Acetal-thiol Click-like Reaction: Facile and Efficient Synthesis of Dynamic Dithioacetals and Recyclable Polydithioacetals.

Dithioacetals are heavily used in organic, material and medical chemistries, and exhibit huge potential to synthesize degradable or recyclable polymers. However, the current synthetic approaches of dithioacetals and polydithioacetals are overwhelmingly dependent on external catalysts and organic solvents. Herein, we disclose a catalyst- and solvent-free acetal-thiol click-like reaction for synthesizing dithioacetals and polydithioacetals. High conversion, higher than acid catalytic acetal-thiol reaction, can be achieved. High universality was confirmed by monitoring the reactions of linear and cyclic acetals (including renewable bio-sourced furan-acetal) with aliphatic and aromatic thiols, and the reaction mechanism of monomolecular nucleophilic substitution (SN1) and auto-protonation (activation) by thiol was clarified by combining experiments and density functional theory computation. Subsequently, we utilize this reaction to synthesize readily recyclable polydithioacetals. By simple heating and stirring, linear polydithioacetals with w of ~110 kDa were synthesized from acetal and dithiol, and depolymerization into macrocyclic dithioacetal and repolymerization into polydithioacetal can be achieved; through reactive extrusion, a semi-interpenetrating polymer dynamic network with excellent mechanical properties and continuous reprocessability was prepared from poly(vinyl butyral) and pentaerythritol tetrakis(3-mercaptopropionate). This green and high-efficient synthesis method for dithioacetals and polydithioacetals is beneficial to the sustainable development of chemistry.

Exploration and structure-activity relationship research of benzenesulfonamide derivatives as potent TRPV4 inhibitors for treating acute lung injury.

RN-9893, a TRPV4 antagonist identified by Renovis Inc., showcased notable inhibition of TRPV4 channels. This research involved synthesizing and evaluating three series of RN-9893 analogues for their TRPV4 inhibitory efficacy. Notably, compounds 1b and 1f displayed a 2.9 to 4.5-fold increase in inhibitory potency against TRPV4 (IC50 = 0.71 ± 0.21 µM and 0.46 ± 0.08 µM, respectively) in vitro, in comparison to RN-9893 (IC50 = 2.07 ± 0.90 µM). Both compounds also significantly outperformed RN-9893 in TRPV4 current inhibition rates (87.6 % and 83.2 % at 10 µM, against RN-9893's 49.4 %). For the first time, these RN-9893 analogues were profiled in an in vivo mouse model, where intraperitoneal injections of 1b or 1f at 10 mg/kg notably mitigated symptoms of acute lung injury induced by lipopolysaccharide (LPS). These outcomes indicate that compounds 1b and 1f are promising candidates for acute lung injury treatment.

Pretreatment and analysis techniques development of TKIs in biological samples for pharmacokinetic studies and therapeutic drug monitoring.

Tyrosine kinase inhibitors (TKIs) have emerged as the first-line small molecule drugs in many cancer therapies, exerting their effects by impeding aberrant cell growth and proliferation through the modulation of tyrosine kinase-mediated signaling pathways. However, there exists a substantial inter-individual variability in the concentrations of certain TKIs and their metabolites, which may render patients with compromised immune function susceptible to diverse infections despite receiving theoretically efficacious anticancer treatments, alongside other potential side effects or adverse reactions. Therefore, an urgent need exists for an up-to-date review concerning the biological matrices relevant to bioanalysis and the sampling methods, clinical pharmacokinetics, and therapeutic drug monitoring of different TKIs. This paper provides a comprehensive overview of the advancements in pretreatment methods, such as protein precipitation (PPT), liquid-liquid extraction (LLE), solid-phase extraction (SPE), micro-SPE (µ-SPE), magnetic SPE (MSPE), and vortex-assisted dispersive SPE (VA-DSPE) achieved since 2017. It also highlights the latest analysis techniques such as newly developed high performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS) methods, capillary electrophoresis (CE), gas chromatography (GC), supercritical fluid chromatography (SFC) procedures, surface plasmon resonance (SPR) assays as well as novel nanoprobes-based biosensing techniques. In addition, a comparison is made between the advantages and disadvantages of different approaches while presenting critical challenges and prospects in pharmacokinetic studies and therapeutic drug monitoring.

