Determination of 23 related analytes in bone marrow fluid and hippocampus of C57BL/6 mice following cerebral ischemia-reperfusion injury by HPLC-FLD.

The study of brain diseases has long been of interest to researchers worldwide, and stroke is the third leading cause of death that threatens human health. At the same time, cerebral ischemia-reperfusion injury is closely associated with high rates of disability and mortality. The conditions of the 6-aminoquinolyl N-hydroxysccinimidyl carbamate method for the derivatization of amino acids in the bone marrow fluid and hippocampus of C57BL/6 mice with cerebral ischemia-reperfusion injury were explored and optimized, such as the column temperature, concentration of derivatization reagents and mobile phase concentration. The mobile phase consisted of 20 mm sodium acetate solution (phosphoric acid to adjust pH 5.0) and 60% acetonitrile solution at a flow rate of 1 ml min-1 . The 23 analytes were separated and determined in a gradient elution procedure; the correlation coefficient r was >0.9990 in the range 0.1-8.0 µg ml-1 . The results showed that the content of relevant analytes was significantly changed in the cerebral ischemia-reperfusion injury model, and the method was suitable for the simultaneous determination of 23 amino acids in the bone marrow fluid and hippocampus of C57BL/6 mice.

Comparison of magnetic resonance imaging and computed tomography in the diagnosis of acute pancreatitis: a systematic review and meta-analysis of diagnostic test accuracy studies.

Background: Acute pancreatitis (AP) is characterized by acute onset, rapid development, and poor prognosis. Timely diagnosis and identification of the cause are the key to formulating the clinical program and improving the prognosis. There were several studies on this topic but the results varied. This study systematically evaluated and analyzed reports on the comparison of magnetic resonance imaging (MRI) and computed tomography (CT) for the diagnosis of AP in recent years, providing evidence for clinical diagnosis and treatment. Methods: The databases of PubMed, Web of Science, The Cochrane Library, China National Knowledge Infrastructure (CNKI), and Wanfang Data were searched for literature on MRI and CT in the diagnosis of AP. After evaluating the articles and extracting the data, the software RevMan 5.4 and Stata 16.0 were used for meta-analysis. Results: A total of 9 articles were included in the selection, with a total of 566 patients having undergone diagnosis. Meta-analysis showed that for MRI, the diagnostic sensitivity was 92%, 95% confidence interval (CI): 85% to 96%; specificity was 74%, 95% CI: 50% to 89%; positive likelihood ratio was 3.5, 95% CI: 1.6 to 8.0; negative likelihood ratio was 0.11, 95% CI: 0.05 to 0.24; diagnostic odds ratio (DOR) was 32, 95% CI: 7 to 136; and the area under the curve (AUC) value was 0.93, 95% CI: 0.90 to 0.95. For CT, the diagnostic sensitivity was 73%, 95% CI: 55% to 85%; specificity was 64%, 95% CI: 42% to 82%; positive likelihood ratio was 2.0, 95% CI: 1.1 to 3.6; negative likelihood ratio was 0.43, 95% CI: 0.24 to 0.76; DOR was 5, 95% CI: 2 to 14; and the AUC value was 0.74, 95% CI: 0.70 to 0.78. The AUC value of MRI was significantly greater than CT (Z=3.684, P=0.023). Discussion: In the diagnosis of AP, MRI is more sensitive, specific, and accurate than CT, and can be used as the first choice for the diagnosis of AP.

A bacteriophage-based, highly efficacious, needle and adjuvant-free, mucosal COVID-19 vaccine

The authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected and effective in preventing COVID-19, but do not induce efficient mucosal immunity, or prevent viral transmission. We developed a bacteriophage T4-based, multicomponent, needle and adjuvant-free, mucosal vaccine by engineering spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of T4-COVID vaccine induced higher virus neutralization antibody titers against multiple variants, balanced Th1/Th2 antibody and cytokine responses, stronger CD4+ and CD8+ T cell immunity, and higher secretory IgA titers in sera and bronchoalveolar lavage with no effect on the gut microbiota, compared to vaccination of mice intramuscularly. The vaccine is stable at ambient temperature, induces apparent sterilizing immunity, and provides complete protection against original SARS-CoV-2 strain and its Delta variant with minimal lung histopathology. This mucosal vaccine is an excellent candidate for boosting immunity of immunized and/or as a second-generation vaccine for the unimmunized population.

