Determination of cotinine and 3-hydroxynicotinine in human serum by liquid chromatography-tandem mass spectrometry and its application.

In this study, we developed a method for determining cotinine and 3-hydroxycotinine in human serum and established a methodology for an in-depth study of tobacco exposure and health. After the proteins in the human serum samples were precipitated with acetonitrile, they were separated on a ZORBAX SB-Phenyl column with a mobile phase of methanol encompassing 0.3% formic acid-water encompassing 0.15% formic acid. The measurement was performed on an API5500 triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Cotinine, 3-hydroxycotinine, and cotinine-d3 isotope internal standards were held for 2.56 minutes, 1.58 minutes, and 2.56 minutes, respectively. In serum, the linear range was 0.05 to 500 ng·mL-1 for cotinine and 0.50 to 1250 ng·mL-1 for 3-hydroxycotinine. The lower limit of quantification (LLOQ) was 0.05 ng·mL-1 and 0.5 ng·mL-1 for cotinine and 3-hydroxycotinine, respectively. The intra-day and inter-day relative standard deviations were <11%, and the relative errors were within ±â€…7%. Moreover, the mean extraction recoveries of cotinine and 3-hydroxycotinine were 98.54% and 100.24%, respectively. This method is suitable for the rapid determination of cotinine and 3-hydroxycotinine in human serum because of its rapidity, sensitivity, strong specificity, and high reproducibility. The detection of cotinine levels in human serum allows for the identification of the cutoff value, providing a basis for differentiation between smoking and nonsmoking populations.

Progress in the treatment of central retinal artery occlusion / 国际眼科杂志(Guoji Yanke Zazhi)

Central retinal artery occlusion(CRAO)refers to occlusion of the central retinal artery(CRA), which acts as the primary blood supply to the inner neurosensory retina, and leads to an acute loss of vision and permanent visual disability. The natural history of visual prognosis in CRAO is generally poor. Despite a variety of treatment options have been studied, such as ocular massage, anterior chamber paracentesis, hyperbaric oxygen therapy(HBOT)and intra-arterial infusion of tissue plasminogen activator(tPA), but there is currently no evidence-based management strategies for the treatment of CRAO. Furthermore, the efficacy of all available managements is debatable and many have uncertain risks. This review will offer a summary of the currently known treatment options for CRAO and probe into their safety and efficacy on the prognosis of CRAO.

Reprogramming Megakaryocytes for Controlled Release of Platelet-like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential.

In this study, we reported that novel single-chain fusion proteins linking thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq) or to α-subunit s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-Gαq or the SC-TP-Gαs to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we tested their dual functions-binding to ligands and directly linking to different signaling pathways within a single polypeptide chain-using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed that the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.

Latest advancements in the study of the relationship between NSAIDs and three prostaglandin E2 synthases.

Tweetable abstract This work describes novel evidence of the relationship between NSAIDs and three prostaglandin E2 synthases.

Design, synthesis and characterization of lead compounds as anti-inflammatory drugs targeting mPGES-1 via enzymelink screening.

Aim: The objective of this study was to synthesize and validate a set of compounds that selectively inhibit mPGES-1, with the potential to be developed into a novel anti-inflammatory drug. Methods: The synthesized compounds were characterized using 1H NMR spectroscopy and LC-MS to confirm their structure. Cellular and enzymatic assays were used to demonstrate their inhibitory activity on prostaglandin E2 production. Results: Docking studies revealed that compounds containing fluoro-, chloro- and methyl- groups displayed strong inhibitory activity against prostaglandin E2. The inhibitory activity of synthesized trimethyl and trifluoro was further validated using enzymatic and cell migration assays. Conclusion: The findings demonstrated that the synthesized compounds possess significant potential as a new generation of nonsteroidal anti-inflammatory drugs that selectively target mPGES-1 with fewer side effects.

On the role of training model of general hospital specialized college in the standardized residency training / 中华医学教育探索杂志

This paper takes the establishment of the College of Basic Gynecology and Obstetrics in the First Affiliated Hospital of Sun Yat-sen University as an example, and through reviewing its establishment background, organizational system (including organizational structure, regulation construction, platform construction and teacher construction) and curriculum system (including curriculum goals and principles, curriculum format, specific curriculum settings, assessment system and evaluation feedback system), so that we can understand the development of the Basic College of Obstetrics and Gynecology and its role in the standardized residency training. This general hospital specialized college model focuses on training comprehensive, professional, applied medical talents and clinically competent physicians, which plays an important role in the standardized residency training, and improves the theoretical knowledge and technical skills of the residents. Running of the college has been widely recognized by peers, This college model is worthy of further promotion.

