CASTOR1 phosphorylation predicts poor survival in male patients with KRAS-mutated lung adenocarcinoma.

BACKGROUND: Lung cancer, a leading global cause of cancer-related mortality, necessitates enhanced prognostic markers for improved treatment outcomes. We have previously shown a tumor suppressive role of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1), which is targeted for degradation upon phosphorylation at S14 (pCASTOR1) in multiple types of cancer. This study focuses on the predictive value of pCASTOR1 in lung adenocarcinoma (LUAD) patients with KRAS mutations. RESULTS: Employing a newly developed pCASTOR1 specific antibody, we found that tumor cells exhibited significantly elevated pCASTOR1 scores compared to non-tumor cells (P < 0.05). Higher pCASTOR1 scores predicted poorer overall survival (OS) (HR = 3.3, P = 0.0008) and relapse-free survival (RFS) (HR = 3.0, P = 0.0035) in male patients with KRAS mutations. pCASTOR1 remained an independent predictor for OS (HR = 4.1, P = 0.0047) and RFS (HR = 3.5, P = 0.0342) after controlling for other factors. Notably, in early-stage LUAD, elevated pCASTOR1 scores were associated with significantly worse OS (HR = 3.3, P = 0.0176) and RFS (HR = 3.1, P = 0.0277) in male patients with KRAS mutations, akin to late-stage patients. CONCLUSION: Elevated pCASTOR1 scores serve as biomarkers predicting poorer OS and RFS in male LUAD patients with KRAS mutations, offering potential clinical utility in optimizing treatment strategies for this subgroup.

A new classification method for gestational diabetes mellitus: a study on the relationship between abnormal blood glucose values at different time points in oral glucose tolerance test and adverse maternal and neonatal outcomes in pregnant women with gestational diabetes mellitus.

Background: Gestational diabetes mellitus (GDM) can lead to various adverse pregnancy outcomes for both mothers and infants, including gestational hypertension, premature rupture of membranes, preterm birth, macrosomia, large for gestational age (LGA) infants, and neonatal hypoglycemia. Previous studies have mainly focused on the overall risk of GDM for adverse maternal and neonatal outcomes, but there has been limited research specifically investigating the relationship between different patterns of abnormal oral glucose tolerance test (OGTT) results and adverse maternal and neonatal outcomes. Objective: The study aimed to analyze the maternal and neonatal outcomes among GDM women with different OGTT patterns and to explore a new classification method capable of stratifying GDM into high-risk (GDM-HR) and low-risk subtypes based on OGTT results. Study Design: We conducted a retrospective cohort study at the Women's Hospital, School of Medicine, Zhejiang University, spanning from November 1, 2015, to April 30, 2018. During the study period, a total of 3268 cases of GDM were enrolled. Based on the results of the OGTT, these GDM cases were classified into 7 subtypes, and the composition ratio of each subtype and their maternal and neonatal outcomes were analyzed. Innovatively, we proposed to categorize GDM-HR (characterized by elevated fasting blood glucose [FBG] levels, including T0, T0+1, T0+2, and T0+1+2) and low-risk GDM (GDM-LR, without elevated FBG, including T1, T2, and T1+2) and compared the maternal and neonatal outcomes between the two subtypes. Results: (1) In this cohort of 3268 GDM cases, the composition ratios of the 7 GDM subtypes were as follows: T0 (7.9%, n=260), T1 (24.2%, n=791), T2 (27.4%, n=897), T0+1 (5.4%, n=175), T0+2 (1.7%, n=56), T1+2 (26.2%, n=855), and T0+1+2 (7.2%, n=234). (2) GDM subtypes with elevated FBG levels (GDM-HR) exhibit more severe adverse prognostic outcomes compared to those without elevated FBG levels (GDM-LR). (3) Multiple logistic regression analysis revealed that compared to the GDM-LR group, the GDM-HR group showed increased fetal birth weight (by approximately 150 grams), and had higher rates of cesarean section (adjusted odds ratio [aOR]: 1.45, 95% confidence interval [CI]: 1.19-1.76), hypertensive disorders of pregnancy (aOR: 1.78, 95% CI: 1.35-2.35), preterm birth (aOR: 1.59, 95% CI: 1.17-2.16), macrosomia (aOR: 2.66, 95% CI: 2.07-3.43), LGA infants (aOR: 2.46, 95% CI: 2.05-2.97), and neonatal hypoglycemia (aOR: 2.00, 95% CI: 1.37-2.91). Partial correlation analysis shows a positive correlation between fetal birth weight and FBG levels, with r=0.222, P<.001. Multiple linear regression indicates that for every 1 mmol/L increase in FBG, the fetal weight is estimated to increase by approximately 188 grams. Conclusion: The composition ratio of GDM subtypes with elevated FBG (GDM-HR) is relatively low within GDM cases, yet it presents with a higher risk of adverse outcomes compared to subtypes without elevated FBG (GDM-LR), warranting increased attention from obstetricians. Applying this new classification method in clinical practice enables better differentiation and individualized management of GDM.

