF-γ as a critical mediator of CD8+ T-cell-NSC cross talk and a potential node for therapeutic intervention in SCI.

Surgical site wound infection, and other postoperative problems after coronary artery bypass grafting in subjects with chronic obstructive pulmonary disease: A meta-analysis.

We performed a meta-analysis to evaluate the effect of chronic obstructive pulmonary disease on surgical site wound infection, and other postoperative problems after coronary artery bypass grafting. A systematic literature search up to April 2022 was performed and 37 444 subjects with coronary artery bypass grafting at the baseline of the studies; 4320 of them were with the chronic obstructive pulmonary disease, and 33 124 were without chronic obstructive pulmonary disease. Odds ratio (OR), and mean difference (MD) with 95% confidence intervals (CIs) were calculated to assess the effect of chronic obstructive pulmonary disease on surgical site wound infection, and other postoperative problems after coronary artery bypass grafting using the dichotomous, and contentious methods with a random or fixed-effect model. The chronic obstructive pulmonary disease subjects had a significantly higher surgical site wound infection (OR, 1.27; 95% CI, 1.01-1.60, P = 0.04), respiratory failure (OR, 1.84; 95% CI, 1.55-2.18, P < 0.001), mortality (OR, 1.61; 95% CI, 1.37-1.89, P < 0.001), pneumonia (OR, 2.30; 95% CI, 1.97-2.68, P < 0.001), pleural effusion (OR, 1.78; 95% CI, 1.12-2.83, P = 0.02), stroke (OR, 1.99; 95% CI, 1.17-3.36, P = 0.01), and length of intensive care unit stay (MD, 0.73; 95% CI, 0.19-1.26, P = 0.008) after coronary artery bypass grafting compared with subjects without chronic obstructive pulmonary disease. However, chronic obstructive pulmonary disease subjects did not show any significant difference in length of hospital stay (MD, 0.83; 95% CI, -0.01 to 1.67, P = 0.05), and pneumothorax (OR, 1.59; 95% CI, 0.98-2.59, P = 0.06) after coronary artery bypass grafting compared with subjects without chronic obstructive pulmonary disease. The chronic obstructive pulmonary disease subjects had a significantly higher surgical site wound infection, respiratory failure, mortality, pneumonia, pleural effusion, stroke, and length of intensive care unit stay, and no significant difference in length of hospital stay, and pneumothorax after coronary artery bypass grafting compared with subjects without chronic obstructive pulmonary disease. The analysis of outcomes should be with caution because of the low sample size of 1 out of 11 studies in the meta-analysis and a low number of studies in certain comparisons.

Single-cell RNA sequencing analysis reveals the relationship of bone marrow and osteopenia in STZ-induced type 1 diabetic mice.

INTRODUCTION: Type 1 diabetes (T1D) is a multifactorial autoimmune disease. Broad knowledge about the genetics, epidemiology and clinical management of T1D has been achieved, but understandings about the cell varieties in the bone marrow during T1D remain limited. OBJECTIVES: We aimed to present a profile of the bone marrow cells and reveal the relationship of bone marrow and osteopenia in streptozotocin (STZ)-induced T1D mice. METHODS: The whole bone marrow cells from the femurs and tibias of healthy (group C) and STZ-induced T1D mice (group D) were collected for single-cell RNA sequencing analysis. Single-cell flow cytometry and immunohistochemistry were performed to confirm the proportional changes among bone marrow neutrophils (BM-neutrophils) (Cxcr2+, Ly6g+) and B lymphocytes (Cd19+). X-ray and micro-CT were performed to detect bone mineral density. The correlation between the ratio of BM-neutrophils/B lymphocytes and osteopenia in STZ-induced T1D mice was analyzed by nonparametric Spearman correlation analysis. RESULTS: The bone marrow cells in groups C and D were divided into 12 clusters, and 249 differentially expressed genes were found. The diversity of CD45+ immune cells between groups C and D were greatly affected: the proportion of BM-neutrophils showed a significant increase while the proportion of B lymphocytes in group D showed a significant decrease. X-ray and micro-CT analyses confirmed that osteopenia occurred in group D mice. In addition, the results of single-cell flow cytometry and correlation analysis showed that the ratio of BM-neutrophils/B lymphocytes negatively correlated with osteopenia in STZ-induced T1D mice. CONCLUSION: A single-cell RNA sequencing analysis revealed the profile and heterogeneity of bone marrow immune cells in STZ-induced T1D mice for the first time. The ratio of BM-neutrophils/B lymphocytes negatively correlated with osteopenia in STZ-induced T1D mice, which may enhance understanding for treating T1D and preventing T1D-induced osteopenia.

