TNFSF14+ natural killer cells prevent spontaneous abortion by restricting leucine-mediated decidual stromal cell senescence.

In preparation for a potential pregnancy, the endometrium of the uterus changes into a temporary structure called the decidua. Senescent decidual stromal cells (DSCs) are enriched in the decidua during decidualization, but the underlying mechanisms of this process remain unclear. Here, we performed single-cell RNA transcriptomics on ESCs and DSCs and found that cell senescence during decidualization is accompanied by increased levels of the branched-chain amino acid (BCAA) transporter SLC3A2. Depletion of leucine, one of the branched-chain amino acids, from cultured media decreased senescence, while high leucine diet resulted in increased senescence and high rates of embryo loss in mice. BCAAs induced senescence in DSCs via the p38 MAPK pathway. In contrast, TNFSF14+ decidual natural killer (dNK) cells were found to inhibit DSC senescence by interacting with its ligand TNFRSF14. As in mice fed high-leucine diets, both mice with NK cell depletion and Tnfrsf14-deficient mice with excessive uterine senescence experienced adverse pregnancy outcomes. Further, we found excessive uterine senescence, SLC3A2-mediated BCAA intake, and insufficient TNFRSF14 expression in the decidua of patients with recurrent spontaneous abortion. In summary, this study suggests that dNK cells maintain senescence homeostasis of DSCs via TNFSF14/TNFRSF14, providing a potential therapeutic strategy to prevent DSC senescence-associated spontaneous abortion.

IFI16-dependent STING signaling is a crucial regulator of anti-HER2 immune response in HER2+ breast cancer.

Relapse to anti-HER2 monoclonal antibody (mAb) therapies, such as trastuzumab in HER2+ breast cancer (BC), is associated with residual disease progression due to resistance to therapy. Here, we identify interferon-γ inducible protein 16 (IFI16)-dependent STING signaling as a significant determinant of trastuzumab responses in HER2+ BC. We show that down-regulation of immune-regulated genes (IRG) is specifically associated with poor survival of HER2+, but not other BC subtypes. Among IRG, IFI16 is identified as a direct target of EZH2, the underexpression of which leads to deficient STING activation and downstream CXCL10/11 expression in response to trastuzumab treatment. Dual inhibition of EZH2 and histone deacetylase (HDAC) significantly activates IFI16-dependent immune responses to trastuzumab. Notably, a combination of a novel histone methylation inhibitor with an HDAC inhibitor induces complete tumor eradication and long-term T cell memory in a HER2+ BC mouse model. Our findings demonstrate an epigenetic regulatory mechanism suppressing the expression of the IFI16-CXCL10/11 signaling pathway that provides a survival advantage to HER2+ BC to confer resistance to trastuzumab treatment.

eccDB: a comprehensive repository for eccDNA-mediated chromatin contacts in multi-species.

SUMMARY: We developed the eccDB database to integrate available resources for extrachromosomal circular DNA (eccDNA) data. eccDB is a comprehensive repository for storing, browsing, searching, and analyzing eccDNAs from multispecies. The database provides regulatory and epigenetic information on eccDNAs, with a focus on analyzing intrachromosomal and interchromosomal interactions to predict their transcriptional regulatory functions. Moreover, eccDB identifies eccDNAs from unknown DNA sequences and analyzes the functional and evolutionary relationships of eccDNAs among different species. Overall, eccDB offers web-based analytical tools and a comprehensive resource for biologists and clinicians to decipher the molecular regulatory mechanisms of eccDNAs. AVAILABILITY AND IMPLEMENTATION: eccDB is freely available at http://www.xiejjlab.bio/eccDB.