Establishment of an HPLC Method for Determination of Coumarin-3-Carboxylic Acid Analogues in Rat Plasma and a Preliminary Study on Their Pharmacokinetics.

A simple high performance liquid chromatography (HPLC) method was developed and validated for the determination of coumarin-3-carboxylic acid analogues (C3AA) in rat plasma and a preliminary study on pharmacokinetics. Ferulic acid (FA) was used as the internal standard substance, and coumarin-3-carboxylic acid (C3A) was used as a substitute for quantitative C3AA. After protein precipitation with methanol, the satisfactory separation was achieved on an ODS2 column when the temperature was maintained at 30 ± 2°C. The correlation coefficient r in the C3A linear equation is equal to 0.9990. Pharmacokinetic parameters for t1/2, Tmax, Cmax, area under the curve (AUC)0-t, average residence time (MRT), apparent volume of distribution (V z/F) and clearance (Cl/F) were 1.89 ± 0.03 h, 0.39 ± 0.14 h, 1.81 ± 0.10 g· mL-1 ·h, 7.88 ± 0.24 g·mL-1·h, 3.23 ± 0.14 h, 0.43 ± 0.03 (mg·kg-1)·(g·mL-1)-1·h-1, respectively. The high performance liquid chromatography-photo diode array detector (HPLC-PDA) method established in this study can be used to separate and determine the content of C3AA in plasma of rats after 60% ethanol extraction by gavage. The plasma concentration-time curve and pharmacokinetic parameters reflect the absorption of C3AA in rat blood after oral administration to some extent.

The variation in the levels of 18 amino acids in the cortex and plasma of cerebral ischemia C57BL/6 mice.

Emerging evidence suggests that amino acid (AA) neurotransmitters play important roles in the pathophysiological processes of cerebral ischemia. In this work, an HPLC with fluorescence detection (HPLC-FLR) method was developed for the simultaneous determination of 18 AAs in the cortex and plasma after cerebral ischemia in mice. The ischemia model was prepared by bilateral common carotid artery occlusion, and then the cortex and plasma of the sham, ischemia, and naringenin groups were collected. Based on the protein precipitation method, a simple and effective sample preparation method was developed. The treated sample contained minimal proteins and lipids. The analysis of the sample was performed by the proposed HPLC-FLR method in combination with o-phthalaldehyde. The results showed a statistically significant increase in excitatory AAs (aspartic acid and glutamic acid), inhibitory AAs (glycine and 4-aminobutyric acid), phenylalanine, citrulline, isoleucine, and leucine levels, and a decrease of glutathione and phenylalanine levels when compared with the sham group in the cortex. Besides, the administration of naringenin had significant effects on excitatory AAs, inhibitory AA (glycine), glutamine, tyrosine, phenylalanine, and leucine levels when compared with the sham group in the cortex. These findings could be utilized in studying and clarifying the mechanisms of ischemia.

A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

A "universal" vaccine design platform that can rapidly generate multiplex vaccine candidates is critically needed to control future pandemics. Here, using SARS-CoV-2 pandemic virus as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates were engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include: expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers is found to be the most potent vaccine candidate in mouse and rabbit models. Without any adjuvant, this vaccine stimulated robust immune responses, both TH1 and TH2 IgG subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new type of nanovaccine design framework might allow rapid deployment of effective phage-based vaccines against any emerging pathogen in the future.

Simultaneous determination of thirteen substances related to NAFLD in mouse brain tissue using 3-aminobutyric acid as internal standard by HPLC-FLD.

Disorders of certain branched-chain amino acids may be associated with the occurrence and development of non-alcoholic fatty liver disease. Measurement of related branched-chain amino acid levels could provide a reference for the clinical and scientific research of the non-alcoholic fatty liver disease. An established HPLC-FLD method was used to quantify aspartic acid, glutamate, glutamine, glycine, taurine, tyrosine, 4-amino butanoic acid, tryptophan, methionine, valine, phenylalanine, isoleucine and leucine in mouse brain tissue. Brain tissue samples mixed with internal standard (3-aminobutyric acid) were processed, then derivatized with 2-O-phthaldialdehyde, and finally separated on an ODS2 column through gradient elution at a flow rate of 1.0 ml·min-1 . The excitation and emission wavelengths were set at 340 and 455 nm, respectively. The mobile phase A was 100% methanol and the mobile phase B consisted of 30 mmol·L-1 sodium acetate (pH 6.8). The injection volume was 20 µl and the single run time was 45 min. Several parameters, accuracy, precision, and stability, were verified and the results showed the established method had good sensitivity and resolution for all of the 13 compounds and internal standard in mouse brain.