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OBJECTIVES: To study the effects and mechanisms of tetramethylpyrazine (TMP) on tumor necrosis factor-α (TNF-α)-induced inflammatory injury in human coronary artery endothelial cells (HCAEC). METHODS: HCAEC were randomly divided into four groups: the control group (no treatment), the model group (treated with TNF-α, 50 ng/mL for 24 hours), the TMP group (pre-treated with TMP, 80 µg/mL for 12 hours followed by TNF-α treatment for 24 hours), and the SIRT1 inhibitor group (pre-treated with TMP and the specific SIRT1 inhibitor EX527 for 12 hours followed by TNF-α treatment for 24 hours). Cell viability was assessed using the CCK-8 method, lactate dehydrogenase (LDH) activity was measured using an LDH assay kit, reactive oxygen species (ROS) levels were observed using DCFH-DA staining, expression of pyroptosis-related proteins was detected by Western blot, and SIRT1 expression was analyzed using immunofluorescence staining. RESULTS: Compared to the control group, the model group showed decreased cell viability, increased LDH activity, ROS level and expression of pyroptosis-related proteins, and decreased SIRT1 expression (P<0.05). Compared to the model group, the TMP group exhibited increased cell viability, decreased LDH activity, ROS level and expression of pyroptosis-related proteins, and increased SIRT1 expression (P<0.05). In comparison to the TMP group, the SIRT1 inhibitor group showed decreased cell viability, increased LDH activity, ROS level and expression of pyroptosis-related proteins, and decreased SIRT1 expression (P<0.05). CONCLUSIONS: TMP may attenuate TNF-α-induced inflammatory injury in HCAEC, which is associated with the inhibition of pyroptosis and activation of the SIRT1 signaling pathway.

Real-world effectiveness of influenza vaccine against medical-attended influenza infection during 2023/24 season in Ili Kazakh Autonomous Prefecture, China: A test-negative, case-control study.

In the post-COVID-19 pandemic era, influenza virus infections continuously lead to a global disease burden. Evaluating vaccine effectiveness against influenza infection is crucial to inform vaccine design and vaccination strategy. In this study, we recruited 1120 patients with influenza-like illness (ILI) who attended fever clinics of 4 sentinel hospitals in the Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China, from January 1 to April 7, 2024. Using a test-negative design, we estimated influenza vaccine effectiveness (VE) of 54.7% (95% CrI: 23.7, 73.1) against medical-attended influenza infection, with 62.3% (95% CrI: 29.3, 79.8) against influenza A, and 51.2% (95% CrI: 28.7, 83.0) against influenza B. Despite the moderate VE estimated in this study, influenza vaccination remains the most important approach to prevent influenza at the community level.

The profile of oral microbiome in Chinese elderly population associated with aging and systemic health status.

OBJECTIVE: The health of oral cavity is considered as an important indicator of aging. Oral microbiota is highly associated with the oral health, while the variation of oral microbiome in elderly population and characteristic microbes associated with aging remain unclear. SUBJECTS AND METHODS: In this study, 130 elderly subjects were recruited and divided into 3 groups according to their age: Stage I group (65 ≤ years < 70), Stage II group (70 ≤ years < 75), and Stage III group (75 ≤ years < 80). Their physiological indices were analyzed with using Illumina MiSeq platform and the oral microbiome was determined by high-throughput sequencing. RESULTS: Along with aging, the level of fasting blood glucose, systolic pressure and monocytes are significantly increased. No significant difference was detected on the whole structure of the oral microbiome among groups. While using Metastats and Spearman's correlation analysis, specific bacteria were identified as potential age- or health index-related bacterial genera including Fusobacterium, Parvimonas, Porphyromonas, Aminobacter, Collinsella, Clostridium and Acinetobacter. CONCLUSION: Our study revealed that the composition structure of salivary microbiota in elderly population was relatively stable while specific bacteria were correlated with age and health status, which is promising to be served as health indicators of the elderly after further exploration.