Regulating effects of Neiguan(PC6)and Gongsun(SP4)on hypothalamic-pituitary-adrenal axis in rats with functional dyspepsia / 针灸推拿医学（英文版）

Objective:To investigate the effects and the possible mechanisms of Neiguan(PC6)and Gongsun(SP4)on the hypothalamic-pituitary-adrenal(HPA)axis in rats with functional dyspepsia(FD),thus to provide a theoretical basis for the clinical application of the Eight Confluent Points.Methods:Forty specific-pathogen-free Sprague-Dawley rats were divided into a blank group,a model group,an electroacupuncture(EA)group,and a Western medicine group by the random number table method,with 10 rats in each group.Rats in the blank group did not receive modeling or intervention.Rats in the other three groups were subjected to the FD with mood disorder model using the compound etiology modeling method.After the successful modeling,rats in the model group did not receive any interventions,rats in the Western medicine group received deanxit and mosaprid intervention,and those in the EA group received EA intervention on the ipsilateral Neiguan(PC6)and Gongsun(SP4)for 21 d.The sugar-water consumption rate was measured before the experiment and before and after interventions to assess the emotional status.The gastric emptying rate was measured after interventions to assess the gastrointestinal dynamics.The expression levels of hypothalamic corticotropin-releasing hormone(CRH),pituitary adrenocorticotropic hormone(ACTH),and adrenal corticosterone(CORT)were measured by enzyme-linked immunosorbent assay.Results:Compared with the blank group,the sugar-water consumption rate and the gastric emptying rate were decreased(P<0.01),and the hypothalamic CRH,pituitary ACTH,and adrenal CORT expression levels were increased(P<0.01)in the model group.Compared with the model group,the sugar-water consumption rate and the gastric emptying rate were significantly increased(P<0.01),while the expression levels of hypothalamic CRH,pituitary ACTH,and adrenal CORT were significantly decreased(P<0.01)in the EA group and the Western medicine group.The differences between the EA group and the Western medicine group were not statistically significant(P>0.05).Conclusion:The Eight Confluent Points Neiguan(PC6)and Gongsun(SP4)can improve the mood and gastrointestinal dynamics in FD rats,which may be achieved by down-regulating the hypothalamic CRH,pituitary ACTH,and adrenal CORT,as well as by correcting the HPA axis hyperfunction.

Effect of liver cirrhosis complicated with thrombocytopenia on perioperative period / 中华消化外科杂志

Objective:To investigate the effect of liver cirrhosis complicated with thrombocytopenia on perioperative period.Methods:The retrospective and descriptive study was conducted. The clinical data of 75 cirrhosis patients complicated with thrombocytopenia who were admitted to the Fuyang City Second People's Hospital from July 2020 to February 2022 were collec-ted. There were 56 males and 19 females, aged (58±11)years. Observation indicators: (1) preoperative conditions; (2) intraoperative and postoperative conditions. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the one way ANOVA. Pairwise comparison was conducted using the LSD- t test. Count data were descri-bed as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test or Fisher exact probability. Comparison of ordinal data was analyzed using the rank sum test. Results:(1) Preoperative conditions. Of 75 cirrhosis patients complicated with thrombocytopenia, there were significant differences in the preoperative Child-Pugh grading and platelet count between patients with mild, moderate, and severe thrombocytopenia ( P<0.05). Results of further analysis showed that compared with patients with mild thrombocytopenia, patients with moderate and severe thrombocytopenia had significantly lower preoperative platelet count ( P<0.05). Compared with patients with moderate thrombocytopenia, patients with severe thrombocytopenia had significantly lower preoperative platelet count ( P<0.05). (2) Intraoperative and postoperative conditions. Of 75 cirrhosis patients complicated with thrombocytopenia, there were significant differences in the surgical anesthesia methods, operation time, postoperative bleeding, duration of hospital stay between patients with mild, moderate, and severe thrombocytopenia ( P<0.05). Results of further analysis showed that compared with patients with mild or moderate thrombocytopenia, patients with severe thrombocytopenia had a higher proportion of general anesthesia during surgery ( P<0.05) and compared with patients with mild thrombocytopenia, patients with severe thrombocytopenia significantly increased operation time and duration of hospital stay ( P<0.05). Of the 6 patients with severe thrombocytopenia, 3 of 5 cases with intraoperative platelet transfusion and 1 case without intraoperative platelet transfusion experienced postoperative bleeding, respectively, showing no significant difference between them ( P>0.05). Four patients with postoperative bleeding were successfully stopped bleeding after treatment and there was no death in the 75 patients. Conclusions:Patients with severe thrombocytopenia have longer operation time and duration of hospital stay, and higher proportion of general anesthesia than those with mild or moderate thrombocytopenia. Perioperative platelet transfusion cannot reduce the risk of postoperative bleeding.

