Single-cell dissection of cervical cancer reveals key subsets of the tumor immune microenvironment.

The tumor microenvironment (TME) directly determines patients' outcomes and therapeutic efficiencies. An in-depth understanding of the TME is required to improve the prognosis of patients with cervical cancer (CC). This study conducted single-cell RNA and TCR sequencing of six-paired tumors and adjacent normal tissues to map the CC immune landscape. T and NK cells were highly enriched in the tumor area and transitioned from cytotoxic to exhaustion phenotypes. Our analyses suggest that cytotoxic large-clone T cells are critical effectors in the antitumor response. This study also revealed tumor-specific germinal center B cells associated with tertiary lymphoid structures. A high-germinal center B cell proportion in patients with CC is predictive of improved clinical outcomes and is associated with elevated hormonal immune responses. We depicted an immune-excluded stromal landscape and established a joint model of tumor and stromal cells to predict CC patients' prognosis. The study revealed tumor ecosystem subsets linked to antitumor response or prognosis in the TME and provides information for future combinational immunotherapy.

Tapered chiral nanoparticles as broad-spectrum thermally stable antivirals for SARS-CoV-2 variants.

The incessant mutations of viruses, variable immune responses, and likely emergence of new viral threats necessitate multiple approaches to novel antiviral therapeutics. Furthermore, the new antiviral agents should have broad-spectrum activity and be environmentally stable. Here, we show that biocompatible tapered CuS nanoparticles (NPs) efficiently agglutinate coronaviruses with binding affinity dependent on the chirality of surface ligands and particle shape. L-penicillamine-stabilized NPs with left-handed curved apexes display half-maximal inhibitory concentrations (IC50) as low as 0.66 pM (1.4 ng/mL) and 0.57 pM (1.2 ng/mL) for pseudo-type SARS-CoV-2 viruses and wild-type Wuhan-1 SARS-CoV-2 viruses, respectively, which are about 1,100 times lower than those for antibodies (0.73 nM). Benefiting from strong NPs-protein interactions, the same particles are also effective against other strains of coronaviruses, such as HCoV-HKU1, HCoV-OC43, HCoV-NL63, and SARS-CoV-2 Omicron variants with IC50 values below 10 pM (21.8 ng/mL). Considering rapid response to outbreaks, exposure to elevated temperatures causes no change in the antiviral activity of NPs while antibodies are completely deactivated. Testing in mice indicates that the chirality-optimized NPs can serve as thermally stable analogs of antiviral biologics complementing the current spectrum of treatments.

The molecular characteristics could supplement the staging system of pT2/T3N0M0 esophageal squamous cell carcinoma: a translational study based on a cohort with over 20 years of follow-up.

OBJECTIVE: This study aimed to construct a model based on 23 enrolled molecules to evaluate prognoses of pT2/3N0M0 esophageal squamous cell carcinoma (ESCC) patients with up to 20 years of follow-up. METHODS: The lasso-Cox model was used to identify the candidate molecule. A nomogram was conducted to develop the survival model (molecular score, MS) based on the molecular features. Cox regression and Kaplan-Meier analysis were used in this study. The concordance index (C-index) was measured to compare the predicted ability between different models. The primary endpoint was overall survival (OS). RESULTS: A total of 226 patients and 23 proteins were enrolled in this study. Patients were classified into high-risk (MS-H) and low-risk (MS-L) groups based on the MS score of 227. The survival curves showed that the MS-L cohort had better 5-year and 10-year survival rates than the MS-H group (5-year OS: 51.0% vs. 8.0%; 10-year OS: 45.0% vs. 5.0%, all p < 0.001). Furthermore, multivariable analysis confirmed MS as an independent prognostic factor after eliminating the confounding factors (Hazard ratio 3.220, p < 0.001). The pT classification was confirmed to differentiate ESCC patients' prognosis (Log-rank: p = 0.029). However, the combination of pT and MS could classify survival curves evidently (overall p < 0.001), which showed that the prognostic prediction efficiency was improved significantly by the combination of the pT and MS than by the classical pT classification (C-index: 0.656 vs. 0.539, p < 0.001). CONCLUSIONS: Our study suggested an MS for significant clinical stratification of T2/3N0M0 ESCC patients to screen out subgroups with poor prognoses. Besides, the combination of pT staging and MS could predict survival more accurately for this cohort than the pT staging system alone.

