Development of an Integrated Disposable Device for SARS-CoV-2 Nucleic Acid Extraction and Detection.

Objective: To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. Results: The precision of the syringe transfer volume was 19.2 ± 1.9 µL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov-2 nucleic acid extraction yield through the device was 7.10 × 10 6 copies/mL, while a commercial kit yielded 2.98 × 10 6 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion: The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).

[Construction and validation of a nomogram for predicting the prognosis of patients with septic shock in department of emergency medicine].

OBJECTIVE: To construct a nomogram model for predicting the 28-day mortality of patients with septic shock in the emergency medicine department and to validate the predictive efficacy. METHODS: Based on the database of the emergency medicine department of Chu Hsien-I Memorial Hospital of Tianjin Medical University, Tianjin Medical University General Hospital and the Second Hospital of Tianjin Medical University, the data of 913 patients with septic shock admitted to the emergency medicine department from January 2017 to October 2020 were collected, including baseline demographic information and clinical characteristics, laboratory indices, and the main endpoints (28-day mortality). The patients were divided into a training set and a validation set based on simple random sampling. All significant variables from the one-way binary Logistic regression analysis of the training set were included in the multivariate Logistic regression analysis to analyze the risk factors for 28-day mortality in patients with septic shock and to construct a column-line graphical model. The predictive efficacy of the nomogram model was assessed using calibration curves and receiver operator characteristic curve (ROC curve). RESULTS: A total of 860 patients with septic shock meeting the criteria were finally enrolled, including 472 in the training set and 388 in the validation set. The 28-day mortalities were 52.5% (248/472) and 54.1% (210/388) for the training and validation sets, respectively. In the training set, age, respiratory rate (RR), the levels of C-reactive protein (CRP), D-dimer, white blood cell count (WBC), neutrophil count (NEU), neutrophil/lymphocyte ratio (NLR), monocyte/lymphocyte ratio (MLR), mean platelet volume (MPV), and platelet count (PLT) in the death group were significantly higher than those in the survival group, and the levels of base remaining (BE), lymphocyte count (LYM), hemoglobin (Hb) and the proportion of chronic obstructive pulmonary diseases (COPD) were significantly lower than those in the survival group (all P < 0.05). Multifactorial Logistic regression analysis showed that NLR [odds ratio (OR) = 0.023 0, 95% confidence interval (95%CI) was -0.204 4 to 0.113 0], MPV (OR = 0.179 8, 95%CI was -0.877 6 to 0.172 7), Hb (OR = 0.007 8, 95%CI was 0.010 3 to 0.040 8), procalcitonin (PCT; OR = 1.957 0, 95%CI was 1.243 0 to 3.081 0), and D-dimer (OR = 0.000 1, 95%CI was -0.000 4 to 0.000 1) were independent predictors of 28-day mortality in patients with septic shock in the emergency department (all P < 0.05). A column-line graph model was established based on the above variables, and the ROC curves showed that the area under the ROC curve (AUC) of the nomogram model in the training set and validation set for predicting the 28-day mortality of patients with septic shock was 0.907 (95%CI was 0.864 to 0.940) and 0.822 (95%CI was 0.781 to 0.863), respectively. The calibration curves showed good agreement between the predicted and observed results for both the training and validation sets. CONCLUSIONS: The nomogram model constructed based on NLR, MPV, Hb, PCT and D-dimer has significant clinical value in predicting the 28-day mortality of patients with septic shock in the emergency medicine department.

FTIR determination of the degree of molar substitution for hydroxypropyl chitosan.

We developed and validated a novel Fourier transform infrared (FTIR) method to determine the degree of molar substitution (MS) for hydroxypropyl chitosan (HPCS) using nuclear magnetic resonance (1H NMR) as a reference, and investigated the factors influencing the MS assay. Through extensive screening of integration methods for candidate bands in the FTIR spectrum of HPCS using 20 HPCS samples with degrees of acetylation (DA) ranging from 0.003 to 0.139, we found that when using band area at 2970 cm-1 as a probe integral, the MS values obtained via the 1H NMR method exhibited linear correlations (R2 > 0.98) with at least 16 integral ratios derived from their FTIR spectra. The optimal reference bands with high reliability are located at 3440 cm-1 and 1415 cm-1, with R2 exceeding 0.99 and a MS range of 0.17-1.92. The band at 2875 cm-1 is less affected by the trace moisture present in HPCS samples than the others. The results of the method validation demonstrated a mean recovery of 98.9 ± 2.8 % and an RSD below 10 %, suggesting a simple, robust, and highly accurate and precise method. This method could be extendable for the determination of the MS of insoluble HPCS derivatives and other hydroxypropylated polysaccharides.