Salidroside Inhibits Lipopolysaccharide-ethanol-induced Activation of Proinflammatory Macrophages via Notch Signaling Pathway.

Activation of macrophages is a key event for the pathogenesis of various inflammatory diseases. Notch signaling pathway recently has been found to be a critical pathway in the activation of proinflammatory macrophages. Salidroside (Sal), one of main bioactive components in Rhodiola crenulata (Hook. F. et Thoms) H. ohba, reportedly possesses anti-inflammatory activity and ameliorates inflammation in alcohol-induced hepatic injury. However, whether Sal regulates the activation of proinflammatory macrophages through Notch signaling pathway remains unknown. The present study investigated the effects of Sal on macrophage activation and its possible mechanisms by using both alcohol and lipopolysaccharide (LPS) to mimic the microenvironment of alcoholic liver. Detection of THP-1-derived macrophages exhibited that Sal could significantly decrease the expression of tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1ß) and IL-6 in the macrophages at both mRNA and protein levels. Furthermore, Sal significantly suppressed NF-κB activation via Notch-Hes signaling pathway in a dose-dependent manner. Moreover, in the microenvironment of alcoholic liver, the expression of Notch-dependent pyruvate dehydrogenase phosphatase 1 (PDP1) was elevated, and that of M1 gene expression [inducible NO synthase (NOS2)] was up-regulated. These changes could all be effectively ameliorated by Sal. The aforementioned findings demonstrated that Sal could inhibit LPS-ethanol-induced activation of proinflammatory macrophages via Notch signaling pathway.

Simultaneous Extraction and Determination of Eighteen Fluoroquinolone, Tetracycline and Sulfonamide Antibiotics from Soils Using Solid-phase Extraction and Liquid Chromatography Tandem Mass Spectrometry / 分析化学

An analytical method was developed for the simultaneous extraction and determination of eighteen fluoroquinolones (FQs), tetracyclines (TCs) and sulfonamides (SAs) antibiotics from soils using solid phaseextraction and liquid chromatography tandem mass spectrometry.Soil sample was firstly extracted by phosphate buffer at pH 3 in combination with 50% of organic modifier acetonitrile, then purified and concentrated by SAX and HLB column.Qualitative and quantitative analysis were carried out for the analyte under the MRM mode after the chromatography separation on Kromasil C_(18)(250 mm x4.6 mm, 5 μm) column.The range of recoveries (in percent) for FQs, TCs, SAs, in the soil matrix was 67.20%-88.98%, 62.23%-85.36%, 55.76%-97.37% with 1.1%-17.2% of relative standard deviation respectively in two different concentra tions.The limits of quantification (LOQ, S/N = 3) were 3.36-8.88 jig/kg, 0.56-0.91 μg/kg and 0.07-1.85 μg/kg for FQs, TCs and SAs, respectively.This method was successfully used to detect 18 anti biotics in 6 soil samples with different land types in Tianjin.Results showed some of the antibiotics in the arable soil were detected, with concentrations of 1.72-119.57 μg/kg.

Relationship between benign prostatic hyperplasia and chronic prostatitis / 中华泌尿外科杂志

Objective To investigate the relationship between benign prostatic hyperplasia (BPH) andchronic prostatitis(CP). Methods Three hundred BPH patients were studied, aged from 51 to 96 (aver-age 72). All patients were divided into 3 groups (Ⅰ°、Ⅱ°and Ⅲ°)according to result of digital rectal examina-tion, which include 85 cases , 139 cases and 76 cases respectively. The incidence of CP among 3 groups were compared and analyzed. Results Two hundreds and thirty-five of the 300 cases with BPH were accompa-nied with CP(77.7%). Among the 233 cases, 53 cases were in Ⅰ degree BPH group (53 / 85, 62.4% ), 113 cases were in Ⅱ degree BPH group (113/139, 81.3%), 67 cases were in Ⅲ degree BPH group (67/76, 88.2%). Conclusions Many BPH patients were accompanied by CP. The prostate size and the inflamma-tion of prostate were positive correlated. The effect of anti-inflammatory treatment in Ⅰ degree and Ⅱ degreeBPH patients was better than Ⅲ degree BPH patients.