[Prenatal ultrasonographic manifestations and genetic diagnosis of nine fetuses with 7q11.23 duplication syndrome].

OBJECTIVE: To analyze ultrasonographic manifestations and genetic etiology of nine fetuses with 7q11.23 duplication syndrome. METHODS: Ultrasonographic finding, pregnancy outcome and follow-up of nine fetuses detected at the Prenatal Diagnosis Center of the Third Affiliated Hospital of Zhengzhou University from January 2017 to December 2021 were retrospectively analyzed. RESULTS: The fetuses were found to harbor a duplication in the 7q11.23 region by chromosomal microarray analysis (CMA). Among these, five had shown ventriculomegaly, including four syndromic and one non-syndromic. For the remainders, one had ventricular septal defect and mild tricuspid regurgitation, one had echogenic intracardiac focus, whilst another two were normal. Five couples had accepted parental verification, and the results confirmed that the 7q11.23 duplication carried by their fetuses were de novo in origin. Following genetic counseling, seven couples had opted to terminate their pregnancies. Two fetuses were delivered at full term, and follow-up had found no abnormalities. CONCLUSION: Prenatal ultrasonographic manifestations of fetuses with 7q11.23 duplication syndrome are variable. CMA can provide assistance for their diagnosis and genetic counseling.

Novel Scaffold Agonists of the α2A Adrenergic Receptor Identified via Ensemble-Based Strategy.

The α2A adrenergic receptor (α2A-AR) serves as a critical molecular target for sedatives and analgesics. However, α2A-AR ligands with an imidazole ring also interact with an imidazoline receptor as well as other proteins and lead to undesirable effects, motivating us to develop more novel scaffold α2A-AR ligands. For this purpose, we employed an ensemble-based ligand discovery strategy, integrating long-term molecular dynamics (MD) simulations and virtual screening, to identify new potential α2A-AR agonists with novel scaffold. Our results showed that compounds SY-15 and SY-17 exhibited significant biological effects in the preliminary evaluation of protein kinase A (PKA) redistribution assays. They also reduced levels of intracellular cyclic adenosine monophosphate (cAMP) in a dose-dependent manner. Upon treatment of the cells with 100 µM concentrations of SY-15 and SY-17, there was a respective decrease in the intracellular cAMP levels by 63.43% and 53.83%. Subsequent computational analysis was conducted to elucidate the binding interactions of SY-15 and SY-17 with the α2A-AR. The binding free energies of SY-15 and SY-17 calculated by MD simulations were -45.93 and -71.97 kcal/mol. MD simulations also revealed that both compounds act as bitopic agonists, occupying the orthosteric site and a novel exosite of the receptor simultaneously. Our findings of integrative computational and experimental approaches could offer the potential to enhance ligand affinity and selectivity through dual-site occupancy and provide a novel direction for the rational design of sedatives and analgesics.

[Genetic diagnosis and analysis of eight cases with central 22q11.2 deletion syndrome].

