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Abstract Glycosylphosphatidylinositol (GPI) acyltransferase is crucial for the synthesis of GPI-anchored proteins. Targeting the fungal glycosylphosphatidylinositol acyltransferase GWT1 by manogepix is a promising antifungal strategy. However, the inhibitory mechanism of manogepix remains unclear. Here, we present cryo-EM structures of yeast GWT1 bound to the substrate (palmitoyl-CoA) and inhibitor (manogepix) at 3.3 Å and 3.5 Å, respectively. GWT1 adopts a unique fold with 13 transmembrane (TM) helixes. The palmitoyl-CoA inserts into the chamber among TM4, 5, 6, 7, and 12. The crucial residues (D145 and K155) located on the loop between TM4 and TM5 potentially bind to the GPI precursor, contributing to substrate recognition and catalysis, respectively. The antifungal drug, manogepix, occupies the hydrophobic cavity of the palmitoyl-CoA binding site, suggesting a competitive inhibitory mechanism. Structural analysis of resistance mutations elucidates the drug specificity and selectivity. These findings pave the way for the development of potent and selective antifungal drugs targeting GWT1.

Abstract The subcortical maternal complex (SCMC) is essential for safeguarding female fertility in mammals. Assembled in oocytes, the SCMC maintains the cleavage of early embryos, but the underlying mechanism remains unclear. Here, we report that 14-3-3, a multifunctional protein, is a component of the SCMC. By resolving the structure of the 14-3-3-containing SCMC, we discover that phosphorylation of TLE6 contributes to the recruitment of 14-3-3. Mechanistically, during maternal-to-embryo transition, the SCMC stabilizes 14-3-3 protein and contributes to the proper control of CDC25B, thus ensuring the activation of the maturation-promoting factor and mitotic entry in mouse zygotes. Notably, the SCMC establishes a conserved molecular link with 14-3-3 and CDC25B in human oocytes/embryos. This study discloses the molecular mechanism through which the SCMC regulates the cell cycle in early embryos and elucidates the function of the SCMC in mammalian early embryogenesis.

Abstract Spermiogenesis, the complex transformation of haploid spermatids into mature spermatozoa, relies on precise spatiotemporal regulation of gene expression at the post-transcriptional level. The mechanisms underlying this critical process remain incompletely understood. Here, we identify centrosomal protein 112 (CEP112) as an essential regulator of mRNA translation during this critical developmental process. Mutations in CEP112 are discovered in oligoasthenoteratospermic patients, and Cep112-deficient male mice recapitulate key phenotypes of human asthenoteratozoospermia. CEP112 localizes to the neck and atypical centrioles of mature sperm and forms RNA granules during spermiogenesis, enriching target mRNAs such as Fsip2, Cfap61, and Cfap74. Through multi-omics analyses and the TRICK reporter assay, we demonstrate that CEP112 orchestrates the translation of target mRNAs. Co-immunoprecipitation and mass spectrometry identify CEP112’s interactions with translation-related proteins, including hnRNPA2B1, EEF1A1, and EIF4A1. In vitro, CEP112 undergoes liquid-liquid phase separation, forming condensates that recruit essential proteins and mRNAs. Moreover, variants in patient-derived CEP112 disrupt phase separation and impair translation efficiency. Our results suggest that CEP112 mediates the assembly of RNA granules through liquid-liquid phase separation to control the post-transcriptional expression of fertility-related genes. This study not only clarifies CEP112’s role in spermatogenesis but also highlights the role of phase separation in translational regulation, providing insights into male infertility and suggesting potential therapeutic targets.

