Based case based learning and flipped classroom as a means to improve international students' active learning and critical thinking ability.

BACKGROUND: International student education has become an important part of higher education and an important symbol to measure the level of higher education. To change the traditional teaching model, here we introduced a combination of Case-Based Learning (CBL)and Flipped Classroom (FC) into the pathophysiology course for international students. This study aimed to explore whether the active learning ability and critical thinking ability of international students can be improved, based on this new teaching model, improving the innovation ability of teachers' team and students' attitude to the reform. METHODS: The two chapters of Cardiac Insufficiency and Apoptosis in Pathophysiology are designed as a CBL + FC teaching method. Distribute the Self-assessment Scale on Active Learning and Critical Thinking (SSACT) and satisfaction questionnaire to international students to evaluate teaching reform based on CBL + FC. RESULTS: Compared with the traditional classroom, the online flipped classroom based on CBL has significantly improved the learning enthusiasm, as these students are required to independently complete literature review, actively participate in classroom teaching, learn to use multiple learning strategies, and collaborate with other students to complete PowerPoint (PPT)production. At the same time, the students' ability to raise problems and solve problems has been greatly improved by analyzing clinical cases; By consulting the literature, the theoretical knowledge learned can be better applied to clinical analysis. The results of the satisfaction survey also show that international students are more likely to accept the flipped classroom teaching mode. CONCLUSIONS: This teaching mode will stimulate the learning motivation of international students, enhance teaching attraction and increase teaching interaction; At the same time, the CBL + FC teaching method can strengthen the evaluation of international students' in and out of class and online learning, enhance students' active learning ability and critical thinking ability, promote the development of personalized learning, and integrate with international medical education.

Hypermethylation of the FOXP3 gene regulates Tregs immunodysregulation in chronic idiopathic thrombocytopenic purpura.

BACKGROUND: Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by a breakdown of immune tolerance; in ITP, the body's immune system mistakenly attacks and destroys platelets. This study aims to investigate the role and underlying mechanisms of FOXP3 in chronic ITP. METHODS: Flow cytometry was used to detect the proportion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) in CD4+CD25+ T lymphocytes from 20 patients with chronic ITP (CITP), 20 acute ITP (AITP) controls, and 20 healthy individuals.CD4+CD25+ Treg cells were isolated from peripheral blood of patients with CITP using magnetic beads and then treated with phosphate-buffered saline solution or decitabine (a methylation inhibitor) for 48 h. The levels of interleukin-2 (IL-2), IL-10, and transforming growth factor-beta1 (TGF-ß1) in the plasma and CD4+CD25+ Treg cells were assessed by Enzyme-linked-immunosorbent serologic assay and quantitative real-time polymerase chain reaction (qRT-PCR). FOXP3 level was measured by qRT-PCR and Western blot analysis. Methylation-specific PCR (MS-PCR) was adopted to detect the status of FOXP3 methylation. RESULTS: The number of Treg cells and the contents of IL-2, IL-10, and TGF-ß1 decreased in patients with CITP, compared to the AITP control group and normal group. FOXP3 expression was reduced and FOXP3 methylation increased in patients with CITP, compared to the AITP control group and normal group. Hypermethylation of FOXP3 promoter led to decrease in FOXP3 level in Treg cells. Inhibition of FOXP3 promoter hypermethylation promoted the secretion of IL-2, IL-10, and TGF-ß1 in Treg cells. CONCLUSION: The number of Treg cells in CITP patients decreased, and the hypermethylation of FOXP3 promoter led to reduction of its expression in Treg cells, thus affecting the immune functioning of Treg cells.

Ultrasonic-assisted preparation of SBS modified asphalt: Cavitation bubble numerical simulation and rheological properties.