[Phosphorylated modification of genistein and their interaction with lysozyme].

A series of genistein's phosphates were synthesized through a simplified Atherton-Todd reaction and the structures of the phosphates were determined by electrospray ionization mass spectrometry (ESI-MS) and NMR. In case of monophosporyl derivatives, NMR spectra show that substitutions occur at the 7-position of genistein but not at its 4' and 5-position. Moreover, the non-covalent complexes of lysozyme with the four new genistein phosphates were detected by ESI-MS.

[Spatial-temporal distribution of glycogen synthase kinase 3beta and adenomatous polyposis coli protein are involved in the injury and repair of airway epithelial cells induced by scratching].

To investigate the roles of glycogen synthase kinase 3beta (GSK3beta) and adenomatous polyposis coli (APC) protein in wound repair of airway epithelial cells (AECs), we established a wound model of airway epithelium in vitro. Then the following tests were undertaken: (1) Western blot was used to detect the levels of total GSK3beta and phosphorylated GSK3beta in human bronchial epithelial (16HBE) cells; (2) The localizations of APC protein was observed by using immunofluorescence technique; (3) Immunoprecipitation was used to investigate the relationship between APC protein and GSK3beta during the repair of 16HBE cells. The results were as follows: (1) The level of phosphorylated GSK3beta increased 0.5 h after scratching (P<0.05), reached a maximum at 6 h (P<0.05), and maintained until 12 h, while the total level of GSK3beta remained constant; (2) Results of immunofluorescence study showed that APC protein clustered with tubulin in the region of the migrating leading cells 6 h after scratching, which was dissimilar with that in the cells 0 h after scratching; (3) GSK3beta and APC protein were immunoprecipitated and analysed on SDS-PAGE. We found that GSK3beta and APC protein were precipitated, indicating that the two proteins existed in a complex. After scratching, dissociation of the two proteins occurred. Taken together, we conclude that scratching caused a decrease in phosphorylation of GSK3beta, and that reduced phosphorylation of GSK3beta promoted APC protein to bind to the plus ends of microtubules and stabilize the growing ends. These observations suggest that APC protein and GSK3beta may synergistically play an important role in the repair of airway epithelium.

Glycogen synthase kinase 3beta induces cell cycle arrest in a cyclin D1-dependent manner in human lung adenocarcinoma cell line A549.

The effect of glycogen synthase kinase 3beta (GSK3beta) has been repeatedly implicated in cell proliferation, but studies on the effect of GSK3beta in different cell lines with different stimuli have drawn different conclusions. To investigate the direct effect of GSK3beta on cell growth in human lung adenocarcinoma cell line A549, we changed its activity by transient transfection with two kinds of GSK3beta mutant plasmids, constitutively active form S9A-GSK3beta and dominant negative form KM-GSK3beta. Twenty-four hours later, cell counting, flow cytometry and Western blot detection were made respectively. The results showed that enhancing GSK3beta activity caused a decrease in cell number, as well as a higher percentage of cells at G(1) phase. Further, the expression of cyclin D1 was down-regulated by GSK3beta. Taken together, our observations suggest that GSK3beta may induce G(1) cell cycle arrest in a cyclin D1-dependent fashion and therefore possibly plays a growth-inhibitory role in A549 cells.

Phosphorylated modification of genistein and their interaction with lysozyme / 药学学报

A series of genistein's phosphates were synthesized through a simplified Atherton-Todd reaction and the structures of the phosphates were determined by electrospray ionization mass spectrometry (ESI-MS) and NMR. In case of monophosporyl derivatives, NMR spectra show that substitutions occur at the 7-position of genistein but not at its 4' and 5-position. Moreover, the non-covalent complexes of lysozyme with the four new genistein phosphates were detected by ESI-MS.