In-situ stress is a core factor controlling dynamic gas disasters in coal mines. Existing in-situ stress models are primarily based on the elastic properties of coal-rock formations and are not suitable for viscoelastic coal media. There is a need to establish an in-situ stress distribution model suitable for viscoelastic coal properties. Coal seams, especially tectonic coal, exhibit significant creep and stress relaxation characteristics. In this study, a Fractional Maxwell stress relaxation mathematical model was developed for viscoelastic coal reservoirs. The stress component of the extended Eaton model was improved to account for the stress relaxation in viscoelastic coal seams and an in-situ stress distribution model was established for viscoelastic coal seams. The results indicate that over geological time scales, viscoelasticity significantly affects the differential stress in coal seams. The degree of coal body creep and stress relaxation increases with the increase in viscosity coefficient and fractional order factor. As the elastic modulus decreases, the Poisson’s ratio, tectonic ratio, and gas pressure increase. The stress characteristics of coal seams exhibit a trend of hydrostatic pressure state distribution. Among them, the elastic modulus and Poisson's ratio contribute significantly to the difference between vertical principal stress and horizontal stress. The tectonic ratio contributes significantly to the difference between horizontal stresses. Gas pressure contributes equally to all three principal stresses. The stress relaxation effect can alter the relative magnitudes of maximum and minimum horizontal principal stresses and reduce stress anisotropy. Based on the critical stress model, a reduction in friction coefficient and an increase in gas pressure were similarly observed in coal seams under different in-situ stress conditions, leading to an approach towards hydrostatic pressure distribution. The mechanical properties of tectonic coal are more conducive to approaching hydrostatic pressure states over geological time scales. The application of the in-situ stress model established in this study validated the field measurements and provided new insights into gas enrichment and permeability reduction in tectonic coal.

Several fungal pathogens cause root rot of common bean, among which Fusarium spp. are the most common pathogens causing Fusarium root rot (FRR) worldwide. FRR has been becoming an increasingly severe disease of common bean in China, but the species of Fusarium spp. have remained unclear. Thus, this study was performed to identify the pathogen causing common bean root rot in Liangcheng County, Inner Mongolia, China. Nineteen Fusarium-like isolates were obtained after pathogen isolation and purification. The pathogenicity test indicated that eight isolates caused severe disease symptoms on common bean, while 11 other isolates were not pathogenic. The eight pathogenic isolates, FCL1–FCL8, were identified as Fusarium cuneirostrum by morphological characterization and phylogenetic analysis using partial sequences of EF-1α, ITS, 28S, and IGS regions. Host range test showed that the representative F. cuneirostrum isolate FCL3 was also pathogenic to mung bean, while not pathogenic to adzuki bean, chickpea, cowpea, faba bean, pea, and soybean. Moreover, 50 common bean and 50 mung bean cultivars were screened for resistance to FRR, and seven highly resistant or resistant cultivars of common bean were identified, while no resistant cultivars of mung bean were screened. This study revealed that F. cuneirostrum was one of common bean FRR pathogens in Inner Mongolia and it could induce mung bean root rot as well. To our knowledge, this is the first report of F. cuneirostrum causing FRR of common bean in China.

Chickpea (Cicer arietinum L.) is an important legume crop worldwide. An emerging disease, basal stem rot with obvious wilt symptoms, was observed in the upper part of chickpea plants during the disease survey in Qiubei County of Yunnan Province. Three fungal isolates (ZD36-1, ZD36-2, and ZD36-3) were obtained from the diseased tissue of chickpea plants collected from the field. Those isolates were morphologically found to be similar to Diaporthe aspalathi. Molecular sequence analyses of multiple gene regions (ITS, tef1, tub2, cal, and his3) indicated that the three isolates showed a high identity with D. aspalathi. Pathogenicity and host range tests of the isolates were performed on the original host chickpea and eight other legume crops. The isolates were strongly pathogenic to chickpea and appeared highly pathogenic to soybean, cowpea, and mung bean; moderated or mild pathogenic to adzuki bean and common bean; however, the isolates did not cause symptoms on grass pea (Lathyrus sativus). Diaporthe aspalathi was previously reported as a main pathogen causing the southern stem canker in soybean. To our knowledge, this is the first report of D. aspalathi inducing basal stem rot on chickpea worldwide.

