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In brief: PLCZ1 mutations are related to total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), characterised by abnormal oocyte oscillations. The novel PLCZ1 compound heterozygous mutations reported by this study were associated with TFF after ICSI, with one of the mutations indicating a gene dosage effect. Abstract: Oocyte activation failure is thought to be one of the main factors for total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), which could be induced by abnormal calcium oscillations. Phospholipase C zeta (PLCZ), a sperm factor, is associated with Ca2+ oscillations in mammalian oocytes. To date, some mutations in PLCZ1 (the gene that encodes PLCZ) have been linked to TFF, as demonstrated by the observed reduction in protein levels or activity to induce Ca2+ oscillations. In this study, normozoospermic males whose sperms exhibited TFF after ICSI and their families were recruited. First, mutations in the PLCZ1 sequence were identified by whole exome sequencing and validated using Sanger sequencing. Then, the locations of PLCZ1/PLCZ and the transcript and protein levels in the sperm of the patients were studied. Subsequently, in vitro function analysis and in silico analysis were performed to investigate the function-structure correlation of mutations identified in PLCZ1 using western blotting, immunofluorescence, RT-qPCR, and molecular simulation. Ca2+ oscillations were detected after cRNA microinjection into MII mouse oocytes to investigate calcium oscillations induced by abnormal PLCZ. Five variants with compound heterozygosity were identified, consisting of five new mutations and three previously reported mutations distributed across the main domains of PLCZ, except the EF hands domain. The transcript and protein levels decreased to varying degrees among all detected mutations in PLCZ1 when transfected in HEK293T cells. Among these, mutations in M138V and R391* of PLCZ were unable to trigger typical Ca2+ oscillations. In case 5, aberrant localisation of PLCZ in the sperm head and an increased expression of PLCZ in the sperm were observed. In conclusion, this study enhances the potential for genetic diagnosis of TFF in clinics and elucidates the possible relationship between the function and structure of PLCZ in novel mutations.

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BACKGROUND: Adverse drug reactions (ADRs), which are the phenotypic manifestations of clinical drug toxicity in humans, are a major concern in precision clinical medicine. A comprehensive evaluation of ADRs is helpful for unbiased supervision of marketed drugs and for discovering new drugs with high success rates. OBJECTIVE: In current practice, drug safety evaluation is often oversimplified to the occurrence or nonoccurrence of ADRs. Given the limitations of current qualitative methods, there is an urgent need for a quantitative evaluation model to improve pharmacovigilance and the accurate assessment of drug safety. METHODS: In this study, we developed a mathematical model, namely the Adverse Drug Reaction Classification System (ADReCS) severity-grading model, for the quantitative characterization of ADR severity, a crucial feature for evaluating the impact of ADRs on human health. The model was constructed by mining millions of real-world historical adverse drug event reports. A new parameter called Severity_score was introduced to measure the severity of ADRs, and upper and lower score boundaries were determined for 5 severity grades. RESULTS: The ADReCS severity-grading model exhibited excellent consistency (99.22%) with the expert-grading system, the Common Terminology Criteria for Adverse Events. Hence, we graded the severity of 6277 standard ADRs for 129,407 drug-ADR pairs. Moreover, we calculated the occurrence rates of 6272 distinct ADRs for 127,763 drug-ADR pairs in large patient populations by mining real-world medication prescriptions. With the quantitative features, we demonstrated example applications in systematically elucidating ADR mechanisms and thereby discovered a list of drugs with improper dosages. CONCLUSIONS: In summary, this study represents the first comprehensive determination of both ADR severity grades and ADR frequencies. This endeavor establishes a strong foundation for future artificial intelligence applications in discovering new drugs with high efficacy and low toxicity. It also heralds a paradigm shift in clinical toxicity research, moving from qualitative description to quantitative evaluation.

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BACKGROUND: Liquid chromatography-mass spectrometry (LC-MS)-based quantitative phosphoproteomics has been widely used to detect thousands of protein phosphorylation modifications simultaneously from the biological specimens. However, the complicated procedures for analyzing phosphoproteomics data has become a bottleneck to widening its application. METHODS: Here, we develop PhosMap, a versatile and scalable tool to accomplish phosphoproteomics data analysis. A standardized phosphorylation data format was created for data analyses, from data preprocessing to downstream bioinformatic analyses such as dimension reduction, differential phosphorylation analysis, kinase activity, survival analysis, and so on. For better usability, we distribute PhosMap as a Docker image for easy local deployment upon any of Windows, Linux, and Mac system. RESULTS: The source code is deposited at https://github.com/BADD-XMU/PhosMap. A free PhosMap webserver (https://huggingface.co/spaces/Bio-Add/PhosMap), with easy-to-follow fashion of dashboards, is curated for interactive data analysis. CONCLUSIONS: PhosMap fills the technical gap of large-scale phosphorylation research by empowering researchers to process their own phosphoproteomics data expediently and efficiently, and facilitates better data interpretation.