OBJECTIVE: To explore the pregnancy outcome and postpartum clinical phenotype of LCR22B/C~D central 22q11.2 deletion syndrome. METHODS: For fetuses diagnosed with central 22q11.2 deletion by chromosomal microarray analysis (CMA) at the Prenatal Diagnosis Center of the Third Affiliated Hospital of Zhengzhou University from January 2019 to April 2022, their prenatal imaging, parental CMA verification, pregnancy outcomes and postpartum clinical phenotype were analyzed. RESULTS: Eight cases of central 22q11.2 deletion syndrome were included, including six cases with LCR22B~D 22q11.2 deletions and two with LCR22C~D 22q11.2 deletions. Among the six cases with LCR22B~D type 22q11.2 deletions, three had shown cardiovascular malformations (right aortic arch, ventricular septal defect, mild tricuspid regurgitation), one had shown urinary defect (right kidney heterotopia). Two cases with LCR22C~D 22q11.2 deletions showed nonspecific ultrasonographic findings, including oligohydramnios with growth restriction and nuchal skin thickening. The CMA verification showed that six cases were inherited from their parents, and five couples had chosen to continue with the pregnancy. Postpartum follow-up showed that the physical and intellectual development of all children were normal. One couple had opted to terminate the pregnancy considering the ectopic fetal right kidney. Two remaining cases had decided to terminate their pregnancies without parental verification. CONCLUSION: The central 22q11.2 deletion syndrome of the LCR22B/C~D type is different from the classical types. Its genetic information mainly comes from parents. Prenatal imaging has mainly shown cardiovascular and urinary abnormalities. Postnatal growth and intellectual development have been normal. Therefore, the couples should be provided with suffice prenatal genetic counseling.

Atlas of Cell Repertoire Within Neointimal Lesions Is Metabolically Altered in Hypertensive Rats.

BACKGROUND: High blood pressure has been suggested to accelerate vascular injury-induced neointimal formation and progression. However, little is known about the intricate relationships between vascular injury and hypertension in the context of arterial remodeling. METHODS: Single-cell RNA-sequencing analysis was used to depict the cell atlas of carotid arteries of Wistar Kyoto rats and spontaneously hypertensive rats with or without balloon injury. RESULTS: We found that hypertension significantly aggravated balloon injury-induced arterial stenosis. A total of 36 202 cells from carotid arteries with or without balloon injury were included in single-cell RNA-sequencing analysis. Cell composition analysis showed that vascular injury and hypertension independently induced distinct aortic cell phenotypic alterations including immune cells, endothelial cells (ECs), and smooth muscle cells. Specifically, our data showed that injury and hypertension-induced specific EC phenotypic alterations, and revealed a transition from functional ECs to hypermetabolic, and eventually dysfunctional ECs in hypertensive rats upon balloon injury. Importantly, our data also showed that vascular injury and hypertension-induced different smooth muscle cell phenotypic alterations, characterized by deferential expression of synthetic signatures. Interestingly, pathway analysis showed that dysregulated metabolic pathways were a common feature in monocytes/macrophages, ECs, and smooth muscle cells in response to injury and hypertension. Functionally, we demonstrate that inhibition of mitochondrial respiration significantly ameliorated injury-induced neointimal formation in spontaneously hypertensive rats. CONCLUSIONS: This study provides the cell landscape changes of the main aortic cell phenotypic alterations in response to injury and hypertension. Our findings suggest that targeting cellular mitochondrial respiration could be a novel therapeutic for patients with hypertension undergoing vascular angioplasty.

A novel missense variant in OTUD5 causes X-linked multiple congenital anomalies-neurodevelopmental syndrome.

BACKGROUND: The OTUD5 gene encodes a deubiquitinating enzyme (DUB) of the OTU family. Variants of OTUD5 are associated with X-linked multiple congenital anomalies-neurodevelopmental syndrome (MCAND). The case described in this study expands the clinical and molecular spectrum of OTUD5. METHODS: Trio-based clinical exome sequencing (trio-CES) was performed on a Chinese boy with a clinical phenotype and both of his parents. Sanger sequencing was employed for validation of the variant detected. RESULTS: The patient presented with characteristic facial features, intellectual disability, motor/language/cognitive, and global developmental delays, limb contractures, and kidney abnormalities, and trio-CES identified a de novo missense variant, c.1305T>A, of the OTUD5 gene. DISCUSSION: We describe OTUD5 gene variation in the Chinese population, with the first report of this variant. Additionally, we provide a comprehensive summary of all published cases of MCAND to date, in order to elucidate the primary clinical features of the syndrome and the variability in phenotype severity. This case expands the genetic and clinical phenotypic spectrum of OTUD5-associated MCAND.