PfENT1 is a promising antimalarial drug target. Here, authors report cryo-EM structures of PfENT1 that, together with biochemical work, suggests PfENT1 is an inosine transporter and describe the inhibitory mechanism of the endofacial inhibitor, GSK4.

Background: Hypertension affects over 1 billion people globally and is the top risk factor of cardiovascular morbidity and mortality. Wuweijiangyasan (WWJYS), as an empirical prescription, has stable depressurization effects. This study investigated the chemical composition and pharmacodynamic effects of WWJYS in regulating the blood pressure (BP), emotion, and blood lipid of spontaneous hypertensive rats, and further explored the depressurization mechanism of WWJYS. Materials and Methods: This study used network pharmacology to identify the origins and predict targets of WWJYS, and artificial intelligence-based molecular docking is used to further predict targets and mechanisms. The chemical constituents of WWJYS were analyzed and identified by ultra high-performance liquid chromatography–mass spectrometry (MS)/MS. Results: In the WWJYS group, the systolic BP level significantly was decreased, and the HR was stable. The irritability became stable after the 5-week treatment compared with the model group (P < 0.05). Rats' rotation tolerance time increased after 2-weeks stabilization. Compared with the model group, angiotensin-converting enzyme 2 protein and mRNA of the WWJYS group increased significantly (P < 0.05). Network pharmacology collected 64 compounds and identified 22 potential targets of WWJYS for antihypertensive activity. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that WWJYS might regulate smooth muscle cells, affect inflammatory response and improve endothelial function through multiple pathways. The molecular docking study further supported that the target proteins have good combinations with the main active components of WWJYS. Conclusions: The data indicated that WWJYS had significant depressurization, analgesic, and sedative, as well as lipid-lowering effects, and the depressurization mechanism of WWJYS may function in multiple signal pathways, especially in improving blood vessel function and intervening inflammation.

White mold of pea caused by Sclerotinia sclerotiorum is a common disease in China. However, we discovered that the diverse Sclerotinia species could cause white mold on pea plants in Chongqing and Sichuan of China during recent disease surveys. Thus, the objective of this study was to confirm the causal agents from diseased pea plants. The obtained isolates of white mold from Chongqing and Sichuan were identified by morphological characters and molecular characterization to determine the pathogen species, and their pathogenicity was confirmed on pea through completing Koch's postulates. Fungal isolates of Sclerotinia-like were obtained from diseased plants or sclerotia. Based on morphological characteristics and molecular characterization, 30 isolates were identified to three species, six isolates as S. minor, seven as S. sclerotiorum, and 17 as S. trifoliorum. In pathogenicity tests on pea cultivars Zhongwan 4 and Longwan 1, all 30 isolates caused typical symptoms of white mold on the inoculated plants, and the inoculated pathogens were re-isolated from the diseased plants. This study confirmed that white mold of pea was caused by three Sclerotinia species, S. sclerotiorum, S. minor and S. trifoliorum in Chongqing and Sichuan. It is the first report that S. minor and S. trifoliorum cause white mold of pea in Southwest China.

Steroid 5α-reductase 2 (SRD5A2), a testosterone metabolism enzyme, is implicated in human disease. Structural and biochemical analyses of PbSRD5A, a bacterial homolog, reveal SRD5A2 substrate binding pocket and provide framework for the design of new drugs targeting this enzyme.

