Visible-Light-Mediated Three-Component Strategy for the Synthesis of Isoxazolines and Isoxazoles.

Isoxazolines and isoxazoles commonly serve as core structures of many therapeutic agents and natural products. However, the metal-free and catalysis-free strategy for the synthesis of these privileged motifs at room temperature remains a challenging task. Herein, we report a three-component strategy to afford diverse isoxazolines and isoxazoles via [3 + 2] cycloadditions of in situ-formed nitronates and olefins/alkynes under visible-light irradiation.

Evolutionary Identification of the Requirement of the Second Intracellular Loop for the Constitutive Activity of Melanocortin-4 Receptors.

Melanocortin-4 receptor (MC4R) functions as a crucial neuroendocrine G protein-coupled receptor (GPCR) in the central nervous system of mammals, displaying agonist-independent constitutive activity that is mainly determined by its N-terminal domain. We previously reported that zebrafish MC4R exhibited a much higher basal cAMP level in comparison to mammalian MC4Rs. However, the functional evolution of constitutive activities in chordate MC4Rs remains to be elucidated. Here we cloned and compared the constitutive activities of MC4Rs from nine vertebrate species and showed that the additive action of the N-terminus with the extracellular region or transmembrane domain exhibited a combined pharmacological effect on the MC4R constitutive activity. In addition, we demonstrated that four residues of F149, Q156, V163, and K164 of the second intracellular loop played a vital role in determining MC4R constitutive activity. This study provided novel insights into functional evolution and identified a key motif essential for constitutive modulation of MC4R signaling.

METTL14-mediated lncRNA XIST silencing alleviates GDM progression by facilitating trophoblast cell proliferation and migration via the miR-497-5p/FOXO1 axis.

Gestational diabetes mellitus (GDM), a prevalent complication during the gestation period, has been linked to impaired proliferation and migration of trophoblasts causing placental maldevelopment. We previously found that lncRNA X-inactive specific transcript (XIST) played an essential role in GDM progression. Here, we investigated the precise biological functions as well as the upstream and downstream regulatory mechanisms of XIST in GDM. We found that XIST and forkhead box O1 (FOXO1) were conspicuously upregulated and miR-497-5p and methyltransferase-like 14 (METTL14) were downregulated in the placentas of GDM patients. XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells. METTL14 inhibited XIST expression through m6A methylation modification. XIST overexpression abrogated the positive effect of METTL14 overexpression on HG-cultured HTR8/SVneo cell progression. MiR-497-5p and FOXO1 are downstream regulatory genes of XIST in HTR8/SVneo cells. Reverse experiments illustrated that XIST mediated HTR8/SVneo cell functions by regulating the miR-497-5p/FOXO1 axis. Additionally, XIST silencing augmented glucose tolerance and alleviated fetal detrimental changes in GDM rats. To conclude, METTL14-mediated XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells via the miR-497-5p/FOXO1 axis, thereby alleviating GDM progression in rats.

Visible-light-induced [3+2] cycloadditions of donor/donor diazo intermediates with alkenes to achieve (spiro)-pyrazolines and pyrazoles.

To date, [3 + 2] cycloadditions of diazo esters with alkynes or alkenes have been a robust tool to generate pyrazoles and pyrazolines. However, methods capable of generating donor/donor diazo species from readily available N-tosylhydrazones to furnish [3 + 2] cycloadditions, remain elusive. Herein, we describe the first visible-light-induced [3 + 2] cycloadditions of donor/donor diazo precursors with alkenes to afford pyrazoles and novel (spiro)pyrazolines bearing a quaternary center. This protocol shows a tolerable substrate scope covering versatile carbonyl compounds and alkenes. Late-stage functionalization of bioactive molecules, one-pot approach, and gram-scale synthesis have also been introduced successfully to prove the practicability. At last, mechanistic experiments and DFT studies suggested the formation of non-covalent interactions enabling the activation of N-tosylhydrazones and the formation of the donor/donor diazo intermediates.

Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results.

Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 .

A Study of the Fluid Intake, Hydration Status, and Health Effects among Pregnant Women in Their Second Trimester in China: A Cross-Sectional Study.