The effectiveness of intervention in hepatitis C patients and improvement in their referral rate.

BACKGROUND: To investigate the effectiveness of the intervention with critical value management and push short messaging service (SMS), and to determine improvement in the referral rate of patients with positive hepatitis C antibody (anti-HCV). METHODS: No intervention was done for patients with positive anti-HCV screening results from 1 January 2015 to 31 October 2021. Patients with positive anti-HCV results at our hospital from 1 November 2021 to 31 July 2022 were informed vide critical value management and push SMS. For inpatients, a competent physician was requested to liaise with the infectious disease physician for consultation, and patients seen in the OPD (outpatient department) were asked to visit the liver disease clinic. The Chi-square correlation test, one-sided two-ratio test and linear regression were used to test the relationship between intervention and referral rate. RESULTS: A total of 638,308 cases were tested for anti-hepatitis C virus (HCV) in our hospital and 5983 of them were positive. 51.8% of the referred patients were aged 18-59 years and 10.8% were aged ≥75 years. The result of Chi-square correlation test between intervention and referral was p = .0000, p < .05. One-sided two-ratio test was performed for statistics of pre-intervention referral rate (p1) and post-intervention referral rate (p2). Normal approximation and Fisher's exact test for the results obtained were 0.000, p < .05, and the alternative hypothesis p1 - p2 < 0 was accepted. The linear regression equation was referral = 0.1396 × intervention + 0.3743, and the result model p = 8.79e - 09, p < .05. The model was significant, and the coefficient of intervention was 0.1396. CONCLUSIONS: The interventions of critical value management and push SMS were correlated with the referral rate of patients with positive anti-HCV.

A pharynx-to-brain axis controls pharyngeal inflammation-induced anxiety.

Anxiety is a remarkably common condition among patients with pharyngitis, but the relationship between these disorders has received little research attention, and the underlying neural mechanisms remain unknown. Here, we show that the densely innervated pharynx transmits signals induced by pharyngeal inflammation to glossopharyngeal and vagal sensory neurons of the nodose/jugular/petrosal (NJP) superganglia in mice. Specifically, the NJP superganglia project to norepinephrinergic neurons in the nucleus of the solitary tract (NTSNE). These NTSNE neurons project to the ventral bed nucleus of the stria terminalis (vBNST) that induces anxiety-like behaviors in a murine model of pharyngeal inflammation. Inhibiting this pharynx→NJP→NTSNE→vBNST circuit can alleviate anxiety-like behaviors associated with pharyngeal inflammation. This study thus defines a pharynx-to-brain axis that mechanistically links pharyngeal inflammation and emotional response.

FGF17 protects cerebral ischemia reperfusion-induced blood-brain barrier disruption via FGF receptor 3-mediated PI3K/AKT signaling pathway.

Maintaining blood-brain barrier (BBB) integrity is critical components of therapeutic approach for ischemic stroke. Fibroblast growth factor 17 (FGF17), a member of FGF8 superfamily, exhibits the strongest expression throughout the wall of all major arteries during development. However, its molecular action and potential protective role on brain endothelial cells after stroke remains unclear. Here, we observed reduced levels of FGF17 in the serum of patients with ischemic stroke, as well as in the brains of mice subjected to middle cerebral artery occlusion (MCAO) injury and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain microvascular endothelial cells (bEnd.3) cells. Moreover, treatment with exogenous recombinant human FGF17 (rhFGF17) decreased infarct volume, improved neurological deficits, reduced Evans Blue leakage and upregulated the expression of tight junctions in MCAO-injured mice. Meanwhile, rhFGF17 increased cell viability, enhanced trans-endothelial electrical resistance, reduced sodium fluorescein leakage, and alleviated reactive oxygen species (ROS) generation in OGD/R-induced bEnd.3 cells. Mechanistically, the treatment with rhFGF17 resulted in nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear accumulation and upregulation of heme oxygenase-1 (HO-1) expression. Additionally, based on in-vivo and in-vitro research, rhFGF17 exerted protective effects against ischemia/reperfusion (I/R) -induced BBB disruption and endothelial cell apoptosis through the activation of the FGF receptor 3/PI3K/AKT signaling pathway. Overall, our findings indicated that FGF17 may hold promise as a novel therapeutic strategy for ischemic stroke patients.

