VCP/p97 UFMylation stabilizes BECN1 and facilitates the initiation of autophagy.

Macroautophagy/autophagy is essential for the degradation and recycling of cytoplasmic materials. The initiation of this process is determined by phosphatidylinositol-3-kinase (PtdIns3K) complex, which is regulated by factor BECN1 (beclin 1). UFMylation is a novel ubiquitin-like modification that has been demonstrated to modulate several cellular activities. However, the role of UFMylation in regulating autophagy has not been fully elucidated. Here, we found that VCP/p97 is UFMylated on K109 by the E3 UFL1 (UFM1 specific ligase 1) and this modification promotes BECN1 stabilization and assembly of the PtdIns3K complex, suggesting a role for VCP/p97 UFMylation in autophagy initiation. Mechanistically, VCP/p97 UFMylation stabilizes BECN1 through ATXN3 (ataxin 3)-mediated deubiquitination. As a key component of the PtdIns3K complex, stabilized BECN1 facilitates assembly of this complex. Re-expression of VCP/p97, but not the UFMylation-defective mutant, rescued the VCP/p97 depletion-induced increase in MAP1LC3B/LC3B protein expression. We also showed that several pathogenic VCP/p97 mutations identified in a variety of neurological disorders and cancers were associated with reduced UFMylation, thus implicating VCP/p97 UFMylation as a potential therapeutic target for these diseases. Abbreviation: ATG14:autophagy related 14; Baf A1:bafilomycin A1;CMT2Y: Charcot-Marie-Toothdisease, axonal, 2Y; CYB5R3: cytochromeb5 reductase 3; DDRGK1: DDRGK domain containing 1; DMEM:Dulbecco'smodified Eagle's medium;ER:endoplasmic reticulum; FBS:fetalbovine serum;FTDALS6:frontotemporaldementia and/or amyotrophic lateral sclerosis 6; IBMPFD1:inclusion bodymyopathy with early-onset Paget disease with or withoutfrontotemporal dementia 1; LC-MS/MS:liquid chromatography tandem mass spectrometry; MAP1LC3B/LC3B:microtubule associated protein 1 light chain 3 beta; MS: massspectrometry; NPLOC4: NPL4 homolog, ubiquitin recognition factor;PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3;PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K:phosphatidylinositol 3-kinase; RPL26: ribosomal protein L26; RPN1:ribophorin I; SQSTM1/p62: sequestosome 1; UBA5: ubiquitin likemodifier activating enzyme 5; UFC1: ubiquitin-fold modifierconjugating enzyme 1; UFD1: ubiquitin recognition factor in ERassociated degradation 1; UFL1: UFM1 specific ligase 1; UFM1:ubiquitin fold modifier 1; UFSP2: UFM1 specific peptidase 2; UVRAG:UV radiation resistance associated; VCP/p97: valosin containingprotein; WT: wild-type.

Mechanism and endoscopic-treatment-induced evolution of biliary non-anastomotic stricture after liver transplantation revealed by single-cell RNA sequencing.

BACKGROUND: Biliary complications, especially non-anastomotic stricture (NAS), are the main complications after liver transplantation. Insufficient sampling and no recognized animal models obstruct the investigation. Thus, the mechanisms and alterations that occur during endoscopic treatment (ET) of NAS remain unclear. METHODS: Samples were obtained with endoscopic forceps from the hilar bile ducts of NAS patients receiving continuous biliary stent implantation after diagnosis. Retrospective analysis of multiple studies indicated that the duration of ET for NAS was approximately 1-2 years. Thus, we divided the patients into short-term treatment (STT) and long-term treatment (LTT) groups based on durations of less or more than 1 year. Samples were subjected to single-cell RNA sequencing. Transcriptomic differences between STT and normal groups were defined as the NAS mechanism. Similarly, alterations from STT to LTT groups were regarded as endoscopic-treatment-induced evolution. RESULTS: In NAS, inflammation and immune-related pathways were upregulated in different cell types, with nonimmune cells showing hypoxia pathway upregulation and immune cells showing ATP metabolism pathway upregulation, indicating heterogeneity. We confirmed a reduction in bile acid metabolism-related SPP1+ epithelial cells in NAS. Increases in proinflammatory and profibrotic fibroblast subclusters indicated fibrotic progression in NAS. Furthermore, immune disorders in NAS were exacerbated by an increase in plasma cells and dysfunction of NK and NKT cells. ET downregulated multicellular immune and inflammatory responses and restored epithelial and endothelial cell proportions. CONCLUSIONS: This study reveals the pathophysiological and genetic mechanisms and evolution of NAS induced by ET, thereby providing preventive and therapeutic insights into NAS. HIGHLIGHTS: For the first time, single-cell transcriptome sequencing was performed on the bile ducts of patients with biliary complications. scRNA-seq analysis revealed distinct changes in the proportion and phenotype of multiple cell types during Nonanastomotic stricture (NAS) and endoscopic treatment. A reduction in bile acid metabolism-related SPP1+ epithelial cells and VEGFA+ endothelial cells, along with explosive infiltration of plasma cells and dysfunction of T and NK cells in NAS patients. SPP1+ macrophages and BST2+ T cells might serve as a surrogate marker for predicting endoscopic treatment.