Resveratrol suppresses microglial activation and promotes functional recovery of traumatic spinal cord via improving intestinal microbiota.

Spinal cord injury (SCI) can change the intestinal microbiota pattern and corresponding metabolites, which in turn affect the prognosis of SCI. Among many metabolites, short-chain fatty acids (SCFAs) are critical for neurological recovery after SCI. Recent research has shown that resveratrol exerts anti-inflammatory properties. But it is unknown if the anti-inflammatory properties of resveratrol are associated with intestinal microbiota and metabolites. We thus investigate the alteration in gut microbiota and the consequent change of SCFAs following resveratrol treatment. The SCI mouse models with retention of gut microbiota (donor) and depletion of gut microbiota (recipient) were established. Fecal microbiota transplantation from donors to recipients was performed with intragastrical administration. Spinal cord tissues of mice were examined by H&E, Nissl, and immunofluorescence stainings. The expressions of the inflammatory profile were examined by qPCR and cytometric bead array. Fecal samples of mice were collected and analyzed with 16S rRNA sequencing. The results showed that resveratrol inhibited the microglial activation and promoted the functional recovery of SCI. The analysis of intestinal microbiota and metabolites indicated that SCI caused dysbiosis and the decrease in butyrate, while resveratrol restored microbiota pattern, reversed intestinal dysbiosis, and increased the concentration of butyrate. Both fecal supernatants from resveratrol-treated donors and butyrate suppressed the expression of pro-inflammatory genes in BV2 microglia. Our result demonstrated that fecal microbiota transplantation from resveratrol-treated donors had beneficial effects on the functional recovery of SCI. One mechanism of resveratrol effects was to restore the disrupted gut microbiota and butyrate.

Chitosan/Calcium-Coated Ginsenoside Rb1 Phosphate Flower-like Microparticles as an Adjuvant to Enhance Immune Responses.

Infectious bursal disease (IBD) is a highly contagious immunocompromising disorder that caused great economic losses in the poultry industry. The field-level control over IBD is primarily via vaccination. The development of a highly effective IBV vaccine has drawn great attention worldwide. Chitosan/Calcium Phosphate (CS/CaP) nanoparticle was a newly developed effective biological delivery system for drug and antigen. Ginsenoside Rb1 is one of the main bioactive components of ginseng root extract, which has antioxidant, anti-inflammatory and immunological enhancement effects. Until now, the combined effect of CS/CaP and ginsenoside Rb1 on the chicken immune response had remained unknown. In this study, the GRb1 and IL-4 were encapsulated into Calcium phosphate and chitosan core structure nanoparticles microspheres (GRb1/IL-4@CS/CaP), and the effect of a newly developed delivery system on an infectious bursal disease virus (IBDV) attenuated vaccine was further evaluated. The results demonstrated that GRb1/IL-4@CS/CaP treatment could induce the activation of chicken dendritic cells (DCs), with the upregulated expression of MHCII and CD80, and the increased production of IL-1ß and TNF-α. Importantly, GRb1/IL-4@CS/CaP could trigger a higher level of IBDV-specific IgG and a higher ratio of IgG2a/IgG1 than the traditional adjuvant groups, promoting the production of cytokine, including IFN-γ, TNF-α, IL-4, IL-6, IL-1α, and IL-1ß, in chicken serum after 28 d and 42 d post-vaccine. Taken in all, GRb1/IL-4@CS/CaP could elicit prolonged vigorous immune responses for IBDV attenuated vaccine in chicken, which might provide an effective adjuvant system for avian vaccine development.

