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Esophageal squamous precancerous lesions (ESPL) are the precursors of esophageal squamous cell carcinoma (ESCC) including low-grade and high-grade intraepithelial neoplasia. Due to the absence of molecular indicators, which ESPL will eventually develop into ESCC and thus should be treated is not well defined. Indicators, for predicting risks of ESCC at ESPL stages, are an urgent need. We perform spatial whole-transcriptome atlas analysis, which can eliminate other tissue interference by sequencing the specific ESPL regions. In this study, the expression of TAGLN2 significantly increases, while CRNN expression level decreases along the progression of ESCC. Additionally, TAGLN2 protein level significantly increases in paired after-progression tissues compared with before-progression samples, while CRNN expression decreases. Functional studies suggest that TAGLN2 promotes ESCC progression, while CRNN inhibits it by regulating cell proliferation. Taken together, TAGLN2 and CRNN are suggested as candidate indicators for the risk of ESCC at ESPL stages. Understanding the molecular changes in the transition from esophageal squamous precancerous lesions (ESPL) to esophageal squamous cell carcinoma (ESCC) remains essential. Here, the authors analyze ESPL samples using spatial transcriptomics and reveal expression changes in TAGLN2 and CRNN during progression to ESCC.

The transcription factor p63 guards genome integrity in the female germline, and its mutations have been reported in patients with premature ovarian insufficiency (POI). However, the precise contribution of the TP63 gene to the pathogenesis of POI needs to be further determined. Here, in 1,030 Chinese patients with POI, we identified 6 heterozygous mutations of the TP63 gene that impaired the C-terminal transactivation-inhibitory domain (TID) of the TAp63α protein and resulted in tetramer formation and constitutive activation of the mutant proteins. The mutant proteins induced cell apoptosis by increasing the expression of apoptosis-inducing factors in vitro. We next introduced a premature stop codon and selectively deleted the TID of TAp63α in mice and observed rapid depletion of the p63+/ΔTID mouse oocytes through apoptosis after birth. Finally, to further verify the pathogenicity of the mutation p.R647C in the TID that was present in 3 patients, we generated p63+/R647C mice and also found accelerated oocyte loss, but to a lesser degree than in the p63+/ΔTID mice. Together, these findings show that TID-related variants causing constitutive activation of TAp63α lead to POI by inducing oocyte apoptosis, which will facilitate the genetic diagnosis of POI in patients and provide a potential therapeutic target for extending female fertility.

Transcriptomic and metabolic profiles of tumor cells and stromal cells in oral squamous cell carcinoma (OSCC)-derived from oral submucosal fibrosis (OSF) (ODSCC) have been reported. However, the complex intercellular regulatory network within the tumor immunosuppressive microenvironment (TISME) in ODSCC remains poorly elucidated. Here, we utilized single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) data from GEO database and multiple immunofluorescence staining (mIF) to reveal distinctive TISME of ODSCC. Results found that compared to OSCC without OSF history (NODSCC), OSCC derived from OSF (ODSCC) showed a significant increase in exhausted CD8 + T and Treg cells (Ro/e > 1, p  < 0.05) and a decrease in cytotoxic T (CTL) (Ro/e < 1). ODSCC enriched in more Inhibin subunit beta A + Macrophages (INHBA + Mac) and Proinflammatory Cancer-associated Fibroblast (iCAF) versus NODSCC. INHBA + Mac possessed strongest immune-suppressive functions, evidenced by highest immune checkpoint scores, lowest MHC scores and highest expression of SPP1 among macrophages. Moreover, INHBA + Mac in ODSCC presented stronger immune-suppressive functions than that in NODSCC. iCAF differentially highly expressed INHBA and enriched in immune-related pathways and collagen/ECM pathways across CAF subsets, and possessed stronger immune-suppressive functions, as shown by up-regulated gene expression of TDO2, IDO1 and DUSP4 in ODSCC versus in NODSCC. Furthermore, INHBA expression was higher in ODSCC than in NODSCC ( p  < 0.01). The classic OSF-inducing molecule arecoline significantly increases the expression of INHBA ( p  < 0.0001) in vitro experiments stimulating THP-1 cells. ST analysis revealed a close co-location of INHBA + Mac, iCAF and Treg and SpaGene identified INHBA-ACVR1/ACVR2A/ACVR2B interaction regions overlapping with distribution of three types of cells. Collectively, ODSCC shows a more severe TISME and potentially poorer sensitivity to immunotherapy than NODSCC. The increased INHBA + Mac and iCAF in ODSCC are associated with the observed more severe TISME. The upregulated INHBA in ODSCC and its interaction with INHBA-ACVR1/ACVR2A/ACVR2B may mediate the modulation effect of INHBA + Mac and iCAF on Treg differentiation and functionality.