Optimizing Ex Vivo CAR-T Cell-Mediated Cytotoxicity Assay through Multimodality Imaging.

CAR-T cell-based therapies have demonstrated remarkable efficacy in treating malignant cancers, especially liquid tumors, and are increasingly being evaluated in clinical trials for solid tumors. With the FDA's initiative to advance alternative methods for drug discovery and development, full human ex vivo assays are increasingly essential for precision CAR-T development. However, prevailing ex vivo CAR-T cell-mediated cytotoxicity assays are limited by their use of radioactive materials, lack of real-time measurement, low throughput, and inability to automate, among others. To address these limitations, we optimized the assay using multimodality imaging methods, including bioluminescence, impedance tracking, phase contrast, and fluorescence, to track CAR-T cells co-cultured with CD19, CD20, and HER2 luciferase reporter cancer cells in real-time. Additionally, we varied the ratio of CAR-T cells to cancer cells to determine optimal cytotoxicity readouts. Our findings demonstrated that the CAR-T cell group effectively attacked cancer cells, and the optimized assay provided superior temporal and spatial precision measurements of ex vivo CAR-T killing of cancer cells, confirming the reliability, consistency, and high throughput of the optimized assay.

Using machine learning to predict the risk of short-term and long-term death in acute kidney injury patients after commencing CRRT.

BACKGROUND: The mortality rate and prognosis of short-term and long-term acute kidney injury (AKI) patients who undergo continuous renal replacement therapy (CRRT) are different. Setting up risk stratification tools for both short-term and long-term deaths is highly important for clinicians. METHOD: A total of 1535 AKI patients receiving CRRT were included in this study, with 1144 from the training set (the Dryad database) and 391 from the validation set (MIMIC IV database). A model for predicting mortality within 10 and 90 days was built using nine different machine learning (ML) algorithms. AUROC, F1-score, accuracy, sensitivity, specificity, precision, and calibration curves were used to assess the predictive performance of various ML models. RESULTS: A total of 420 (31.1%) deaths occurred within 10 days, and 1080 (68.8%) deaths occurred within 90 days. The random forest (RF) model performed best in both predicting 10-day (AUROC: 0.80, 95% CI: 0.74-0.84; accuracy: 0.72, 95% CI: 0.67-0.76; F1-score: 0.59) and 90-day mortality (AUROC: 0.78, 95% CI: 0.73-0.83; accuracy: 0.73, 95% CI: 0.69-0.78; F1-score: 0.80). The importance of the feature shows that SOFA scores are rated as the most important risk factor for both 10-day and 90-day mortality. CONCLUSION: Our study, utilizing multiple machine learning models, estimates the risk of short-term and long-term mortality among AKI patients who commence CRRT. The results demonstrated that the prognostic factors for short-term and long-term mortality are different. The RF model has the best prediction performance and has valuable potential for clinical application.

Interactions between toll-like receptors signaling pathway and gut microbiota in host homeostasis.

BACKGROUND: Toll-like receptors (TLRs) are a family of fundamental pattern recognition receptors in the innate immune system, constituting the first line of defense against endogenous and exogenous antigens. The gut microbiota, a collection of commensal microorganisms in the intestine, is a major source of exogenous antigens. The components and metabolites of the gut microbiota interact with specific TLRs to contribute to whole-body immune and metabolic homeostasis. OBJECTIVE: This review aims to summarize the interaction between the gut microbiota and TLR signaling pathways and to enumerate the role of microbiota dysbiosis-induced TLR signaling pathways in obesity, inflammatory bowel disease (IBD), and colorectal cancer (CRC). RESULTS: Through the recognition of TLRs, the microbiota facilitates the development of both the innate and adaptive immune systems, while the immune system monitors dynamic changes in the commensal bacteria to maintain the balance of the host-microorganism symbiosis. Dysbiosis of the gut microbiota can induce a cascade of inflammatory and metabolic responses mediated by TLR signaling pathways, potentially resulting in various metabolic and inflammatory diseases. CONCLUSION: Understanding the crosstalk between TLRs and the gut microbiota contributes to potential therapeutic applications in related diseases, offering new avenues for treatment strategies in conditions like obesity, IBD, and CRC.