Plasmodium falciparum, the deadliest causal agent of malaria, caused more than half of the 229 million malaria cases worldwide in 2019. The emergence and spreading of frontline drug-resistant Plasmodium strains are challenging to overcome in the battle against malaria and raise urgent demands for novel antimalarial agents. The P. falciparum formate-nitrite transporter (PfFNT) is a potential drug target due to its housekeeping role in lactate efflux during the intraerythrocytic stage. Targeting PfFNT, MMV007839 was identified as a lead compound that kills parasites at submicromolar concentrations. Here, we present 2 cryogenic-electron microscopy (cryo-EM) structures of PfFNT, one with the protein in its apo form and one with it in complex with MMV007839, both at 2.3 Å resolution. Benefiting from the high-resolution structures, our study provides the molecular basis for both the lactate transport of PfFNT and the inhibition mechanism of MMV007839, which facilitates further antimalarial drug design.

Abstract Background Drug resistance in China is becoming a more and more serious issue. Infection by drug-resistant bacteria has become a major disease that seriously threatens the health of Chinese people and affects national medical finance. Therefore, it is of great scientific and clinical significance to actively carry out research on the prevention and treatment of infections by multi-drug resistant organisms (MDRO). Previous studies by the authors suggested that patients with hospital-acquired pneumonia caused by MDRO mostly showed the pathological state of “insufficient healthy Qi and internal accumulation of pathogenic Qi” and “acute deficiency syndrome” mainly characterized by Qi deficiency. Buzhong Yiqi decoction is a famous classic prescription in traditional Chinese medicine (TCM) for treating internal damage fever. This study intends to provide an evidence-based rationale for Buzhong Yiqi decoction in treating MDRO hospital-acquired pneumonia by conducting a multi-center randomized controlled clinical study. Methods/design This study is designed to be a multi-center randomized controlled study in which patients are assigned randomly into control (standard therapy) and trial (standard therapy plus Buzhong Yiqi decoction) groups. The patients will be selected from the emergency department and the ICU inpatient department of five study sites and will all be diagnosed with MDRO hospital-acquired pneumonia and meet the inclusion criteria. Forty patients are to be enrolled in each study site, resulting in a total of 200 patients in the study. The treatment course is 28 days. Discussion In this study: (1) the theory of “acute Qi deficiency” in MDRO hospital-acquired pneumonia is put forward for the first time, and the basic theories of TCM are further improved; (2) a multi-center randomized controlled clinical study will be performed for the first time with Buzhong Yiqi decoction, the classic prescription for reinforcing healthy Qi and eliminating pathogenic Qi, providing a reliable evidence-based rationale for the treatment of MDRO pulmonary infection with TCM; (3) the clinical application and modern disease spectrum of Buzhong Yiqi decoction is expanded, and the scientific notion of “treating different diseases with the same method” is enriched further. Trial registration China Clinical Trial Registry, ChiCTR1900022429. Registered on April 11, 2019. http://www.chictr.org.cn/listbycreater.aspx.

Fuyuan Xingnao decoction (FYXN), a traditional Chinese formula comprised of seven herbs, has been utilized to treat diabetes mellitus complicated with cerebral infarction (DMCI) for years. Yet, its protective and regulatory mechanism is poorly understood. The aim of the study is to investigate the effects of FYXN on DMCI in vitro and in vivo, as well as its mechanism in angiogenesis. For in vivo experiments, FYXN was administered to DMCI rats with streptozotocin (STZ) injection-induced diabetes. Then middle cerebral artery occlusion (MCAO) was conducted and the cerebral cortex sections of the rats were obtained. The ultrastructure of cerebral microvessels and new vessel density of ischemic penumbra were evaluated by the transmission electron microscopy (TEM) assay and immunohistochemistry, respectively. Protein and mRNA expression levels of Rab1/AT1R in cortex were assayed by Western blotting and real-time fluorescence quantitative real-time polymerase chain reaction (RT-qPCR). In vitro, FYXN serum was produced in rats on the fourth day 2 h after the last FYXN administration. Green fluorescence was observed after transfection with lentivirus packaged Rab1-WT or siRNA for 24 h. The activity of brain microvascular endothelial cells (BMECs) treated with sera from these rats was tested by MTT assay and Transwell assays, respectively. The expression of AT1R on the cell membrane and endoplasmic reticulum of BMECs was evaluated by immunofluorescence staining. Protein expression levels of signaling molecules in the Rab1/AT1R pathways were also detected. Results showed that in vivo, FYXN treatment significantly intensified CD31 staining in the cortical areas and enhanced the mRNA and protein levels of AT1R, Ang II, Rab1a, Rab1b and VEGF expression in ischemic cerebral cortex tissues. In vitro, the expression levels of AT1R, Ang II, Rab1a, Rab1b and VEGF in the cerebral infarction model group were significantly higher than those in the control group, with further increases after administration of FYXN drug serum. FYXN promoted the proliferation and migration of BMECs by activating the Rab1/AT1R signaling pathway. In conclusion, FYXN exerts a protective effect against DMCI by promoting angiogenesis via the Rab1/AT1R pathway, which provides strong evidence for the therapeutic effect of FYXN on DMCI.