SBS (styrene-butadiene-styrene block copolymer) is currently the most widely used asphalt modifier, and SBS modified asphalt is usually prepared by high-speed shearing. This paper combines the cavitation effect of ultrasonic to assist in the preparation of SBS modified asphalt, and conducts numerical simulation and rheological properties research on the cavitation bubbles in the molten SBS modified asphalt fluid. The cavitation bubbles in the modified asphalt fluid will expand and contract as the pressure changes inside and outside the bubbles. When the cavitation bubble is compressed to the minimum and the pressure inside the bubble reaches 1.94 × 105Pa, the direction of the velocity vector near the cavitation bubble will change with the expansion and compression of the bubble. The expansion-contraction process of a single cavitation bubble can release 6.41 × 10-7J of energy, thus breaking the long bonds in asphalt and generating a large number of free radicals react with the unsaturated C = C bonds in the SBS molecules. According to the preparation process of modified asphalt, the influence of ultrasonic wave on rheological property of modified asphalt was studied through experiments. The results show that ultrasonic treatment can enhance the elasticity of asphalt and improve the temperature sensitivity of asphalt. With the increase of ultrasonic treatment time, the anti-rutting deformation ability of SBS modified asphalt is greatly improved. At the same temperature, the recovery rate of asphalt also increases with the increase of ultrasonic treatment time, and the non-recoverable compliance (Jnr) decreases Combined with the numerical simulation of cavitation bubbles, the ultrasonic process is added to asphalt production, which is of great significance for the green production of modified asphalt and the improvement of the rheological properties of modified asphalt.

Spatiotemporal single-cell analysis decodes cellular dynamics underlying different responses to immunotherapy in colorectal cancer.

Expanding the efficacy of immune checkpoint blockade (ICB) in colorectal cancer (CRC) presses for a comprehensive understanding of treatment responsiveness. Here, we analyze multiple sequential single-cell samples from 22 patients undergoing PD-1 blockade to map the evolution of local and systemic immunity of CRC patients. In tumors, we identify coordinated cellular programs exhibiting distinct response associations. Specifically, exhausted T (Tex) or tumor-reactive-like CD8+ T (Ttr-like) cells are closely related to treatment efficacy, and Tex cells show correlated proportion changes with multiple other tumor-enriched cell types following PD-1 blockade. In addition, we reveal the less-exhausted phenotype of blood-associated Ttr-like cells in tumors and find that their higher abundance suggests better treatment outcomes. Finally, a higher major histocompatibility complex (MHC) II-related signature in circulating CD8+ T cells at baseline is linked to superior responses. Our study provides insights into the spatiotemporal cellular dynamics following neoadjuvant PD-1 blockade in CRC.

Enhancement of Pulmonary Function and Reduction of Complications Through EIT-Guided Yoga Breathing Exercise After Esophagectomy.

BACKGROUND This study aimed to investigate the impact of EIT-guided yoga breathing training on postoperative pulmonary complications (PPCs) for esophageal cancer patients. MATERIAL AND METHODS Total of 62 patients underwent radical resections of esophageal cancer. Esophageal cancer patients were randomized to the standard care group, or the intervention group receiving an additional complete breathing exercise under the guidance of EIT in AICU. Following extubation after the esophagectomy, pulmonary functions were evaluated by EIT with center of ventilation (CoV), dependent silent spaces (DSS), and non-dependent silent spaces (NSS). RESULTS Sixty-one older esophageal cancer patients (31 in the Control group and 30 in the EIT group) were included in the final analysis. Forty-four patients experienced pulmonary complications after esophagectomy, 27 (87.1%) in the Control group and 17 (36.7%) in the EIT group (RR, 0.42 (95% CI: 0.26, 0.69). The most common pulmonary complication was pleural effusion, with an incidence of 30% in the EIT group and 74.2% in the Control group, with RR of 0.40 (95% CI: 0.23, 0.73). Time for the first pulmonary complication was significantly longer in the EIT group than in the Control group (hazard ratio, HR, 0.43; 95% CI 0.21 to 0.87; P=0.019). Patients in the EIT group had significantly higher scores in CoV, DSS, and NSS than in the Control group. CONCLUSIONS Guided by EIT, the addition of the postoperative breathing exercise to the standardized care during AICU could further improve pulmonary function, and reduce postoperative pulmonary complications after esophagectomy.