GAS-Luc2 Reporter Cell Lines for Immune Checkpoint Drug Screening in Solid Tumors.

Recent studies highlight the integral role of the interferon gamma receptor (IFNγR) pathway in T cell-mediated cytotoxicity against solid but not liquid tumors. IFNγ not only directly facilitates tumor cell death by T cells but also indirectly promotes cytotoxicity via myeloid phagocytosis in the tumor microenvironment. Meanwhile, full human ex vivo immune checkpoint drug screening remains challenging. We hypothesized that an engineered gamma interferon activation site response element luciferase reporter (GAS-Luc2) can be utilized for immune checkpoint drug screening in diverse ex vivo T cell-solid tumor cell co-culture systems. We comprehensively profiled cell surface proteins in ATCC's extensive collection of human tumor and immune cell lines, identifying those with endogenously high expression of established and novel immune checkpoint molecules and binding ligands. We then engineered three GAS-Luc2 reporter tumor cell lines expressing immune checkpoints PD-L1, CD155, or B7-H3/CD276. Luciferase expression was suppressed upon relevant immune checkpoint-ligand engagement. In the presence of an immune checkpoint inhibitor, T cells released IFNγ, activating the JAK-STAT pathway in GAS-Luc2 cells, and generating a quantifiable bioluminescent signal for inhibitor evaluation. These reporter lines also detected paracrine IFNγ signaling for immune checkpoint-targeted ADCC drug screening. Further development into an artificial antigen-presenting cell line (aAPC) significantly enhanced T cell signaling for superior performance in these ex vivo immune checkpoint drug screening platforms.

Face presentation at term: incidence, risk factors and influence on maternal and neonatal outcomes.

OBJECTIVES: The incidence, diagnosis, management and outcome of face presentation at term were analysed. METHODS: A retrospective, gestational age-matched case-control study including 27 singletons with face presentation at term was conducted between April 2006 and February 2021. For each case, four women who had the same gestational age and delivered in the same month with vertex position and singletons were selected as the controls (control group, n = 108). Conditional logistic regression was used to assess the risk factors of face presentation. The maternal and neonatal outcomes of the face presentation group were followed up. RESULTS: The incidence of face presentation at term was 0.14‰. After conditional logistic regression, the two factors associated with face presentation were high parity (adjusted odds ratio [aOR] 2.76, 95% CI 1.19-6.39)] and amniotic fluid index > 18 cm (aOR 2.60, 95% CI 1.08-6.27). Among the 27 cases, the diagnosis was made before the onset of labor, during the latent phase of labor, during the active phase of labor, and during the cesarean section in 3.7% (1/27), 40.7% (11/27), 11.1% (3/27) and 44.4% (12/27) of cases, respectively. In one case of cervical dilation with a diameter of 5 cm, we innovatively used a vaginal speculum for rapid diagnosis of face presentation. The rate of cesarean section and postpartum haemorrhage ≥ 500 ml in the face presentation group was higher than that of the control group (88.9% vs. 13.9%, P < 0.001, and 14.8% vs. 2.8%, P = 0.024), but the Apgar scores were similar in both sets of newborns. Among the 27 cases of face presentation, there were three cases of adverse maternal and neonatal outcomes, including one case of neonatal right brachial plexus injury and two cases of severe laceration of the lower segment of the uterus with postpartum haemorrhage ≥ 1000 ml. CONCLUSIONS: Face presentation was rare. Early diagnosis is difficult, and thus easily neglected. High parity and amniotic fluid index > 18 cm are risk factors for face presentation. An early diagnosis and proper management of face presentation could lead to good maternal and neonatal outcomes.

Ling-Gui-Zhu-Gan decoction ameliorates nonalcoholic fatty liver disease via modulating the gut microbiota.