Proteogenomic characterization of cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a highly heterogeneous cancer with limited understanding and few effective therapeutic approaches. We aimed at providing a proteogenomic CCA characterization to inform biological processes and treatment vulnerabilities. APPROACH AND RESULTS: Integrative genomic analysis with functional validation uncovered biological perturbations downstream of driver events including DPCR1 , RBM47 mutations, SH3BGRL2 copy number alterations, and FGFR2 fusions in CCA. Proteomic clustering identified three subtypes with distinct clinical outcomes, molecular features, and potential therapeutics. Phosphoproteomics characterized targetable kinases in CCA, suggesting strategies for effective treatment with CDK and MAPK inhibitors. Patients with CCA with HBV infection showed increased antigen processing and presentation (APC) and T cell infiltration, conferring a favorable prognosis compared with those without HBV infection. The characterization of extrahepatic CCA recommended the feasible application of vascular endothelial-derived growth factor inhibitors. Multiomics profiling presented distinctive molecular characteristics of the large bile duct and the small bile duct of intrahepatic CCA. The immune landscape further revealed diverse tumor immune microenvironments, suggesting immune subtypes C1 and C5 might benefit from immune checkpoint therapy. TCN1 was identified as a potential CCA prognostic biomarker, promoting cell growth by enhancing vitamin B12 metabolism. CONCLUSIONS: We characterized the proteogenomic landscape of 217 CCAs with 197 paired normal adjacent tissues and identified their subtypes and potential therapeutic targets. The multiomics analyses with other databases and some functional validations have indicated strategies regarding the clinical, biological, and therapeutic approaches to the management of CCA.

N6-Methyladenosine-mediated phase separation suppresses NOTCH1 expression and promotes mitochondrial fission in diabetic cardiac fibrosis.

BACKGROUND: N6-methyladenosine (m6A) modification of messenger RNA (mRNA) is crucial for liquid-liquid phase separation in mammals. Increasing evidence indicates that liquid-liquid phase separation in proteins and RNAs affects diabetic cardiomyopathy. However, the molecular mechanism by which m6A-mediated phase separation regulates diabetic cardiac fibrosis remains elusive. METHODS: Leptin receptor-deficient mice (db/db), cardiac fibroblast-specific Notch1 conditional knockout (POSTN-Cre × Notch1flox/flox) mice, and Cre mice were used to induce diabetic cardiac fibrosis. Adeno-associated virus 9 carrying cardiac fibroblast-specific periostin (Postn) promoter-driven small hairpin RNA targeting Alkbh5, Ythdf2, or Notch1, and the phase separation inhibitor 1,6-hexanediol were administered to investigate their roles in diabetic cardiac fibrosis. Histological and biochemical analyses were performed to determine how Alkbh5 and Ythdf2 regulate Notch1 expression in diabetic cardiac fibrosis. NOTCH1 was reconstituted in ALKBH5- and YTHDF2-deficient cardiac fibroblasts and mouse hearts to study its effects on mitochondrial fission and diabetic cardiac fibrosis. Heart tissue samples from patients with diabetic cardiomyopathy were used to validate our findings. RESULTS: In mice with diabetic cardiac fibrosis, decreased Notch1 expression was accompanied by high m6A mRNA levels and mitochondrial fission. Fibroblast-specific deletion of Notch1 enhanced mitochondrial fission and cardiac fibroblast proliferation and induced diabetic cardiac fibrosis in mice. Notch1 downregulation was associated with Alkbh5-mediated m6A demethylation in the 3'UTR of Notch1 mRNA and elevated m6A mRNA levels. These elevated m6A levels in Notch1 mRNA markedly enhanced YTHDF2 phase separation, increased the recognition of m6A residues in Notch1 mRNA by YTHDF2, and induced Notch1 degradation. Conversely, epitranscriptomic downregulation rescues Notch1 expression, resulting in the opposite effects. Human heart tissues from patients with diabetic cardiomyopathy were used to validate the findings in mice with diabetic cardiac fibrosis. CONCLUSIONS: We identified a novel epitranscriptomic mechanism by which m6A-mediated phase separation suppresses Notch1 expression, thereby promoting mitochondrial fission in diabetic cardiac fibrosis. Our findings provide new insights for the development of novel treatment approaches for patients with diabetic cardiac fibrosis.

ROS-induced oxidative stress is a major contributor to sperm cryoinjury.