Adaptive Selection of Cis-regulatory Elements in the Han Chinese.

Cis-regulatory elements have an important role in human adaptation to the living environment. However, the lag in population genomic cohort studies and epigenomic studies, hinders the research in the adaptive analysis of cis-regulatory elements in human populations. In this study, we collected 4,013 unrelated individuals and performed a comprehensive analysis of adaptive selection of genome-wide cis-regulatory elements in the Han Chinese. In total, 12.34% of genomic regions are under the influence of adaptive selection, where 1.00% of enhancers and 2.06% of promoters are under positive selection, and 0.06% of enhancers and 0.02% of promoters are under balancing selection. Gene ontology enrichment analysis of these cis-regulatory elements under adaptive selection reveals that many positive selections in the Han Chinese occur in pathways involved in cell-cell adhesion processes, and many balancing selections are related to immune processes. Two classes of adaptive cis-regulatory elements related to cell adhesion were in-depth analyzed, one is the adaptive enhancers derived from neanderthal introgression, leads to lower hyaluronidase level in skin, and brings better performance on UV-radiation resistance to the Han Chinese. Another one is the cis-regulatory elements regulating wound healing, and the results suggest the positive selection inhibits coagulation and promotes angiogenesis and wound healing in the Han Chinese. Finally, we found that many pathogenic alleles, such as risky alleles of type 2 diabetes or schizophrenia, remain in the population due to the hitchhiking effect of positive selections. Our findings will help deepen our understanding of the adaptive evolution of genome regulation in the Han Chinese.

Dysfunctional Mitochondria Clearance in Situ: Mitophagy in Obesity and Diabetes-Associated Cardiometabolic Diseases.

Several mitochondrial dysfunctions in obesity and diabetes include impaired mitochondrial membrane potential, excessive mitochondrial reactive oxygen species generation, reduced mitochondrial DNA, increased mitochondrial Ca2+ flux, and mitochondrial dynamics disorders. Mitophagy, specialized autophagy, is responsible for clearing dysfunctional mitochondria in physiological and pathological conditions. As a paradox, inhibition and activation of mitophagy have been observed in obesity and diabetes-related heart disorders, with both exerting bidirectional effects. Suppressed mitophagy is beneficial to mitochondrial homeostasis, also known as benign mitophagy. On the contrary, in most cases, excessive mitophagy is harmful to dysfunctional mitochondria elimination and thus is defined as detrimental mitophagy. In obesity and diabetes, two classical pathways appear to regulate mitophagy, including PTEN-induced putative kinase 1 (PINK1)/Parkin-dependent mitophagy and receptors/adapters-dependent mitophagy. After the pharmacologic interventions of mitophagy, mitochondrial morphology and function have been restored, and cell viability has been further improved. Herein, we summarize the mitochondrial dysfunction and mitophagy alterations in obesity and diabetes, as well as the underlying upstream mechanisms, in order to provide novel therapeutic strategies for the obesity and diabetes-related heart disorders.

Somatosensory cortex and central amygdala regulate neuropathic pain-mediated peripheral immune response via vagal projections to the spleen.

Pain involves neuroimmune crosstalk, but the mechanisms of this remain unclear. Here we showed that the splenic T helper 2 (TH2) immune cell response is differentially regulated in male mice with acute versus chronic neuropathic pain and that acetylcholinergic neurons in the dorsal motor nucleus of the vagus (AChDMV) directly innervate the spleen. Combined in vivo recording and immune cell profiling revealed the following two distinct circuits involved in pain-mediated peripheral TH2 immune response: glutamatergic neurons in the primary somatosensory cortex (GluS1HL)→AChDMV→spleen circuit and GABAergic neurons in the central nucleus of the amygdala (GABACeA)→AChDMV→spleen circuit. The acute pain condition elicits increased excitation from GluS1HL neurons to spleen-projecting AChDMV neurons and increased the proportion of splenic TH2 immune cells. The chronic pain condition increased inhibition from GABACeA neurons to spleen-projecting AChDMV neurons and decreased splenic TH2 immune cells. Our study thus demonstrates how the brain encodes pain-state-specific immune responses in the spleen.

Graded diagnosis of Helicobacter pylori infection using hyperspectral images of gastric juice.