Cyclosporine A protects podocytes by regulating transgelin in adriamycin-induced podocyte injury.

INTRODUCTION: The calcineurin inhibitor cyclosporine A (CsA) has been shown to effectively reduce proteinuria. However, its precise mechanism is still not fully understood. Our previous study showed that CsA reduced proteinuria by directly stabilizing the foot process (FP) cytoskeletal structure via cofilin-1, suggesting that synaptopodin, a podocyte-specific actin protein, is not the sole target of CsA in podocytes. METHODS: In this study, we established an adriamycin (ADR)-induced nephropathy rat model and a cultured podocyte injury model. We employed Western blotting and immunofluorescence techniques to assess the expression and distribution of transgelin, KLF-4, nephrin, and synaptopodin. RESULTS: We observed a significant increase in proteinuria levels accompanied by loss of normal FP structure in the ADR-induced nephropathy rat model. The levels of the actin cross-linking protein transgelin were increased significantly, while those of the podocyte-specific molecules nephrin and synaptopodin were decreased in vivo. Treatment with CsA effectively reduced proteinuria while restoring FP effacement stability in ADR-induced nephropathy models, and restoring the expression of transgelin, nephrin, and synaptopodin both in vivo and in vitro. Furthermore, CsA treatment dose-dependently decreased transgelin levels while significantly increasing KLF-4 expression in injured podocytes. In addition, CsA failed to downregulate transgelin when KLF-4 was specifically knocked down. CONCLUSION: Our findings suggest that CsA protects against podocyte injury by downregulating abnormally high levels of transgelin via upregulation of KLF-4 expression.

Screening and characterization of a novel linear B-cell epitope on orf virus F1L protein.

Background: Orf, also known as contagious ecthyma (CE), is an acute, contagious zoonotic disease caused by the orf virus (ORFV). The F1L protein is a major immunodominant protein on the surface of ORFV and can induce the production of neutralizing antibodies. Methods: The prokaryotic expression system was used to produce the recombinant F1L protein of ORFV, which was subsequently purified and used to immunize mice. Positive hybridoma clones were screened using an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity and specificity of the monoclonal antibody (mAb) were verified through Western blot and indirect immunofluorescence (IFA). The linear antigenic epitope specific to the mAb was identified through Western blot, using truncated F1L proteins expressed in eukaryotic cells. A multiple sequence alignment of the ORFV reference strains was performed to evaluate the degree of conservation of the identified epitope. Results: After three rounds of subcloning, a mAb named Ba-F1L was produced. Ba-F1L was found to react with both the exogenously expressed F1L protein and the native F1L protein from ORFV-infected cells, as confirmed by Western blot and IFA. The mAb recognized the core epitope 103CKSTCPKEM111, which is highly conserved among various ORFV strains, as shown by homologous sequence alignment. Conclusion: The mAb produced in the present study can be used as a diagnostic reagent for detecting ORFV and as a basic tool for exploring the mechanisms of orf pathogenesis. In addition, the identified linear epitope may be valuable for the development of epitope-based vaccines.

Silencing CK19 regulates ferroptosis by affecting the expression of GPX4 and ACSL4 in oral squamous cell carcinoma in vivo and in vitro.