Increased extracellular free water (FW) is considered to provide better pathophysiological information than conventional diffusion tensor imaging (DTI) metrics. The cholinergic brain network is a key hub for cognitive function, and microstructural changes detected by free water imaging in this system may be associated with cognitive impairment in Alzheimer's disease (AD). However, the specific impact of FW changes in the cholinergic brain network on cognitive domains across the AD continuum and their diagnostic value remain unclear. Here, we investigated the basal forebrain cholinergic free water alterations based on free water-corrected diffusion tensor imaging in healthy controls (  = 36), amnestic mild cognitive impairment (aMCI;  = 31), the AD group (  = 33). The cholinergic basal forebrain subregions were divided into the Broca diagonal band (Ch1-3) and the Meynert basal nucleus (Ch4). The cognitive domains performance was measured using the Montreal Cognitive Assessment (MoCA). Additionally, we evaluated the diagnostic value of free water fraction (FWf) within the cholinergic system. FWf in the bilateral Ch1-3 and Ch4 regions increased with age, and was significantly higher in aMCI and AD (  < 0.001). In AD, the FWf within Ch4 was correlated with total MoCA score (  = -0.42,  = 0.015), especially with visual spatial/executive (  = -0.47,  = 0.006) and orientation deficits (  = -0.38,  = 0.029). No significant correlations were found in the aMCI group. ROC curve analysis showed that FWf within the cholinergic brain network had high diagnostic efficacy for AD versus HC (AUC = 0.958, 95% CI = 0.909-1.00), and moderate diagnostic efficacy for aMCI versus HC (AUC = 0.795, 95% CI = 0.685-0.905) and aMCI versus AD (AUC = 0.719, 95% CI = 0.589-0.850). FW imaging captures microstructural damage in the cholinergic brain network across the entire AD continuum. These changes occur early in aMCI but selectively affect domain-specific cognition in the later stages of AD, possibly through cholinergic network dysfunction. Our results highlight the potential of free water imaging as a biomarker for cognitive decline.

Although COVID-19 has become a major challenge to global health, there are currently no efficacious agents for effective treatment. Cytokine storm syndrome (CSS) can lead to acute respiratory distress syndrome (ARDS), which contributes to most COVID-19 mortalities. Research points to interleukin 6 (IL-6) as a crucial signature of the cytokine storm, and the clinical use of the IL-6 inhibitor tocilizumab shows potential for treatment of COVID-19 patient. In this study, we challenged wild-type and adenovirus-5/human angiotensin-converting enzyme 2-expressing BALB/c mice with a combination of polyinosinic-polycytidylic acid and recombinant SARS-CoV-2 spike-extracellular domain protein. High levels of TNF-α and nearly 100 times increased IL-6 were detected at 6 h, but disappeared by 24 h in bronchoalveolar lavage fluid (BALF) following immunostimulant challenge. Lung injury observed by histopathologic changes and magnetic resonance imaging at 24 h indicated that increased TNF-α and IL-6 may initiate CSS in the lung, resulting in the continual production of inflammatory cytokines. We hypothesize that TNF-α and IL-6 may contribute to the occurrence of CSS in COVID-19. We also investigated multiple monoclonal antibodies (mAbs) and inhibitors for neutralizing the pro-inflammatory phenotype of COVID-19: mAbs against IL-1α, IL-6, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and inhibitors of p38 and JAK partially relieved CSS; mAbs against IL-6, TNF-α, and GM-CSF, and inhibitors of p38, extracellular signal-regulated kinase, and myeloperoxidase somewhat reduced neutrophilic alveolitis in the lung. This novel murine model opens a biologically safe, time-saving avenue for clarifying the mechanism of CSS/ARDS in COVID-19 and developing new therapeutic drugs.

