AIM: To investigate the signaling mechanisms involved in the priming effects of lipopolysaccharide(LPS) on heat killed Staphylococcus aureus(HKSa)-induced nitric oxide(NO) production in macrophages.METHODS: Murine macrophage RAW264.7 was used in the experiment.Griess reagent was used to measure the content of nitrite in culture medium.Real-time PCR and Western blot was utilized to examine the mRNA and protein levels of toll-like receptor 2(TLR2),respectively.Dual luciferase reporter assay was used to assess the transcriptional activity of nuclear factor of activated T cells(NF-AT).RESULTS: The RAW264.7 cells pretreated with LPS for 24 h significantly enhanced NO production induced by HKSa,suggesting that LPS primed the macrophages and increased the reactivity of the cells to HKSa.LPS increased the mRNA and protein levels of TLR2 in a dose-dependent manner in RAW264.7 cells.The RAW264.7 cells pretreated with LPS enhanced NO production induced by peptidoglycan,one of the specific ligand of TLR2.The priming effect of LPS on HKSa-induced NO production was partly blocked by TLR2 neutralizing antibody.LPS significantly enhanced the transcriptional activity of NF-AT,which was inhibited by BAPTA/AM(a cell-permeable cytosolic calcium chelator) and cyclosporine A(CsA,an inhibitor for calcineurin).Both BAPTA/AM and CsA inhibited the priming effect of LPS on HKSa-induced NO production in RAW264.7 cells.CONCLUSION: The present study confirms the priming effect of LPS on the reactivity of RAW264.7 cells to HKSa.Pattern recognition receptor TLR2 and calcium/calcineurin/NF-AT signaling pathway may be involved in the priming process initiated by LPS.

Objective To evaluate the extremity function of patients with intracerebral hemorrhage (ICH)using short-latency somatosensory evoked potentials (SEPs) and a modified intracerebral hemorrhage (MICH) scale.Methods On admission, SEP was applied in the examination of 61 patients with ICH. P40 latency and the amplitude of posterior tibial nerve potentials in both the healthy and affected extremities were measured. Abnormalities were classified based on the margin of lower extremity SEP latency and the main waveform changes. MICH was measured simultaneously to prepare a prognosis. The modified Rankin scale (MRS) score was assessed 3 months after the attack as well.Results Compared with the healthy side, there were significant differences in posterior tibial nerve P40 latency and amplitude on the affected side among patients with ICH. P40 latency and MRS scores on the affected side 3 months after the attack were positively correlated. On the unaffected side, P40 amplitude and the MRS score 3 months after the attack were negatively correlated. The MICH score on admission and the MRS score 3 months after the attack were positively correlated. Based on MRS scores (MRS≥4 indicating a poor prognosis), the predictive sensitivities for a poor prognosis of SEP and the MICH scale in patients with ICH were 80.77% and 84.61% respectively, while the specificities were 62.68% and 88.57% respectively, and the accordance rates were 70.5% and 86.9% respectively. Conclusions SEP and the MICH scale are closely correlated with the prognosis for extremity function in patients with ICH.The combination of SEP with the MICH scale might be helpful in predicting the prognosis of the patients with ICH.

Objective To analyze the clinical characteristics and risk factors of patients with confirmed venous thromboembolism (VTE) in order to improve recognition of VTE, and reduce the rate of missed diagnosis and wrong diagnosis. Methods A retrospectively review was performed for 205 patients diagnosed with VTE confirmed by CT pulmonary angiography (CTPA), radionuclide pulmonary ventilation/perfusion (V/Q) imaging, lower extremity deep vein ultrasound or venography in the First Affiliated Hospital of Bengbu Medical College from January 2009 to December 2018. The clinical manifestations, laboratory examination results, imaging results, treatment and prognosis of patients diagnosed with VTE were analyzed. The clinical possibility was assessed by pulmonary thromboembolism (PTE) simplified Wells score and deep venous thrombosis (DVT) Wells score. 130 non-VTE patients admitted in the same period were enrolled as controls, and the risk factors of VTE were screened by multivariate Logistic regression analysis. Results Among 205 VTE patients, 14 cases had incomplete data, 2 cases were complicated with other diseases deteriorated, 2 cases were excluded because of economic reasons, 10 cases abandoned treatment because of serious illness, and finally 177 cases were included in the analysis. The main clinical symptoms of VTE patients were chest tightness (36.16%), followed by chest pain (29.94%), dyspnea (29.38%) and hemoptysis (24.29%). Swelling or tenderness of unilateral/bilateral lower extremities (38.98%) and lung moist rale (20.90%) were the most common signs. ST-T changes were the main changes in electrocardiogram (ECG, 49.15%), followed by SⅠQⅢTⅢ or QⅢTⅢ changes (35.03%). Only 5.65% of the patients had plasma D-dimer less than 0.5 mg/L. 31.07% (55/177) patients had normal arterial blood gas results. Of the 177 VTE patients, 175 were diagnosed as PTE by CTPA, with bilateral/multi-lobar pulmonary artery embolism and its branches being the main type [44.57% (78/175)]. Two cases were diagnosed as PTE by V/Q imaging. Among them, 112 cases were received lower extremity deep venous ultrasound or lower extremity deep venography, 51 cases were diagnosed as lower extremity DVT, with thrombosis of popliteal and above vein as common [68.63% (35/51)]. The clinical possibility assessment showed that 67.23% (119/177) patients might have PTE (PTE simplified Wells score greater than or equal to 2), 38.98% (69/177) patients might have lower extremity DVT (DVT Wells score greater than or equal to 2). Multivariate Logistic regression analysis showed that operation less than 4 weeks [odds ratio (OR) = 5.503, 95% confidence interval (95%CI) = 1.577-19.206, P = 0.007], trauma or fracture less than 3 months (OR = 6.771, 95%CI = 1.510-30.370, P = 0.012), VTE history (OR = 0.072, 95%CI =0.009-0.549, P = 0.011) were independent risk factors for VTE occurrence. Thrombolytic therapy was administered in 13 cases while anticoagulant therapy alone was prescribed in 164 cases. 176 patients recovered, while 1 case died. Conclusions VTE clinical manifestations are not specific. Patients with risk factors should be vigilant, be strengthen with diagnostic awareness, paid attention to the evaluation of clinical possibilities. Timely thrombolytic or anticoagulant treatment after diagnosis, can improve the survival rate.