Numerous studies have supported that nonalcoholic fatty liver disease (NAFLD) is highly associated with gut microbiota dysbiosis. Ling-Gui-Zhu-Gan decoction (LG) has been clinically used to treat NAFLD, but the underlying mechanism remains unknown. This study investigated the therapeutic effect and mechanisms of LG in mice with NAFLD induced by a high-fat diet (HD). An HD-induced NAFLD mice model was established to evaluate the efficacy of LG followed by biochemical and histopathological analysis. Metagenomics, metabolomics, and transcriptomics were used to explore the structure and metabolism of the gut microbiota. LG significantly improved hepatic function and decreased lipid droplet accumulation in HD-induced NAFLD mice. LG reversed the structure of the gut microbiota that is damaged by HD and improved intestinal barrier function. Meanwhile, the LG group showed a lower total blood bile acids (BAs) concentration, a shifted BAs composition, and a higher fecal short-chain fatty acids (SCFAs) concentration. Furthermore, LG could regulate the hepatic expression of genes associated with the primary BAs biosynthesis pathway and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Our study suggested that LG could ameliorate NAFLD by altering the structure and metabolism of gut microbiota, while BAs and SCFAs are considered possible mediating substances. IMPORTANCE: Until now, there has still been no study on the gut microbiota and metabolomics of Ling-Gui-Zhu-Gan decoction (LG) in nonalcoholic fatty liver disease (NAFLD) mouse models. Our study is the first to report on the reshaping of the structure and metabolism of the gut microbiota by LG, as well as explore the potential mechanism underlying the improvement of NAFLD. Specifically, our study demonstrates the potential of gut microbial-derived short-chain fatty acids (SCFAs) and blood bile acids (BAs) as mediators of LG therapy for NAFLD in animal models. Based on the results of transcriptomics, we further verified that LG attenuates NAFLD by restoring the metabolic disorder of BAs via the up-regulation of Fgf15/FXR in the ileum and down-regulation of CYP7A1/FXR in the liver. LG also reduces lipogenesis in NAFLD mice by mediating the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which then contributes to reducing hepatic inflammation and improving intestinal barrier function to treat NAFLD.

Protective role of forsythoside B in Kawasaki disease-induced cardiac injury: Inhibition of pyroptosis via the SIRT1-NF-κB-p65 signaling pathway.

Kawasaki disease (KD), an acute exanthematous febrile pediatric illness involving systemic non-specific inflammatory reactions in small- and medium-sized arteries, poses a significant risk of coronary artery and myocardial inflammatory injury. Developing new KD treatments with improved safety and fewer side-effects is highly desirable. Forsythoside B (FTS-B), extracted from the Forsythia suspensa plant, exerts anti-inflammatory activity by inhibiting NF-κB, which is regulated by SIRT1, the reduced expression of which is strongly associated with cardiovascular disease. However, it has yet to be established whether FTS-B influences KD-related inflammatory damage. In this study, we investigated the effects of FTS-B on inflammation in cellular and murine models of KD. Our findings revealed that KD is associated with cardiac dysfunction and inflammatory injury to myocardial and human coronary artery endothelial cells (HCAECs), resulting in a pyroptosis-feedback loop. Both cellular and KD models were characterized by reduced SIRT1 expression and increased NF-κB p65 expression. Contrastingly, the rates of pyroptosis in both murine model myocardial tissues and HCAECs were significantly alleviated in response to FTS-B treatment. Also in both models, we detected an increase of SIRT1 expression and a decrease in the expression of p65. Further examination of the protective mechanism of FTS-B using the SIRT1-specific inhibitor, EX 527, revealed that this inhibitor blocked the palliative effects of FTS-B on inflammatory injury-induced pyroptosis. These results highlight the potential utility of the SIRT1-NF-κB-p65 pathway as a therapeutic target for KD treatment and demonstrate that FTS-B can alleviate KD-induced cardiac and HCAEC inflammatory injury via inhibition of pyroptosis.

Knowledge, attitudes, and practices towards Kawasaki disease from caregivers of children with Kawasaki disease: a cross-sectional study.

