Discovery of a Highly Potent Oxysterol Receptor GPR183 Antagonist Bearing the Benzo[d]thiazole Structural Motif for the Treatment of Inflammatory Bowel Disease (IBD).

Accumulating evidence has demonstrated a critical pathological role of oxysterol receptor GPR183 in various inflammatory and autoimmune diseases, including inflammatory bowel disease (IBD). However, the currently reported GPR183 antagonists are very limited and not qualified for in vivo studies due to their inferior druglike properties. Herein, we conducted a structural elaboration focusing on improving its PK and safety profile based on a reference antagonist NIBR189. Of note, compound 33, bearing an aminobenzothiazole motif, exhibited reduced hERG inhibition, improved PK properties, and robust antagonistic activity (IC50 = 0.82 nM) with high selectivity against GPR183. Moreover, compound 33 displayed strong in vitro antimigration and anti-inflammatory activity in monocytes. Oral administration of compound 33 effectively improved the pathological symptoms of DSS-induced experimental colitis. All of these findings demonstrate that compound 33 is a novel and promising GPR183 antagonist suitable for further investigation to treat IBD.

Selective Electrochemical Halogenation of Functionalized Quinolone.

This work describes an effective C3-H halogenation of quinoline-4(1H)-ones under electrochemical conditions, in which potassium halides serve as both halogenating agents and electrolytes. The protocol provides expedient access to different halogenated quinoline-4(1H)-ones with unique regioselectivity, broad substrate scope, and gram-scale synthesis employing convenient, environmentally friendly electrolysis, in an undivided cell. Mechanism studies have shown that halogen radicals can promote the activation of N-H bonds in quinolones.

Activation of orphan receptor GPR132 induces cell differentiation in acute myeloid leukemia.

Blocked cellular differentiation is a critical pathologic hallmark of acute myeloid leukemia (AML). Here, we showed that genetic activation of the orphan GPCR GPR132 significantly induced cell differentiation of AML both in vitro and in vivo, indicating that GPR132 is a potential trigger of myeloid differentiation. To explore the therapeutic potential of GPR132 signaling, we screened and validated a natural product 8-gingerol (8GL) as a GPR132 agonist. Notably, GPR132 activation by 8GL promoted differentiation and reduced colony formation in human AML cell lines with diverse genetic profiles. Mechanistic studies revealed that 8GL treatment inhibits the activation of the mammalian target of rapamycin (mTOR), a regulator of AML cell differentiation blockade, via activating GPR132-Gs-PKA pathway. We further showed that the combination of 8GL and an mTOR inhibitor synergistically elicited AML cell differentiation in vitro. Importantly, 8GL alone or in combination with an mTOR inhibitor remarkably impaired tumor growth and extended mouse survival in an AML xenograft model accompanied by enhanced cell differentiation. Notably, genetic or pharmacological activation of GPR132 triggered the differentiation of human primary AML cells. In summary, this study demonstrated that activation of orphan GPR132 represents a potential strategy for inducing myeloid differentiation in AML patients.

Extracellular vesicle IL-32 promotes the M2 macrophage polarization and metastasis of esophageal squamous cell carcinoma via FAK/STAT3 pathway.