STUDY QUESTION: What is the mechanism behind cryoinjury in human sperm, particularly concerning the interplay between reactive oxygen species (ROS) and autophagy, and how does it subsequently affect sperm fate? SUMMARY ANSWER: The freeze-thaw operation induces oxidative stress by generating abundant ROS, which impairs sperm motility and activates autophagy, ultimately guiding the sperm toward programmed cell death such as apoptosis and necrosis, as well as triggering premature capacitation. WHAT IS KNOWN ALREADY: Both ROS-induced oxidative stress and autophagy are thought to exert an influence on the quality of frozen-thawed sperm. STUDY DESIGN, SIZE, DURATION: Overall, 84 semen specimens were collected from young healthy fertile males, with careful quality evaluation. The specimens were split into three groups to investigate the ROS-induced cryoinjury: normal control without any treatment, sperm treated with 0.5 mM hydrogen peroxide (H2O2) for 1 h, and sperm thawed following cryopreservation. Samples from 48 individuals underwent computer-assisted human sperm analysis (CASA) to evaluate sperm quality in response to the treatments. Semen samples from three donors were analyzed for changes in the sperm proteome after H2O2 treatment, and another set of samples from three donors were analyzed for changes following the freeze-thaw process. The other 30 samples were used for fluorescence-staining and western blotting. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm motility parameters, including progressive motility (PR %) and total motility (PR + NP %), were evaluated using the CASA system on a minimum of 200 spermatozoa. The proteomic profiles were determined with label-free mass spectrometry (MS/MS) and protein identification was performed via ion search against the NCBI human database. Subsequently, comprehensive bioinformatics was applied to detect significant proteomic changes and functional enrichment. Fluorescence-staining and western blot analyses were also conducted to confirm the proteomic changes on selected key proteins. The ROS level was measured using 2',7'-dichlorodihydrofluorescein diacetate labeling and the abundance of bioactive mitochondria was determined by evaluating the inner mitochondrial membrane potential (MMP) level. Molecular behaviors of sequestosome-1 (p62 or SQSTM1) and microtubule-associated proteins 1A/1B light chain 3 (LC3) were monitored to evaluate the state of apoptosis in human sperm. Fluorescent probes oxazole yellow (YO-PRO-1) and propidium iodide (PI) were utilized to monitor programmed cell death, namely apoptosis and necrosis. Additionally, gradient concentrations of antioxidant coenzyme Q10 (CoQ10) were introduced to suppress ROS impacts on sperm. MAIN RESULTS AND THE ROLE OF CHANCE: The CASA analysis revealed a significant decrease in sperm motility for both the H2O2-treatment and freeze-thaw groups. Fluorescence staining showed that high ROS levels were produced in the treated sperm and the MMPs were largely reduced. The introduction of CoQ10 at concentrations of 20 and 30 µM resulted in a significant rescue of progressive motility (P < 0.05). The result suggested that excessive ROS could be the major cause of sperm motility impairment, likely by damaging mitochondrial energy generation. Autophagy was significantly activated in sperm when they were under oxidative stress, as evidenced by the upregulation of p62 and the increased conversion of LC3 as well as the upregulation of several autophagy-related proteins, such as charged multivesicular body protein 2a, mitochondrial import receptor subunit TOM22 homolog, and WD repeat domain phosphoinositide-interacting protein 2. Additionally, fluorescent staining indicated the occurrence of apoptosis and necrosis in both H2O2-treated sperm and post-thaw sperm. The cell death process can be suppressed when CoQ10 is introduced, which consolidates the view that ROS could be the major contributor to sperm cryoinjury. The freeze-thaw process could also initiate sperm premature capacitation, demonstrated by the prominent increase in tyrosine phosphorylated proteins, verified with anti-phosphotyrosine antibody and immunofluorescence assays. The upregulation of capacitation-related proteins, such as hyaluronidase 3 and Folate receptor alpha, supported this finding. LARGE SCALE DATA: The data underlying this article are available in the article and its online supplementary material. LIMITATIONS, REASONS FOR CAUTION: The semen samples were obtained exclusively from young, healthy, and fertile males with progressive motility exceeding 60%, which might overemphasize the positive effects while possibly neglecting the negative impacts of cryoinjury. Additionally, the H2O2 treatment conditions in this study may not precisely mimic the oxidative stress experienced by sperm after thawing from cryopreservation, potentially resulting in the omission of certain molecular alterations. WIDER IMPLICATIONS OF THE FINDINGS: This study provides substantial proteomic data for a comprehensive and deeper understanding of the impact of cryopreservation on sperm quality. It will facilitate the design of optimal protocols for utilizing cryopreserved sperm to improve applications, such as ART, and help resolve various adverse situations caused by chemotherapy, radiotherapy, and surgery. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Major Innovation Project of Research Institute of National Health Commission (#2022GJZD01-3) and the National Key R&D Program of China (#2018YFC1003600). All authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