The environmental sources of benzophenones: Distribution, pretreatment, analysis and removal techniques.

Benzophenones (BPs) have wide practical applications in real human life due to its presence in personal care products, UV-filters, drugs, food packaging bags, etc. It enters the wastewater by daily routine activities such as showering, impacting the whole aquatic system, then posing a threat to human health. Due to this fact, the monitoring and removal of BPs in the environment is quite important. In the past decade, various novel analytical and removal techniques have been developed for the determination of BPs in environmental samples including wastewater, municipal landfill leachate, sewage sludge, and aquatic plants. This review provides a critical summary and comparison of the available cutting-edge pretreatment, determination and removal techniques of BPs in environment. It also focuses on novel materials and techniques in keeping with the concept of "green chemistry", and describes on challenges associated with the analysis of BPs, removal technologies, suggesting future development strategies.

Discovery of highly potent and selective KRASG12C degraders by VHL-recruiting PROTACs for the treatment of tumors with KRASG12C-Mutation.

Although several covalent KRASG12C inhibitors have made great progress in the treatment of KRASG12C-mutant cancer, their clinical applications are limited by adaptive resistance, motivating novel therapeutic strategies. Through drug design and structure optimization, a series of highly potent and selective KRASG12C Proteolysis Targeting Chimeras (PROTACs) were developed by incorporating AMG510 and VHL ligand VH032. Among them, degrader YN14 significantly inhibited KRASG12C-dependent cancer cells growth with nanomolar IC50 and DC50 values, and ＞ 95 % maximum degradation (Dmax). Molecular dynamics (MD) simulation showed that YN14 induced a stable KRASG12C: YN14: VHL ternary complex with low binding free energy (ΔG). Notably, YN14 led to tumor regression with tumor growth inhibition (TGI%) rates more than 100 % in the MIA PaCa-2 xenograft model with well-tolerated dose-schedules. We also found that KRASG12C degradation exhibited advantages in overcoming adaptive KRASG12C feedback resistance over KRASG12C inhibition. Furthermore, combination of RTKs, SHP2, or CDK9 inhibitors with YN14 exhibited synergetic efficacy in KRASG12C-mutant cancer cells. Overall, these results demonstrated that YN14 holds exciting prospects for the treatment of tumors with KRASG12C-mutation and boosted efficacy could be achieved for greater clinical applications via drug combination.

L-type calcium ion channel-mediated activation of autophagy in vascular smooth muscle cells via thonningianin A (TA) alleviates vascular calcification in type 2 diabetes mellitus.

Vascular calcification (VC) is associated with increased morbidity and mortality, especially among people with type 2 diabetes mellitus (T2DM). The pathogenesis of vascular calcification is incompletely understood, and until now, there have been no effective therapeutics for vascular calcification. The L-type calcium ion channel in the cell membrane is vital for Ca2+ influx. The effect of L-type calcium ion channels on autophagy remains to be elucidated. Here, the natural compound thonningianin A (TA) was found to ameliorate vascular calcification in T2DM via the activation of L-type calcium ion channels. The results showed that TA had a concentration-dependent ability to decrease the transcriptional and translational expression of the calcification-related proteins runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteopontin (OPN) (P < 0.01) via ATG7-dependent autophagy in ß-glycerophosphate (ß-GP)- and high glucose (HG)-stimulated primary mouse aortic smooth muscle cells (MASMCs) and alleviate aortic vascular calcification in VitD3-stimulated T2DM mice. However, nifedipine, an inhibitor of L-type calcium ion channels, reversed TA-induced autophagy and Ca2+ influx in MASMCs. Molecular docking analysis revealed that TA was located in the hydrophobic pocket of Cav1.2 α1C and was mainly composed of the residues Ile, Phe, Ala and Met, which confirmed the efficacy of TA in targeting the L-type calcium channel of Cav1.2 on the cell membrane. Moreover, in an in vivo model of vascular calcification in T2DM mice, nifedipine reversed the protective effects of TA on aortic calcification and the expression of the calcification-related proteins RUNX2, BMP2 and OPN (P < 0.01). Collectively, the present results reveal that the activation of cell membrane L-type calcium ion channels can induce autophagy and ameliorate vascular calcification in T2DM. Thonningianin A (TA) can target and act as a potent activator of L-type calcium ion channels. Thus, this research revealed a novel mechanism for autophagy induction via L-type calcium ion channels and provided a potential therapeutic for vascular calcification in T2DM.