BACKGROUND: Metastasis is the leading cause of mortality in human cancers, including esophageal squamous cell carcinoma (ESCC). As a pro-inflammatory cytokine, IL-32 was reported to be a poor prognostic factor in many cancers. However, the role of IL-32 in ESCC metastasis remains unknown. METHODS: ESCC cells with ectopic expression or knockdown of IL-32 were established and their effects on cell motility were detected. Ultracentrifugation, Transmission electron microscopy and Western blot were used to verify the existence of extracellular vesicle IL-32 (EV-IL-32). Coculture assay, immunofluorescence, flow cytometry, and in vivo lung metastasis model were performed to identify how EV-IL-32 regulated the crosstalk between ESCC cells and macrophages. RESULTS: Here, we found that IL-32 was overexpressed and positively correlated to lymph node metastasis of ESCC. IL-32 was significantly higher in the tumor nest compared with the non-cancerous tissue. We found that IL-32ß was the main isoform and loaded in EV derived from ESCC cells. The shuttling of EV-IL-32 derived from ESCC cells into macrophages could promote the polarization of M2 macrophages via FAK-STAT3 pathway. IL-32 overexpression facilitated lung metastasis and was positively correlated with the proportion of M2 macrophages in tumor microenvironment. CONCLUSIONS: Taken together, our results indicated that EV-IL-32 derived from ESCC cell line could be internalized by macrophages and lead to M2 macrophage polarization via FAK-STAT3 pathway, thus promoting the metastasis of ESCC. These findings indicated that IL-32 could serve as a potential therapeutic target in patients with ESCC.

Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology.

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

A Novel Small Molecular Prostaglandin Receptor EP4 Antagonist, L001, Suppresses Pancreatic Cancer Metastasis.

Metastatic pancreatic cancer remains a major clinical challenge, emphasizing the urgent need for the exploitation of novel therapeutic approaches with superior response. In this study, we demonstrate that the aberrant activation of prostaglandin E2 (PGE2) receptor 4 (EP4) is a pro-metastatic signal in pancreatic cancer. To explore the therapeutic role of EP4 signaling, we developed a potent and selective EP4 antagonist L001 with single-nanomolar activity using a panel of cell functional assays. EP4 antagonism by L001 effectively repressed PGE2-elicited cell migration and the invasion of pancreatic cancer cells in a dose-dependent manner. Importantly, L001 alone or combined with the chemotherapy drug gemcitabine exhibited remarkably anti-metastasis activity in a pancreatic cancer hepatic metastasis model with excellent tolerability and safety. Mechanistically, EP4 blockade by L001 abrogated Yes-associated protein 1 (YAP)-driven pro-metastatic factor expression in pancreatic cancer cells. The suppression of YAP's activity was also observed upon L001 treatment in vivo. Together, these findings support the notions that EP4-YAP signaling axis is a vital pro-metastatic pathway in pancreatic cancer and that EP4 inhibition with L001 may deliver a therapeutic benefit for patients with metastatic pancreatic cancer.

Tryptophan 2,3-dioxygenase 2 controls M2 macrophages polarization to promote esophageal squamous cell carcinoma progression via AKT/GSK3ß/IL-8 signaling pathway.

Tryptophan 2,3-dioxygnease 2 (TDO2) is specific for metabolizing tryptophan to kynurenine (KYN), which plays a critical role in mediating immune escape of cancer. Although accumulating evidence demonstrates that TDO2 overexpression is implicated in the development and progression of multiple cancers, its tumor-promoting role in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we observed that TDO2 was overexpressed in ESCC tissues and correlated significantly with lymph node metastasis, advanced clinical stage, and unfavorable prognosis. Functional experiments showed that TDO2 promoted tumor cell proliferation, migration, and colony formation, which could be prevented by inhibition of TDO2 and aryl hydrocarbon receptor (AHR). Further experimentation demonstrated that TDO2 could promote the tumor growth of KYSE150 tumor-bearing model, tumor burden of C57BL/6 mice with ESCC induced by 4-NQO, enhance the expression of phosphorylated AKT, with subsequent phosphorylation of GSK3ß, and polarization of M2 macrophages by upregulating interleukin-8 (IL-8) to accelerate tumor progression in the tumor microenvironment (TME). Collectively, our results discovered that TDO2 could upregulate IL-8 through AKT/GSK3ß to direct the polarization of M2 macrophages in ESCC, and suggested that TDO2 could represent as an attractive therapeutic target and prognostic marker to ESCC.

Follow-up study on COVID-19 survivors one year after discharge from hospital.