Assessment of the Endophytic Fungal Composition of Lactobacillus plantarum and Enterococcus faecalis-Fermented Astragalus membranaceus Using Single-Molecule, Real-Time Sequencing Technology.

Endophytic fungus represents microorganisms existing within the healthy plant organs, which can significantly influence metabolic product production in plants, a process with great research value and broad prospects for development. To investigate the effect of fermentation with probiotic cultures on the endophytic fungal diversity and composition of Astragalus membranaceus, we used single-molecular, real-time sequencing (Pacific Biosciences) for 18S ribosomal RNA (rRNA) sequencing. The results showed that the endophytic fungi of A. membranaceus mainly belonged to Aspergillus, Penicillium, Cystofilobasidium, Candida, Guehomyces, and Wallemia. Furthermore, the endophytic fungal diversity and abundance of A. membranaceus were more variable after fermentation with Enterococcus faecium and/or Lactobacillus plantarum. Our data lays a solid and comprehensive foundation for further exploration of endophytic fungi from A. membranaceus as potential sources of functional compounds.

Single-cell transcriptome analysis reveals the immune heterogeneity and the repopulation of microglia by Hif1α in mice after spinal cord injury.

Neuroinflammation is regarded as a vital pathological process in spinal cord injury (SCI), which removes damaged tissue, secretes cytokines, and facilitates regeneration. Repopulation of microglia has been shown to favor recovery from SCI. However, the origin and regulatory factors of microglia repopulation after SCI remain unknown. Here, we used single-cell RNA sequencing to portray the dynamic transcriptional landscape of immune cells during the early and late phases of SCI in mice. B cells and migDCs, located in the meninges under physiological conditions, are involved in immune surveillance. Microglia quickly reduced, and peripheral myeloid cells infiltrated three days-post-injury (dpi). At 14 dpi, microglia repopulated, myeloid cells were reduced, and lymphocytes infiltrated. Importantly, genetic lineage tracing of nestin+ and Cx3cr1+ cells in vivo showed that the repopulation of microglia was derived from residual microglia after SCI. We found that residual microglia regress to a developmental growth state in the early stages after SCI. Hif1α promotes microglial proliferation. Conditional ablation of Hif1α in microglia causes larger lesion sizes, fewer axon fibers, and impaired functional recovery in the late stages after SCI. Our results mapped the immune heterogeneity in SCI and raised the possibility that targeting Hif1α may help in axon regeneration and functional recovery after SCI.

Sesquiterpenes and Monoterpenes from the Leaves and Stems of Illicium simonsii and Their Antibacterial Activity.

Two undescribed ether derivatives of sesquiterpenes, 1-ethoxycaryolane-1, 9ß-diol (1) and 2-ethoxyclovane-2ß, 9α-diol (3), and one new monoterpene glycoside, p-menthane-1α,2α,8-triol-4-O-ß-D-glucoside (5), were obtained, together with eight known compounds from the stems and leaves of I. simonsii. Their structures were elucidated by spectroscopic methods. Compounds 1-11 were evaluated for their potency against Staphylococcus aureus and clinical methicillin-resistant S. aureus (MRSA). Among them, compound 3 was weakly active against S. aureus (MIC = 128 µg/mL), and compounds 6 and 7 exhibited good antibacterial activity against S. aureus and MRSA (MICs = 2-8 µg/mL). A primary mechanism study revealed that compounds 6 and 7 could kill bacteria by destroying bacterial cell membranes. Moreover, compounds 6 and 7 were not susceptible to drug resistance development.