Objective:To investigate the relationship between inherited dysplasminogenemia and cerebral infarction (CI) by phenotype and gene mutation analysis of 2 inherited dysplasminogenemia pedigrees.Methods:Retrospective analysis was carried out on clinical data of 2 patients diagnosed with CI who were treated in the Department of Neurology, the First Affiliated Hospital of Wenzhou Medical University in January and March 2021, and peripheral venous blood samples were collected from proband 1 and his family members (8 subjects, 4 generations in total) and proband 2 and her family members (5 subjects of 3 generations in total), and their plasminogen (PLG) activity (PLG:A), protein C activity, protein S activity, antithrombin activity and the content of PLG antigen (PLG: Ag), fibrinogen, D-dimer and fibrinogen degradation products were measured for definite diagnosis. All 19 exons,5′ and 3′ untranslated regions of PLG were amplified with polymerase chain reaction, and the amplification products were analyzed by direct DNA sequencing. The results were compared with human PLG reference sequences published in the National Center for Biotechnology Information database using Chromas software to find the mutation sites, and confirmed by reverse sequencing.Results:Both of the 2 patients with confirmed CI had a young onset, and PLG: A was reduced to 21% in the proband 1 and to about 50% in 4 family members; PLG: A was reduced to about 50% in the proband 2 and 2 family members; PLG:Ag and the above tests were essentially normal in both probands and family members. Gene analysis showed that the proband 1 had the homozygous mutation of c.1858G>A in exon 15, the 4 family members of the proband 1, proband 2 and her 2 family members had the heterozygous mutation of c.1858G>A in exon 15, which resulted in a mutation of alanine at position 620 in PLG to threonine (p.Ala620Thr).Conclusions:The decrease of PLG:A was caused by the p.Ala620Thr missense mutation of PLG gene. Proband having CI may be related to the inhibition of fibrinolytic function in the organism due to the p.Ala620Thr missense mutation.

Objective To observe changes of spatial learning-memory in rats with chronic hypoxic hypercapnia and the effect of gingkgo biloba extra. Methods After established the rat model of chronic hypoxic hypercapnia,seventy-two rats were randomly divided into four groups normal control (NC),hypoxic-hypercapnia 4-week (4HH),hypoxic-hy-percapnia 4-week+gingkgo biloba extra (EGb)high dose(100 mg/kg)group[4HH+EGb(H)] and hypoxic-hypercapnia 4-week+EGb low dose (50 mg/kg) group[4HH+EGb(L)]. Praxiology in rats was asessed by the Morris water maze and step down test. Results The spatial learning-memory in rats exposed to chronic hypoxic-hypercapnia 4-week(4HH group)were displayed significant impairment in their performance,the longer mean escape latencies and swim path dis tances,the more error times. 4HH+EGb(H) and 4HH+EGb(L)groups shortened the reaction time of leaning, pro longed the latent time of memory, reduced times of mistakes. Conclusions EGb can enhance the capacity of learning-memory in the rats exposed chronic hypoxic hypercapnia.