Fusarium wilt (FW), caused by Fusarium oxysporum f. sp. pisi (Fop), has always been an important disease affecting pea production and causing severe yield losses in most pea-growing areas worldwide. Growing resistant pea cultivars is the most economical and effective method for controlling the disease. In this study, firstly, 21 Fusarium oxysporum isolates were identified as races 1 and 5 of Fop based on morphological and molecular characteristics, and the disease reactions of seven pea differential cultivars. Then, a detailed resistance evaluation strategy was established and validated by a death rate score, disease index, and percentage of leaves showing symptoms for each individual plant. Finally, a 1311 pea germplasm collection including 740 accessions from China and 571 accessions aboard or unknown sources was evaluated for resistance to a representative isolate PF22b of Fop race 5, and the results showed that 28 accessions and 164 accessions were highly resistant (HR) and resistant (R), respectively. Among these resistance accessions, 13 HR and 44 R accessions were collected from 19 provinces in China, most of which came from Sichuan, Tibet, and Gansu Provinces. The 15 HR and 120 R accessions were collected in 10 countries outside China or unknown sources, the majority of which came from the United States, Australia, and Russia. The findings would provide important information for using resistance pea cultivars to control Fusarium wilt. Incorporating these resistance accessions into breeding programs will contribute to improving the Fop resistance of pea cultivars.

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.

Powdery mildew is one of the severe diseases on common bean in Southwestern China, but the identity of the pathogen inciting this disease is unclear. The objective of this study was to identify the causal agent of common bean powdery mildew and to screen resistant cultivars. The pathogen was identified through morphological identification, molecular phylogenetic analysis, and pathogenicity tests. Resistance of common bean cultivars was evaluated by artificial inoculation at the seedling stage. The common bean powdery mildew isolate CBPM1 was obtained after pathogen isolation and purification. Morphological identification confirmed that the isolate CBPM1 belonged to the Oidium subgenus Pseudoidium and germinated Pseudoidium-type germ tubes. Molecular phylogenetic analysis showed that the isolate CBPM1 and Erysiphe vignae isolates from different hosts were clustered into a distinct group. The pathogenicity and host range tests revealed that the isolate CBPM1 was strongly pathogenic to common bean, multiflora bean, lablab bean, cowpea, and mung bean, but not to soybean, adzuki bean, pea, faba bean, chickpea, lentil, pumpkin, and cucumber. In addition, 54 common bean cultivars were identified for resistance to powdery mildew, and 15 were resistant or segregant. Based on the morphological, molecular and pathogenic characteristics, the causal agent of common bean powdery mildew was identified as E. vignae. This is the first time E. vignae has been confirmed on common bean. Cultivars with different resistance levels were screened, and these cultivars could be used for disease control or the breeding of new resistant cultivars.