POLE2 knockdown suppresses lymphoma progression via downregulating Wnt/ß-catenin signaling pathway.

Lymphoma is the most common malignant tumor arising from immune system. Recently, DNA polymerase epsilon subunit 2 (POLE2) was identified to be a tumor promotor in a variety of malignant tumors. However, the biological role of POLE2 in lymphoma is still largely unclear. In our present study, the expression patterns of POLE2 in lymphoma tissues were identified by immunohistochemistry (IHC) staining of human tissue microarray. Cell viability was determined by CCK-8 assay. Cell apoptosis and cycle distribution were evaluated by Annexin V and PI staining, respectively. Cell migration was analyzed by transwell assay. Tumor growth in vivo was observed by a xenograft model of mice. The potential signaling was explored by human phospho-kinase array and immunoblotting. POLE2 was significantly upregulated in human lymphoma tissues and cells. POLE2 knockdown attenuated the proliferation, migration capabilities of lymphoma cells, as well as induced cell apoptosis and cycle arrest. Moreover, POLE2 depletion impaired the tumor growth in mice. Furthermore, POLE2 knockdown apparently inhibited the activation of ß-Catenin and downregulated the expression of Wnt/ß-Catenin signaling-related proteins. POLE2 knockdown suppressed the proliferation and migration of lymphoma cells by inhibiting Wnt/ß-Catenin signaling pathway. POLE2 may serve as a novel therapeutic target for lymphoma.

Sleep Deprivation Promotes Endothelial Inflammation and Atherogenesis by Reducing Exosomal miR-182-5p.

BACKGROUND: Insufficient or disrupted sleep increases the risk of cardiovascular disease, including atherosclerosis. However, we know little about the molecular mechanisms by which sleep modulates atherogenesis. This study aimed to explore the potential role of circulating exosomes in endothelial inflammation and atherogenesis under sleep deprivation status and the molecular mechanisms involved. METHODS: Circulating exosomes were isolated from the plasma of volunteers with or without sleep deprivation and mice subjected to 12-week sleep deprivation or control littermates. miRNA array was performed to determine changes in miRNA expression in circulating exosomes. RESULTS: Although the total circulating exosome levels did not change significantly, the isolated plasma exosomes from sleep-deprived mice or human were a potent inducer of endothelial inflammation and atherogenesis. Through profiling and functional analysis of the global microRNA in the exosomes, we found miR-182-5p is a key exosomal cargo that mediates the proinflammatory effects of exosomes by upregulation of MYD88 (myeloid differentiation factor 88) and activation of NF-ĸB (nuclear factor kappa-B)/NLRP3 pathway in endothelial cells. Moreover, sleep deprivation or the reduction of melatonin directly decreased the synthesis of miR-182-5p and led to the accumulation of reactive oxygen species in small intestinal epithelium. CONCLUSIONS: The findings illustrate an important role for circulating exosomes in distant communications, suggesting a new mechanism underlying the link between sleep disorder and cardiovascular disease.

Establishment and application of a gold nanoparticle-based immunochromatographic test strip for the detection of avian leukosis virus P27 antigen in egg white samples.