PURPOSE: To examine the knowledge, attitudes, and practices (KAP) of caregivers of children with Kawasaki disease toward Kawasaki disease. METHODS: This cross-sectional study was conducted at four hospitals in China from March 2023 to June 2023. The KAP scores were evaluated using a self-designed questionnaire (Cronbach's α = 0.840; KMO = 0.7381). Correlations between dimension scores were evaluated by Pearson correlation analysis. A structural equation model (SEM) was used to examine the relationships among factors. RESULTS: Of 643 surveyed, 49.50% were male caregivers. The mean knowledge, attitude, and practice scores were 7.12 ± 2.34 (possible range, 0-11), 29.23 ± 5.67 (possible range, 12-60), and 21.57 ± 5.34 (possible range, 6-30). Knowledge correlated with attitude (r = 0.172, P < 0.001) and practice (r = 0.280, P < 0.001). Attitude was significantly related to practice (r = 0.598, P < 0.001). SEM showed knowledge had a positive effect on attitudes (ß = 0.581, P < 0.001) and practices (ß = 0.786, P < 0.001). In addition, attitudes also positively affected practices (ß = 0.554, P < 0.001). Occupation type (ß = 0.598, P = 0.025) and monthly per capita income (ß=-0.750, P = 0.020) had different effects on attitudes, while monthly per capita income also had negative effects on practices (ß=-0.410, P = 0.021). CONCLUSION: Caregivers of children with Kawasaki disease have moderate knowledge and unfavorable attitudes but proactive practices toward this disease. The results could help design an educational intervention to improve KAP, which could translate into better patient management and outcomes. TRIAL REGISTRATION: Not applicable.

Activation of glucocorticoid receptor signaling inhibits KSHV-induced inflammation and tumorigenesis.

IMPORTANCE: Kaposi's sarcoma (KS) is the most common cancer in HIV-infected patients caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Hyperinflammation is the hallmark of KS. In this study, we have shown that KSHV mediates hyperinflammation by inducing IL-1α and suppressing IL-1Ra. Mechanistically, KSHV miRNAs and vFLIP induce hyperinflammation by activating the NF-κB pathway. A common anti-inflammatory agent dexamethasone blocks KSHV-induced hyperinflammation and tumorigenesis by activating glucocorticoid receptor signaling to suppress IL-1α and induce IL-1Ra. This work has identified IL-1-mediated inflammation as a potential therapeutic target and dexamethasone as a potential therapeutic agent for KSHV-induced malignancies.

Activation of glucocorticoid receptor signaling inhibits KSHV-induced inflammation and tumorigenesis.

Hyperinflammation is the hallmark of Kaposi's sarcoma (KS), the most common cancer in AIDS patients caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. However, the role and mechanism of induction of inflammation in KS remain unclear. In a screening for inhibitors of KSHV-induced oncogenesis, over half of the identified candidates were anti-inflammatory agents including dexamethasone functions by activating glucocorticoid receptor (GR) signaling. Here, we examined the mechanism mediating KSHV-induced inflammation. We found that numerous inflammatory pathways were activated in KSHV-transformed cells. Particularly, interleukin-1 alpha (IL-1α) and IL-1 receptor antagonist (IL-1Ra) from the IL-1 family were the most induced and suppressed cytokines, respectively. We found that KSHV miRNAs mediated IL-1α induction while both miRNAs and vFLIP mediated IL-1Ra suppression. Furthermore, GR signaling was inhibited in KSHV-transformed cells, which was mediated by vFLIP and vCyclin. Dexamethasone treatment activated GR signaling, and inhibited cell proliferation and colony formation in soft agar of KSHV-transformed cells but had a minimal effect on matched primary cells. Consequently, dexamethasone suppressed the initiation and growth of KSHV-induced tumors in mice. Mechanistically, dexamethasone suppressed IL-1α but induced IL-1Ra expression. Treatment with recombinant IL-1α protein rescued the inhibitory effect of dexamethasone while overexpression of IL-1Ra caused a weak growth inhibition of KSHV-transformed cells. Furthermore, dexamethasone induced IκBα expression resulting in inhibition of NF-κB pathway and IL-1α expression. These results reveal an important role of IL-1 pathway in KSHV-induced inflammation and oncogenesis, which can be inhibited by dexamethasone-activated GR signaling, and identify IL-1-mediated inflammation as a potential therapeutic target for KSHV-induced malignancies.

Molecular and immune signatures, and pathological trajectories of fatal COVID-19 lungs defined by in situ spatial single-cell transcriptome analysis.