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The impairment of sperm maturation is one of the major pathogenic factors in male subfertility, a serious medical and social problem affecting millions of global couples. Regrettably, the existing research on sperm maturation is slow, limited, and fragmented, largely attributable to the lack of a global molecular view. To fill the data gap, we newly established a database, namely the Sperm Maturation Database (SperMD, http://bio-add.org/SperMD ). SperMD integrates heterogeneous multi-omics data (170 transcriptomes, 91 proteomes, and five human metabolomes) to illustrate the transcriptional, translational, and metabolic manifestations during the entire lifespan of sperm maturation. These data involve almost all crucial scenarios related to sperm maturation, including the tissue components of the epididymal microenvironment, cell constituents of tissues, different pathological states, and so on. To the best of our knowledge, SperMD could be one of the limited repositories that provide focused and comprehensive information on sperm maturation. Easy-to-use web services are also implemented to enhance the experience of data retrieval and molecular comparison between humans and mice. Furthermore, the manuscript illustrates an example application demonstrated to systematically characterize novel gene functions in sperm maturation. Nevertheless, SperMD undertakes the endeavor to integrate the islanding omics data, offering a panoramic molecular view of how the spermatozoa gain full reproductive abilities. It will serve as a valuable resource for the systematic exploration of sperm maturation and for prioritizing the biomarkers and targets for precise diagnosis and therapy of male subfertility.

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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.

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Genes have the ability to produce transcript variants that perform specific cellular functions. However, accurately detecting all transcript variants remains a long-standing challenge, especially when working with poorly annotated genomes or without a known genome. To address this issue, we have developed a new computational method, TransIntegrator, which enables transcriptome-wide detection of novel transcript variants. For this, we determined 10 Illumina sequencing transcriptomes and a PacBio full-length transcriptome for consecutive embryo development stages of amphioxus, a species of great evolutionary importance. Based on the transcriptomes, we employed TransIntegrator to create a comprehensive transcript variant library, namely iTranscriptome. The resulting iTrancriptome contained 91 915 distinct transcript variants, with an average of 2.4 variants per gene. This substantially improved current amphioxus genome annotation by expanding the number of genes from 21 954 to 38 777. Further analysis manifested that the gene expansion was largely ascribed to integration of multiple Illumina datasets instead of involving the PacBio data. Moreover, we demonstrated an example application of TransIntegrator, via generating iTrancriptome, in aiding accurate transcriptome assembly, which significantly outperformed other hybrid methods such as IDP-denovo and Trinity. For user convenience, we have deposited the source codes of TransIntegrator on GitHub as well as a conda package in Anaconda. In summary, this study proposes an affordable but efficient method for reliable transcriptomic research in most species.

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Macrophages are heterogeneous and play critical roles in development and disease, but their diversity, function, and specification remain inadequately understood during human development. We generated a single-cell RNA sequencing map of the dynamics of human macrophage specification from PCW 4-26 across 19 tissues. We identified a microglia-like population and a proangiogenic population in 15 macrophage subtypes. Microglia-like cells, molecularly and morphologically similar to microglia in the CNS, are present in the fetal epidermis, testicle, and heart. They are the major immune population in the early epidermis, exhibit a polarized distribution along the dorsal-lateral-ventral axis, and interact with neural crest cells, modulating their differentiation along the melanocyte lineage. Through spatial and differentiation trajectory analysis, we also showed that proangiogenic macrophages are perivascular across fetal organs and likely yolk-sac-derived as microglia. Our study provides a comprehensive map of the heterogeneity and developmental dynamics of human macrophages and unravels their diverse functions during development.

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Small molecule inhibitors of the bromodomain and extraterminal domain (BET) family proteins have emerged as promising options not only for the treatment of multiple cancers but also for disturbing the process of sperm maturation with potential for use as viable contraceptive targets. In this study, we find that the BET family inhibitor NHWD870 and BRDT can bind well in vitro through bioinformatics software prediction and protein binding inhibition experiments. NHWD870 can produce a good contraceptive effect through animal experiments in vivo, and the fertility can be restored to normal after drug withdrawal. Transcriptomics and proteomics results suggest that NHWD870 affects pathways related to spermatogenesis and maturation, further contributing to the male infertility phenotype. Our results show that NHWD870 can induce a complete and reversible contraceptive effect in mice, which is stronger than that of JQ1 and its synthesized derivatives. This study is expected to eventually lead to clinical trials.