Avian leukemia is an infectious tumorous disease of chickens caused by subgroup A of the avian leukemia virus (ALV-A), which mainly causes long-term viremia, slow growth, immune suppression, decreased production performance, multi-tissue tumors, and even death. The infection rate of this disease is very high in chicken herds in China, causing huge economic losses to the poultry industry every year. We successfully expressed the specific antigen protein of ALV (P27) through recombinant protein technology and screened a pair of highly sensitive monoclonal antibodies (mAbs) through mouse immunity, cell fusion, and antibody pairing. Based on this pair of antibodies, we established a dual antibody sandwich ELISA and gold nanoparticle immunochromatographic strip (AuNP-ICS) detection method. In addition, the parameters of the dual antibody sandwich ELISA and AuNP-ICS were optimized under different reaction conditions, which resulted in the minimum detection limits of 0.2 ng mL-1 and 1.53 ng ml-1, respectively. Commonly available ELISA and AuNP-ICS products on the market were compared, and we found that our established immune rapid chromatography had higher sensitivity. This established AuNP-ICS had no cross-reactivity with Influenza A (H1N1), Influenza A (H9N2), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), Listeria monocytogenes listeriolysin (LLO), and Staphylococcal enterotoxin SED or SEC. Finally, the established AuNP-ICS was used to analyze 35 egg samples, and the results showed 5 positive samples and 30 negative samples. The AuNP-ICS rapid detection method established by our group had good specificity, high sensitivity, and convenience, and could be applied to the clinical sample detection of ALV-A.

Nonalcoholic Fatty Liver Disease Development in Male Mice upon Exposure to Flubendiamide.

Flubendiamide (FLU), a widely used diamide insecticide, has been observed to potentiate adipogenesis in 3T3-L1 preadipocytes in vitro. Whether exposure to FLU disrupts hepatic lipid homeostasis in mammals and induces visceral obesity, however, remains unclear. The aim of this study was to assess the effects of FLU when administered orally to male C57BL/6J mice under normal diet (ND) and high-fat diet (HFD) conditions. FLU accumulated at higher levels in the tissues of the HFD group than those of the ND group, indicating that an HFD contributed to the accumulation of lipophilic pesticides in vivo. Notably, FLU (logP = 4.14) is highly lipophilic and easily accumulates in fat. Exposure to FLU had opposing effects on the lipid metabolism of the liver in the ND and HFD groups. Liver triacylglycerol levels in the ND group were reduced, while those in the HFD group were increased, resulting in more severe hepatic steatosis. More lipid accumulation was also observed in HepG2 cells exposed to FLU. Changes in hepatic lipid deposition in vivo occurred as the enhanced transcriptional regulation of the genes involved in lipid uptake, de novo lipogenesis, and fatty acid ß-oxidation (FAO). Moreover, an excessive increase in FAO caused oxidative stress, which in turn exacerbated the inflammation of the liver. This study revealed the disruptive effect of FLU exposure on hepatic lipid homeostasis, which may facilitate the triggering of nonalcoholic fatty liver disease in HFD-fed mice.

Microstructure Evolution of Reactive Polyurethane Films During In Situ Polyaddition and Film-Formation Processes.

Due to the advantages of low energy consumption, no air and water pollutions, the reactive polyurethane films (RPUFs) are replacing the solvated and waterborne PUFs nowadays, which significantly promotes the green and low-carbon production of PU films. However, the microstructure evolution and in situ film-formation mechanism of RPUFs in solvent-free media are still unclear. Herein, according to time-temperature equivalence principle, the in situ polyaddition and film-formation processes of RPUFs generated by the typical polyaddition of diisocyanate terminated prepolymer (component B) and polyether glycol (component A) are thoroughly investigated at 25 °C. According to the temporal change of viscosity, the RPUFs gradually transfer from liquid to gel and finally to solid state. Further characterizing the molecular weight, hydrogen bonds, crystallinity, gel content, and phase images, the polyaddition and film-formation processes can be divided into three stages as 1) chain extension and microcrystallization; 2) gelation and demicrocrystallization; 3) microphase separation and film-formation. This work promotes the understanding of the microstructure evolution and film-formation mechanism of RPUFs, which can be used as the theoretical guidance for the controllable preparation of high-performance products based on RPUFs.

Serum metabolomic profiling of patients with liver cirrhosis at different stages.