Mitofilin in cardiovascular diseases: Insights into the pathogenesis and potential pharmacological interventions.

The impact of mitochondrial dysfunction on the pathogenesis of cardiovascular disease is increasing. However, the precise underlying mechanism remains unclear. Mitochondria produce cellular energy through oxidative phosphorylation while regulating calcium homeostasis, cellular respiration, and the production of biosynthetic chemicals. Nevertheless, problems related to cardiac energy metabolism, defective mitochondrial proteins, mitophagy, and structural changes in mitochondrial membranes can cause cardiovascular diseases via mitochondrial dysfunction. Mitofilin is a critical inner mitochondrial membrane protein that maintains cristae structure and facilitates protein transport while linking the inner mitochondrial membrane, outer mitochondrial membrane, and mitochondrial DNA transcription. Researchers believe that mitofilin may be a therapeutic target for treating cardiovascular diseases, particularly cardiac mitochondrial dysfunctions. In this review, we highlight current findings regarding the role of mitofilin in the pathogenesis of cardiovascular diseases and potential therapeutic compounds targeting mitofilin.

Devosia lacusdianchii sp. nov., an attached bacterium inhibited by metabolites from its symbiotic Microcystis.

A novel alphaproteobacterial strain JXJ CY 41T was isolated from a culture mass of Microcystis, collected from Lake Dianchi, south-west, China. Strain JXJ CY 41T was gram-strain-negative, aerobic, motile, with rod-shaped cells (0.4-1.0 × 1.7-3.5 µm). It was positive for catalase and starch hydrolysis, negative for oxidase and hydrolysis of Tweens (20, 40, and 80). Growth occurred at 10-44 °C, pH 5.0-10.0, and 0-5.0% (w/v) NaCl. Major fatty acids included C16:0 (28.1%), 11-methyl C18:1 ω7c (36.7%) and C18:1 ω7c (20.8%). Q10 was the sole ubiquinone. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, glycolipid, and an unidentified lipid. The DNA G + C content was 63.1%. Its 16S rRNA gene sequence showed high similarities with Devosia oryziradicis G19T (99.5%; not validly published), D. yakushimensis Yak96BT (98.3%) and D. ginsengisoli Gsoil 520T (98.1%), and less than 98.1% similarities with other members of the genus Devosia. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain JXJ CY 41T and its 5 closest similar strains were 19.9-24.1% and 75.7-80.5%, respectively. Based on the data above, strain JXJ CY 41T was identified as a novel species of the genus Devosia, for which the epithet Devosia lacusdianchii sp. nov. was proposed. The type strain is JXJ CY 41T (= KCTC 72812T = CGMCC 1.17502T). Strain JXJ CY 41T exhibited different interactions with Microcystis aeruginosa FACHB-905 (Maf) under different conditions, and Maf could control the bacterial cellular density by secreting unknown specific chemical compounds according to its nutritional requirements.

The mechanism and therapeutic potential of lncRNA MIR497HG/miR-16-5p axis in breast cancer.