Erythrocyte membrane-associated protein inhibiting Th17 cell differentiation via IL-6 / STAT3 / ROR-γt signaling pathway in experimental autoimmune encephalomyelitis mice / 解剖学报

Objective To explore the effect of exogenous and endogenous erythrocyte membrane-associated protein (ERMAP) on helper T cell 17 (Th17) cell differentiation through interleukin 6 / signal transducers and activators of transcription 3 / retionoid-related orphan nuclear receptor-γt(IL-6 / STAT3 / ROR-γt) signal pathway in the mouse model of experimental autoimmune encephalomyelitis (EAE) . Methods Using flow cytometry to verify the function of ERMAP-Ig fusion protein at different concentrations; Agarose gel electrophoresis was performed to identify ERMAP knockout mice. Flow cytometry was performed to detect the effect of ERMAP-Ig fusion protein on Th17 cell differentiation in vitro. Forty 6-week-old normal C57BL / 6 mice were randomly divided into 2 groups to establish EAE models, control-Ig and ERMAP-Ig groups, with 20 mice in each group; Clinical scores were recorded; Flow cytometry was performed to detect Th17 cell differentiation in EAE mice in vivo. Forty 6-week-old identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models. Identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models, ERMAP

Discovery of the mechanisms of acupuncture in the treatment of migraine based on functional magnetic resonance imaging and omics / 医学前沿

Migraine is one of the most prevalent and disabling neurological disease, but the current pharmacotherapies show limited efficacy and often accompanied by adverse effects. Acupuncture is a promising complementary therapy, but further clinical evidence is needed. The influence of acupuncture on migraine is not an immediate effect, and its mechanism remains unclear. This study aims to provide further clinical evidence for the anti-migraine effects of acupuncture and explore the mechanism involved. A randomized controlled trial was performed among 10 normal controls and 38 migraineurs. The migraineurs were divided into blank control, sham acupuncture, and acupuncture groups. Patients were subjected to two courses of treatment, and each treatment lasted for 5 days, with an interval of 1 day between the two courses. The effectiveness of treatment was evaluated using pain questionnaire. The functional magnetic resonance imaging (fMRI) data were analyzed for investigating brain changes induced by treatments. Blood plasma was collected for metabolomics and proteomics studies. Correlation and mediation analyses were performed to investigate the interaction between clinical, fMRI and omics changes. Results showed that acupuncture effectively relieved migraine symptoms in a way different from sham acupuncture in terms of curative effect, affected brain regions, and signaling pathways. The anti-migraine mechanism involves a complex network related to the regulation of the response to hypoxic stress, reversal of brain energy imbalance, and regulation of inflammation. The brain regions of migraineurs affected by acupuncture include the lingual gyrus, default mode network, and cerebellum. The effect of acupuncture on patients' metabolites/proteins may precede that of the brain.

Wnt7a / β-catenin / autoimmune regulator signaling pathway involved in the occurrence of type 1 diabetes / 解剖学报

[Abstract] Objective To investigate the relationship between the expression of Wnt signaling pathway, autoimmune regulator (AIRE) and type 1 diabetes (T1D) tissue specific antigen (TSAs) insulin 2(Ins2) and glutamic acid decarboxybase(GAD67) in thymus and the occurrence of T1D in NOD/ Ltj mice with spontaneous type 1 diabetes (T1D). Methods Sixty female NOD/ Ltj mice were divided into three groups: 3 weeks group, 16 weeks non-onset group and 16 weeks onset group. Two consecutive non-fasting blood glucose levels ≥ 11. 1 mmol/ L were considered as the occurrence of T1D. Pancreatic HE staining was used to observe the occurrence of islet inflammation. Anti-Ins and CD45 immunohistochemical staining showed islet cells or infiltrating inflammatory cells. The protein levels and mRNA expressions of Wnt7a, -catenin, AIRE, Ins and GAD67 in thymus were detected by Western blotting and Real-time PCR. The proportion of T cells in thymus was analyzed by flow cytometry. Results 1. With the occurrence of T1D, the islet structure was destroyed, a large number of lymphocytes infiltrated, and the remaining islet cells were reduced. A large number of CD45