The fluid intake and hydration status during pregnancy may influence the health outcomes of both the mother and the fetus. However, there are few studies related to this. The aim of the present study was to investigate fluid intake behaviors among pregnant women in their second trimester, to evaluate their hydration status and pregnancy complications, and to further explore the association of fluid intake and the amniotic fluid index (AFI). Participants' total fluid intake (TFI) levels were determined using a 7-day 24 h fluid intake questionnaire. The levels of water intake from food were not recorded or measured. Morning urine samples were collected, and both urine osmolality levels and urine specific gravity (USG) were tested to evaluate their hydration status. Fasting blood samples were also collected and measured for osmolality and complete blood count (CBC). A total of 324 participants completed the study. They were divided into four groups based on quartiles of TFI, including participants with lower (LFI1 and LFI2) and higher (HFI1 and HFI2) fluid intake levels. The median TFI was 1485 mL, and the median values of the four groups with different TFI levels were 1348, 1449, 1530, and 1609 mL, respectively. Only 3.4% of the participants attained the recommended value following an adequate water intake (1.7 L) level for pregnant women in China. Plain water was the main TFI resource (78.8~100.00%), and differences in the plain water intake levels among the four groups were evident (χ2 = 222.027, p < 0.05). The urine osmolality decreased sequentially with increasing TFI values from the LFI1 to HFI2 group, and significant differences in the urine osmolality levels among the four groups were evident (p < 0.05). Meanwhile, the percentage of dehydrated participants decreased from 26.8% in the LFI1 group to 0.0% in the HFI2 group (χ2 = 131.241, p < 0.05). Participants with higher TFI values had higher AFI values (χ2 = 58.386, all p < 0.05), and moderate-intensity correlations were found between TFI and urine osmolality, hydration status, and AFI (all p < 0.05). A large proportion of the participants had insufficient TFIs during the second trimester of pregnancy, and a proportion of the participants were dehydrated. The preliminary analysis showed that the AFI was correlated with the TFI during the second trimester of pregnancy. A sufficient TFI is necessary for pregnant women to improve their hydration status and may have effects on their health. The results can provide appropriate scientific references for the development of beneficial recommendations concerning adequate water intake levels for pregnant women in China.

TRAF3-EWSR1 signaling axis acts as a checkpoint on germinal center responses.

The formation of germinal centers (GCs) is crucial for humoral immunity and vaccine efficacy. Constant stimulation through microbiota drives the formation of constitutive GCs in Peyer's patches (PPs), which generate B cells that produce antibodies against gut antigens derived from commensal bacteria and infectious pathogens. However, the molecular mechanism that regulates this persistent process is poorly understood. We report that Ewing Sarcoma Breakpoint Region 1 (EWSR1) is a brake to constitutive GC generation and immunoglobulin G (IgG) production in PPs, vaccination-induced GC formation, and IgG responses. Mechanistically, EWSR1 suppresses Bcl6 upregulation after antigen encounter, thereby negatively regulating induced GC B cell generation and IgG production. We further showed that tumor necrosis factor receptor-associated factor (TRAF) 3 serves as a negative regulator of EWSR1. These results established that the TRAF3-EWSR1 signaling axis acts as a checkpoint for Bcl6 expression and GC responses, indicating that this axis is a therapeutic target to tune GC responses and humoral immunity in infectious diseases.

Clinical Study of Anti-PD-1 Immunotherapy Combined with Gemcitabine Chemotherapy in Multiline Treatment of Advanced Pancreatic Cancer.