Despite intensive studies during the last 3 years, the pathology and underlying molecular mechanism of coronavirus disease 2019 (COVID-19) remain poorly defined. In this study, we investigated the spatial single-cell molecular and cellular features of postmortem COVID-19 lung tissues using in situ sequencing (ISS). We detected 10 414 863 transcripts of 221 genes in whole-slide tissues and segmented them into 1 719 459 cells that were mapped to 18 major parenchymal and immune cell types, all of which were infected by SARS-CoV-2. Compared with the non-COVID-19 control, COVID-19 lungs exhibited reduced alveolar cells (ACs) and increased innate and adaptive immune cells. We also identified 19 differentially expressed genes in both infected and uninfected cells across the tissues, which reflected the altered cellular compositions. Spatial analysis of local infection rates revealed regions with high infection rates that were correlated with high cell densities (HIHD). The HIHD regions expressed high levels of SARS-CoV-2 entry-related factors including ACE2, FURIN, TMPRSS2 and NRP1, and co-localized with organizing pneumonia (OP) and lymphocytic and immune infiltration, which exhibited increased ACs and fibroblasts but decreased vascular endothelial cells and epithelial cells, mirroring the tissue damage and wound healing processes. Sparse nonnegative matrix factorization (SNMF) analysis of niche features identified seven signatures that captured structure and immune niches in COVID-19 tissues. Trajectory inference based on immune niche signatures defined two pathological routes. Trajectory A primarily progressed with increased NK cells and granulocytes, likely reflecting the complication of microbial infections. Trajectory B was marked by increased HIHD and OP, possibly accounting for the increased immune infiltration. The OP regions were marked by high numbers of fibroblasts expressing extremely high levels of COL1A1 and COL1A2. Examination of single-cell RNA-seq data (scRNA-seq) from COVID-19 lung tissues and idiopathic pulmonary fibrosis (IPF) identified similar cell populations consisting mainly of myofibroblasts. Immunofluorescence staining revealed the activation of IL6-STAT3 and TGF-ß-SMAD2/3 pathways in these cells, likely mediating the upregulation of COL1A1 and COL1A2 and excessive fibrosis in the lung tissues. Together, this study provides a spatial single-cell atlas of cellular and molecular signatures of fatal COVID-19 lungs, which reveals the complex spatial cellular heterogeneity, organization, and interactions that characterized the COVID-19 lung pathology.

Preparation of thrombin-loaded calcium alginate microspheres with dual-mode imaging and study on their embolic properties in vivo.

Transcatheter arterial embolization (TAE) has played a huge role in the interventional treatment of organ bleeding and accidental bleeding. Choosing bio-embolization materials with good biocompatibility is an important part of TAE. In this work, we prepared a calcium alginate embolic microsphere using high-voltage electrostatic droplet technology. The microsphere simultaneously encapsulated silver sulfide quantum dots (Ag2S QDs) and barium sulfate (BaSO4), and fixed thrombin on its surface. Thrombin can achieve an embolic effect while stopping bleeding. The embolic microsphere has good near-infrared two-zone (NIR-II) imaging and X-ray imaging effects, and the luminous effect of NIR-II is better than that of X-rays. This breaks the limitations of traditional embolic microspheres that only have X-ray imaging. And the microspheres have good biocompatibility and blood compatibility. Preliminary application results show that the microspheres can achieve a good embolization effect in the ear arteries of New Zealand white rabbits, and can be used as an effective material for arterial embolization and hemostasis. This work realizes the clinical embolization application of NIR-II combined with X-ray multimodal imaging technology in biomedical imaging, achieving complementary advantages and excellent results, more suitable for studying biological changes and clinical applications.

SARS-CoV-2 infection of kidney tissues from severe COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests diverse clinical pathologies involving multiple organs. While the respiratory tract is the primary SARS-CoV-2 target, acute kidney injury is common in COVID-19 patients, displaying as acute tubular necrosis (ATN) resulting from focal epithelial necrosis and eosinophilia, glomerulosclerosis, and autolysis of renal tubular cells. However, whether any renal cells are infected by SARS-CoV-2 and the mechanism involved in the COVID-19 kidney pathology remain unclear. METHODS: Kidney tissues obtained at autopsy from four severe COVID-19 patients and one healthy subject were examined by hematoxylin and eosin staining. Indirect immunofluorescent antibody assay was performed to detect SARS-CoV-2 spike protein S1 and nonstructural protein 8 (NSP8) together with markers of different kidney cell types and immune cells to identify the infected cells. RESULTS: Renal parenchyma showed tissue injury comprised of ATN and glomerulosclerosis. Positive staining of S1 protein was observed in renal parenchymal and tubular epithelial cells. Evidence of viral infection was also observed in innate monocytes/macrophages and NK cells. Positive staining of NSP8, which is essential for viral RNA synthesis and replication, was confirmed in renal parenchymal cells, indicating the presence of active viral replication in the kidney. CONCLUSIONS: In fatal COVID-19 kidneys, there are SARS-CoV-2 infection, minimally infiltrated innate immune cells, and evidence of viral replication, which could contribute to tissue damage in the form of ATN and glomerulosclerosis.