An accurate and non-invasive diagnosis of the clinical stage is critical for effectively managing liver cirrhosis. This study aimed to identify serum metabolite biomarkers and clinical features that may reliably predict high-risk cirrhosis. This cross-sectional study recruited 94 cirrhotic patients (70 for identification cohort, 24 for validation cohort) from Minhang Hospital Affiliated with Fudan University between 2018 and 2021, who were analyzed by targeted quantitative metabolomics technique. Baseline clinical characteristics were collected, and different stage cirrhosis classification was performed according to the presence or absence of decompensated events. Potential metabolite biomarkers were screened, and a model for predicting the decompensation stage was created. Finally, the incidence of decompensated outcomes was analyzed. A total of 560 metabolites were detected in the identification cohort. Indole-3-propionic acid (IPA) was the most significantly decreased metabolic biomarker in the decompensated group (P<0.01, |log2FC| >2), having the strongest correlation with hyaluronic acid (r=-0.50, P<0.01). It also performed well for differentiating decompensated cirrhosis with an area under the curve (AUC) of 0.79(0.75 at internal validation). Another diagnostic model consisting of indole-3-propionic acid, hemoglobin, and albumin showed better predictive performance with an AUC of 0.97 (0.91 at internal validation). Also, 31 (44.29%) patients developed decompensated events at a median follow-up of 22.76±15.24 months. The cumulative incidence of decompensated events based on IPA subgroups (IPA <39.67ng/ml and ≥39.67ng/ml) showed a significant difference (P<0.01). "Indole-3-propionic acid" and a diagnostic model of hemoglobin and albumin can non-invasively identify cirrhotic populations at risk for decompensation, aiding in future management of liver cirrhosis.

Mining and rational design of psychrophilic catalases using metagenomics and deep learning models.

A complete catalase-encoding gene, designated soiCat1, was obtained from soil samples via metagenomic sequencing, assembly, and gene prediction. soiCat1 showed 73% identity to a catalase-encoding gene of Mucilaginibacter rubeus strain P1, and the amino acid sequence of soiCAT1 showed 99% similarity to the catalase of a psychrophilic bacterium, Pedobacter cryoconitis. soiCAT1 was identified as a psychrophilic enzyme due to the low optimum temperature predicted by the deep learning model Preoptem, which was subsequently validated through analysis of enzymatic properties. Experimental results showed that soiCAT1 has a very narrow range of optimum temperature, with maximal specific activity occurring at the lowest test temperature (4 °C) and decreasing with increasing reaction temperature from 4 to 50 °C. To rationally design soiCAT1 with an improved temperature range, soiCAT1 was engineered through site-directed mutagenesis based on molecular evolution data analyzed through position-specific amino acid possibility calculation. Compared with the wild type, one mutant, soiCAT1S205K, exhibited an extended range of optimum temperature ranging from 4 to 20 °C. The strategies used in this study may shed light on the mining of genes of interest and rational design of desirable proteins. KEY POINTS: • Numerous putative catalases were mined from soil samples via metagenomics. • A complete sequence encoding a psychrophilic catalase was obtained. • A mutant psychrophilic catalase with an extended range of optimum temperature was engineered through site-directed mutagenesis.

Lamin A/C and Vimentin as a Coordinated Regulator during Amoeboid Migration in Microscale Confined Microenvironments.

Cell migration occurs in confined microenvironments, which plays a vital role in the process of tumor metastasis. However, it is challenging to study their behaviors in vivo. Here we developed a cell squeeze system that can be scaled down to micrometers to mimic native physical confined microenvironments, wherein degrees of surface adhesion and mechanical constraints could be manipulated in order to investigate cell-migrating behaviors. Based on the microscale cell squeeze system, we found the synergistic role of lamin A/C and vimentin in cell transition and migration under strong confinement. The dynamic variations in lamin A/C and vimentin expression establish a positive feedback loop in response to confinement, effectively promoting amoeboid migration by modulating nuclear deformability while ensuring cell viability. This work shed light on modulating cell response to microenvironments by altering the expression of lamin A/C and/or vimentin, which may be a more efficient way of inhibiting cancer metastasis.