OBJECTIVE: To evaluate the long-term consequences of COVID-19 survivors one year after recovery, and to identify the risk factors associated with abnormal patterns in chest imaging manifestations or impaired lung function. METHODS: COVID-19 patients were recruited and prospectively followed up with symptoms, health-related quality of life, psychological questionnaires, 6-minute walking test, chest computed tomography (CT), pulmonary function tests, and blood tests. Multivariable logistic regression models were used to evaluate the association between the clinical characteristics and chest CT abnormalities or pulmonary function. RESULTS: Ninety-four patients with COVID-19 were recruited between January 16 and February 6, 2021. Muscle fatigue and insomnia were the most common symptoms. Chest CT scans were abnormal in 71.28% of participants. The results of multivariable regression showed an increased odds in age. Ten patients had diffusing capacity of the lung for carbon monoxide (DLCO) impairment. Urea nitrogen concentration on admission was significantly associated with impaired DLCO. IgG levels and neutralizing activity were significantly lower compared with those in the early phase. CONCLUSIONS: One year after hospitalization for COVID-19, a cohort of survivors were mainly troubled with muscle fatigue and insomnia. Pulmonary structural abnormalities and pulmonary diffusion capacities were highly prevalent in surviving COVID-19 patients. It is necessary to intervene in the main target population for long-term recovery.

Evaluation of the curative effects of Bailing capsules for treating chronic obstructive pulmonary disease: A protocol for systematic review and meta-analysis.

BACKGROUND: The goal of the present study is to evaluate the efficacy and safety of Bailing capsules, which is a traditional Chinese drug that can improve lung functionality when used to treat chronic obstructive pulmonary disease (COPD) patients. METHODS: A comprehensive search will be performed on the following primary electronic databases: PubMed, EMBASE, Cochrane Library, Chinese National Knowledge Infrastructure, and WanFang database. A search of secondary sources includes reference lists of included studies. Two pairs of review authors will screen and scrutinize selected articles. This study will analyze continuous data as mean differences and dichotomous data as odds ratios, both with 95% confidence intervals. A sensitivity analysis will also be conducted to evaluate the stableness of the outcomes. RevMan 5.3 software was adopted to accomplish all the statistical analysis. RESULTS: The results obtained in this research shall be published in a peer-reviewed journal. CONCLUSION: Based on the interpretations of the results, useful conclusions will be presented. These conclusions will offer additional insights with useful evidence to assess whether it is viable to use Bailing capsules as an effective and safety treatment option for COPD. ETHICS AND DISSEMINATION: The present work does not involve any humans or animals; therefore, ethical approval is not needed. SYSTEMATIC REVIEW REGISTRATION: March 26, 2021.osf.io/kvgbu. (https://osf.io/kvgbu/).

Follow-up study of the pulmonary function and related physiological characteristics of COVID-19 survivors three months after recovery.

BACKGROUND: The long-term pulmonary function and related physiological characteristics of COVID-19 survivors have not been studied in depth, thus many aspects are not understood. METHODS: COVID-19 survivors were recruited for high resolution computed tomography (HRCT) of the thorax, lung function and serum levels of SARS-CoV-2 IgG antibody tests 3 months after discharge. The relationship between the clinical characteristics and the pulmonary function or CT scores were investigated. FINDINGS: Fifty-five recovered patients participated in this study. SARS-CoV-2 infection related symptoms were detected in 35 of them and different degrees of radiological abnormalities were detected in 39 patients. Urea nitrogen concentration at admission was associated with the presence of CT abnormalities (P = 0.046, OR 7.149, 95% CI 1.038 to 49.216). Lung function abnormalities were detected in 14 patients and the measurement of D-dimer levels at admission may be useful for prediction of impaired diffusion defect (P = 0.031, OR 1.066, 95% CI 1.006 to 1.129). Of all the subjects, 47 of 55 patients tested positive for SARS-CoV-2 IgG in serum, among which the generation of Immunoglobulin G (IgG) antibody in female patients was stronger than male patients in infection rehabilitation phase. INTERPRETATION: Radiological and physiological abnormalities were still found in a considerable proportion of COVID-19 survivors without critical cases 3 months after discharge. Higher level of D-dimer on admission could effectively predict impaired DLCO after 3 months discharge. It is necessary to follow up the COVID-19 patients to appropriately manage any persistent or emerging long-term sequelae. FUNDING: Key Scientific Research Projects of Henan Higher Education Institutions.