The Crystal Structure and Molecular Docking of CYP76AH3 in the Tanshinone Biosynthesis Pathway / 中国生物化学与分子生物学报

Salvia miltiorrhiza is widely used in the treatment of the angina pectoris, coronary heart disease and myocardial infarction. CYP76AH3 is the key P450 enzyme, locating at the branch point of the tanshinone synthesis pathway. The crystal structural study and key amino acid analysis are of great significance for synthetic biology study on tanshinone. But it is always a challenge for scientists to carry out protein purification, crystallization and crystal structural studies on transmembrane type Ⅱ P450 enzymes. In this study, the prokaryotic expression plasmid was generated, and the high-purity target protein was purified. CYP76AH3 was successfully crystallized, and the crystal structure was solved.After docking range was determined by Cavityplus analysis, molecular docking with Discovery Studio was conducted. The docking result indicated that Gly298 and Asp294 had hydrogen bond interaction with the substrate, while Phe479, Leu367 and Leu293 had hydrophobic interaction with the substrate.In addition, the effect of mutations at the key amino acids on the protein structure stability was predicted throung point mutation simulation. This study would provide a target for protein engineering of CYP76AH3 and lay a foundation for the study of synthesis biology on tanshinones.

Engineering 'Enzymelink' for screening lead compounds to inhibit mPGES-1 while maintaining prostacyclin synthase activity.

Aim: This study investigated our Enzymelinks, COX-2-10aa-mPGES-1 and COX-2-10aa-PGIS, as cellular cross-screening targets for quick identification of lead compounds to inhibit inflammatory PGE2 biosynthesis while maintaining prostacyclin synthesis. Methods: We integrated virtual and wet cross-screening using Enzymelinks to rapidly identify lead compounds from a large compound library. Results: From 380,000 compounds virtually cross-screened with the Enzymelinks, 1576 compounds were identified and used for wet cross-screening using HEK293 cells that overexpressed individual Enzymelinks as targets. The top 15 lead compounds that inhibited mPGES-1 activity were identified. The top compound that specifically inhibited inflammatory PGE2 biosynthesis alone without affecting COX-2 coupled to PGI2 synthase (PGIS) for PGI2 biosynthesis was obtained. Conclusion: Enzymelink technology could advance cyclooxygenase pathway-targeted drug discovery to a significant degree.

Research progress of JMJD3 in Parkinson's disease / 中国药理学通报

Parkinson's disease is a common neurodegenerative disease in middle-aged and elderly people, in which the pathogenic factors are not yet clear. Genetics, dietary habits, environmental toxins, immunological abnormalities, inflammation and oxidative stress response, apoptosis, and mitochondrial dysfunctions which caused by a variety of physiological and biogenic changes are likely to exacerbate the occurrence of Parkinson's disease. In recent years, studies have shown that the activity of microglia is closely related to Parkinson's disease, and that the active microglia can promote the release of inflammatory factors, while the differentiation of dopamine neurons in the substantial nigra of midbrain area is also closely related to Parkinson's disease. As a histone H3K27me3 demethylase, JMJD3 is involved and affects the activity of microglia, which can regulate the polarization of microglia as well and affect the survival of dopaminergic neurons in the mesencephalon. This provides new methods and strategies for treating Parkinson' s disease. This paper summarizes the structure and function of JMJD3, as well as its role in neuro-inflammation mediated by microglia and its effect on neurons, and explores the functions and related research progress of JMJD3 in Parkinson's disease.

Changes of gut microbiota in experimental model rats with hyperuricemia-induced mild cognitive impairment / 中国药理学通报

Aim To explore changes of gut microbiota in experimental model rats with hyperuricemia-induced mild cognitive impairment. Methods The experimental rats were provided with a diet containing 2% W/W uric acid (UA) and 2% W/W oxonic acid for period ranging from 1 day to 12 weeks. Morris water maze, blood biochemical indexes and other auxiliary models were used to evaluate the experimental animal model. Changes of gut microbiota were studied by 16S rDNA high-throughput sequencing. Results Compared with control group, the amount of UA of rats was significantly elevated. In addition, the rats showed significantly reduced spatial learning and memory in an escape latency and probe trial. The diversity of gut microbiota in model rats changed, and the relative abundances of 14 species of gut microbiota changed markedly. The abundance of Butyrivibrio, Erysipelotrichia, Erysipelotrichales, Erysipelotrichaceae, Erysipelatoclostridium, Lachnoclostridium 5, Anaeroplasmatales, Anaeroplasmataceae and Anaeroplasma in model mice increased, while that of Dorea, RuminococcaceaeUCG_005, Butyricicoccus, Tyzzerella 3 and Parasutterella decreased. Conclusions The continuous increase of UA can regulate gut microbiota, and the imbalance of gut microbiota is closely related to the development of mild cognitive impairment, which may be one of the mechanisms of hyperuricemia-induced mild cognitive impairment.