Objective: This study aimed to investigate the efficacy and safety of anti-PD-1 immunotherapy combined with gemcitabine chemotherapy in multiline treatment of advanced pancreatic cancer. Methods: A retrospective analysis was performed on the clinical data of 32 patients with advanced pancreatic cancer treated with sintilimab regimen from January 2019 to December 2021 in our hospital. All patients were followed up until death or April 2022, in the form of outpatient, in-hospital review, or telephone follow-up. Follow-up content included routine blood, liver and kidney functions, tumor markers, plain or enhanced abdominal CT, and abdominal MRI examinations. Clinical efficacy was evaluated according to mRECIST criteria, and the severity of adverse effects was evaluated according to American Institute for Cancer Research (AICR) Standard Term for Adverse Events, Version 5.0. Results: During treatment, the dosage of sintilimab was halved in 2 patients due to adverse reactions. All patients were treated with sintilimab for 1~10 times, with an average of 6 ± 4 times. The total response rate (ORR) and disease control rate (DCR) were 6.25% and 12.50% and 25.00% and 37.50%, respectively, after 1 and 3 months of treatment. The mean follow-up time of 32 patients was 1-12 months, and the median follow-up time was 4 ± 3 months. By the end point of follow-up, a total of 25 patients died, and the median progression-free survival (PFS) was 3.8 (95% CI (1.85-5.63)) months. The median overall survival (OS) was 5.1 months (95% CI (3.63~7.68). After treatment, the levels of tumor markers CA125, CEA and CA199 were partly decreased compared with those before treatment (all P < 0.001). After treatment, the blood routine indexes d-dimer, CRP (C-reactive protein), NLR (neutral granulocyte to lymphocyte ratio), and MLR (monocyte to lymphocyte ratio) decreased compared with those before treatment. In 32 patients with advanced pancreatic cancer, the adverse reactions with an incidence more than 10% included fatigue, rash, hypothyroidism, hyperuricemia, and renal insufficiency. Only 2 patients showed grade 3 fatigue symptom, and all the others showed no adverse reactions of grades 3~5. In this study, all patients' adverse reactions were relieved after symptomatic treatment. Conclusion: Gemcitabine chemotherapy in multiline treatment of advanced pancreatic cancer with sintilimab can achieve certain clinical benefits without serious adverse reactions.

Dapl1 controls NFATc2 activation to regulate CD8+ T cell exhaustion and responses in chronic infection and cancer.

CD8+ T cells are central mediators of immune responses against infections and cancer. Here we identified Dapl1 as a crucial regulator of CD8+ T cell responses to cancer and infections. Dapl1 deficiency promotes the expansion of tumour-infiltrating effector memory-like CD8+ T cells and prevents their functional exhaustion, coupled with increased antitumour immunity and improved efficacy of adoptive T cell therapy. Dapl1 controls activation of NFATc2, a transcription factor required for the effector function of CD8+ T cells. Although NFATc2 mediates induction of the immune checkpoint receptor Tim3, competent NFATc2 activation prevents functional exhaustion of CD8+ T cells. Interestingly, exhausted CD8+ T cells display attenuated NFATc2 activation due to Tim3-mediated feedback inhibition; Dapl1 deletion rescues NFATc2 activation and thereby prevents dysfunction of exhausted CD8+ T cells in chronic infection and cancer. These findings establish Dapl1 as a crucial regulator of CD8+ T cell immunity and a potential target for cancer immunotherapy.

Myeloid cell TBK1 restricts inflammatory responses.

Proinflammatory cytokine production by innate immune cells plays a crucial role in inflammatory diseases, but the molecular mechanisms controlling the inflammatory responses are poorly understood. Here, we show that TANK-binding kinase 1 (TBK1) serves as a vital regulator of proinflammatory macrophage function and protects against tissue inflammation. Myeloid cell-conditional Tbk1 knockout (MKO) mice spontaneously developed adipose hypertrophy and metabolic disorders at old ages, associated with increased adipose tissue M1 macrophage infiltration and proinflammatory cytokine expression. When fed with a high-fat diet, the Tbk1-MKO mice also displayed exacerbated hepatic inflammation and insulin resistance, developing symptoms of nonalcoholic steatohepatitis. Furthermore, myeloid cell-specific TBK1 ablation exacerbates inflammation in experimental colitis. Mechanistically, TBK1 functions in macrophages to suppress the NF-κB and MAP kinase signaling pathways and thus attenuate induction of proinflammatory cytokines, particularly IL-1ß. Ablation of IL-1 receptor 1 (IL-1R1) eliminates the inflammatory symptoms of Tbk1-MKO mice. These results establish TBK1 as a pivotal anti-inflammatory mediator that restricts inflammation in different disease models.

Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination.