Highlights A simple and convenient graphene bio-interface was designed by using multifunctional nano-denatured bovine serum albumin (nano-dBSA) film. Highly sensitive cancer biomarker detection in diluted serum at the femtogram per milliliter level was achieved using the nano-dBSA functionalized graphene field-effect transistor. A simple, convenient, and highly sensitive bio-interface for graphene field-effect transistors (GFETs) based on multifunctional nano-denatured bovine serum albumin (nano-dBSA) functionalization was developed to target cancer biomarkers. The novel graphene–protein bioelectronic interface was constructed by heating to denature native BSA on the graphene substrate surface. The formed nano-dBSA film served as the cross-linker to immobilize monoclonal antibody against carcinoembryonic antigen (anti-CEA mAb) on the graphene channel activated by EDC and Sulfo-NHS. The nano-dBSA film worked as a self-protecting layer of graphene to prevent surface contamination by lithographic processing. The improved GFET biosensor exhibited good specificity and high sensitivity toward the target at an ultralow concentration of 337.58 fg mL −1 . The electrical detection of the binding of CEA followed the Hill model for ligand–receptor interaction, indicating the negative binding cooperativity between CEA and anti-CEA mAb with a dissociation constant of 6.82 × 10 −10  M. The multifunctional nano-dBSA functionalization can confer a new function to graphene-like 2D nanomaterials and provide a promising bio-functionalization method for clinical application in biosensing, nanomedicine, and drug delivery.

Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some studies discovered that DUBs inhibit or enhance viral infection by various mechanisms, there is lack of information on the role of DUBs in virus regulation, which needs to be further investigated. Immunoblotting, real-time polymerase chain reaction, in vivo / in vitro deubiquitination, protein immunoprecipitation, immunofluorescence, and co-localization biological techniques were employed to examine the effect of ubiquitin-specific protease 3 (USP3) on APOBEC3G (A3G) stability and human immunodeficiency virus (HIV) replication. To analyse the relationship between USP3 and HIV disease progression, we recruited 20 HIV-infected patients to detect the levels of USP3 and A3G in peripheral blood and analysed their correlation with CD4 + T-cell counts. Correlation was estimated by Pearson correlation coefficients (for parametric data). The results demonstrated that USP3 specifically inhibits HIV-1 replication in an A3G-dependent manner. Further investigation found that USP3 stabilized 90% to 95% of A3G expression by deubiquitinating Vif-mediated polyubiquitination and blocking its degradation in an enzyme-dependent manner. It also enhances the A3G messenger RNA (mRNA) level by binding to A3G mRNA and stabilizing it in an enzyme-independent manner. Moreover, USP3 expression was positively correlated with A3G expression ( r  = 0.5110) and CD4 + T-cell counts ( r  = 0.5083) in HIV-1-infected patients. USP3 restricts HIV-1 viral infections by increasing the expression of the antiviral factor A3G. Therefore, USP3 may be an important target for drug development and serve as a novel therapeutic strategy against viral infections.

Objective The detection kit for plasma Chitinase-3-like Protein 1 was developed using the magnetic bead chemiluminescence method, in order to investigate the diagnostic value of DD, FDP, CHI3L1, AFP-L3 and PIVKA-II in hepatocellular carcinoma. Method The CHI3L1 detection kit was developed using the chemiluminescence method. The luminescence value obtained from the chemiluminescence analyzer was utilized for sensitive detection of CHI3L1, and the performance of the kit was evaluated accordingly. Moreover, this study enrolled 200 patients with hepatocellular carcinoma who were treated at the Oncology Department of the Affiliated Hospital of Jiangnan University between August 2022 and November 2023 as study subjects, while 100 healthy individuals undergoing physical examinations during the same period served as a control group. The plasma CHI3L1 levels in these subjects were measured using our institute’s developed kit. Simultaneously, DD, FDP, AFP-L3, and PIVKA-II levels were assessed in all subjects to investigate their relationship with general pathology in patients with hepatocellular carcinoma. Additionally, ROC curves were generated to evaluate both single and combined detections’ diagnostic efficacy for hepatocellular carcinoma. Result The serological index changes of DD, FDP, AFP-L3, PIVKA-II, and CHI3L1 were not associated with patient gender. The concentrations of AFP-L3 and PIVKA-II in the 45–59 age group were significantly higher than in other groups (P < 0.05). Additionally, DD, CHI3L1, and PIVKA-II levels were markedly elevated in patients with tumors > 5 cm, medium-to-high differentiation, nerve invasion, lymph node metastasis, or distant metastasis. In advanced liver cancer (stages III–IV), DD, FDP, and CHI3L1 concentrations were significantly higher than in early-stage patients (stages I–II). For single diagnostic analysis, the AUC for CHI3L1 was 0.923, while the combined AUC for all five indices was 0.961, indicating greater diagnostic value when used together. The CHI3L1 chemiluminescence detection kit had a minimum detection limit of 1.50 ng/mL, with precision and accuracy within 10%, and R > 0.99. Compared to a clinical reference kit, the correlation coefficient (R) was 0.994, meeting clinical performance evaluation criteria. Conclusion The CHI3L1 chemiluminescence kit developed meets clinical requirements. CHI3L1 can be used as an indicator for early screening of liver cancer, and the detection value of combined five indicators DD, FDP, AFP-L3, PIVKA-II and CHI3L1 is higher than that of single detection.