To analyze the mechanism of how interfering with the cytokeratin 19 (CK19) pathway via the ferroptosis pathway affects tumor biological behaviors in the process of oral squamous cell carcinoma (OSCC) development. TCGA was used to analyze the expression of CK19 in pan-cancer and head and neck squamous cell carcinoma (HNSC) and to explore the ferroptosis-related genes related to HNSC. The effect of silencing CK19 on the migration ability of HSC-4 cells was verified by wound healing and migration assay. HSC-4 cells with silencing of CK19 and tumor-bearing nude mouse model were constructed. RT-qPCR, immunofluorescence and western blot were used to analyze the expression of ferroptosis-related genes. CK19 is highly expressed in human OSCC and nude mice. The migration ability of cells in the CK19-silenced group was lower than that of the control group. In vivo and in vitro, CK19 was negatively correlated with the expression of ACSL4 and positively correlated with the expression of GPX4. Compared with the control group, GPX4 expression was down-regulated and ACSL4 expression was up-regulated in the CK19-silenced group. Silencing CK19 also increased intracellular Fe2+ content and MDA content. Silencing CK19 can affect the expression of GPX4 and ACSL4 to regulate ferroptosis and at the same time increase the content of MDA, Fe2+ and ROS levels, thereby activating the regulation of ferroptosis pathway in the development of OSCC.

Ligand exchange induced crystal structure and morphology evolution of copper-tin-sulfur binary and ternary compounds.

In this manuscript, a simple one-pot heat-up method has been used to prepare multi-component copper-tin-sulfur nanomaterials, including binary Cu1.94S, ternary Cu4SnS4, and Cu1.94S/Cu4SnS4 nanocrystals by varying the reaction temperature, reaction time, and the type of copper source. Post-synthetic ligand exchange (LE) has further been introduced to replace the long-chain ligands originating from 1-dodecanethiol. It has been found that the LE process not only changes the surface ligands but also significantly affects the crystal structure and optical properties of nanocrystals. After LE, the crystal structures of Cu1.94S and Cu4SnS4 transformed to Cu7S4 and Cu3SnS4, respectively, with the Cu1.94S/Cu4SnS4 nanocrystals showing the same trend. This phenomenon could be ascribed to the loss of Cu+ originating from the strong complexation of Cu+ and ammonia with the formation of [Cu(NH3)n]2+ ions under aerobic conditions. Proton nuclear magnetic resonance (1H NMR) has been used to characterize the ligands on the surface before and after LE, which further demonstrated that the -SH was replaced during LE. Meanwhile, the band gaps of the obtained nanocrystals after LE show an obvious shift in the near-infrared region due to the evolution of crystal structures. This study will provide useful guidance for the LE of nanocrystals and the application of copper-based sulfide nanomaterials in optoelectronics and other fields.

Tumor-Derived Exosomes Promote Tumor Growth Through Modulating Microvascular Hemodynamics in a Human Ovarian Cancer Xenograft Model.

OBJECTIVE: Abnormal tumor vascular network contributes to aberrant blood perfusion and reduced oxygenation in tumors, which lead to poor efficacy of chemotherapy and radiotherapy. We aimed to explore the effects of the tumor-derived exosomes (TDEs) and C188-9 (a small molecule inhibitor of signal transducer and activator of transcription 3, STAT3) on tumor microvascular hemodynamics and determine which blood flow oscillations for various frequency intervals are responsible for these changes. METHODS: Microvascular hemodynamics parameters were recorded using a PeriFlux 6000 EPOS system in tumor surface in a nude mouse subcutaneous xenograft model. Oscillations of laser Doppler flowmetry (LDF) signal were investigated by wavelet transform analysis. RESULTS: TDEs facilitated tumor growth at least partially was associated with increasing blood flow in smaller vessels with lower speed and decreasing the blood flow at larger vessels with higher speed. Lower oxyhemoglobin saturation (SO2) on tumor surface was aggravated by TDEs, and C188-9 treatment significantly alleviated this decrease. Wavelet transform spectral analysis revealed that TDEs increased the amplitude of oscillations in four frequency intervals related to endothelial (NO-dependent and -independent), myogenic and neurogenic activities, and C188-9 had no effect on this increase. CONCLUSIONS: TDEs facilitated tumor growth partially was associated with increasing blood flow in distributing vessels, reducing blood perfusion in larger vessels, and lowering SO2 on tumor surface. Enhanced vascular smooth muscle, endothelial and neurogenic activities occurred in tumor superficial zone.

TG-Net: Using text prompts for improved skin lesion segmentation.