Proteomic analysis of exosomes derived from fatty hepatocytes of grass carp.

Exosomes participate in intercellular communication by carrying proteins, messenger RNA, microRNAs, and non-coding RNA. Fatty liver is a common phenomenon in farmed fish, but there has been little study of fatty hepatocytes-derived exosomes. Here, we successfully isolated exosomes from hepatocytes of grass carp, named Exos (hepatocytes-derived exosomes) and OA-Exos (fatty hepatocytes-derived exosomes), from which 617 differentially expressed proteins were identified using liquid chromatography tandem mass spectrometry. Of these, 320 proteins were promoted and 297 proteins were restrained, which were gathered in biological processes and cellular components (cellular processes, cells, and intracellular structures). The results of kyoto encyclopedia of genes and genomes (KEGG) analysis revealed that the differential expression proteins were gathered in "carbohydrate transport and metabolism", "translation, ribosomal structure and biogenesis", "posttranslational modification, protein turnover, chaperones", and "intracellular trafficking, secretion, and vesicular transport". In addition, five differentially expressed exosomal proteins were further confirmed by parallel reaction monitoring, including 2-phospho-D-glycerate hydrolyase, cytochrome b5, fatty acid-binding protein domain-containing protein, metallothionein, and malate dehydrogenas, which were downregulated. These findings provided evidence that exosomes derived from fatty hepatocytes of grass carp may be biomarkers for the early diagnosis, treatment, and prevention of fatty liver in fishery development.

Simultaneous Covalent Modification of K-Ras(G12D) and K-Ras(G12C) with Tunable Oxirane Electrophiles.

Owing to their remarkable pharmaceutical properties compared to those of noncovalent inhibitors, the development of targeted covalent inhibitors (TCIs) has emerged as a powerful method for cancer treatment. The K-Ras mutant, which is prevalent in multiple cancers, has been confirmed to be a crucial drug target in the treatment of various malignancies. However, although the K-Ras(G12D) mutation is present in up to 33% of K-Ras mutations, no covalent inhibitors targeting K-Ras(G12D) have been developed to date. The relatively weak nucleophilicity of the acquired aspartic acid (12D) residue in K-Ras may be the reason for this. Herein, we present the first compound capable of covalently engaging both K-Ras(G12D) and K-Ras(G12C) mutants. Proteome profiling revealed that this compound effectively conjugates with G12C and G12D residues, modulating the protein functions in situ. These findings offer a unique pathway for the development of novel dual covalent inhibitors.

Loss to follow-up of minorities, adolescents, and young adults on clinical trials: A report from the Children's Oncology Group.

BACKGROUND: The increasing number of childhood cancer survivors necessitates continued follow-up to monitor for long-term complications. Inequities in loss to follow-up for patients enrolled on pediatric clinical trials have not been well studied. METHODS: This was a retrospective study of 21,084 patients residing in the United States enrolled on phase 2/3 and phase 3 Children's Oncology Group (COG) trials between January 1, 2000 and March 31, 2021. Rates of loss to follow-up to COG were evaluated using log-rank tests and multivariable Cox proportional hazards regression models with adjusted hazard ratios (HRs). Demographic characteristics included age at enrollment, race, ethnicity, and zip code level socioeconomic data. RESULTS: Adolescent and young adult (AYA) patients 15-39 years old at diagnosis had an increased hazard of loss to follow-up compared to patients 0-14 years old (HR, 1.89; 95% confidence interval (CI), 1.76-2.02). In the overall cohort, non-Hispanic Blacks were found to have an increased hazard of loss to follow-up compared to non-Hispanic Whites (HR, 1.56; 95% CI, 1.43-1.70). Among AYAs, the highest loss to follow-up rates were among non-Hispanic Blacks (69.8% ± 3.1%), patients on germ cell tumor trials (78.2% ± 9.2%), and patients living in zip codes with a median household income ≤150% of the federal poverty line at diagnosis (66.7% ± 2.4%). CONCLUSIONS: AYAs, racial and ethnic minority patients, and those living in lower socioeconomic status areas had the highest rates of loss to follow-up among clinical trial participants. Targeted interventions are warranted to ensure equitable follow-up and improved assessment of long-term outcomes. PLAIN LANGUAGE SUMMARY: Little is known about disparities in loss to follow-up for pediatric cancer clinical trial participants. In this study, we found that participants who were adolescents and young adults when treated, those who identified as a racial and/or ethnic minority, or those residing in areas with lower socioeconomic status at diagnosis were associated with higher rates of loss to follow-up. As a result, the ability to assess their long-term survival, treatment-related health conditions, and quality of life is hindered. These findings suggest the need for targeted interventions to improve long-term follow-up among disadvantaged pediatric clinical trial participants.