The experimental class is a very important part of medical research graduate students in teaching practice. The lack of attention to experimental teaching, the unclear teaching purpose, and the disconnection between teaching content and practical application are the most important problems in current teaching practice. In this study, we propose to emphasize the experimental teaching objectives and experimental records to enhance students' initiative and standardize rigor; increase the training of advanced experimental equipment to improve students' hands-on ability; enrich teaching methods and means to improve teaching conditions; cultivate students' scientific thinking to enhance Student's experimental operating skills. The measures help comprehensively cultivate postgraduates' innovation and practical ability, and have great significance for medical research work.

Aminoglycosides are broad-spectrum antibacterials to treat bacterial infections, especially gram-negative bacteria infections. However, aminoglycosides are losing efficacy because of the increase in antibiotic resistance and their inherent toxicity, attracting more interests in developing new aminoglycosides. Several clinically used aminoglycosides are mainly exerted by inhibition of protein synthesis through binding to bacterial rRNA. The bacterial ribosome RNA is the most currently exploited RNA drug target. Identification of new compounds that target RNAs is indispensable to fight with the growing threat that bacteria pose to human safety. In this work, we used carbohydrate microarrays to probe interactions of low molecular weight ligands with RNAs and proteins. Carbohydrate microarrays, comprising hundreds to thousands of different glycan structures on surfaces in a spatially discrete pattern, are sensitive and versatile tools to study the interactions between biological macromolecules. Herein, aminoglycosides have been immobilized onto the modified glass microscope slides and their interactions with RNAs and proteins are then measured through the labeled fluorescence. The results displayed that microarray can be used to detect the binding of aminoglycosides with three types of target molecules, including the small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), the large group I ribozyme RNA (approximately 400 nucleotide) and certain proteins (toxicity-causing enzymes, such as DNA polymerase and phospholipase C). For rRNA A-site mimics, the fluorescence intensities of 16S rRNA is stronger than that of 18S rRNA, illustrating that as a screen technique, the microarray method can not only determine the binding affinity to RNA but also detect the specific binding to bacterial rRNA mimic. The ability to screen group I ribozyme RNA can be helpful to the discovery of new RNA therapeutic targets. Binding of immobilized aminoglycosides to toxicity-causing proteins (DNA polymerase and phospholipase C) is a new method to study of aminoglycoside toxicity. These studies lay the foundation for rapid identification of new RNA-binding ligands with strong and specific binding affinity for their desired targets.

Abnormally activated CDK9 participates in the super-enhancer mediated transcription of short-lived proteins required for cancer cell survival. Targeting CDK9 has shown potent anti-tumor activity in clinical trials among different cancers. However, the study and knowledge on drug resistance to CDK9 inhibitors are very limited. In this study, we established an AML cell line with acquired resistance to a highly selective CDK9 inhibitor BAY1251152. Through genomic sequencing, we identified in the kinase domain of CDK9 a mutation L156F, which is also a coding SNP in the CDK9 gene. By knocking in L156F into cancer cells using CRISPR/Cas9, we found that single CDK9 L156F could drive the resistance to CDK9 inhibitors, not only ATP competitive inhibitor but also PROTAC degrader. Mechanistically, CDK9 L156F disrupts the binding with inhibitors due to steric hindrance, further, the mutation affects the thermal stability and catalytic activity of CDK9 protein. To overcome the drug resistance mediated by the CDK9-L156F mutation, we discovered a compound, IHMT-CDK9-36 which showed potent inhibition activity both for CDK9 WT and L156F mutant. Together, we report a novel resistance mechanism for CDK9 inhibitors and provide a novel chemical scaffold for the future development of CDK9 inhibitors.

Angiogenesis is an essential process in tumor growth, invasion and metastasis. VEGF receptor 2 (VEGFR2) inhibitors targeting tumor angiogenic pathway have been widely used in the clinical cancer treatment. However, most of currently used VEGFR2 kinase inhibitors are multi-target inhibitors which might result in target-associated side effects and therefore limited clinical toleration. Highly selective VEGFR inhibitors are still highly demanded from both basic research and clinical application point of view. Here we report the discovery and characterization of a novel VEGFR2 inhibitor (CHMFL-VEGFR2-002), which exhibited high selectivity among structurally closed kinases including PDGFRs, FGFRs, CSF1R, etc. CHMFL-VEGFR2-002 displayed potent inhibitory activity against VEGFR2 kinase in the biochemical assay (IC = 66 nmol/L) and VEGFR2 autophosphorylation in cells (ECs ∼100 nmol/L) as well as potent anti-proliferation effect against VEGFR2 transformed BaF3 cells (GI = 150 nmol/L). In addition, CHMFL-VEGFR2-002 also displayed good anti-angiogenesis efficacy and exhibited good PK (pharmacokinetics) profile with bioavailability over 49% and anti-angiogenesis efficacy in both zebrafish and mouse models without apparent toxicity. These results suggest that CHMFL-VEGFR2-002 might be a useful research tool for dissecting new functions of VEGFR2 kinase as well as a potential anti-angiogenetic agent for the cancer therapy.