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Metastasis is the main fatal cause of colorectal cancer (CRC). Although enormous efforts have been made to date to identify biomarkers associated with metastasis, there is still a huge gap to translate these efforts into effective clinical applications due to the poor consistency of biomarkers in dealing with the genetic heterogeneity of CRCs. In this study, a small cohort of eight CRC patients was recruited, from whom we collected cancer, paracancer, and normal tissues simultaneously and performed whole-exome sequencing. Given the exomes, a novel statistical parameter LIP was introduced to quantitatively measure the local invasion power for every somatic and germline mutation, whereby we affirmed that the innate germline mutations instead of somatic mutations might serve as the major driving force in promoting local invasion. Furthermore, via bioinformatic analyses of big data derived from the public zone, we identified ten potential driver variants that likely urged the local invasion of tumor cells into nearby tissue. Of them, six corresponding genes were new to CRC metastasis. In addition, a metastasis resister variant was also identified. Based on these eleven variants, we constructed a logistic regression model for rapid risk assessment of early metastasis, which was also deployed as an online server, AmetaRisk (http://www.bio-add.org/AmetaRisk). In summary, we made a valuable attempt in this study to exome-wide explore the genetic driving force to local invasion, which provides new insights into the mechanistic understanding of metastasis. Furthermore, the risk assessment model can assist in prioritizing therapeutic regimens in clinics and discovering new drug targets, and thus substantially increase the survival rate of CRC patients.

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Recurrent metastasis is a major fatal cause of breast cancer. Regretfully, the driving force and the molecular beneath have not been fully illustrated yet. In this study, a cohort of breast cancer patients with locoregional metastasis was recruited. For them, we collected the matched samples of the primary tumor and metastatic tumor, and then we determined the mutation profiles with whole-exome sequencing (WES). On basis of the profiles, we identified a list of deleterious variants in eight susceptible genes. Of them, filamin A (FLNA) was considered a potential driver gene of metastasis, and its low expression could enhance 5 years' relapse survival rate by 15%. To prove the finding, we constructed a stable FLNA knockout tumor cell line, which manifested that the cell abilities of proliferation, migration, and invasion were significantly weakened in response to the gene knockout. Subsequently, xenograft mouse experiments further proved that FLNA knockout could inhibit local or distal metastasis. Putting all the results together, we consolidated that FLNA could be a potential driver gene to metastasis of breast cancer, in particular triple-negative breast cancer. Additional experiments also suggested that FLNA might intervene in metastasis via the regulation of MMP-1 expression. In summary, this study demonstrates that FLNA may play as a positive regulator in cancer proliferation and recurrence. It provides new insight into breast cancer metastasis and suggests a potential new therapeutic target for breast cancer therapy.

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The treatment of autism spectrum disorder (ASD) is one of the most difficult challenges in neurodevelopmental diseases, because of the unclear pathogenesis research and low brain-lesion targeting efficiency. Besides, maternal immune activation has been reported as the most mature and widely used model of ASD and aspirin-triggered lipoxin A4 is a potent anti-inflammatory mediator being involved in the resolution of neuroinflammation in ASD. Therefore, an aspirin encapsulated cascade drug delivery system (Asp@TMNPs) is established, which can successively target the blood-brain barrier (BBB) and microglial cells and response to the acid microenvironment in lysosome. As a result, the mitochondrial oxidative stress, DNA damage, and inflammation of microglial cells are prominently alleviated. After the treatment of Asp@TMNPs, the social interaction, stereotype behavior, and anxious condition of ASD mice are notably improved and the activation of microglial cells is inhibited. Overall, this system successively penetrates the BBB and targets microglial cells, therefore, it significantly enhances the intracephalic drug accumulation and improves anti-neuroinflammatory efficacy of aspirin, providing a promising strategy for ASD treatment.

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The mechanisms shaping the amino acids recruitment pattern into the proteins in the early life history presently remains a huge mystery. In this study, we conducted genome-wide analyses of amino acids usage and genetic codons structure in 7270 species across three domains of life. The carried-out analyses evidenced ubiquitous usage bias of amino acids that were likely independent from codon usage bias. Taking advantage of codon usage bias, we performed pseudotime analysis to re-determine the chronological order of the species emergence, which inspired a new species relationship by tracing the imprint of codon usage evolution. Furthermore, the multidimensional data integration showed that the amino acids A, D, E, G, L, P, R, S, T and V might be the first recruited into the last universal common ancestry (LUCA) proteins. The data analysis also indicated that the remaining amino acids most probably were gradually incorporated into proteogenesis process in the course of two long-timescale parallel evolutionary routes: I→F→Y→C→M→W and K→N→Q→H. This study provides new insight into the origin of life, particularly in terms of the basic protein composition of early life. Our work provides crucial information that will help in a further understanding of protein structure and function in relation to their evolutionary history.