The next generation of EGFR inhibitors: a patenting perspective of PROTACs based EGFR degraders.

INTRODUCTION: Abnormal expression of epidermal growth factor receptor (EGFR) contributes to tumor development, especially in non-small cell lung cancer (NSCLC). Although multiple inhibitors have been developed to target diverse EGFR mutations and several have been approved, the inevitable drug resistance and side effect remain a challenge, which motivates novel strategies. Proteolysis-targeting chimeras (PROTACs) have been gaining momentum for their potential as novel therapeutics for human diseases by triggering protein degradation. To date, various potent and specific EGFR PROTACs have been discovered and some of them have entered clinical trials. AREAS COVERED: This review provides an overview of EGFR degraders in patents from 2016 to 2022. It provides an update of the discovery strategies, chemical structures, and molecular profiling of all available EGFR PROTACs. SciFinder, PubMed, Web of Science, EPO, and CNIPA databases were used for searching the literature and patents for EGFR PROTACs. EXPERT OPINION: By employing the PROTAC technology, highly potent and selective EGFR degraders based on four generation EGFR inhibitors have been developed, which offer a new strategy to target EGFR mutations and overcome the drug resistance. Despite the satisfactory result in vitro and in vivo studies, their therapeutic value awaits more rigorous preclinical testing and clinical investigation.

Oxidative stress signaling in the pathogenesis of diabetic cardiomyopathy and the potential therapeutic role of antioxidant naringenin.

Diabetes mellitus (DM) is one of the most prevalent metabolic disorders that poses a global threat to human health. It can lead to complications in multiple organs and tissues, owing to its wide-ranging impact on the human body. Diabetic cardiomyopathy (DCM) is a specific cardiac manifestation of DM, which is characterized by heart failure in the absence of coronary heart disease, hypertension and valvular heart disease. Given that oxidative stress is a key factor in the pathogenesis of DCM, intervening to mitigate oxidative stress may serve as a therapeutic strategy for managing DCM. Naringenin is a natural product with anti-oxidative stress properties that can suppress oxidative damage by regulating various oxidative stress signaling pathways. In this review, we address the relationship between oxidative stress and its primary signaling pathways implicated in DCM, and explores the therapeutic potential of naringenin in DCM.

ATP promotes resident CD34+ cell migration mainly through P2Y2-Stim1-ERK/p38 pathway.