BACKGROUND: Breast cancer has become a major public health problem in the current society, and its incidence rate ranks the first among Chinese female malignant tumors. This paper once again confirmed the efficacy of lncRNA in tumor regulation by introducing the mechanism of the diagnosis of breast cancer by the MIR497HG/miR-16-5p axis. METHODS: The abnormal expression of MIR497HG in breast cancer was determined by RT-qPCR method, and the correlation between MIR497HG expression and clinicopathological characteristics of breast cancer patients was analyzed via Chi-square test. To understand the diagnostic potential of MIR497HG in breast cancer by drawing the receiver operating characteristic curve (ROC). The overexpressed MIR497HG (pcDNA3.1-MIR497HG) was designed and constructed to explore the regulation of elevated MIR497HG on biological function of BT549 and Hs 578T cells through Transwell assays. Additionally, the luciferase gene reporter assay and Pearson analysis evaluated the targeting relationship of MIR497HG to miR-16-5p. RESULTS: MIR497HG was decreased in breast cancer and had high diagnostic function, while elevated MIR497HG inhibited the migration and invasion of BT549 and Hs 578T cells. In terms of functional mechanism, miR-16-5p was the target of MIR497HG, and MIR497HG reversely regulated the miR-16-5p. miR-16-5p mimic reversed the effects of upregulated MIR497HG on cell biological function. CONCLUSIONS: In general, MIR497HG was decreased in breast cancer, and the MIR497HG/miR-16-5p axis regulated breast cancer tumorigenesis, providing effective insights for the diagnosis of patients.

Chromosome 1q21 Aberrations Are Poor Prognostic Factors for Newly Diagnosed Multiple Myeloma Patients.

BACKGROUND: Chromosome 1q21 aberrations are one of the most common cytogenetic abnormalities in patients with multiple myeloma (MM). However, the prognostic value remains controversial. This study aimed to determine the prognostic value of numerical abnormalities of chromosome 1q21 for newly diagnosed patients with MM patients in Chinese population. METHODS: We retrospectively analyzed 629 patients with newly diagnosed MM who received the detection of chromosome 1q21 by fluorescence in situ hybridization in China. RESULTS: Among 629 patients, 309 (49.1%) had 1q21 abnormalities, of which 187 (29.7%) had three copies and 122 (19.4%) had four or more copies. Patients with two copies of 1q21 had a significantly longer median overall survival (OS) than those with three copies or ≥4 copies and also had longer progression-free survival (PFS). However, patients with three or ≥4 copies had similar OS and PFS. Univariate Cox proportional hazards regression analyses determined that 1q21 aberrations are associated with shorter OS and PFS. 1q21 aberrations are also independent poor prognostic factors for OS and PFS in multivariable analyses. Del(17p), t(4;14), and t(14;16) are common high-risk cytogenetic abnormalities (HRCAs) in patients with MM. Patients with 1q21+ alone or 1q21+ combined with HRCAs had shorter OS and PFS than patients without cytogenetic abnormalities. Patients with 1q21+ and t(11;14) also had shorter PFS but had similar OS than patients without cytogenetic abnormalities. CONCLUSION: Our study showed that chromosome 1q21 aberrations are poor prognostic factors for newly diagnosed patients with MM.

DNA-encoded chemistry technology yields expedient access to SARS-CoV-2 Mpro inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [Ki] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (Ki = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (Ki = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.

Identification of novel biomarkers of ferroptosis involved in keloid based on bioinformatics analysis.

Keloid is a fibroproliferative disease of unknown aetiology, which has a significant impact the quality of life of patients. Ferroptosis plays an important role in the occurrence and development of fibrosis, but there is still a lack of research related to keloids. The objective of this work was to identify the hub genes related to ferroptosis in keloid to better understand the keloid process. The microarray data (GSE7890 GSE145725, and GSE44270) (23 keloid and 22 normal fibroblast) were analysed via the gene expression comprehensive database (GEO). Only GSE7890 met the FerrDB database. Cell cycle and pathway analysis were performed with gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to differentially expressed genes (DEG). The differential genes were confirmed in other GEO datasets (GSE145725 and GSE44270), and multi-fibrosis-gene correlation analysed. To validate these hub genes, quantitative real-time PCR (qRT-PCR) was conducted. A total of 581 DEGs were screened, with 417 genes down-regulated and 164 genes up-regulated, with 11 ferroptosis genes significantly up-regulated in both keloid and normal tissue, and 6 genes are consistent with our findings and are associated with multiple fibrosis genes. The qRT-PCR results and tissues of normal skin and keloid agreed with our predictions. Our findings provide new evidence for the ferroptosis-related molecular pathways and biomarker of keloid.