Transcriptomics and Metabolomics for Co-Exposure to a Cocktail of Neonicotinoids and the Synergist Piperonyl Butoxide.

Here, the transcriptomics and metabolomics on a model of exposure to a cocktail of neonicotinoids (Neo) containing seven commercial compounds and a synergist piperonyl butoxide (PBO) were established. The results showed that Neo and PBO disrupted mRNA and metabolite levels in a dose-dependent manner. Neo caused tryptophan pathway-related neurotoxicity, reduced lipolysis, and promoted fat mass accumulation in the liver, while PBO induced an increase in inflammatory factors and damage to intercellular membranes. Co-exposure enhanced Neo-induced liver steatosis, focal necrosis, and oxidative stress by inhibiting oxidative phosphorylation (OXPHOS). Furthermore, diglycerides and metabolic biomarkers demonstrated that the activation of insulin signaling is associated with restricted OXPHOS, which commonly leads to a high risk of non-alcoholic fatty liver disease (NAFLD) and Alzheimer's disease (AD) as the result of over-synthesis of lipids, low energy supply, and high thermogenesis. The study demonstrates that chronic disease can be induced by Neo and the synergist PBO at the molecular level.

MT1M regulates gastric cancer progression and stemness by modulating the Hedgehog pathway protein GLI1.

Gastric cancer (GC) is a highly prevalent and aggressive malignancy with a poor prognosis. Recent evidence suggested that metallothionein 1 M (MT1M) may play a critical role in cancer development, progression, and drug resistance; however, its role in GC remains largely unknown. In this study, we investigated the expression and function of MT1M in GC both in vitro and in vivo. We found that MT1M expression was significantly downregulated in GC tissues and cell lines. Decreased expression of MT1M was associated with worse clinical prognosis, particularly in patients treated with 5-fluorouracil. Low expression of MT1M was indicative of poor overall survival (OS, HR 0.56 [95% CI 0.37-0.84], P < 0.005), first progression survival (FP, HR 0.54 [95% CI 0.36-0.79], P < 0.005), and post-progression survival (PPS, HR 0.65 [95% CI 0.45-0.94], P < 0.05). We also demonstrated that overexpression of MT1M inhibited cell proliferation and induced apoptosis in GC cells and in tumor xenografts, and it improved chemosensitivity to 5-fluorouracil. Furthermore, we found that MT1M overexpression could inhibit stem cell characteristics by targeting GLI1 and affecting GLI1 ubiquitination. Collectively, these findings indicated that MT1M may act as a tumor suppressor in GC and could serve as a potential therapeutic target to attenuate stemness and chemotherapy resistance of GC.

An initial study on the predictive value using multiple MRI characteristics for Ki-67 labeling index in glioma.