Automatic skin segmentation is an efficient method for the early diagnosis of skin cancer, which can minimize the missed detection rate and treat early skin cancer in time. However, significant variations in texture, size, shape, the position of lesions, and obscure boundaries in dermoscopy images make it extremely challenging to accurately locate and segment lesions. To address these challenges, we propose a novel framework named TG-Net, which exploits textual diagnostic information to guide the segmentation of dermoscopic images. Specifically, TG-Net adopts a dual-stream encoder-decoder architecture. The dual-stream encoder comprises Res2Net for extracting image features and our proposed text attention (TA) block for extracting textual features. Through hierarchical guidance, textual features are embedded into the process of image feature extraction. Additionally, we devise a multi-level fusion (MLF) module to merge higher-level features and generate a global feature map as guidance for subsequent steps. In the decoding stage of the network, local features and the global feature map are utilized in three multi-scale reverse attention modules (MSRA) to produce the final segmentation results. We conduct extensive experiments on three publicly accessible datasets, namely ISIC 2017, HAM10000, and PH2. Experimental results demonstrate that TG-Net outperforms state-of-the-art methods, validating the reliability of our method. Source code is available at https://github.com/ukeLin/TG-Net.

Manure enriched with nitrogen derived from high-protein food waste in a large dining facility.

Food waste (FW) from large dining facility has been a pressing environmental challenge in China recently. This study developed an innovative species-specific feeding strategy for producing pigeon meat and excellent manure from FW. Adding FW to the feed of pigeons significantly increased their feed intake and promoted their growth although the pigeons showed a strong aversion to the FW. We produced a "super manure" with exceptionally high nitrogen (N) content (mean = 10.77 % on a dry basis, 8.04-12.57 %, n = 264) by feeding slowly-growing pigeon species (Columba livia vs. and Caoge Huzhou 11) with protein-high commercial feed and FW. A significant negative relationship between the N and carbon (C) contents in the pigeon manure was found, with C depletion higher than N depletion. Furthermore, the N content in the anaerobic composting (AnC) manure was 29.16 % higher than that in the FW. Fourier transform infrared (FT-IR) analysis and stable isotopes δ13C and δ15N in the manure clearly identified the transformations of nutrients during pigeon feeding and the AnC process. This study opens a path for producing N-high manure using protein-high food waste.

Genetic variation in the aquaporin TONOPLAST INTRINSIC PROTEIN 4;3 modulates maize cold tolerance.

Cold stress is a major abiotic stress that threatens maize (Zea mays L.) production worldwide. Understanding the molecular mechanisms underlying cold tolerance is crucial for breeding resilient maize varieties. Tonoplast intrinsic proteins (TIPs) are a subfamily of aquaporins in plants. Here, we report that TIP family proteins are involved in maize cold tolerance. The expression of most TIP genes was responsive to cold stress. Overexpressing TIP2;1, TIP3;2 or TIP4;3 reduced the cold tolerance of maize seedlings, while loss-of-function mutants of TIP4;3 exhibited enhanced cold tolerance. Candidate gene-based association analysis revealed that a 328-bp transposon insertion in the promoter region of TIP4;3 was strongly associated with maize cold tolerance. This transposon insertion conferred cold tolerance by repressing TIP4;3 expression through increased methylation of its promoter region. Moreover, TIP4;3 was found to suppress stomatal closure and facilitate reactive oxygen species (ROS) accumulation under cold stress, thereby inhibiting the expression of cold-responsive genes, including DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR 1 (DREB1) genes and a subset of peroxidase genes, ultimately attenuating maize cold tolerance. This study thus elucidates the mechanism underlying TIP-mediated cold tolerance and identifies a favourable TIP4;3 allele as a potential genetic resource for breeding cold-tolerant maize varieties.

Generation and Functional Verification of Hypoxia-sensitive Chimeric Antigen Receptor-T Cells.

Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating solid tumors remains challenging, as exemplified by the safety concerns that arise when CAR-T cells attack normal cells expressing the target antigens. Researchers have explored various approaches to enhance the tumor selectivity of CAR-T cell therapy. One representative strategy along this line is the construction of hypoxia-sensitive CAR-T cells, which are designed by fusing an oxygen-dependent degradation domain to the CAR moiety and are strategized to attain high CAR expression only in a hypoxic environment-the tumor microenvironment (TME). This paper presents a protocol for the generation of such CAR-T cells and their functional characterization, including methods to analyze the changes in CAR expression and killing capacity in response to different oxygen levels established by a mobile incubator chamber. The constructed CAR-T cells are anticipated to demonstrate CAR expression and cytotoxicity in an oxygen-sensitive manner, thus supporting their capability to distinguish between hypoxic TME and normoxic normal tissues for selective activation.

Intensive Surveillance of Porcine-Rhesus Kidney Xenotransplant Using Different Ultrasound Techniques.

BACKGROUND: Significant progress has been made in kidney xenotransplantation in the past few years, and this field is accelerating towards clinical translation. Therefore, surveillance of the xenograft with appropriate tools is of great importance. Ultrasonography has been widely used in kidney allotransplantation and served as an economical and non-invasive method to monitor the allograft. However, questions remain whether the ultrasonographic criteria established for human kidney allograft could also be applied in xenotransplantation. METHODS: In the current study, we established a porcine-rhesus life sustaining kidney xenotransplantation model. The xenograft underwent intensive surveillance using gray-scale, colorful Doppler ultrasound as well as 2D shear wave elastography. The kidney growth, blood perfusion, and cortical stiffness were measured twice a day. These parameters were compared with the clinical data including urine output, chemistry, and pathological findings. RESULTS: The observation continued for 16 days after transplantation. Decline of urine output and elevated serum creatinine were observed on POD9 and biopsy proven antibody-mediated rejection was seen on the same day. The xenograft underwent substantial growth, with the long axis length increased by 32% and the volume increased by threefold at the end of observation. The resistive index of the xenograft arteries elevated in response to rejection, together with impaired cortical perfusion, while the peak systolic velocity (PSV) was not compromised. The cortical stiffness also increased along with rejection. CONCLUSION: In summary, the ultrasound findings of kidney xenograft shared similarities with those in allograft but possessed some unique features. A modified criteria needs to be established for further application of ultrasound in kidney xenotransplantation.

Low-Density Lipoprotein Cholesterol, Cardiovascular Disease Risk, and Mortality in China.

Importance: Limited evidence supports the association between low-density lipoprotein cholesterol (LDL-C) and mortality across different atherosclerotic cardiovascular disease (ASCVD) risk stratifications. Objective: To explore the associations between LDL-C levels and mortality and to identify the optimal ranges of LDL-C with the lowest risk of mortality in populations with diverse ASCVD risk profiles. Design, Setting, and Participants: The ChinaHEART project is a prospective cohort study that recruited residents aged 35 to 75 years from 31 provinces in mainland China between November 2014 and December 2022. Participants were categorized into low-risk, primary prevention, and secondary prevention cohorts on the basis of their medical history and ASCVD risk. Data analysis was performed from December 2022 to October 2023. Main Outcomes and Measures: The primary end point was all-cause mortality, and secondary end points included cause-specific mortality. Mortality data were collected from the National Mortality Surveillance System and Vital Registration. The association between LDL-C levels and mortality was assessed by using Cox proportional hazard regression models with various adjusted variables. Results: A total of 4 379 252 individuals were recruited, and 3 789 025 (2 271 699 women [60.0%]; mean [SD] age, 56.1 [10.0] years) were included in the current study. The median (IQR) LDL-C concentration was 93.1 (70.9-117.3) mg/dL overall at baseline. During a median (IQR) follow-up of 4.6 (3.1-5.8) years, 92 888 deaths were recorded, including 38 627 cardiovascular deaths. The association between LDL-C concentration and all-cause or cardiovascular disease (CVD) mortality was U-shaped in both the low-risk cohort (2 838 354 participants) and the primary prevention cohort (829 567 participants), whereas it was J-shaped in the secondary prevention cohort (121 104 participants). The LDL-C levels corresponding to the lowest CVD mortality were 117.8 mg/dL in the low-risk group, 106.0 mg/dL in the primary prevention cohort, and 55.8 mg/dL in the secondary prevention cohort. The LDL-C concentration associated with the lowest all-cause mortality (90.9 mg/dL vs 117.0 mg/dL) and CVD mortality (87 mg/dL vs 114.6 mg/dL) were both lower in individuals with diabetes than in individuals without diabetes in the overall cohort. Conclusions and Relevance: This study found that the association between LDL-C and mortality varied among different ASCVD risk cohorts, suggesting that stricter lipid control targets may be needed for individuals with higher ASCVD risk and those with diabetes.

Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts.

Introduction: G protein-coupled bile acid receptor (TGR5), the first G protein-coupled receptor for bile acids identified, is capable of activating a variety of intracellular signaling pathways after interacting with bile acids. TGR5 plays an important role in multiple physiological processes and is considered to be a potential target for the treatment of various metabolic diseases, including type 2 diabetes. Evidence has emerged that genetic deletion of TGR5 results in an increase in basal urine output, suggesting that it may play a critical role in renal water and salt reabsorption. The present study aims to elucidate the effect and mechanism of TGR5 activation on urine concentration. Methods: Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-h urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry and immunofluorescence were performed to examine the cellular location of proteins. The cultured primary medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1 h, followed by 12-h treatment of LCA and INT-777. Luciferase reporter assays were used to detect the effect of CREB on the gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA-protein interactions. Results: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in a cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB-binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays. Conclusion: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect.

SIRT1-regulated ROS generation activates NMDAR2B phosphorylation to promote central sensitization and allodynia in a male chronic migraine rat model.

Background: Central sensitization is one of the pivotal pathological mechanisms in chronic migraine (CM). Silent information regulator 1 (SIRT1) was shown to be involved in CM, but its specific mechanism is unclear. Reactive oxygen species (ROS) are increasingly regarded as important signaling molecules in several models of pain. However, studies about the role of ROS in the central sensitization of CM model are rare. We thus explored the specific process of SIRT1 involvement in the central sensitization of CM, focusing on the ROS pathway. Methods: Inflammatory soup was repeatedly administered to male Sprague-Dawley rats to establish a CM model. The SIRT1 expression level in trigeminal nucleus caudalis (TNC) tissues was assessed by qRT-PCR and Western blotting analysis. The levels of ROS were detected by a Tissue Reactive Oxygen Detection Kit, DHE staining, and the fluorescence signal intensity of 8-OHdG. A ROS scavenger (tempol), a SIRT1 activator (SRT1720), a SIRT1 inhibitor (EX527), and a mitochondrial fission inhibitor (Mdivi-1) were used to investigate the specific molecular mechanisms involved. NMDAR2B, CGRP, ERK, and mitochondrial fission-related protein were evaluated by Western blotting, and the CGRP level in frozen sections of the TNC was detected via immunofluorescence staining. Results: After repeated inflammatory soup infusion and successful establishment of the CM rat model, SIRT1 expression was found to be significantly reduced, accompanied by elevated ROS levels. Treatment with Tempol, SRT1720, or Mdivi-1 alleviated allodynia and reduced the increase in NMDAR2B phosphorylation and CGRP and ERK phosphorylation in the CM rat. In contrast, EX527 had the opposite effect in CM rat. SRT1720 and EX527 decreased and increased ROS levels, respectively, in CM rats, and tempol reversed the aggravating effect of EX527 in CM rats. Furthermore, the regulatory effect of SIRT1 on ROS may include the involvement of the mitochondrial fission protein DRP1. Conclusion: The results indicate the importance of SIRT1 in CM may be due to its role in regulating the production of ROS, which are involved in modulating central sensitization in CM. These findings could lead to new ideas for CM treatment with the use of SIRT1 agonists and antioxidants.