Extracellular adenosine triphosphate (ATP) is one of the most abundant biochemical constitutes within the stem cell microenvironment and is postulated to play critical roles in cell migration. However, it is unclear whether ATP regulates the cell migration of CD34+ vascular wall-resident stem/progenitor cells (VW-SCs) and participates in angiogenesis. Therefore, the biological mechanisms of cell migration mediated by ATP was determined by in vivo subcutaneous matrigel plug assay, ex vivo aortic ring assay, in vitro transwell migration assay, and other molecular methods. In the present study, ATP dose-dependently promoted CD34+ VW-SCs migration, which was more obviously attenuated by inhibiting or knocking down P2Y2 than P2Y6. Furthermore, it was confirmed that ATP potently promoted the migration of resident CD34+ cells from cultured aortic artery rings and differentiation into endothelial cells in matrigel plugs by using inducible lineage tracing Cd34-CreERT2; R26-tdTomato mice, whereas P2Y2 and P2Y6 blocker greatly inhibited the effect of ATP. In addition, ATP enhanced the protein expression of stromal interaction molecule 1 (STIM1) on cell membrane, blocking the calcium release-activated calcium (CRAC) channel with shSTIM1 or BTP2 apparently inhibited ATP-evoked intracellular Ca2+ elevation and channel opening, thereby suppressing ATP-driven cell migration. Moreover, extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 and p38 inhibitor SB203580 remarkably inhibited ERK and p38 phosphorylation, cytoskeleton rearrangement, and subsequent cell migration. Unexpectedly, it was found that knocking down STIM1 greatly inhibited ATP-triggered ERK/p38 activation. Taken together, it was suggested that P2Y2 signaled through the CRAC channel mediated Ca2+ influx and ERK/p38 pathway to reorganize the cytoskeleton and promoted the migration of CD34+ VW-SCs.NEW & NOTEWORTHY In this study, we observed that the purinergic receptor P2Y2 is critical in the regulation of vascular wall-resident CD34+ cells' migration. ATP could activate STIM1-mediated extracellular Ca2+ entry by triggering STIM1 translocation to the plasma membrane, and knockdown of STIM1 prevented ERK/p38 activation-mediated cytoskeleton rearrangement and cell migration.

Bioinspired yeast-based ß-glucan system for oral drug delivery.

Oral drug delivery is the preferred route of drug administration for patients, especially those who need long-term medication. Recently, bioinspired drug delivery systems have emerged for the oral delivery of various therapeutics. Among them, the yeast-based ß-glucan system is a novel and promising platform, for oral administration that can overcome the biological barriers of the harsh gastrointestinal environment. Remarkably, the yeast-based ß-glucan system not only protects the drug through the harsh gastrointestinal environment but also achieves targeted therapeutic effects by specifically recognizing immune cells, especially macrophages. Otherwise, it exhibits immunomodulatory properties. Based on the pleasant characteristics of the yeast-based ß-glucan system, they are widely used in various macrophage-related diseases for oral administration. In this review, we introduced the structure and function of yeast-based ß-glucan. Subsequently, we further summarized the current preparation methods of yeast-based ß-glucan carriers and the strategies for preparing yeast-based ß-glucan drug delivery systems. In addition, we focus on discussing the applications of ß-glucan drug delivery systems in various diseases. Finally, the current challenges and future perspectives of the ß-glucan drug delivery system are introduced.

Evaluation of the clinical utility of extended non-invasive prenatal testing in the detection of chromosomal aneuploidy and microdeletion/microduplication.