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Members of the genus Bougainvillea are rich sources of natural dyes, pigments, and traditional medicines. They are also commonly used as ornamentals in roadside landscape construction. However, the horticultural development of Bougainvillea flowers with extended growth periods and coloration is not always feasible. One reason is limited molecular knowledge and no genomic information for Bougainvillea. Here, we compiled an integrative transcriptome of all expressed transcripts for Bougainvillea × buttiana Miss Manila by integrating 20 Illumina-sequencing RNA transcriptomes. The integrative transcriptome consisted of 97,623 distinct transcripts. Of these, 47,006 were protein-coding, 31,109 were non-coding, and 19,508 were unannotated. In addition, we affirmed that the integrative transcriptome could serve as a surrogate reference to the genome in aiding accurate transcriptome assembly. For convenience, we curated the integrative transcriptome database for Bougainvillea, namely InTransBo, which can be freely accessed at http://www.bio-add.org/InTransBo/index.jsp . To the best of our knowledge, the present study is the most comprehensive genomic resource for Bougainvillea up-to-date. The integrative transcriptome helps fill the genomic gap and elucidate the transcriptional nature of Bougainvillea. It may also advance progress in the precise regulation of flowering in horticulture. The same strategy can be readily applied toward the systematic exploration of other plant species lacking complete genomic information.

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Sperm fibrous sheath (FS) is closely related to sperm maturation, capacitation and motility, and A-kinase anchor protein 4 (AKAP4) is the most abundant protein in sperm FS. Previous studies found incomplete sperm FSs and abnormal flagella in Akap4 knockout mice. Meanwhile, it was reported that the partial deletion in AKAP4 is highly relevant to the dysplasia of the FS in an infertile man, and so far, there is no report about male infertility caused by hemizygous AKAP4 variant. Furthermore, the specific mechanisms of how the variant is relevant to the phenotype remain elusive. In this study, we investigated three multiple morphological abnormalities of the sperm flagella-affected men from three independent families (including one consanguine family) carried hemizygous c.C1285T variant in AKAP4. The patients carried this variant, which showed dysplastic sperm FS, and the protein expression of AKAP4 was decreased in flagella, which was further confirmed in HEK-293T cells in vitro. In addition, the co-localization and interaction between AKAP4 and glutamine-rich protein 2 (QRICH2) on the molecular level were identified by immunofluorescence and co-immunoprecipitation (CO-IP). The hemizygous c.1285C > T variant in AKAP4 induced decreased protein expression of QRICH2 in spermatozoa. These results suggested that the normal expression of AKAP4 is required for maintaining the expression of QRICH2 and the decreased protein expression of AKAP4 and QRICH2，as well as the interaction between them induced by the hemizygous variant of AKAP4 caused dysplastic fibrous sheath, which eventually led to reduced sperm motility and male infertility.

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BACKGROUND: Segmental duplication (SD) regions are distinct targets for aneuploidy detection owing to the virtual elimination of amplification bias. The difficulty of searching SD sequences for assay design has hampered their applications. METHODS: We developed a computational program, ChAPDes, which integrates SD searching, refinement, and design of specific PCR primer/probe sets in a pipeline to remove most of the manual work. The generated primer/probe sets were first tested in a multiplex multicolour melting curve analysis for the detection of five common aneuploidies. The primer/probe sets were then tested in a digital PCR assay for the detection of trisomy 21. Finally, a digital PCR protocol was established to quantify maternal plasma DNA sequences for the non-invasive prenatal detection of fetal trisomy 21. FINDINGS: ChAPDes could output 21,772 candidate primer/probe sets for trisomy 13, 18, 21 and sex chromosome aneuploidies within 2 working days. Clinical evaluation of the multiplex multicolour melting curve analysis involving 463 fetal genomic DNA samples revealed a sensitivity of 100% and specificity of 99.64% in comparison with the reference methods. Using the established digital PCR protocol, we correctly identified two trisomy 21 fetuses and thirteen euploid foetuses from the maternal plasma samples. INTERPRETATION: The combination of ChAPDes with digital PCR detection could facilitate the use of SD as potential biomarkers for the non-invasive prenatal testing of fetal chromosomal aneuploidies. FUNDING: None.