A Novel Age-Related Circular RNA Circ-ATXN2 Inhibits Proliferation, Promotes Cell Death and Adipogenesis in Rat Adipose Tissue-Derived Stromal Cells.

Adipose tissue-derived stromal cells are promising candidates investigating the stem cell-related treatment. However, their proportion and utility in the human body decline with time, rendering stem cells incompetent to complete repair processes in vivo. The involvement of circRNAs in the aging process is poorly understood. Rat subcutaneous adipose tissue from 10-week-old and 27-month-old rats were used for hematoxylin and eosin (H and E) staining, TUNEL staining, and circRNA sequencing. Rat adipose tissue-derived stromal cells were cultured and overexpressed with circ-ATXN2. Proliferation was examined using xCELLigence real-time cell analysis, EdU staining, and cell cycle assay. Apoptosis was induced by CoCl2 and examined using flow cytometry. RT-PCR assay and Oil Red O staining were used to measure adipogenesis at 48 h and 14 days, respectively. H and E staining showed that the diameter of adipocytes increased; however, the number of cells decreased in old rats. TUNEL staining showed that the proportion of apoptotic cells was increased in old rats. A total of 4,860 and 4,952 circRNAs was detected in young and old rats, respectively. Among them, 67 circRNAs exhibited divergent expression between the two groups (fold change ≥2, p ≤ 0.05), of which 33 were upregulated (49.3%) and 34 were downregulated (50.7%). The proliferation of circ-ATXN2-overexpressing cells decreased significantly in vitro, which was further validated by xCELLigence real-time cell analysis, EdU staining, and cell cycle assay. Overexpression of circ-ATXN2 significantly increased the total apoptotic rate from 5.78 ± 0.46% to 11.97 ± 1.61%, early apoptotic rate from 1.76 ± 0.22% to 5.50 ± 0.66%, and late apoptosis rate from 4.02 ± 0.25% to 6.47 ± 1.06% in adipose tissue-derived stromal cells. Furthermore, in circ-ATXN2-overexpressing cells, RT-PCR assay revealed that the expression levels of adipose differentiation-related genes PPARγ and CEBP/α were increased and the Oil Red O staining assay showed more lipid droplets. Our study revealed the expression profile of circRNAs in the adipose tissue of old rats. We found a novel age-related circular RNA-circ-ATXN2-that inhibits proliferation and promotes cell death and adipogenesis in rat adipose tissue-derived stromal cells.

Improved strategy for jet-in-air cell sorting with high purity, yield, viability, and genome stability.

Flow cytometric sorting is a vital tool in biological research and clinical diagnostics. Theoretically, a high-speed jet-in-air sorter is a fluorescent-activated cell sorting sorter that ideally processes cells with high purity, yield, and viability. However, high-speed jet-in-air sorting is a complex process due to its inherent requirements for high fluidic stability and electronic and timing precision. Here, we report that an additional manual correction of drop delay leads to improved cell yield. Adding 2% FBS to the loading buffer had no significant effect on the fate of sorted cells in 4 h. However, the addition of a suitable concentration of FBS/BSA in the collecting buffer resulted in a notable increase in cell count and proliferation and a significant decrease in cell apoptosis for cell lines and primary cells. Moreover, the level of gene expression remained steady in the 5% FBS collecting buffer. In summary, here we demonstrate techniques that can be easily followed to refine sorted yields of healthy cells.

HMGB1 Activates Myeloid Dendritic Cells by Up-Regulating mTOR Pathway in Systemic Lupus Erythematosus.