BACKGROUND AND PURPOSE: Ki-67 labeling index (LI) is an important indicator of tumor cell proliferation in glioma, which can only be obtained by postoperative biopsy at present. This study aimed to explore the correlation between Ki-67 LI and apparent diffusion coefficient (ADC) parameters and to predict the level of Ki-67 LI noninvasively before surgery by multiple MRI characteristics. METHODS: Preoperative MRI data of 166 patients with pathologically confirmed glioma in our hospital from 2016 to 2020 were retrospectively analyzed. The cut-off point of Ki-67 LI for glioma grading was defined. The differences in MRI characteristics were compared between the low and high Ki-67 LI groups. The receiver operating characteristic (ROC) curve was used to estimate the accuracy of each ADC parameter in predicting the Ki-67 level, and finally a multivariate logistic regression model was constructed based on the results of ROC analysis. RESULTS: ADCmin, ADCmean, rADCmin, rADCmean and Ki-67 LI showed a negative correlation (r = - 0.478, r = - 0.369, r = - 0.488, r = - 0.388, all P < 0.001). The Ki-67 LI of low-grade gliomas (LGGs) was different from that of high-grade gliomas (HGGs), and the cut-off point of Ki-67 LI for distinguishing LGGs from HGGs was 9.5%, with an area under the ROC curve (AUROC) of 0.962 (95%CI 0.933-0.990). The ADC parameters in the high Ki-67 group were significantly lower than those in the low Ki-67 group (all P < 0.05). The peritumoral edema (PTE) of gliomas in the high Ki-67 LI group was higher than that in the low Ki-67 LI group (P < 0.05). The AUROC of Ki-67 LI level assessed by the multivariate logistic regression model was 0.800 (95%CI 0.721-0.879). CONCLUSIONS: There was a negative correlation between ADC parameters and Ki-67 LI, and the multivariate logistic regression model combined with peritumoral edema and ADC parameters could improve the prediction ability of Ki-67 LI.

Vaccinia virus induces EMT-like transformation and RhoA-mediated mesenchymal migration.

The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.

3D sheep rumen epithelial structures driven from single cells in vitro.

Ruminants play a vital economic role as livestock, providing high-quality protein for humans. At present, 3D-cultured ruminant abomasum and intestinal organoids have been successfully established to study host and pathogen interaction. The rumen is a unique digestive organ of ruminants that occupies 70% of the volume of the digestive tract and its microbiota can decompose lignocellulose to support animal growth. Here we report a method for culturing rumen epithelial organoids. We found that single rumen epithelial cells form self-organized 3D structures representative of typical stratified squamous epithelium, which is similar to rumen epithelium. EGF, Noggin, Wnt3a, IGF-1, and FGF-10 significantly enhanced the seeding efficiency of organoids. Moreover, the inclusion of CHIR-99021, A83-01, SB202190, and Y-27632 is crucial for organoid formation and maintenance. Importantly, we demonstrate that rumen epithelial cells retain their ability to form organoids after passage, cryopreservation, and resuscitation. The rumen epithelial organoids express rumen cell type-specific genes, uptake fatty acids, and generate 2D cultures. In summary, our data demonstrate that it is feasible to establish organoids from single rumen epithelial cells, which is a novel in vitro system that may reduce the use of experimental animals.

Combining manipulation of integration loci and secretory pathway on expression of an Aspergillus niger glucose oxidase gene in Trichoderma reesei.

Trichoderma reesei (T. reesei) is well-known for its excellent ability to secret a large quantity of cellulase. However, unlike the endogenous proteins, little is known about the molecular mechanisms governing heterologous protein production. Herein, we focused on the integration loci and the secretory pathway, and investigated their combinatorial effects on heterologous gene expression in T. reesei using a glucose oxidase from Aspergillus niger as a model protein. Integration in the cel3c locus was more efficient than the cbh1 locus in expressing the AnGOx by increasing the transcription of AnGOx in the early stage. In addition, we discovered that interruption of the cel3c locus has an additional effect by increasing the expression of the secretory pathway component genes. Accordingly, overexpressing three secretory pathway component genes, that were snc1, sso2, and rho3, increased AnGOx expression in the cbh1 transformant but not in the cel3c transformant.