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BACKGROUND: Drug-induced glaucoma (DIG) is a kind of serious adverse drug reaction that can cause irreversible blindness. Up-to-date, the molecular mechanism of DIG largely remains unclear yet due to the medical complexity of glaucoma onset. METHODS: In this study, we conducted data mining of tremendous historical adverse drug events and genome-wide drug-regulated gene signatures to identify glaucoma-associated drugs. Upon these drugs, we carried out serial network analyses, including the weighted gene co-expression network analysis (WGCNA), to illustrate the gene interaction network underlying DIG. Furthermore, we applied pathogenic risk assessment to discover potential biomarker genes for DIG. RESULTS: As the results, we discovered 13 highly glaucoma-associated drugs, a glaucoma-related gene network, and 55 glaucoma-susceptible genes. These genes likely played central roles in triggering DIGs via an integrative mechanism of phototransduction dysfunction, intracellular calcium homeostasis disruption, and retinal ganglion cell death. Further pathogenic risk analysis manifested that a panel of nine genes, particularly OTOF gene, could serve as potential biomarkers for early-onset DIG prognosis. CONCLUSIONS: This study elucidates the possible molecular basis underlying DIGs systematically for the first time. It also provides prognosis clues for early-onset glaucoma and thus assists in designing better therapeutic regimens.

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Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrate that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses reveal key residues that govern the interaction between PTP-MEG2 and its substrate, a peptide containing the phosphorylated NSF-pY83 site, specify PTP-MEG2 substrate selectivity, and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface reveals that PTP-MEG2 controls the fusion pore opening through NSF independent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we uncover that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 sites. Further structural and biochemical analyses confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies thus provide mechanistic insights in complex exocytosis processes.

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PingTang No.5 capsule (PT5), a modified Traditional Chinese Medicine (TCM) formula of Zexie Decoction, is used to treat patients with lipid metabolism disorders in our hospital. The present study was designed to investigate the mechanisms of PT5 in treating non-alcoholic fatty liver disease (NAFLD). PT5 information including ingredients, pharmacological properties, and potential targets was obtained from TCM databases. The candidate targets of PT5 were predicted by network pharmacological analysis, and the possible pathway and mechanism were obtained from DAVID database, followed by experimental validation in NAFLD mice model treated with PT5. Total 328 compounds were selected using the threshold oral bioactivity (OB) > 30% or drug-likeness (DL) > 0.1 of pharmacology characteristic, and 1033 candidate targets obtained to construct the network analysis. The 113 targets were selected from the intersection between candidate targets of PT5 and NAFLD relative gene. These targets were evaluated in diabetic complications, cancer, Hepatitis B, Fluid shear stress and atherosclerosis, and TNF signaling pathway. TNF-α was the important factor in protein interaction analysis of STRING and involved in the lipid regulation and oxidative stress in NAFLD. When administrated to the NAFLD mice, PT5 reduced weight, blood fatty acids, decreased the adipocyte size, and improved the metabolism. Besides, the molecular verification of lipid metabolism increased and oxidative stress reduced that interpreted the mechanism of PT5 preventing liver cell from lipid accumulation and injury of NAFLD. These results presented PT5 have the potential therapy as an alternative treatment for NAFLD.

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Collectively called zooxanthellae, photosynthetic dinoflagellates in the family Symbiodiniaceae are typical endosymbionts that unequivocally mediate coral responses to environmental changes. Symbiodiniaceae are genetically diverse, encompassing at least nine phylogenetically distinct genera (clades A-I). The ribosomal internal transcribed spacer 2 (ITS2) region is commonly utilized for determining Symbiodiniaceae diversity within clades. However, ITS2 is often inadvertently interpreted together with the tailing part of the ribosomal RNA genes (5.8S and 28S or equivalent), leading to unresolved taxonomy and equivocal annotations. To overcome this hurdle, we mined in GenBank and expert reference databases for ITS2 sequences of Symbiodiniaceae having explicit boundaries with adjacent rRNAs. We profiled a Hidden Markov Model of the ITS2-proximal 5.8S-28S rRNA interaction, which was shown to facilitate the delimitation of Symbiodiniaceae ITS2 from GenBank, while considerably reducing sequence ambiguity and redundancy in reference databases. The delineation of ITS2 sequences unveiled intra-clade sequence diversity and inter-clade secondary structure conservation. We compiled the clean data into a non-redundant database that archives the largest number of Symbiodiniaceae ITS2 sequences known to date with definite genotype/subclade representations and well-defined secondary structures. This database provides a fundamental reference catalog for consistent and precise genotyping of Symbiodiniaceae and a tool for automated annotation of user-supplied sequences.

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Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.

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