The Protective Effects of Up-Regulating Prostacyclin Biosynthesis on Neuron Survival in Hippocampus.

Cellular arachidonic acid (AA), an unsaturated fatty acid found ubiquitously in plasma membranes, is metabolized to different prostanoids, such as prostacyclin (PGI2) and prostaglandin E2 (PGE2), by the three-step reactions coupling the upstream cyclooxygenase (COX) isoforms (COX-1 and COX-2) with the corresponding individual downstream synthases. While the vascular actions of these prostanoids are well-characterized, their specific roles in the hippocampus, a major brain area for memory, are poorly understood. The major obstacle for its understanding in the brain was to mimic the biosynthesis of each prostanoid. To solve the problem, we utilized Single-Chain Hybrid Enzyme Complexes (SCHECs), which could successfully control cellular AA metabolites to the desired PGI2 or PGE2. Our in vitro studies suggested that neurons with higher PGI2 content and lower PGE2 content exhibited survival protection and resistance to Amyloid-ß-induced neurotoxicity. Further extending to an in vivo model, the hybrid of PGI2-producing transgenic mice and Alzheimer's disease (AD) mice showed restored long-term memory. These findings suggested that the vascular prostanoids, PGI2 and PGE2, exerted significant regulatory influences on neuronal protection (by PGI2), or damage (by PGE2) in the hippocampus, and raised a concern that the wide uses of aspirin in cardiovascular diseases may exert negative impacts on neurodegenerative protection. Graphic Abstract Our study intended to understand the crosstalk of prostanoids in the hippocampus, a major brain area impacted in AD, by using hybrid enzymes to redirect the synthesis of prostanoids to PGE2 and PGI2, respectively. Our data indicated that during inflammation, the vascular mediators, PGI2 and PGE2, exerted significant regulatory influences on neuronal protection (by PGI2), or damage (by PGE2) in the hippocampus. These findings also raised a concern that the widely uses of non-steroidal anti-inflammatory drugs in cardiovascular diseases may exert negative impacts on neurodegenerative protection.

Acute changes in colonic PGE2 levels as a biomarker of efficacy after treatment of the Pirc (F344/NTac-Apc am1137) rat with celecoxib.

OBJECTIVE: This study sought to evaluate short-term treatment with COX-2 inhibitors and acute changes in colonic PGE2 levels as predictors of long-term efficacy in a genetic model of colorectal cancer. METHODS: Celecoxib oral suspension (40 mg/kg BID) was dosed to Apc-mutant Pirc (F344/NTac-Apcam1137) rats for 4 days (short-term group), or the equivalent dose of 1500 ppm celecoxib was administered in the diet for 4 months (long-term group). Percent inhibition of colonic PGE2 was calculated, and the reduction in colonic PGE2 was assessed in relation to suppression of adenomatous colon polyps. RESULTS: Colonic mucosa PGE2 was fourfold higher in Pirc than in F344 wild-type rats (21 vs. 5.6 pg/mg epithelial tissue), due at least in part to higher COX-2 expression, and this was confirmed by elevated PGE2-d11 levels in Pirc colonic S9 incubations. In the 4-day study, dose-dependent reductions in PGE2 were observed in colonic epithelium (-33% (P>0.05) and -57% (P=0.0012)), after low- and high-dose celecoxib treatments of 4 mg/kg and 40 mg/kg (bid), respectively. In the 4-month study, 1500 ppm celecoxib suppressed colonic epithelium PGE2 by 43.5%, and tumor multiplicity by 80% (P<0.0015). Suppression of plasma 6-keto PGF1α also was corroborated following long-term treatment with 1500 ppm celecoxib (P<0.05). CONCLUSIONS: Acute changes in colonic mucosa PGE2 provided a rapid means of predicting long-term chemopreventive effects from celecoxib, and might be useful for screening of new COX-2 inhibitor compounds.