BACKGROUND: With the development of whole-genome sequencing technology, non-invasive prenatal testing (NIPT) has been applied gradually to screen chromosomal microdeletions and microduplications that cannot be detected by traditional karyotyping. However, in NIPT, some false positives and false negatives occur. This study aimed to investigate the applicability of extended NIPT (NIPT-PLUS) in the detection of chromosomal aneuploidy and microdeletion/microduplication syndrome (MMS). METHODS: A total of 452 pregnancies that underwent prenatal diagnostic testing (amniocentesis or chorionic villus sampling) by chromosomal microarray analysis (CMA), were screened by NIPT-PLUS from the peripheral blood sample of the pregnant women. The results of the two tested items were compared and analysed. RESULTS: Of the 452 cases, 335 (74.12%) had positive CMA results, and 117 (25.88%) had no abnormal results. A total of 86 cases of trisomy 21, 18 and 13 and sex chromosome aneuploidy (SCA) were detected by CMA and NIPT-PLUS, with a detection rate of 96.51% (83/86). Among them, the detection rates of T18, T13; 47, XXY; 47, XXX and 47 XYY were 100%, and the detection rates of T21 and 45 XO were 96.55% and 90%, respectively. The detection sensitivity of rare chromosomal trisomy (RAT) was 80% (4/5). The positive predictive values of NIPT-PLUS for chromosome aneuploidy T21, T18 and T13 and for SCA and RAT were 90.32%, 87.50%, 25.00%, 88.89% and 50%, respectively. A total of 249 cases (74.32%) of chromosomal MMS were detected by CMA. The detection rate of NIPT-PLUS was 63.86% (159/249), and 90 cases (36.14%) were missed. The larger the MMS fragment, the higher the NIPT-PLUS detection sensitivity. In addition, most small fragments were of maternal origin. CONCLUSION: The comparison between the CMA and NIPT-PLUS techniques shows that NIPT-PLUS has high sensitivity for detecting chromosomal aneuploidy and chromosomal copy number variations (CNVs) with fragments > 5 M. However, the sensitivity of CNV for fragments < 5 M is low, and the missed detection rate is high. Additionally, confined placental mosaicism and foetal mosaicism are the key factors causing false negatives in NIPT-PLUS, while maternal chromosomal abnormalities and confined placental mosaicism are key contributors to false positives, so appropriate genetic counselling is especially important for pregnant women before and after NIPT-PLUS testing.

Discovery of novel exceptionally potent and orally active c-MET PROTACs for the treatment of tumors with MET alterations.

Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC50 values and achieved picomolar DC50 values and >99% of maximum degradation (Dmax) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-METY1230H and c-METD1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.

ATP purinergic receptor signalling promotes Sca-1+ cell proliferation and migration for vascular remodelling.

AIMS: Vascular resident stem cells expressing stem cell antigen-1 (Sca-1+ cells) promote vascular regeneration and remodelling following injury through migration, proliferation and differentiation. The aim of this study was to examine the contributions of ATP signalling through purinergic receptor type 2 (P2R) isoforms in promoting Sca-1+ cell migration and proliferation after vascular injury and to elucidate the main downstream signalling pathways. METHODS AND RESULTS: ATP-evoked changes in isolated Sca-1+ cell migration were examined by transwell assays, proliferation by viable cell counting assays and intracellular Ca2+ signalling by fluorometry, while receptor subtype contributions and downstream signals were examined by pharmacological or genetic inhibition, immunofluorescence, Western blotting and quantitative RT-PCR. These mechanisms were further examined in mice harbouring TdTomato-labelled Sca-1+ cells with and without Sca-1+-targeted P2R knockout following femoral artery guidewire injury. Stimulation with ATP promoted cultured Sca-1+ cell migration, induced intracellular free calcium elevations primarily via P2Y2R stimulation and accelerated proliferation mainly via P2Y6R stimulation. Enhanced migration was inhibited by the ERK blocker PD98059 or P2Y2R-shRNA, while enhanced proliferation was inhibited by the P38 inhibitor SB203580. Femoral artery guidewire injury of the neointima increased the number of TdTomato-labelled Sca-1+ cells, neointimal area and the ratio of neointimal area to media area at 3 weeks post-injury, and all of these responses were reduced by P2Y2R knockdown. CONCLUSIONS: ATP induces Sca-1+ cell migration through the P2Y2R-Ca2+-ERK signalling pathway, and enhances proliferation through the P2Y6R-P38-MAPK signalling pathway. Both pathways are essential for vascular remodelling following injury. Video Abstract.

[Isolation, culture and validation of CD34+ vascular wall-resident stem cells from mice].

Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34+ VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34+ VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34+ VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca2+ release and extracellular Ca2+ entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34+ VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34+ VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca2+ release from endoplasmic reticulum of CD34+ VW-SCs. Store-operated Ca2+ entry (SOCE) was activated by using thapsigargin (TG) applied in Ca2+-free/Ca2+ reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34+ VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.