Research has shown that HMGB1 can activate dendritic cells (DCs), but its molecular mechanisms are not clear. In this study, we reported that the myeloid dendritic cells (mDCs) were activated in the peripheral blood of SLE patients, and the activation of mDCs was associated with the up-regulation of HMGB1 and mTOR. After stimulated by HMGB1, expression of mTOR and its substrates P70S6K and 4EBP1 in dendritic cells increased considerably (P < 0.01). The expression of HLA-DR, CD40, and CD86 on dendritic cells also significantly increased following these stimuli (P < 0.01). In addition, stimulation with HMGB1 enhanced cytokine (IL-1ß, IL-6, and TNF-a) production in dendritic cells. In contrast, the HMGB1-mediated expression of HLA-DR, CD40, and CD86 on dendritic cells and production of IL-1ß, IL-6, and TNF-α were reduced by rapamycin. Rapamycin can inhibit HMGB1-induced activation of mDCs and secretion of pro-inflammatory cytokines. These findings indicated that HMGB1activates mDCs by up-regulating the mTOR pathway in SLE.

Phosphoproteome Profiling Revealed the Importance of mTOR Inhibition on CDK1 Activation to Further Regulate Cell Cycle Progression.

The mammalian target of rapamycin (mTOR) functions as a critical regulator of cell cycle progression. However, the underlying mechanism by which mTOR regulates cell cycle progression remains elusive. In this study, we used stable isotope labeling of amino acids in cell culture with a two-step strategy for phosphopeptide enrichment and high-throughput quantitative mass spectrometry to perform a global phosphoproteome analysis of mTOR inhibition by rapamycin. By monitoring the phosphoproteome alterations upon rapamycin treatment, downregulation of mTOR signaling pathway was detected and enriched. Further functional analysis of phosphoproteome revealed the involvement of cell cycle events. Specifically, the elevated profile of cell cycle-related substrates was observed, and the activation of CDK1, MAPK1, and MAPK3 kinases was determined. Second, pathway interrogation using kinase inhibitor treatment confirmed that CDK1 activation operated downstream from mTOR inhibition to further regulate cell cycle progression. Third, we found that the activation of CDK1 following 4-12 h of mTOR inhibition was accompanied by the activation of the Greatwall-endosulfine complex. In conclusion, we presented a high-confidence phosphoproteome map inside the cells upon mTOR inhibition by rapamycin. Our data implied that mTOR inhibition could contribute to CDK1 activation for further regulating cell cycle progression, which was mediated by the Greatwall-endosulfine complex.

Rapamycin alleviates renal damage in mice with systemic lupus erythematosus through improving immune response and function.

This study aimed to explore the therapeutic effect and mechanism of rapamycin (RAPA) on systemic lupus erythematosus (SLE) in BALB/C mice induced by pristane. The mice were randomly divided into 5 groups (n = 6): control, model, saline, RAPA (1 mg/kg) and RAPA (2 mg/kg). All groups were injected with pristane except control. HE staining revealed 1 mg/kg and 2 mg/kg RAPA treatments obviously alleviated pathological changes in the kidney of SLE mice such as glomeruli enlargement, hyperplasia of mesangial cells, epithelial and endothelial cells, infiltration of inflammatory cells, and edema-like degeneration of renal tubules. Compared with control group, body weights and anti-ribosomal P-protein antibody (ARPA) level of the mice in model group and saline group decreased (P < 0.05), while immune complex deposition and levels of anti-dsDNA antibody, anti-smRNP antibody and urine protein in model group and saline group increased (P < 0.05). However, compared with model group and saline group, body weights of the mice in RAPA (1 mg/kg) group and RAPA (2 mg/kg) group increased (P < 0.05), while immune complex deposition and levels of anti-dsDNA antibody, anti-smRNP antibody, ARPA, and urine protein in RAPA (1 mg/kg) group and RAPA (2 mg/kg) group decreased (P < 0.05). Compared with control group, the proportion of dentritic cells (DC) in the kidney and peripheral blood decreased while the proportion of Th1, Th2 and Th17 cells in the spleen, kidney and peripheral blood increased in model group and saline group (P < 0.05). Compared with model group and saline group, 1 mg/kg and 2 mg/kg RAPA treatments boosted the proportion of DC in the kidney and peripheral blood, reduced the proportion of Th1 and Th17 cells in the spleen, kidney and peripheral blood, and lessened the proportion of Th2 cells in the kidney and peripheral blood (P < 0.05). In conclusion, RAPA alleviated renal damage in SLE mice through improving immune response and function.