Objective: To find the target genes in combination of astragalus root (Astragalus membranaceus) and spindle tree wings (Euonymus alatus (THUNB.) SIEB.) (Qijian Heji) with the function of regulating blood glucose levels in kk mice by real-time fluorescence quantitative PCR (RT-PCR). To achieve the primary exploration on the material basis for Qijian Heji to improve the blood glucose levels of type 2 diabetic kk mice. Methods: We applied RT-PCR to detecting the expression of gene targets in kidney tissues of type 2 diabetes model kk mice. The detected gene targets were predicted by the network pharmacology method in the early stage of the project. Through the observation of the hypoglycemic effect of different ratios of Qijian Heji, the optimal proportion was determined and then used to conduct research of material basis. Results: Continuous administration of Qijian Heji (astragalus root: spindle tree wings = 3:1) for six weeks resulted in significantly lowering blood glucose levels in mice in the fourth week, dropping from 33.3 to 21.9 mmol/L; this lower level was maintained in the fifth to sixth weeks. Blood glucose values in the positive drug group decreased from 33.3 to 21.85 mmol/L in the fourth week and maintained steady decreasing in the fifth and sixth weeks. Conclusion: Qijian Heji, with proportion of 3:1, could significantly reduce the blood glucose values in kk mice, animal experiment results verified the accurate prediction of network pharmacology, and at the same time, it is verified there was a certain therapeutic effect about the combination of the two herbs on reducing the glucose value. Animal experiments and RT-PCR results showed that Qijian Heji conducted a hypoglycemic effect by modulating estrogen receptor (ESR1) and renin (REN) expression.

Objective: To evaluate the efficacy of traditional Chinese herbs on heart failure (HF) using nuclear magnetic resonance (NMR) metabolomics. Methods: Plasma metabolomics was conducted on both patients with HF and healthy controls. The partial least squares model was applied to determine potential metabolic markers and related metabolic pathways of the disease. HF patients with the traditional Chinese syndrome pattern of qi deficiency and blood stasis were divided into two groups, and treated, respectively, with conventional medicine and a combination of conventional and Chinese herbs, aimed at tonifying qi and activating blood. Healthy participants served as a control group for comparison. Results and conclusion: Both conventional and herbal treatments appear to regulate disorders of amino acid and glucose metabolism. Combined treatment appears to be more comprehensive, indicating that herbal and conventional medicines exert their effects through different mechanisms in treating HF. It can be further inferred that herbs that tonify qi and activate blood may regulate different essential metabolites.

Background: Chronic heart failure (CHF) is characterized by insufficient blood supply from heart to meet the body's metabolic demands. Integrating Western and traditional Chinese medicine to treat CHF has proved a validated therapeutic approach. In recent years, metabolomics has been regarded as a potential platform to provide biomarkers for disease-subtypes. Objective: To examine 38 patients, combined NMR plasma metabolomics and traditional Chinese medicine diagnosis in order to identify diagnostic biomarkers for two CHF syndrome subtypes. Methods: After processing the spectra, orthogonal partial least square discriminant analysis was performed, and the contributing NMR signals were analyzed using Y-scrambling statistical validation with good reliability. Results: Plasma metabolic patterns of yin deficiency and yang deficiency patients were clearly discriminated. The yin-deficiency group had increased level of lactate, glycoprotein, lipoprotein and lower levels of glucose, valine and proline. The yang-deficiency group had higher levels of lactate, glycoprotein and pyruvic acid, and lower levels of glucose and lipoprotein. Potential biomarkers of CHF based on the two traditional Chinese medicine syndromes indicated alternative modes of metabolites and metabolic pathways in the disease, e.g. dysfunction of energy utilization and disturbance in fatty acids, amino acids. Conclusion: This study suggests that combining metabolomics with traditional Chinese medicine diagnosis can reveal metabolic signatures for CHF syndrome subtypes. The plasma metabolites identified might be of special clinical relevance for subtypes of CHF, which could lead to further understanding of mechanisms involved and an improvement in personalized treatment for CHF.

The transcription activator-like (TAL) effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats). Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of the DNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5 Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections.