Sepsis refers to the systemic inflammatory response syndrome caused by infection. It is a major clinical problem and cause of death for patients in intensive care units worldwide. The Fat mass and obesity-related protein (FTO) is the primary N6-methyladenosine demethylase. However, the role of FTO in the pathogenesis of inflammatory diseases remains unclear. We herein show that nanoparticle-mediated Fto-siRNA delivery or FTO inhibitor entacapone administration dramatically inhibited macrophage activation, reduced the tissue damage and improved survival in a mouse model of LPS-induced endotoxic shock. Importantly, ablation of FTO could inhibit NLRP3 inflammasome through FoxO1/NF-κB signaling in macrophages. In conclusion, FTO is involved in inflammatory response of LPS-induced septic shock and inhibition of FTO is promising for the treatment of septic shock.

Human trophoblast stem cells (hTSCs) provide a valuable model to study placental development and function. While primary hTSCs have been derived from embryos/early placenta, and transdifferentiated hTSCs from naïve human pluripotent stem cells (hPSCs), the generation of hTSCs from primed PSCs is problematic. We report the successful generation of TSCs from primed hPSCs and show that BMP4 substantially enhances this process. TSCs derived from primed hPSCs are similar to blastocyst-derived hTSCs in terms of morphology, proliferation, differentiation potential, and gene expression. We define the chromatin accessibility dynamics and histone modifications (H3K4me3/H3K27me3) that specify hPSC-derived TSCs. Consistent with low density of H3K27me3 in primed hPSC-derived hTSCs, we show that knockout of H3K27 methyltransferases (EZH1/2) increases the efficiency of hTSC derivation from primed hPSCs. Efficient derivation of hTSCs from primed hPSCs provides a simple and powerful model to understand human trophoblast development, including the pathogenesis of trophoblast-related disorders, by generating disease-specific hTSCs.

Diabetic retinopathy (DR) represents the most typical complication of type 2 diabetes mellitus and one of the most primary oculopathy causing blindness. However, the mechanism of DR remains unknown. RIPK1/RIPK3, as homologous serine/threonine kinases, are key elements in mediating necroptosis and may have functions in DR development. To clarify the relationship between DR and RIPK1/RIPK3, this study established a model of apoptosis using high-glucose induced RGCs, which were treated with 7.5, 19.5, and 35 mM D-glucose for 12, 24, and 48 h, respectively. Subsequently, the expression of RIPK1/RIPK3 was determined and the protective effect of necrostatin-1 on RGCs injury induced by high glucose was explored. The results demonstrated that the expression of RIPK1 and RIPK3 in the cells was increased markedly following 12 h treatment with 19.5 mM D-glucose. Additionally, following an addition of 100 μM necrostatin-1 in 19.5 mM D-glucose medium for RGCs treatment 12 h, the protein expression of RIPK1 and RIPK3 was decreased markedly, and the number of Nissl bodies in cells was increased substantially. The findings of the present study indicated that high glucose could induce the expression of RIPK1/RIPK3, and necrostatin-1 could effectively protect RGCs from D-glucose-induced cell necrosis.

The COVID-19 pandemic poses a global threat to public health and economy. The continuously emerging SARS-CoV-2 variants present a major challenge to the development of antiviral agents and vaccines. In this study, we identified that EK1 and cholesterol-coupled derivative of EK1, EK1C4, as pan-CoV fusion inhibitors, exhibit potent antiviral activity against SARS-CoV-2 infection in both lung- and intestine-derived cell lines (Calu-3 and Caco2, respectively). They are also effective against infection of pseudotyped SARS-CoV-2 variants B.1.1.7 (Alpha) and B.1.1.248 (Gamma) as well as those with mutations in S protein, including N417T, E484K, N501Y, and D614G, which are common in South African and Brazilian variants. Crystal structure revealed that EK1 targets the HR1 domain in the SARS-CoV-2 S protein to block virus-cell fusion and provide mechanistic insights into its broad and effective antiviral activity. Nasal administration of EK1 peptides to hACE2 transgenic mice significantly reduced viral titers in lung and intestinal tissues. EK1 showed good safety profiles in various animal models, supporting further clinical development of EK1-based pan-CoV fusion inhibitors against SARS-CoV-2 and its variants.

Resolving the genomic basis underlying phenotypic variations is a question of great importance in evolutionary biology. However, understanding how genotypes determine the phenotypes is still challenging. Centuries of artificial selective breeding for beauty and aggression resulted in a plethora of colors, long-fin varieties, and hyper-aggressive behavior in the air-breathing Siamese fighting fish (Betta splendens), supplying an excellent system for studying the genomic basis of phenotypic variations. Combining whole-genome sequencing, quantitative trait loci mapping, genome-wide association studies, and genome editing, we investigated the genomic basis of huge morphological variation in fins and striking differences in coloration in the fighting fish. Results revealed that the double tail, elephant ear, albino, and fin spot mutants each were determined by single major-effect loci. The elephant ear phenotype was likely related to differential expression of a potassium ion channel gene, kcnh8. The albinotic phenotype was likely linked to a cis-regulatory element acting on the mitfa gene and the double-tail mutant was suggested to be caused by a deletion in a zic1/zic4 coenhancer. Our data highlight that major loci and cis-regulatory elements play important roles in bringing about phenotypic innovations and establish Bettas as new powerful model to study the genomic basis of evolved changes.

Dysregulations in transcription factors (TFs) and their genetic products play important roles in tumorigenesis, tumor progression and metastasis. However, prognostic value of the transcriptional regulatory networks in different cancers has not been investigated in depth. The purpose of our study was to identify and validate a potential predictive signature that combines TFs and their regulatory products in eight solid tumors. We used bioinformatics analysis to identify MET Transcriptional Regulator (MACC1) and Serine Peptidase Inhibitor Kunitz Type 1 (SPINT1) as candidate TFs with the respective downstream regulatory proteins for patient prognosis in pan-cancer. Subsequent molecular analysis of clinical gastric cancer tissue samples further verified the negative correlation between MACC1 and SPINT1. Further, we showed that mechanistically, MACC1/SPINT1 mediated the pro-HGF proteolysis and c-Met phosphorylation in HGF/c-MET signaling pathway. Kaplan-Meier plots and receiver operating characteristics analysis revealed that the two-gene signature combining MACC1 with SPINT1 was effective in predicting survival in all eight cancer cohorts tested. In conclusion, our study clarified the regulatory relationship between MACC1 and SPINT1 in the context of the HGF/c-MET signaling pathway and determined MACC1/SPINT1 panel as a valuable signature for the prediction of prognosis in patients for multiple solid cancer types.

In mammalian testes, extensive remodeling of the microtubule (MT) and actin cytoskeletons takes place in Sertoli cells across the seminiferous epithelium to support spermatogenesis. However, the mechanism(s) involving regulatory and signaling proteins remains poorly understood. Herein, A-kinase anchoring protein 9 (AKAP9, a member of the AKAP multivalent scaffold protein family) was shown to be one of these crucial regulatory proteins in the rat testis. Earlier studies have shown that AKAP9 serves as a signaling platform by recruiting multiple signaling and regulatory proteins to create a large protein complex that binds to the Golgi and centrosome to facilitate the assembly of the MT-nucleating γ-tubulin ring complex to initiate MT polymerization. We further expanded our earlier studies based on a Sertoli cell-specific AKAP9 knockout mouse model to probe the function of AKAP9 by using the techniques of immunofluorescence analysis, RNA interference (RNAi), and biochemical assays on an in vitro primary Sertoli cell culture model, and an adjudin-based animal model. AKAP9 robustly expressed across the seminiferous epithelium in adult rat testes, colocalizing with MT-based tracks, and laid perpendicular across the seminiferous epithelium, and prominently expressed at the Sertoli-spermatid cell-cell anchoring junction (called apical ectoplasmic specialization [ES]) and at the Sertoli cell-cell interface (called basal ES, which together with tight junction [TJ] created the blood-testis barrier [BTB]) stage specifically. AKAP9 knockdown in Sertoli cells by RNAi was found to perturb the TJ-permeability barrier through disruptive changes in the distribution of BTB-associated proteins at the Sertoli cell cortical zone, mediated by a considerable loss of ability to induce both MT polymerization and actin filament bundling. A considerable decline in AKAP9 expression and a disruptive distribution of AKAP9 across the seminiferous tubules was also noted during adjudin-induced germ cell (GC) exfoliation in this animal model, illustrating AKAP9 is essential to maintain the homeostasis of cytoskeletons to maintain Sertoli and GC adhesion in the testis.

Breast cancer is one of the most common malignant tumors in women and is the second leading cause of cancer deaths among women. The tumorigenesis and progression of breast cancer are not well understood. The existing researches have indicated that non-coding RNAs, which mainly include long non-coding RNA (lncRNA) and microRNA (miRNA), have gradually become important regulators of breast cancer. We aimed to screen the differential expression of miRNA and lncRNA in the different breast cancer stages and identify the key non-coding RNA using TCGA data. Based on series test of cluster (STC) analysis, bioinformatics analysis, and negatively correlated relationships, 122 lncRNAs, 67 miRNAs, and 119 mRNAs were selected to construct the regulatory network of lncRNA and miRNA. It was shown that the miR-93/20b/106a/106b family was at the center of the regulatory network. Furthermore, 6 miRNAs, 10 lncRNAs, and 15 mRNAs were significantly associated with the overall survival (OS, log-rank P < 0.05) of patients with breast cancer. Overexpressed miR-93 in MCF-7 breast cancer cells was associated with suppressed expression of multiple lncRNAs, and these downregulated lncRNAs (MESTIT1, LOC100128164, and DNMBP-AS1) were significantly associated with poor overall survival in breast cancer patients. Therefore, the miR-93/20b/106a/106b family at the core of the regulatory network discovered by our analysis above may be extremely important for the regulation of lncRNA expression and the progression of breast cancer. The identified key miRNA and lncRNA will enhance the understanding of molecular mechanisms of breast cancer progression. Targeting these key non-coding RNA may provide new therapeutic strategies for breast cancer treatment and may prevent the progression of breast cancer from an early stage to an advanced stage.