Helicobacter pylori is a potential underlying cause of many diseases. Although the Carbon 13 breath test is considered the gold standard for detection, it is high cost and low public accessibility in certain areas limit its widespread use. In this study, we sought to use machine learning and deep learning algorithm models to classify and diagnose H. pylori infection status. We used hyperspectral imaging system to gather gastric juice images and then retrieved spectral feature information between 400 and 1000 nm. Two different data processing methods were employed, resulting in the establishment of one-dimensional (1D) and two-dimensional (2D) datasets. In the binary classification task, the random forest model achieved a prediction accuracy of 83.27% when learning features from 1D data, with a specificity of 84.56% and a sensitivity of 92.31%. In the ternary classification task, the ResNet model learned from 2D data and achieved a classification accuracy of 91.48%.

Tandem mass spectrometry (MS/MS) molecular networking guided profiling of small molecules from Aquilaria sinensis (Lour.) Gilg leaves and their bioactivity evaluation.

INTRODUCTION: Agarwood, a fragrant resinous wood mainly formed by Aquilaria spp., is used worldwide as a natural fragrance and traditional medicine. A large amount of Aquilaria sinensis (Lour.) Gilg leaves are underutilised during the process of the agarwood industry, and the development of A. sinensis leaves as tea has recently attracted more and more attention. However, the small molecule profile of A. sinensis leaves and their bioactivities has been rarely reported. OBJECTIVE: To conduct a rapid untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of A. sinensis leaves with a molecular networking (MN) strategy and evaluate its antioxidant and antidiabetic value. METHOD: A MN-assisted tandem mass spectrometry (MS/MS) analysis strategy was used to investigate the small molecule profile of A. sinensis leaves. Additionally, the integration of antioxidant and α-glucosidase inhibitory assays with MN analysis was executed to expeditiously characterise the bioactive compounds for potential prospective application. RESULTS: Five main chemical groups including phenol C-glycosides, organic acids, 2-(2-phenylethyl) chromones, benzophenone O-glycosides and flavonoids were rapidly revealed from the A. sinensis leaves. Eighty-one compounds were provisionally identified by comparing their MS/MS fragments with canonical pathways. The featured xanthone C-glycosides and benzophenone C-glycosides were recognised as the primary components of A. sinensis leaves. Several dimers and a trimer of mangiferin were reported firstly in A. sinensis leaves. Furthermore, 17 and 14 potential bioactive molecules were rapidly annotated from antioxidant and α-glucosidase inhibitory fraction, respectively. CONCLUSION: Our findings will help expand the utilisation of A. sinensis leaves and thus promote the high-quality development of agarwood industry.

Molecular mechanisms of noncoding RNA and epigenetic regulation in obesity with consequent diabetes mellitus development.

Diabetes mellitus (DM) and obesity have become two of the most prevalent and challenging diseases worldwide, with increasing incidence and serious complications. Recent studies have shown that noncoding RNA (ncRNA) and epigenetic regulation play crucial roles in the pathogenesis of DM complicated by obesity. Identification of the involvement of ncRNA and epigenetic regulation in the pathogenesis of diabetes with obesity has opened new avenues of investigation. Targeting these mechanisms with small molecules or RNA-based therapies may provide a more precise and effective approach to diabetes treatment than traditional therapies. In this review, we discuss the molecular mechanisms of ncRNA and epigenetic regulation and their potential therapeutic targets, and the research prospects for DM complicated with obesity.

Deciphering tumor-infiltrating dendritic cells in the single-cell era.

Dendritic cells (DCs) serve as a pivotal link connecting innate and adaptive immunity by processing tumor-derived antigens and activating T cells. The advent of single-cell sequencing has revolutionized the categorization of DCs, enabling a high-resolution characterization of the previously unrecognized diversity of DC populations infiltrating the intricate tumor microenvironment (TME). The application of single-cell sequencing technologies has effectively elucidated the heterogeneity of DCs present in the tumor milieu, yielding invaluable insights into their subpopulation structures and functional diversity. This review provides a comprehensive summary of the current state of knowledge regarding DC subtypes in the TME, drawing from single-cell studies conducted across various human tumors. We focused on the categorization, functions, and interactions of distinct DC subsets, emphasizing their crucial roles in orchestrating tumor-related immune responses. Additionally, we delve into the potential implications of these findings for the identification of predictive biomarkers and therapeutic targets. Enhanced insight into the intricate interplay between DCs and the TME promises to advance our comprehension of tumor immunity and, in turn, pave the way for the development of more efficacious cancer immunotherapies.