Characterization of CD103+ CD8+ tissue-resident T cells in esophageal squamous cell carcinoma: may be tumor reactive and resurrected by anti-PD-1 blockade.

Though therapy that promotes anti-tumor response about CD8+ tumor-infiltrating lymphocytes (TILs) has shown great potential, clinical responses to CD8+ TILs immunotherapy vary considerably, largely because of different subpopulation of CD8+ TILs exhibiting different biological characters. To define the relationship between subpopulation of CD8+ TILs and the outcome of antitumor reaction, the phenotype and function of CD103+ CD8+ TILs in esophageal squamous cell carcinoma (ESCC) were investigated. CD103+ CD8+ TILs were presented in ESCC, which displayed phenotype of tissue-resident memory T cells and exhibited high expression of immune checkpoints (PD-1, TIM-3). CD103+ CD8+ TILs were positively associated with the overall survivals of ESCC patients. This population of cells elicited potent proliferation and cytotoxic cytokine secretion potential. In addition, CD103+ CD8+ TILs were elicited potent anti-tumor immunity after anti-PD-1 blockade and were not affected by chemotherapy. This study emphasized the feature of CD103+ CD8+ TILs in immune response and identified potentially new targets in ESCC patients.

Serum miR-21 level: a potential diagnostic and prognostic biomarker for non-small cell lung cancer.

Because lung cancer is the most common cause of cancer death among both men and women, focused efforts are necessary to identify and develop biomarkers that aid in the detection and treatment of this serious disease. Recent research has been aimed at understanding the roles of microRNAs (miRNAs) in tumorigenesis and their utility as cancer biomarkers. Here, miR-21 was investigated as a potential serum biomarker for non-small cell lung cancer (NSCLC). The relative expression level of miR-21 was detected by real-time PCR in the sera of 80 NSCLC patients; sera were also collected from 60 healthy people as a control. The most suitable cut-off value and the prognostic value of serum miR-21 levels were analyzed using a receiver-operating curve. The relative serum miR-21 level in NSCLC patients was significantly higher than that in healthy people (P<0.05). For relative miR-21 expression, the area under the ROC curve was 0.812 (95% CI: 0.736-0.888) with a sensitivity of 73.8% and a specificity of 71.7%, based on a cut-off value of 1.22. NSCLC patients were divided into two groups based on miR-21 expression; those with higher relative expression (≥1.22) had significantly lower survival time than those in the lower expression group (P<0.05). Further, serum miR-21 level and survival time were negatively correlated in NSCLC patients (P<0.05). Thus, miR-21 may be useful as a diagnostic and prognostic indicator of NSCLC.

A toll-like receptor 4 (TLR4) variant is associated with asthma severity.