Inflammasomes are crucial for innate immunity against infections and, when deregulated, also contribute to inflammatory diseases. Here, we identify a critical function of the E3 ubiquitin ligase Peli1 in regulating the activation of NLRP3 inflammasome. Peli1 deficiency impairs induction of interleukin-1ß (IL-1ß) secretion by different NLRP3 inducers, but not by inducers of the Aim2, NLRP1, and NLRC4 inflammasomes. Peli1-deficient mice have alleviated peritonitis induction by alum and display increased resistance to lipopolysaccharide (LPS) endotoxin shock, coupled with decreased serum concentration of IL-1ß. Peli1 is required for NLRP3-induced caspase-1 activation and IL-1ß maturation. Mechanistically, Peli1 conjugates K63 ubiquitin chain to lysine 55 of the inflammasome adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which in turn facilitates ASC/NLRP3 interaction and ASC oligomerization, thereby contributing to inflammasome activation. Peli1 deficiency impairs the ubiquitination of ASC and inhibits inflammasome activation. Our findings establish Peli1 as an important inflammasome regulator and suggest a mechanism by which Peli1 mediates inflammatory responses.

Microglia promote autoimmune inflammation via the noncanonical NF-κB pathway.

Microglia have been implicated in neuroinflammatory diseases, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). We demonstrate that microglia mediate EAE disease progression via a mechanism relying on the noncanonical nuclear factor kB (NF-κB) pathway. Microglia-specific deletion of the noncanonical NF-κB-inducing kinase (NIK) impairs EAE disease progression. Although microglial NIK is dispensable for the initial phase of T cell infiltration into the central nervous system (CNS) and EAE disease onset, it is critical for the subsequent CNS recruitment of inflammatory T cells and monocytes. Our data suggest that following their initial CNS infiltration, T cells activate the microglial noncanonical NF-κB pathway, which synergizes with the T cell-derived cytokine granulocyte-macrophage colony-stimulating factor to induce expression of chemokines involved in the second-wave of T cell recruitment and disease progression. These findings highlight a mechanism of microglial function that is dependent on NIK signaling and required for EAE disease progression.

The clinical effect of aspirin combined with low-molecular-weight heparin in the treatment of severe preeclampsia and the combination's effect on pregnancy outcomes.

OBJECTIVE: To explore the clinical effects of aspirin combined with low-molecular-weight heparin (LMWH) in the treatment of patients with severe preeclampsia and the combination's influence on pregnancy outcomes. METHODS: From October 2018 to June 2020, 104 patients with severe preeclampsia who underwent treatment in our hospital were recruited as the study cohort and divided into two groups according to different treatment scheme each patient underwent. In the research group (RG), the 54 patients were administered aspirin combined with LMWH, and the other 50 patients in the control group (CG) were administered routine treatment. The total effective rates were compared between the two groups. The blood pressure, coagulation function, hemorheology, and renal function indexes were compared before and after the therapy. The Apgar scores of the newborns and the incidences of adverse pregnancy outcomes were measured at 1 and 5 minutes after the births. RESULTS: After the therapy, the systolic blood pressure (SBP) and the diastolic blood pressure (DBP) in the RG were lower than they were in the CG. The PT and APTT in the RG were significantly higher than they were in the CG, and the FIB and D-D were significantly lower than they were in the CG. After the treatment, the hematocrit, the erythrocyte sedimentation rate, and the plasma viscosity in the RG were significantly lower than they were in the CG. The 24 h UP, BUN, UA, and Scr levels in the RG were significantly lower than they were in the CG. The Apgar scores of the newborns in the RG were significantly higher than they were in the CG at 1 min and 5 min after the births. After the therapy, the incidence of adverse pregnancy outcomes in the RG was significantly lower than it was in the CG, and the total effective rate in the RG was significantly higher than it was in the CG. CONCLUSION: Aspirin combined with LMWH can effectively improve the clinical efficacy, the coagulation function, the renal function, and the blood pressure levels, and the combination can reduce adverse pregnancy outcomes in severe preeclampsia patients.

DYRK1a mediates BAFF-induced noncanonical NF-κB activation to promote autoimmunity and B-cell leukemogenesis.