[Guidelines for quality control of human use experience study on traditional Chinese medicine].

National Medical Products Administration released the Special provisions on the administration of registration of traditio-nal Chinese medicine(TCM) in February 2023, encouraging high-quality human use experience(HUE) study in TCM clinical practice to obtain sufficient evidence for TCM registration support. The provisions suggested that the HUE study should meet the relevant requirements and accept the drug registration verification. This paper aims to standardize the HUE study, obtain high-quality HUE data to support registration applications, and promote the standardization of research. In accordance with the relevant laws and regulations of the state and the requirements of the technical guidelines for the HUE study in the drug review center of the National Medical Products Administration, the clinical characteristics of TCM were considered, and the Clinical Evaluation Committee of Traditional Chinese Me-dicine of the Chinese Pharmaceutical Association organized and formulated the Guidelines for quality control of human use experience study on traditional Chinese medicine,including the conditions of medical institutions carrying out HUE study, researchers, sponsors, key information and requirements of pharmacy, research programs, key points of ethical review, requirements of the research implementation process, risk management, and subject protection of HUE study. After several rounds of consultation with experts, a guideline document suitable for supporting drug registration and guiding HUE study on TCM was finally formed.

[Guidelines for post-marketing research on clinical effectiveness of Chinese patent medicines].

Due to the lack of specialized guidance, the post-marketing research on clinical effectiveness of Chinese patent medicines demonstrates varied quality and lacks high-quality evidence, failing to meet the demands of policy-making, clinical decision-making, and industrial decision-making. To address this issue, this project gathered experts in clinical medicine, clinical pharmacy, evidence-based medicine, drug epidemiology, medical ethics, and policy and regulation in China. They referred to the model of international post-marketing research on medicines and developed Guidelines for post-marketing research on clinical effectiveness of Chinese patent medicines under the framework of relevant laws and regulations and technical guidance documents in China. The guidelines were developed with consideration to the characteristics of Chinese patent medicines, China's national conditions, and all the stakeholders including marketing authorization holders, clinical researchers, drug administration, and users. The development of the guidelines followed the requirements for developing group standards set by the China Association of Chinese Medicine. The guidelines fully implement the concept of full life-cycle research, emphasizing the combination of traditional Chinese medicine(TCM) theory, human use experience, and clinical trials and pay attention to the compliance, scientificity, and ethics of research. The guidelines clarify the topic selection and decision-making path of the post-marketing research on effectiveness of Chinese patent medicines through six steps: determining research purpose, analyzing drug characteristics, evaluating research basis, proposing clinical orientation, clarifying research purpose, and implementing classified research. The general principles of research design and implementation were clarified from eight aspects: research type, research objects, sample size, efficacy indicators, bias, missing data, evidence level, and practicality. It focuses on the research on the TCM syndrome-based efficacy evaluation, clinical value-oriented mechanism of action, and the effectiveness of Chinese patent medicines with different routes of administration. The guidelines provide a universal methodological basis for the post-marketing research on clinical effectiveness of Chinese patent medicines.

[Establishment and content validity assessment of evaluation index system for development value of traditional Chinese medicine preparations in medical institutions].