Conserved molecular chaperone PrsA stimulates protective immunity against group A Streptococcus.

Group A Streptococcus (GAS) is a significant human pathogen that poses a global health concern. However, the development of a GAS vaccine has been challenging due to the multitude of diverse M-types and the risk of triggering cross-reactive immune responses. Our previous research has identified a critical role of PrsA1 and PrsA2, surface post-translational molecular chaperone proteins, in maintaining GAS proteome homeostasis and virulence traits. In this study, we aimed to further explore the potential of PrsA1 and PrsA2 as vaccine candidates for preventing GAS infection. We found that PrsA1 and PrsA2 are highly conserved among GAS isolates, demonstrating minimal amino acid variation. Antibodies specifically targeting PrsA1/A2 showed no cross-reactivity with human heart proteins and effectively enhanced neutrophil opsonophagocytic killing of various GAS serotypes. Additionally, passive transfer of PrsA1/A2-specific antibodies conferred protective immunity in infected mice. Compared to alum, immunization with CFA-adjuvanted PrsA1/A2 induced higher levels of Th1-associated IgG isotypes and complement activation and provided approximately 70% protection against invasive GAS challenge. These findings highlight the potential of PrsA1 and PrsA2 as universal vaccine candidates for the development of an effective GAS vaccine.

Fecal Microbiota Transplantation Revealed a Pain-related Gut Microbiota Community in Ovariectomized Mice.

Higher sensitivity to pain is a common clinical symptom in postmenopausal females. The gut microbiota (GM) has recently been identified as participating in various pathophysiological processes and may change during menopause and contribute to multiple postmenopausal symptoms. Here, we investigated the possible correlation between GM alteration and allodynia in ovariectomized (OVX) mice. Results showed that OVX mice exhibited allodynia from 7 weeks after surgery compared with sham-operated (SHAM) mice by comparing pain-related behaviors. Fecal microbiota transplantation (FMT) from OVX mice induced allodynia in normal mice while FMT from SHAM mice alleviated allodynia in OVX mice. Microbiome 16S rRNA sequencing and linear discriminant analysis revealed alteration of the GM after OVX. Furthermore, Spearman's correlation analysis showed associations between pain-related behaviors and genera, and further verification identified the possible pain-related genera complex. Our findings provide new insights into the underlying mechanisms of postmenopausal allodynia, and suggest pain-related microbiota community as a promising therapeutic target. PERSPECTIVE: This article provided the evidence of gut microbiota playing essential roles in postmenopausal allodynia. This work intended to offer a guidance for further mechanism investigation into gut-brain axis and probiotics screening for postmenopausal chronic pain.

The Effect of Calycosin-7-O-ß-D-Glucoside and its Synergistic Augmentation of Cisplatin-induced Apoptosis in SK-OV-3 Cells.

OBJECTIVE: This study aims to examine the synergetic augmentation of calycosin-7-O-ß-D-glucoside (CG) on cisplatin (CDDP) to induce apoptosis of human epithelial ovarian SK-OV-3 cancer cells. METHODS: The SK-OV-3 cells were divided into four groups: control, CDDP monotherapy, CG monotherapy, and combined CDDP and CG treatment. The cell counting kit-8 method detected cell proliferation at different times and under different treatments. Hoechst 33258 staining and annexin V-FITC/propidium iodide double staining methods were used to observe the apoptosis of the SK-OV-3 cells. The caspase-3 enzyme activity detection method, quantitative reverse transcription-polymerase chain reaction, and western blot were used to detect the apoptosis-related factors and the activities of the enzyme in SK-OV-3 cells. RESULTS: The inhibition rates of SK-OV-3 cell proliferation when exposed to 10 µM of CDDP, 50 µM of CG, and a combination of 10 µM of CDDP and 50 µM of CG were 23.2% ± 1.1%, 26.7% ± 2.0%, and 46.7% ± 1.3% after 48 h, respectively. Following the use of the drug combination, the apoptosis rate and caspase-3 enzyme activity were significantly higher than in the single-drug treatment group; the data differences were also significant (p < 0.05). At the protein and ribonucleic acid levels, CG significantly enhanced the effect of CDDP on p53, caspase-3, caspase-9, Bax, and Bcl-2. CONCLUSION: In vitro, CG significantly increases the CDDP-induced apoptosis of the SK-OV-3 cells through the p53 pathway at the cellular level. In addition, using the drugs in combination reduces the toxicity and side effects caused by using CDDP alone.

Unique virulence role of post-translocational chaperone PrsA in shaping Streptococcus pyogenes secretome.

Streptococcus pyogenes (group A Streptococcus, GAS) is a strict human pathogen causing a broad spectrum of diseases and a variety of autoimmune sequelae. The pathogenesis of GAS infection mostly relies on the production of an extensive network of cell wall-associated and secreted virulence proteins, such as adhesins, toxins, and exoenzymes. PrsA, the only extracellular parvulin-type peptidyl-prolyl isomerase expressed ubiquitously in Gram-positive bacteria, has been suggested to assist the folding and maturation of newly exported proteins to acquire their native conformation and activity. Two PrsA proteins, PrsA1 and PrsA2, have been identified in GAS, but the respective contribution of each PrsA in GAS pathogenesis remains largely unknown. By combining comparative proteomic and phenotypic analysis approaches, we demonstrate that both PrsA isoforms are required to maintain GAS proteome homeostasis and virulence-associated traits in a unique and overlapping manner. The inactivation of both PrsA in GAS caused remarkable impairment in biofilm formation, host adherence, infection-induced cytotoxicity, and in vivo virulence in a murine soft tissue infection model. The concordance of proteomic and phenotypic data clearly features the essential role of PrsA in GAS full virulence.

Pilot-scale constructed bypass channel for urban river restoration: the remedial efficiency and the variance in biodiversity.

Like the blood vessels of the cities, urban rivers play a significant role on maintaining the cities' sustainable development. In addition to the prevention of pollutants discharge and improving the river water quality, the restoration of the urban rivers' ecosystem should be well concerned. To fill this gap, a pilot-scale study with constructed bypass channel (CBC) was conducted. The pollutants reduction by the aquatic plants of the CBC was evaluated, and the similarities/differences of the aquatic biodiversity between the CBC and the natural rivers were analyzed. The results indicated that the mean removal efficiency of TP, NH3-N, TN, and COD by the CBC was 66%, 60%, 52%, and 36%, respectively. Chlorophyta, Bacillariophyta, and Cyanophyta were the dominant phytoplankton phyla in the CBC which was in accordance with the studies conducted in the Dongjiang River and the Pearl River. During the study period of about 6 months, the population density and the biomass of the phytoplankton and the zooplankton increased over time. The quality of the influent dominated the aquatic organisms' diversity of the CBC. N-element dominated not only the phytoplankton variability, but also the bacterial species of the CBC. The timber pile and the submerged plant root soil were more suitable for the growth of the functional bacteria; thus, the construction of the river restoration infrastructures should avoid using large-scale cement materials. Overall, the study would improve the understanding of urban river restoration practice and provide guidance for future restoration practice especially from the aquatic ecological perspectives.