B-cell-activating factor (BAFF) mediates B-cell survival and, when deregulated, contributes to autoimmune diseases and B-cell malignancies. The mechanism connecting BAFF receptor (BAFFR) signal to downstream pathways and pathophysiological functions is not well understood. Here we identified DYRK1a as a kinase that responds to BAFF stimulation and mediates BAFF-induced B-cell survival. B-cell-specific DYRK1a deficiency causes peripheral B-cell reduction and ameliorates autoimmunity in a mouse model of lupus. An unbiased screen identified DYRK1a as a protein that interacts with TRAF3, a ubiquitin ligase component mediating degradation of the noncanonical nuclear factor (NF)-κB-inducing kinase (NIK). DYRK1a phosphorylates TRAF3 at serine-29 to interfere with its function in mediating NIK degradation, thereby facilitating BAFF-induced NIK accumulation and noncanonical NF-κB activation. Interestingly, B-cell acute lymphoblastic leukemia (B-ALL) cells express high levels of BAFFR and respond to BAFF for noncanonical NF-κB activation and survival in a DYRK1a-dependent manner. Furthermore, DYRK1a promotes a mouse model of B-ALL through activation of the noncanonical NF-κB pathway. These results establish DYRK1a as a critical BAFFR signaling mediator and provide novel insight into B-ALL pathogenesis.

LncRNA XIST serves as a diagnostic biomarker in gestational diabetes mellitus and its regulatory effect on trophoblast cell via miR-497-5p/FOXO1 axis.

BACKGROUND: Gestational diabetes mellitus (GDM) is increasingly common in pregnancy. This study's purpose was to identify the expression of XIST and manifest the potential mechanism of XIST in GDM. METHODS: Ninety-three patients with GDM and 93 normal pregnant women were included in this investigation. qRT-PCR was conducted to evaluate the expression of miR-497-5p and XIST and the relationship between XIST and fasting blood glucose (FBG) was explored by Pearson assay. The clinical diagnosis of XIST on GDM patients was validated by the receiver operator characteristic (ROC) curve. Cell counting kit-8 (CCK-8) was applied to elucidate cell viability. Luciferase reporter assay was performed to document the relationship among XIST, miR-497-5p, and FOXO1. RESULTS: The expression of XIST was increased in GDM patients and HTR-8/SVneo cell models caused by high glucose (HG). The expression of XIST was associated with the FBG levels and appeared to be a feasible indicator in discriminating GDM patients. The expression of miR-497-5p was prominently reduced in GDM patients and cell models. Inhibition of XIST might alleviate the adverse function of HG on cell viability via sponging miR-497-5p. FOXO1 was proved to be a downstream target gene of miR-497-5p. CONCLUSIONS: Overexpression of XIST and downregulation of miR-497-5p were indicated in this publication. XIST might serve as a promising diagnostic marker for GDM patients. XIST/miR-497-5p/FOXO1 axis played a critical role in the regulation of trophoblast cells.

Three-dimensional distribution and oxidation degree analysis of coal gangue dump fire area: A case study.

Spontaneous combustion of coal gangue dumps poses a significant threat to the health and safety of nearby residents and has adverse effects on the environment. The establishment of measures to extinguish these fires requires information on the three-dimensional characteristics and oxidation degree of the dumps. An acquisition method for the index data was proposed. The temperature and the radon concentration were used as the principal indicators, and the gas concentration was a secondary index for verifying the results. Kriging interpolation was applied to predict the value of the unsampled points. Additionally, the three-dimensional characteristics of the temperature and radon anomalies were determined, thresholds were set, and the changes in the temperature and radon migration were considered to estimate the extent and depth of the fire in the coal gangue dumps. The oxidation degree of the anomalous area was identified according to the critical value of the temperature and radon anomalies. The application of this method in the gangue dump of the Tashan coal mine showed the existence of 17 oxidation areas, covering an area of 31,433 m2, including 4 shallow oxidation areas, 4 deep oxidation areas in coal waste dumps, and 9 medium-deep oxidation areas. According to the decision criterion, 4 areas with relatively high oxidation degree were identified, whereas the remaining sites were low-oxidation areas. Additionally, surface fires and internal fires can be transformed into each other, posing a significant threat. The results obtained from the various data sources were consistent and in agreement with the ground survey results, indicating that the proposed method is effective for the detection of fires in coal gangue dumps.