The development of traditional Chinese medicine(TCM) preparations as an incubator for new drugs in medical institutions has flourished, while an evaluation index system remains to be established for comprehensively assessing the development value of these prescriptions. This study established an item pool through literature research, employed the Delphi method to determine the content of evaluation indexes, and adopted the superiority chart to determine the weight of each index. Two-level evaluation index system for the development value of TCM preparations in medical institutions was established, which included 7 first-level items and 36 se-cond-level items, demonstrating scientific validity. The first-level items(weight) were inheritance(10.61%), effectiveness(23.22%), safety(22.71%), innovation(13.21%), economy(10.00%), suitability(8.57%), and accessibility(11.68%). The top three second-level items in terms of weight distribution were adverse reaction monitoring(6.73%), evidence of therapeutic effect(5.71%), and clinical response rate(4.75%). The bottom three second-level items were production advantages(0.86%), medicinal dosage(0.48%), and medicinal smell or taste(0.18%). The content validity of the established system was assessed, which revealed that the index system was reliable, with the overall and average content validity indexes of 0.47 and 0.90, respectively. Furthermore, the established evaluation index system was used to evaluate six TCM preparations in a city-level hospital of TCM in Sichuan Province, which demonstrated that the system had operability. The results indicate that the evaluation index system is scientific, reliable, and operable, providing a reference for developers to selectively develop TCM preparations in medical institutions. In practical application, the system can be adjusted regarding the index weights according to actual conditions.

Switchable terahertz orbital angular momentum Bessel beams based on spin-decoupled multifunctional reflective metasurfaces.

In this study, switchable terahertz (THz) multi-orbital angular momentum (OAM) Bessel beams (BBs) were developed based on a spin-decoupled reflective multifunctional metasurface (MTS). Switchability was achieved by switching the feed between left-hand circular polarization (LCP), right-hand circular polarization (RCP), and linear polarization (LP) incidences. A switchable physical model was established for calculating the beam direction, OAM mode, polarization, and non-diffractive distance of the outgoing BBs. As an example, a spin-decoupled MTS was designed to generate dual BBs under LCP incidence, which was subsequently switched to RCP or LP for switchability. The outgoing BBs could be switched among three types of beams: Type-1 under LCP incidence (LCP, θL = 40°, φL = 0°, lL = 1, dL = 18 cm) and (RCP, θR = -40°, φR = 0°, lR = -1, dR = 20 cm); Type-2 under RCP incidence (RCP, θR = 40°, φR = 0°, lR = 1, dR = 18 cm) and (LCP, θL = -19°, φL = 0°, lL = 3, dL = 16.4 cm); and Type-3 under LP incidence (LP, θ = 40°, φ = 0°, l = 1, d = 18 cm), (RCP, θR = -40°, φR = 0°, lR = -1, dR = 20 cm) and (LCP, θL = -19°, φL = 0°, lL = 3, dL = 16.4 cm). Compared with previous MTSs, the proposed spin-decoupled MTS has the advantages of switchability among BBs, high non-diffractive distance/aperture size ratio of 15, large beam deflection angle of up to 40°, and high BB conversion efficiency of up to 96%. The simulated results were consistent with those calculated using the physical model, thus validating the physical model. The designed switchable BBs have potential THz near-field applications, such as high-capacity near-field wireless communications, wireless power transfer, high-resolution imaging, non-destructive testing, and speed detection of high-speed rotating objects.

METTL3 boosts mitochondrial fission and induces cardiac fibrosis by enhancing LncRNA GAS5 methylation.

Dysregulated mitochondrial metabolism occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the role of mitochondrial fission is not well appreciated in cardiac fibrosis, which is accompanied by enhanced fibroblast proliferation and migration. We investigated the causes and consequences of mitochondrial fission in cardiac fibrosis using cultured cells, animal models, and clinical samples. Increased METTL3 expression caused excessive mitochondrial fission, resulting in the proliferation and migration of cardiac fibroblasts that lead to cardiac fibrosis. Knockdown of METTL3 suppressed mitochondrial fission, inhibiting fibroblast proliferation and migration for ameliorating cardiac fibrosis. Elevated METTL3 and N6-methyladenosine (m6A) levels were associated with low expression of long non-coding RNA GAS5. Mechanistically, METTL3-mediated m6A methylation of GAS5 induced its degradation, dependent of YTHDF2. GAS5 could interact with mitochondrial fission marker Drp1 directly; overexpression of GAS5 suppressed Drp1-mediated mitochondrial fission, inhibiting cardiac fibroblast proliferation and migration. Knockdown of GAS5 produced the opposite effect. Clinically, increased METTL3 and YTHDF2 levels corresponded with decreased GAS5 expression, increased m6A mRNA content and mitochondrial fission, and increased cardiac fibrosis in human heart tissue with atrial fibrillation. We describe a novel mechanism wherein METTL3 boosts mitochondrial fission, cardiac fibroblast proliferation, and fibroblast migration: METTL3 catalyzes m6A methylation of GAS5 methylation in a YTHDF2-dependent manner. Our findings provide insight into the development of preventative measures for cardiac fibrosis.