Causal associations of gut microbiota and metabolites on sepsis: a two-sample Mendelian randomization study.

Background: Sepsis stands as a dire medical condition, arising when the body's immune response to infection spirals into overdrive, paving the way for potential organ damage and potential mortality. With intestinal flora's known impact on sepsis but a dearth of comprehensive data, our study embarked on a two-sample Mendelian randomization analysis to probe the causal link between gut microbiota and their metabolites with severe sepsis patients who succumbed within a 28-day span. Methods: Leveraging data from Genome-wide association study (GWAS) and combining it with data from 2,076 European descendants in the Framingham Heart Study, single-nucleotide polymorphisms (SNPs) were employed as Instrumental Variables (IVs) to discern gene loci affiliated with metabolites. GWAS summary statistics for sepsis were extracted from the UK Biobank consortium. Results: In this extensive exploration, 93 distinct genome-wide significant SNPs correlated with gut microbial metabolites and specific bacterial traits were identified for IVs construction. Notably, a substantial link between Coprococcus2 and both the incidence (OR of 0.80, 95% CI: 0.68-0.94, P=0.007) and the 28-day mortality rate (OR 0.48, 95% CI: 0.27-0.85, P=0.013) of sepsis was observed. The metabolite α-hydroxybutyrate displayed a marked association with sepsis onset (OR=1.08, 95% CI: 1.02-1.15, P=0.006) and its 28-day mortality rate (OR=1.17, 95% CI: 1.01-1.36, P=0.029). Conclusion: This research unveils the intricate interplay between the gut microbial consortium, especially the genus Coprococcus, and the metabolite α-hydroxybutyrate in the milieu of sepsis. The findings illuminate the pivotal role of intestinal microbiota and their metabolites in sepsis' pathogenesis, offering fresh insights for future research and hinting at novel strategies for sepsis' diagnosis, therapeutic interventions, and prognostic assessments.

Recent positive selection signatures reveal phenotypic evolution in the Han Chinese population.

Characterizing natural selection signatures and relationships with phenotype spectra is important for understanding human evolution and both biological and pathological mechanisms. Here, we identified 24 genetic loci under recent selection by analyzing rare singletons in 3946 high-depth whole-genome sequencing data of Han Chinese. The loci include immune-related gene regions (MHC cluster, IGH cluster, STING1, and PSG), alcohol metabolism-related gene regions (ADH1B, ALDH2, and ALDH3B2), and the olfactory perception gene OR4C16, in which the MHC cluster, ADH1B, and ALDH2 were also identified by TOPMed and WestLake Biobank. Among the signals, the IGH cluster is particularly interesting, in which the favored allele of variant 14_105737776_C_T (rs117518546, IgG1-G396R) promotes immune response, but also increases the risk of an autoimmune disease systemic lupus erythematosus (SLE). It is also surprising that our newly discovered ALDH3B2 evolved in the opposite direction to ALDH2 for alcohol metabolism. Besides monogenic traits, we found that multiple complex traits experienced polygenic adaptation. Particularly, multi-methods consistently revealed that lower blood pressure was favored in natural selection. Finally, we built a database named RePoS (recent positive selection, http://bigdata.ibp.ac.cn/RePoS/) to integrate and display multi-population selection signals. Our study extended our understanding of natural evolution and phenotype adaptation in Han Chinese as well as other populations.

Shielding Effects of Aromatic (Indole) Ring for Structural Analysis.

This review provides a critical analysis of shielding effects induced by an aromatic (indole) ring of small molecules mainly including three members of naturally occurring secondary metabolites asterric acid analogs, diketopiperazines (DKPs) possessing an aromatic or an indole ring, and rubrolides. Empirical rules about the shielding effects induced by an aromatic (indole) ring are classified, based on which some 1H NMR chemical shift values in the A-ring and structures of asterric acid analogs are revised, and the relative configurations of some DKPs possessing an indole ring are also assigned or revised. The empirical rules could provide an efficient and convenient method for NMR data analysis and configuration determination for the three members of small molecules mentioned above.

Brain regulation of gastric dysfunction induced by stress.