Certain risk alleles associated with autoantibody-positive rheumatoid arthritis (RA) have been linked to poorer prognoses. In patients with autoantibody-positive RA, abatacept shows differential efficacy to tumor necrosis factor inhibitors. Our aim was to investigate the relationship between clinical response to abatacept and to adalimumab and presence of risk alleles encoding human leukocyte antigen (HLA)-DRB1 shared epitope (SE) in RA.

Sorafenib is the first-line therapeutic option for advanced hepatocellular carcinoma (HCC). Many patients exhibit a primary resistance (PR) response after initial treatment. In previous studies, compared to acquired resistance, the mechanism of PR is unclear. The present study aimed to evaluate the response of patient samples to sorafenib by patient-derived xenograft (PDX) models, and the differences at the transcriptome level between the sorafenib PR group and the sorafenib sensitive group were analyzed by single-cell sequencing technology. A specific cell cluster may be differentiated by the liver bud hepatic cells, and the JUN transcription factors in this cell cluster were highly activated. The albumin is secreted by other cell clusters, and the cluster stimulates the FcRn complex receptor to activate the HIF pathway and cell proliferation, resulting in a poor response to sorafenib. These findings are validated by both cell communication analysis and experiments. Thus, the current studies provided a novel approach for the treatment of sorafenib-resistant HCC.

Purpose: Choroidal neovascularization (CNV) is the main pathogenic process and a leading cause of severe vision loss in neovascular age-related macular degeneration (AMD). We investigated the antiangiogenic efficacy of dihydroartemisinin (DHA) in an experimental laser-induced CNV mouse model. Methods: After fluorescein angiography confirmed that CNV was induced by laser photocoagulation in C57BL/6J mice, DHA or vehicle was given by intragastric administration once a day. On day 6 and day 12, fluorescein angiography, optic coherence tomography, and flat-mounting analysis were performed to grade CNV leakage, measure CNV thickness and evaluate CNV areas, respectively. Immunofluorescence staining and Western blot analysis were performed to evaluate the expression of NF-κB, VEGF, and VEGFR2. To confirm the safety of intragastric DHA application, changes in retinal morphology and neural cell apoptosis were tested by histopathological examination and TUNEL assay, and retinal function was determined by electroretinogram (ERG). Results: Intragastric administration of DHA significantly suppressed CNV leakage and CNV formation in both thickness and area. Immunofluorescence showed that DHA suppressed VEGFR2 and NF-κB p65 expression in laser-induced lesions. Compared to the normal group, the protein expression of VEGF, VGFER2, NF-κB p65, and NF-κB1 p50 increased significantly in the vehicle group after laser photocoagulation, while it was profoundly inhibited by DHA treatment. In addition, histopathological examination, TUNEL analysis, and ERG test showed no obvious evidence of retinal toxicity caused by DHA. Conclusion: Systemic administration of DHA can effectively inhibit laser-induced CNV formation in mice, which might be due to the suppression of the classic NF-κB signaling pathway and downregulation of VEGFR2 and VEGF expression. The current results suggest that DHA could be a natural potential alternative therapeutic strategy for neovascular AMD.

Idiopathic pulmonary hypertension (IPAH) is a condition that affects various tissues and organs and the metabolic and inflammatory systems. The most prevalent metabolic condition is metabolic syndrome (MS), which involves insulin resistance, dyslipidemia, and obesity. There may be a connection between IPAH and MS, based on a plethora of studies, although the underlying pathogenesis remains unclear. Through various bioinformatics analyses and machine learning algorithms, we identified 11 immune- and metabolism-related potential diagnostic genes (EVI5L, RNASE2, PARP10, TMEM131, TNFRSF1B, BSDC1, ACOT2, SAC3D1, SLA2, P4HB, and PHF1) for the diagnosis of IPAH and MS, and we herein supply a nomogram for the diagnosis of IPAH in MS patients. Additionally, we discovered IPAH's aberrant immune cells and discuss them here.

Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent cryo-ET. Here we describe a precise cryogenic fabrication system, ELI-TriScope, which sets electron (E), light (L) and ion (I) beams at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella. ELI-TriScope uses a commercial dual-beam scanning electron microscope modified to incorporate a cryo-holder-based transfer system and embed an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome with a success rate of ~91% and discovered new in situ structural features of the human centrosome by cryo-ET.

Disheveled-associated activator of morphogenesis 2 (DAAM2) regulates actin polymerization and cell motility. In this study, we investigated the role of DAAM2 in the cytoskeleton and phagocytosis of rat Sertoli cells in vitro and in vivo through siRNA transfection and intratesticular injection. We found that knockdown of DAAM2 significantly attenuated cytoskeletal and tight junction marker expression and reduced the integrity of the Sertoli cell monolayer. In rats, loss of DAAM2 induced disarrangement and deformation of sperms and promoted accumulation of apoptotic sperms in the testis, accompanied by morphological abnormalities in the blood-testis barrier. DAAM2 silencing also reduced the ability of Sertoli cells to engulf apoptotic spermatogenic cells and green fluorescence-labeled beads. RNA sequencing and bioinformatics analysis revealed that phagocytosis and cytoskeleton-related genes and pathways were significantly associated with DAAM2. Our study suggests that DAAM2 may be involved in spermatogenesis possibly by regulating cytoskeleton organization and phagocytosis of Sertoli cells.

Aluminum-activated malate transporters (ALMTs) form an anion channel family that plays essential roles in diverse functions in plants. Arabidopsis ALMT12, also named QUAC1 (quick anion channel 1), regulates stomatal closure in response to environmental stimuli. However, the molecular basis of ALMT12/QUAC1 activity remains elusive. Here, we describe the cryo-EM structure of ALMT12/QUAC1 from Glycine max at 3.5-Å resolution. GmALMT12/QUAC1 is a symmetrical dimer, forming a single electropositive T-shaped pore across the membrane. The transmembrane and cytoplasmic domains are assembled into a twisted two-layer architecture, with their associated dimeric interfaces nearly perpendicular. GmALMT12/QUAC1-mediated currents display rapid kinetics of activation/deactivation and a bell-shaped voltage dependency, reminiscent of the rapid (R)-type anion currents. Our structural and functional analyses reveal a domain-twisting mechanism for malate-mediated activation. Together, our study uncovers the molecular basis for a previously uncharacterized class of anion channels and provides insights into the gating and modulation of the ALMT12/QUAC1 anion channel.

Fishes are the one of the most diverse groups of animals with respect to their modes of sex determination, providing unique models for uncovering the evolutionary and molecular mechanisms underlying sex determination and reversal. Here, we have investigated how sex is determined in a species of both commercial and ecological importance, the Siamese fighting fish Betta splendens.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important target for vaccine and drug development. However, the rapid emergence of variant strains with mutated S proteins has rendered many treatments ineffective. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered that the S protein contains two previously unidentified Cathepsin L (CTSL) cleavage sites (CS-1 and CS-2). Both sites are highly conserved among all known SARS-CoV-2 variants. Our structural studies revealed that CTSL cleavage promoted S to adopt receptor-binding domain (RBD) "up" activated conformations, facilitating receptor-binding and membrane fusion. We confirmed that CTSL cleavage is essential during infection of all emerged SARS-CoV-2 variants (including the recently emerged Omicron variant) by pseudovirus (PsV) infection experiment. Furthermore, we found CTSL-specific inhibitors not only blocked infection of PsV/live virus in cells but also reduced live virus infection of ex vivo lung tissues of both human donors and human ACE2-transgenic mice. Finally, we showed that two CTSL-specific inhibitors exhibited excellent In vivo effects to prevent live virus infection in human ACE2-transgenic mice. Our work demonstrated that inhibition of CTSL cleavage of SARS-CoV-2 S protein is a promising approach for the development of future mutation-resistant therapy.

Epithelial ovarian cancer (EOC) is the leading cause of deaths among patients with gynecologic malignancies. In recent years, cancer stem cells (CSCs) have attracted great attention, which have been regarded as new biomarkers and targets in cancer diagnoses as well as therapies. However, therapeutic failure caused by chemotherapy resistance in late-stage EOC occurs frequently. The 5-year survival rate of patients with EOC remains at about 30%.

Vascular damage is a major consequence of bone fracture. Taohong Siwu decoction (TSD) can raise the expression of vascular endothelial growth factor (VEGF) in fracture healing. However, its molecular mechanism in promoting angiogenesis is still unknown. The aim of this study was to investigate the potential mechanisms of TSD in the regulation of osteo-angiogenesis in fracture healing.

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we have developed a cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 110 nm enough for the subsequent cryo-lamella fabrication. We have successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.