Identification of prognostic and therapeutic biomarkers in type 2 papillary renal cell carcinoma.

BACKGROUND: Papillary renal cell carcinoma (PRCC) can be divided into type 1 (PRCC1) and type 2 (PRCC2) and PRCC2 share a more invasive phenotype and worse prognosis. This study aims to identify potential prognostic and therapeutic biomarkers in PRCC2. METHODS: A cohort from The Cancer Genome Atlas and two datasets from Gene Expression Omnibus were examined. Common differentially expressed genes (DEGs) were screened and potential biomarkers were explored by using Kaplan-Meier method and cox regression analysis. Functional enrichment analysis was utilized to evaluate the potential biological functions. Tumor infiltrating immune cells were estimated by CIBERSORT algorithm. Ninety-two PRCC2 samples from Fudan University Shanghai Cancer Center were obtained, and immunostaining was performed to validate prognostic and therapeutic significance of the potential biomarker. RESULTS: PRCC2 has worse overall survival and shares distinct molecular characteristics from PRCC1. There was significant higher expression level of Targeting protein for Xklp2 (TPX2) in PRCC2 compared with normal tissues. Higher expression level of TPX2 was significantly associated with worse overall survival in PRCC2 and kinesin family genes expression were found significantly elevated in high risk PRCC2. Abundance of tumor infiltrating M1 macrophage was significantly higher in PRCC2 and it was also associated with worse overall survival. In the FUSCC cohort, higher TPX2 expression was significantly correlated with worse overall and progression-free survival. Retrospective analysis indicated that mTOR inhibitor (everolimus) had greater efficacy in the high-risk group than in the low-risk group (overall response rate: 28.6% vs. 16.7%) and that everolimus had greater efficacy than sunitinib in the high-risk group (overall response rate: 28.6% vs. 20%). CONCLUSIONS: TPX2 was a prognostic and therapeutic biomarker in PRCC2. Higher abundance of tumor infiltrating M1 macrophage was significantly associated with worse overall survival in PRCC2. mTOR inhibitors may have good efficacy in patients with high-risk PRCC2.

Molecular cloning, sequence, and expression patterns of DNA damage induced transcript 3 (DDIT3) gene in female yaks (Bos grunniens).

Endoplasmic reticulum stress (ERS) plays an important role in regulating the reproductive process of female mammals, mainly involved in follicular atresia and corpus luteum regression. DNA damage induced transcript 3 (DDIT3) is a marker gene of ERS. The objectives of the present study were to clone and analyze the sequence and tissue expression characteristics of DDIT3 gene in female yaks. By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length 507-bp DDIT3-cDNA, encoding for 168-aa protein. Yak DDIT3 exhibited highest and least identity with that of bison and horse, respectively. Real-time PCR analyses revealed that the expression level of DDIT3 gene in ovary was higher than that in heart, liver, kidney, spleen, lung, uterus and oviduct (p < 0.05). DDIT3 expression level in ovary and uterus during pregnancy was higher than that in follicular phase, luteal phase and fetus stage. DDIT3 was highly expressed in metaphase II oocytes and granulosa cells than that in germinal vesicle and metaphase I oocytes (p < 0.05), respectively. This is the first molecular characterization and expression patterns of DDIT3 gene in female yaks. These results indicated that the DDIT3 gene possibly plays an important role in regulating ovary function and pregnancy maintenance in yaks.