Integrated mRNA-miRNA transcriptome profiling of blood immune responses potentially related to pulmonary fibrosis in forest musk deer.

Background: Forest musk deer (FMD, Moschus Berezovskii) is a critically endangered species world-widely, the death of which can be caused by pulmonary disease in the farm. Pulmonary fibrosis (PF) was a huge threat to the health and survival of captive FMD. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been involved in the regulation of immune genes and disease development. However, the regulatory profiles of mRNAs and miRNAs involved in immune regulation of FMD are unclear. Methods: In this study, mRNA-seq and miRNA-seq in blood were performed to constructed coexpression regulatory networks between PF and healthy groups of FMD. The hub immune- and apoptosis-related genes in the PF blood of FMD were explored through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Further, protein-protein interaction (PPI) network of immune-associated and apoptosis-associated key signaling pathways were constructed based on mRNA-miRNA in the PF blood of the FMD. Immune hub DEGs and immune hub DEmiRNAs were selected for experimental verification using RT-qPCR. Results: A total of 2744 differentially expressed genes (DEGs) and 356 differentially expressed miRNAs (DEmiRNAs) were identified in the PF blood group compared to the healthy blood group. Among them, 42 DEmiRNAs were negatively correlated with 20 immune DEGs from a total of 57 correlations. The DEGs were significantly associated with pathways related to CD molecules, immune disease, immune system, cytokine receptors, T cell receptor signaling pathway, Th1 and Th2 cell differentiation, cytokine-cytokine receptor interaction, intestinal immune network for IgA production, and NOD-like receptor signaling pathway. There were 240 immune-related DEGs, in which 186 immune-related DEGs were up-regulated and 54 immune-related DEGs were down-regulated. In the protein-protein interaction (PPI) analysis of immune-related signaling pathway, TYK2, TLR2, TLR4, IL18, CSF1, CXCL13, LCK, ITGB2, PIK3CB, HCK, CD40, CD86, CCL3, CCR7, IL2RA, TLR3, and IL4R were identified as the hub immune genes. The mRNA-miRNA coregulation analysis showed that let-7d, miR-324-3p, miR-760, miR-185, miR-149, miR-149-5p, and miR-1842-5p are key miRNAs that target DEGs involved in immune disease, immune system and immunoregulation. Conclusion: The development and occurrence of PF were significantly influenced by the immune-related and apoptosis-related genes present in PF blood. mRNAs and miRNAs associated with the development and occurrence of PF in the FMD.

Differential phosphorylation of Ca2+-permeable channel CNGC20 modulates calcium-mediated freezing tolerance in Arabidopsis.

Plants respond to cold stress at multiple levels, including increasing cytosolic calcium (Ca2+) influx and triggering the expression of cold-responsive genes. Here we show that the Ca2+-permeable channel CYCLIC NUCLEOTIDE GATED CHANNEL20 (CNGC20) positively regulates freezing tolerance in Arabidopsis (Arabidopsis thaliana) by mediating cold-induced Ca2+ influx. Moreover, we demonstrate that the leucine-rich repeat receptor-like kinase PLANT PEPTIDE CONTAINING SULFATED TYROSINE1 RECEPTOR (PSY1R) is activated by cold, phosphorylating and enhancing the activity of CNGC20. The psy1r mutant exhibited decreased cold-evoked Ca2+ influx and freezing tolerance. Conversely, COLD-RESPONSIVE PROTEIN KINASE1 (CRPK1), a protein kinase that negatively regulates cold signaling, phosphorylates and facilitates the degradation of CNGC20 under prolonged periods of cold treatment, thereby attenuating freezing tolerance. This study thus identifies PSY1R and CRPK1 kinases that regulate CNGC20 activity and stability, respectively, thereby antagonistically modulating freezing tolerance in plants.