Asthma is a complex airways disease resulting from the input of both biological and environmental factors. Previous studies of single-nucleotide polymorphisms in toll-like receptor 4 (TLR4), which produces a protein involved in regulating T cell populations, have presented conflicting results regarding its role in asthma severity. In the current study, individuals with asthma were genotyped for variants of TLR4, and the genotypes were compared with asthma severity and T cell subpopulations. TLR4 rs11536879 (A>G) and rs1927907 (G>A) genotypes were determined in 350 asthma patients using TaqMan. Asthma severity was graded by clinical symptoms, and blood markers and lung function measures were also collected. T cell subpopulations were identified from peripheral blood by flow cytometry. No significant correlations were observed between genotypes at TLR4 rs11536879 or rs1927907 and eosinophil counts, total serum IgE, serum hypersensitive C-reactive protein, forced expiratory volume in 1 second (FEV1%), or FEV1/forced vital capacity (FVC) in asthma patients (P > 0.05). However, the GG genotype of rs1927907 was correlated with higher asthma severity (P < 0.05). No associations were detected between genotypes at rs11536879 or rs1927907 and CD4(+)CD25(high) regulatory T cell counts in peripheral blood from asthmatic patients (P > 0.05), but the rs1927907 genotype was associated with TLR4 expression on the surface of CD4(+)CD25(high) regulatory T cells (P < 0.05). Therefore, the TLR4 variant rs1927907 appears to be related to asthma severity and TLR4 expression on the surface of CD4(+)CD25(high) regulatory T cells, suggesting the potential influence of TLR4 on T cell population balances.

Bone marrow mesenchymal stem cell transplantation for treatment of emphysemic rats.

OBJECTIVE: To study the efficacy of bone marrow mesenchymal stem cell (MSC) transplantation to treat pulmonary emphysema in rats. METHODS: Sixty rats were randomly divided into control, model and transplantation groups. Each group contained 20 rats. Rat models of emphysema were established via intratracheal instillation of lipopolysaccharide and exposure of model and transplantation groups to smoke. Then, cultured bone marrow MSCs were injected into rats in the transplantation group via the tail vein. Pathological changes of the lung in rats were observed. RESULTS: Emphysemic pathological changes were found in model and transplantation groups, but changes were significantly attenuated after transplantation, compared with that of the model group. The mean alveoli number (MAN) and pulmonary alveolar area (PAA) showed statistically significant differences among the three groups (P < 0.001). The MAN in the transplantation group was higher than that in the model group, but still lower than that in the control group. The PAA in the transplantation group was lower than that in the model group, but higher than that in the control group (P < 0.05). After transplantation, Brdu-positive cells were observed and CK was expressed in a small number of Brdu-positive cells. Brdu- and CK-positive cells were not found in control and model groups. CONCLUSION: Transplantation of bone marrow MSCs can significantly attenuate lung inflammation and pathological changes in emphysemic rats, which may be associated with MSC differentiation into alveolar epithelial cells in recipient lung tissues.

Effects of imidapril treatment on aquaporin-2 expression in the kidneys and excretion in the urine of hypertensive rats.

Renal aquaporin-2 (AQP2) is critical for maintaining water balance and is associated with hypertension. Anti-hypertensive drugs, including imidapril, improve kidney function; however, it remains unclear whether these effects are mediated through the regulation of AQP2. In this study, the effects of imidapril on AQP2 expression in the kidneys and excretion in urine were assessed in hypertensive rats. Hypertension was induced in 24 rats, which were randomized into a control group, treated with water only, and an imidapril treatment group (n=12 per group). Blood and urine samples were collected from all rats to determine blood pressure (BP), serum Na+, urine volume and urine osmolality after 8 weeks of treatment. Molecular and immunological techniques were used to measure the expression of AQP2 in the kidneys. Urine AQP2 concentration was detected by indirect enzyme-linked immunosorbent assay (ELISA). The concentration of plasma arginine vasopressin (AVP), a regulator of AQP2 was detected by radioimmunoassay (RIA). Hypertensive rats treated with imidapril exhibited reduced BP and 24-h urine osmolality, with a concomitant increase in 24-h urine volume, compared with control hypertensive rats (P<0.05). Additionally, the expression of Aqp2 mRNA, detected by RT-PCR, and AQP2 protein, detected by immunohistochemistry and western blotting, in renal tissue significantly decreased (P<0.05). Finally, urine AQP2 concentration increased while plasma AVP concentration decreased following imidapril treatment (P<0.05). These findings indicate that imidapril reduces the expression level of AQP2 in renal tissue and accelerates its excretion.