MCC950 Reduces Neuronal Apoptosis in Spinal Cord Injury in Mice.

BACKGROUND: Traumatic Spinal Cord Injury (SCI) is a severe condition usually accompanied by an inflammatory process that gives rise to uncontrolled local apoptosis and a subsequent unfavorable prognosis. One reason for this unfavorable outcome could be the activation of the NLRP3 inflammasome. OBJECTIVE: MCC950 is a specific inhibitor of NLRP3 that further inhibits the formation of the NLRP3 inflammasome. The purpose of this study was to determine whether the NLRP3 inflammasome was associated with the severity of local apoptosis and whether MCC950 could prevent neuronal apoptosis following SCI. METHODS: In this study, primary cortical neurons were cultured in vitro. With or without pretreatment/ posttreatment with MCC950, neurons were subjected to Oxygen-Glucose Deprivation (OGD) for 2 h and then reperfusion for 20 h. Immunofluorescence was used to determine the expression of NLRP3, ASC, and cleaved caspase-1 in neurons. In vivo, SCI model mice were established with a 5 g weight-drop method. MCC950 was intraperitoneally injected at 0, 2, 4, 6, 8, 10, and 12 days after SCI. Basso Mouse Scale (BMS) scores and footprint assays were used to assess motor function. Paw withdrawal threshold and tail-flick latency were used to assess somatosensory function. H&E, Nissl, and TUNEL staining were used to measure histological changes and apoptosis at 3 days after SCI, and scar formation was observed by Masson staining and GFAP immunohistochemical analysis at 28 days after SCI. RESULTS: Immunofluorescence analysis confirmed that MCC950 inhibited OGD-induced activation of the NLRP3 inflammasome in neurons. Behavioral tests, Masson staining, and GFAP immunohistochemical analysis showed that MCC950-treated mice had improved neuronal functional recovery and reduced scar formation at 28 days after SCI. H&E, Nissl, and TUNEL staining confirmed that there were more living neurons and fewer apoptotic neurons in MCC950-treated mice than control mice at 3 days after SCI. CONCLUSION: These results reveal that MCC950 exerts neuroprotective effects by reducing neuronal apoptosis, preserving the survival of the remaining neurons, attenuating the severity of the damage, and promoting the recovery of motor function after SCI.

Glycyrrhizic acid protects juvenile epileptic rats against hippocampal damage through activation of Sirtuin3.

Glycyrrhizic acid (GA) and Sirtuin3 (Sirt3) were both found to be involved in epilepsy (EP), but their interaction was rarely studied. Herein, we aim to investigate the underlying mechanism of GA with the interaction of Sirt3 in juvenile EP rats. The EP model in juvenile rats was established by lithium chloride-pilocarpine and treated with different concentrations of GA, GA + DMSO or GA + 3-TYP [a selective inhibitor of Sirtuin3 (Sirt3)]. The expression of Sirt3, mitochondrial autophagy-related genes (C-III core 1, COX IV, LC3-I, LC3-II), apoptosis-related genes (Bcl-2, Bax, Caspase-3), glutathione (GSH), superoxide dismutase (SOD), malondialchehyche (MDA) and reactive oxygen species (ROS) as well as mitochondrial membrane potential were subsequently detected. The juvenile EP rats treated with GA showed increased level of C-III core 1 and COX IV, increased LC3-I/LC3-II, GSH and SOD, decreased MDA, increased expression of Sirt3, and Bcl-2, and decreased expression of Bax and Caspase-3. However, inhibition of Sirt3 caused reverse results. Collectively, GA could alleviate hippocampal pathological damage, promote mitochondrial autophagy and reduce oxidative stress in juvenile EP rats through activation of Sirt3. Understanding of these mechanisms may allow devising of novel therapeutics for pediatric EP.