N6-Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections.

Mitogen-activated protein kinases (MAPKs) play critical roles in the induction of numerous cytokines, chemokines, and inflammatory mediators that mobilize the immune system to counter pathogenic infections. Dual-specificity phosphatase 1 (DUSP1) is a member of the dual-specificity phosphatases that inactivates MAPKs through a negative-feedback mechanism. Here, we report that in response to viral and bacterial infections, not only the DUSP1 transcript but also its N6-methyladenosine (m6A) levels rapidly increase together with that of the m6A reader protein YTHDF2, resulting in enhanced YTHDF2-mediated DUSP1 transcript degradation. The knockdown of DUSP1 promotes p38 and Jun N-terminal kinase (JNK) phosphorylation and activation, thus increasing the expression of innate immune response genes, including the interleukin-1ß (IL-1ß), colony-stimulating factor 3 (CSF3), transglutaminase 2 (TGM2), and proto-oncogene tyrosine-protein kinase Src (SRC) genes. Similarly, the knockdown of the m6A eraser ALKBH5 increases the DUSP1 transcript m6A level, resulting in accelerated transcript degradation, the activation of p38 and JNK, and the enhanced expression of IL-1ß, CSF3, TGM2, and SRC. These results demonstrate that m6A and the reader protein YTHDF2 orchestrate optimal innate immune responses during viral and bacterial infections by downregulating the expression of a negative regulator, DUSP1, of the p38 and JNK pathways that are central to innate immune responses against pathogenic infections. IMPORTANCE Innate immunity is central to controlling pathogenic infections and maintaining the homeostasis of the host. In this study, we have revealed a novel mechanism regulating innate immune responses during viral and bacterial infections. We have found that N6-methyladenosine (m6A) and the reader protein YTHDF2 regulate dual-specificity phosphatase 1, a negative regulator of the mitogen-activated protein kinases p38 and JNK, to maximize innate immune responses during viral and bacterial infections. These results provide novel insights into the mechanism regulating innate immunity, which could help in the development of novel approaches for controlling pathogenic infections.

DRR1 promotes neuroblastoma cell differentiation by regulating CREB expression.

BACKGROUND: Neuroblastoma is the most common cancer in infants and the most common extracranial solid tumor in childhood. DRR1 was identified to be downregulated in poorly differentiated ganglion cells from neuroblastoma model mice. However, the roles of DRR1 in neuroblastoma remain largely unclear. METHODS: The neuroblastoma cells were induced to differentiate, and the expression of DRR1 was detected. The expression of the neuroblastoma cell differentiation markers was analyzed in DRR1 shRNA- or DRR1-expressing vector-treated neuroblastoma cells. The downstream genes of DRR1 were screened with ChIP-seq assay. Finally, TNB1 cells were infected with DRR1 shRNA and CREB expressing vector containing lentivirus, and the expression of the cell differentiation markers, cell cycle distribution and tumor growth were analyzed. RESULTS: The expression of DRR1 was increased in differentiated neuroblastoma cells, and downregulation of DRR1 expression inhibited the differentiation of neuroblastoma cells. Further experiments indicated that CREB is a candidate downstream gene of DRR1, and it mediates neuroblastoma cell differentiation. Moreover, overexpression of CREB rescued the effect of DRR1 shRNA on cell differentiation, cell cycle distribution and tumor growth in neuroblastoma. CONCLUSIONS: DRR1-CREB axis modulates the differentiation of neuroblastoma cells and is associated with the outcome of neuroblastoma patients. IMPACT: DRR1 is involved in regulation of the differentiation of neuroblastoma. Binding with actin is essential for DRR1 to regulate neuroblastoma cell differentiation. CREB is a candidate downstream gene of DRR1 in regulating of the differentiation of neuroblastoma.