Therapeutic Drug Monitoring and Pharmacokinetic Analysis of Cyclosporine in a Pediatric Patient with Hemophagocytic Lymphohistiocytosis Complicated by Diabetes Insipidus: A Grand Round.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease. Initial therapy is based on etoposide, dexamethasone, and cyclosporine (CSA). The pharmacokinetics (PKs) of CSA and other drugs are sometimes altered in patients with HLH complicated by diabetes insipidus (DI) but the precise mechanisms remain unknown. METHODS: In this study, the authors present a case of a 4-year-old boy with HLH complicated by DI. CSA concentrations were determined by enzyme multiplied immunoassay technique; noncompartmental PK analysis of the plasma concentration-time data was performed using PKSolver; and linear regression analysis was performed to determine linearity of relationship between urine output and C0 levels of CSA. RESULTS: Although C0 values of CSA were lower than the target levels, the patient was successfully treated and a good clinical outcome was achieved. Linear regression analysis showed a strong negative correlation between urine output and the serum trough concentration (C0) of CSA, pharmacokinetic analysis showed the main PK parameters of CSA as follows: C0, 50.2 mcg/L; peak concentration (Cmax), 723.4 mcg/L; area under the curve0-24, 7478.2 mcg·h/L; clearance, 0.77 L/h/kg, elimination half-life, 5.3 hours, and volume of distribution, 6.0 L/kg. CONCLUSIONS: To the best of the authors' knowledge, this is the first report of the CSA PK profile in a patient with HLH complicated by DI. The authors suppose that a large fluid output and input leads to extensive CSA distribution. These results suggest that the monitoring of the Cmax and area under the curve of CSA might be more clinically and pharmacokinetically significant than that of C0 in patients with HLH complicated by DI. This case highlights the importance of therapeutic drug monitoring and demonstrates PK parameters of CSA in a pediatric patient with HLH complicated by DI.

Integrated Bioinformatics Analysis of Hub Genes and Pathways Associated with a Compression Model of Spinal Cord Injury in Rats.

BACKGROUND Spinal cord injury (SCI) is a serious nervous system condition that can cause lifelong disability. The aim of this study was to identify potential molecular mechanisms and therapeutic targets for SCI. MATERIAL AND METHODS We constructed a weighted gene coexpression network and predicted which hub genes are involved in SCI. A compression model of SCI was established in 45 Sprague-Dawley rats, which were divided into 5 groups (n=9 per group): a sham operation group, and 1, 3, 5, and 7 days post-SCI groups. The spinal cord tissue on the injured site was harvested on 1, 3, 5, and 7 days after SCI and 3 days after surgery in the sham operation group. High-throughput sequencing was applied to investigate the expression profile of the mRNA in all samples. Differentially expressed genes were screened and included in weighted gene coexpression network analysis (WGCNA). Co-expressed modules and hub genes were identified by WGCNA. The biological functions of each module were investigated using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. RESULTS According to the RNA-seq data, a total of 1965 differentially expressed genes were screened, and WGCNA identified 10 coexpression modules and 5 hub genes. Module function analysis revealed that SCI was associated with immune response, cell division, neuron projection development, and collagen fibril organization. CONCLUSIONS Our study revealed dynamic changes in a variety of biological processes following SCI and identified 5 hub genes via WGCNA. These results provide insights into the molecular mechanisms and therapeutic targets of SCI.

Vitamin C: A stem cell promoter in cancer metastasis and immunotherapy.

Vitamin C is an electron donor and is involved in a variety of biochemical reactions in stem cell and cancer stem cell, as well as collagen synthesis and the regulation of hypoxia-inducible factor synthesis, which two affect extracellular matrix remodelling and hence cancer metastasis. Specific doses of vitamin C can stop cancer cell glycolysis and block nitroso synthesis, indicating the potential of vitamin C in cancer treatment. Recent studies preliminary revealed Vitamin C enhance the cancer's immune response to anti PD-L1 therapy through multiple indirect approaches. Herein we reviewed the recent function of vitamin C for further research in sequential aspects of cancer stem cell, extracellular matrix remodeling, cancer metastasis and cancer immunotherapy.

[Exploration of developmental toxicity mechanism of pentachlorophenol using cDNA microarray].

0hpf zebrafish embryos were exposed to 50 microg/L pentachlorophenol(PCP) for 8h in vitro. Total RNA sample was extracted then and hybridized with Affymetrix Zebrafish Genome Array representing approximately 14 900 transcripts. A total of 1 149 transcripts was significantly up-regulated while 501 transcripts were down-regulated. Bioinformatic tools were used for further analysis. The result indicated that genes with significant expression changes were related to molecular functions including antioxidant activity, signal transducer activity, translation regulator activity, transcription regulator activity, et al. Genes regulated by BMP signals, FGF signals, and Nodal signals including smad2, smad5, bmp4, bmp7, flh, n-ras may involve in the developmental toxicity of PCP, with Signal log ratios of 4.6, 2.1, 1.6, 1.0, 1, 1.3, 1.0, respectively. This investigation may provide new biomarkers to further study of the developmental toxicity of PCP.