Coronavirus disease 2019 (COVID-19) is a current global health threat caused by SARS-CoV-2. The innate immune response such as type I IFN (IFN-I) is the first line of host defense against viral infections, whereas SARS-CoV-2 proteins antagonize IFN-I production through distinct mechanisms. Ubiquitin signaling is essential for immunity to restrict pathogen proliferation. Due to its enormous impact on human health and the global economy, intensive efforts have been invested in studying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its interactions with hosts. However, the role of the ubiquitin network in pathogenicity has not yet been explored. Here, we found that ORF9b of SARS-CoV-2 is ubiquitinated on Lys-4 and Lys-40 by unknown E3 ubiquitin ligases and is degraded by the ubiquitin proteasomal system. Importantly, we identified USP29 as a host factor that prevents ORF9b ubiquitination and subsequent degradation. USP29 interacts with the carboxyl end of ORF9b and removes ubiquitin chains from the protein, thereby inhibiting type I interferon (IFN) induction and NF-κB activation. We also found that ORF9b stabilization by USP29 enhanced the virulence of VSV-eGFP and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). Moreover, we observed that the mRNA level of USP29 in SARS-CoV-2 patients was higher than that in healthy people. Our findings provide important evidence indicating that targeting USP29 may effectively combat SARS-CoV-2 infection. IMPORTANCE Coronavirus disease 2019 (COVID-19) is a current global health threat caused by SARS-CoV-2. The innate immune response such as type I IFN (IFN-I) is the first line of host defense against viral infections, whereas SARS-CoV-2 proteins antagonize IFN-I production through distinct mechanisms. Among them, ORF9b inhibits the canonical IκB kinase alpha (IKKɑ)/β/γ-NF-κB signaling and subsequent IFN production; therefore, discovering the regulation of ORF9b by the host might help develop a novel antiviral strategy. Posttranslational modification of proteins by ubiquitination regulates many biological processes, including viral infections. Here, we report that ORF9b is ubiquitinated and degraded through the proteasome pathway, whereas deubiquitinase USP29 deubiquitinates ORF9b and prevents its degradation, resulting in the enhancement of ORF9b-mediated inhibition of IFN-I and NF-κB activation and the enhancement of virulence of VSV-eGFP and SARS-CoV-2 trVLP.

Background The maintenance of genome stability in primordial germ cells (PGCs) is crucial for the faithful transmission of genetic information and the establishment of reproductive reserve. Numerous studies in recent decades have linked the Fanconi anemia (FA) pathway with fertility, particularly PGC development. However, the role of FAAP100, an essential component of the FA core complex, in germ cell development is unexplored. Results We find that FAAP100 plays an essential role in R-loop resolution and replication fork protection to counteract transcription-replication conflicts (TRCs) during mouse PGC proliferation. FAAP100 deletion leads to FA pathway inactivation, increases TRCs as well as cotranscriptional R-loops, and contributes to the collapse of replication forks and the generation of DNA damage. Then, the activated p53 signaling pathway triggers PGC proliferation defects, ultimately resulting in insufficient establishment of reproductive reserve in both sexes of mice. Conclusions Our findings suggest that FAAP100 is required for the resolution of TRCs in PGCs to safeguard their genome stability.