Psychological and physical stressors have been implicated in gastric disorders in humans. The mechanism coupling the brain to the stomach underlying stress-induced gastric dysfunction has remained elusive. Here, we show that the stomach directly receives acetylcholinergic inputs from the dorsal motor nucleus of the vagus (AChDMV), which are innervated by serotonergic neurons in the dorsal raphe nucleus (5-HTDRN). Microendoscopic calcium imaging and multi-tetrode electrophysiological recordings reveal that the 5-HTDRN → AChDMV → stomach circuit is inhibited with chronic stress accompanied by hypoactivate gastric function. Artificial activation of this circuit reverses the gastric dysfunction induced by chronic stress in both male and female mice. Our study demonstrates that this 5-HTDRN → AChDMV → stomach axis drives gastric dysfunction associated with stress, thus providing insights into the circuit basis for brain regulation of the stomach.

A DNA Nano Firework for Imaging and Inhibitor Screening of Flap Endonuclease 1 in Living Cells.

In situ observation of changes in the activity of marker proteins in living cells is crucial for both biomarker-based disease diagnosis and drug screening. Flap endonuclease 1 (FEN1) has been recognized as a broad-spectrum cancer biomarker and therapeutic target. However, simple and reliable methods for in situ studying the FEN1 activity changes in living cells are limited. Here, we introduce a nano firework as a fluorescent sensor to sense and report FEN1 activity changes in living cells through FEN1 recognizing the substrates on the surface of the nano firework to release and restore the fluorescence of the prequenched fluorophores. We verified the high selectivity, anti-interference ability, stability, and quantitative performance of the nano firework in tubes and living cells, respectively. A series of controlled experiments have demonstrated that the nano firework could accurately report changes in FEN1 activity in different cells, enabling "sensors in, results out" in the manner of simple addition to the cell culture medium. Using an in silico molecular docking study and experiments, we also explored the ability of the nano firework for rapid screening of FEN1 inhibitors and found two new candidate compounds myricetrin and neoisoliquritin, which could be used as FEN1 inhibitors for further research. These performances of the nano firework suggest that it can be used in high-throughput screening applications, providing a promising tool for biomarker-based new drug discovery.

Intermolecular Triazene Alkene Cycloaddition via Lewis Base Catalysis: Access to Diverse Trifluoromethylated Pyrazolines.

A novel approach to chemoselective synthesis of biologically important CF3 -subsituted pyrazolines was developed via a Lewis base catalyzed intermolecular triazene cycloaddition reaction of an array of terminal/internal alkenes with CF3 CHN2 . This strategy features a catalytic amount of 1,8-diazabicyclo[5.4.0]undec-7-ene, high yields (up to 95 %), wide substrate scope and excellent functional group tolerance (54 examples). Importantly, we preformed scaffold diversification of a panel of known pharmaceuticals, natural products, and bioactive heterocycles to generate the corresponding pyrazoline derivatives with potential broad bioactivities for further development.

Necroptosis signaling and mitochondrial dysfunction cross-talking facilitate cell death mediated by chelerythrine in glioma.

Glioma is the most common primary malignant brain tumor with poor survival and limited therapeutic options. Chelerythrine (CHE), a natural benzophenanthridine alkaloid, has been reported to exhibit the anti-tumor effects in a variety of cancer cells. However, the molecular target and the signaling process of CHE in glioma remain elusive. Here we investigated the underlying mechanisms of CHE in glioma cell lines and glioma xenograft mice model. Our results found that CHE-induced cell death is associated with RIP1/RIP3-dependent necroptosis rather than apoptotic cell death in glioma cells at the early time. Mechanism investigation revealed the cross-talking between necroptosis and mitochondria dysfunction that CHE triggered generation of mitochondrial ROS, mitochondrial depolarization, reduction of ATP level and mitochondrial fragmentation, which was the important trigger for RIP1-dependent necroptosis activation. Meanwhile, PINK1 and parkin-dependent mitophagy promoted clearance of impaired mitochondria in CHE-incubated glioma cells, and inhibition of mitophagy with CQ selectively enhanced CHE-induced necroptosis. Furthermore, early cytosolic calcium from the influx of extracellular Ca2+ induced by CHE acted as important "priming signals" for impairment of mitochondrial dysfunction and necroptosis. Suppression of mitochondrial ROS contributed to interrupting positive feedback between mitochondrial damage and RIPK1/RIPK3 necrosome. Lastly, subcutaneous tumor growth in U87 xenograft was suppressed by CHE without significant body weight loss and multi-organ toxicities. In summary, the present study helped to elucidate necroptosis was induced by CHE via mtROS-mediated formation of the RIP1-RIP3-Drp1 complex that promoted Drp1 mitochondrial translocation to enhance necroptosis. Our findings indicated that CHE could potentially be further developed as a novel therapeutic strategy for treatment of glioma.