Glycogen synthase kinase 3beta induces cell cycle arrest in a cyclin D1-dependent manner in human lung adenocarcinoma cell line A549 / 生理学报

The effect of glycogen synthase kinase 3beta (GSK3beta) has been repeatedly implicated in cell proliferation, but studies on the effect of GSK3beta in different cell lines with different stimuli have drawn different conclusions. To investigate the direct effect of GSK3beta on cell growth in human lung adenocarcinoma cell line A549, we changed its activity by transient transfection with two kinds of GSK3beta mutant plasmids, constitutively active form S9A-GSK3beta and dominant negative form KM-GSK3beta. Twenty-four hours later, cell counting, flow cytometry and Western blot detection were made respectively. The results showed that enhancing GSK3beta activity caused a decrease in cell number, as well as a higher percentage of cells at G(1) phase. Further, the expression of cyclin D1 was down-regulated by GSK3beta. Taken together, our observations suggest that GSK3beta may induce G(1) cell cycle arrest in a cyclin D1-dependent fashion and therefore possibly plays a growth-inhibitory role in A549 cells.

Spatial-temporal distribution of glycogen synthase kinase 3beta and adenomatous polyposis coli protein are involved in the injury and repair of airway epithelial cells induced by scratching / 生理学报

To investigate the roles of glycogen synthase kinase 3beta (GSK3beta) and adenomatous polyposis coli (APC) protein in wound repair of airway epithelial cells (AECs), we established a wound model of airway epithelium in vitro. Then the following tests were undertaken: (1) Western blot was used to detect the levels of total GSK3beta and phosphorylated GSK3beta in human bronchial epithelial (16HBE) cells; (2) The localizations of APC protein was observed by using immunofluorescence technique; (3) Immunoprecipitation was used to investigate the relationship between APC protein and GSK3beta during the repair of 16HBE cells. The results were as follows: (1) The level of phosphorylated GSK3beta increased 0.5 h after scratching (P<0.05), reached a maximum at 6 h (P<0.05), and maintained until 12 h, while the total level of GSK3beta remained constant; (2) Results of immunofluorescence study showed that APC protein clustered with tubulin in the region of the migrating leading cells 6 h after scratching, which was dissimilar with that in the cells 0 h after scratching; (3) GSK3beta and APC protein were immunoprecipitated and analysed on SDS-PAGE. We found that GSK3beta and APC protein were precipitated, indicating that the two proteins existed in a complex. After scratching, dissociation of the two proteins occurred. Taken together, we conclude that scratching caused a decrease in phosphorylation of GSK3beta, and that reduced phosphorylation of GSK3beta promoted APC protein to bind to the plus ends of microtubules and stabilize the growing ends. These observations suggest that APC protein and GSK3beta may synergistically play an important role in the repair of airway epithelium.

Role of glycogen synthase kinase 3 in squamous differentiation induced by cigarette smoke in porcine tracheobronchial epithelial cells.

Epidemiological evidence suggests that cigarette smoke induces squamous metaplasia in human tracheobronchial epithelium that can progress to lung squamous carcinoma. But it is not well understood how tracheobronchial epithelial cells transduce the signals that mediate cigarette smoke-induced squamous differentiation or squamous metaplasia. In the present study, we found that in vitro cigarette smoke components notably inhibited glycogen synthase kinase 3 (GSK3) and induced the expression of involucrin, a marker of squamous differentiation. The inactivation of GSK3 by two highly selective inhibitors, lithium and SB216763, also significantly enhanced involucrin expression in cultured porcine tracheobronchial epithelial cells (PTBECs). Moreover, we demonstrated that cigarette smoke components significantly promoted activator protein-1 (AP-1) binding activities to the upstream regulatory region of involucrin gene, and similar results were observed by further studies through using GSK3 inhibitors to imitate the effects of cigarette smoke components. Taken together, we conclude that GSK3 is involved in involucrin expression induced by cigarette smoke in PTBEC probably via negatively regulating AP-1 activity, implying a possible mechanism responsible for squamous differentiation induced by cigarette smoke.

[Study on the phosphorylaions reaction between ethyl salicylate and DEPH].

Atherton-Todd reaction has been extensively applied to the synthesis of phosphates and phosphoroamidates. Under Atherton-Todd reaction conditions, dialkyl phosphites were probably transformed into diakyl phosphorochloridates. We have tried to use Atherton-Todd reaction to synthesize phosphate of salicylic-acid. But when the -COOH of salicylic acid was under unprotected, the yield was quite low. It was found that the -COOH possibly participated in the reaction. The competitive reaction between the -COOH and -OH of salicylic acid probably existed. In order to understand the detail mechanism, ethyl-salicylate was selected to be phosphorylated In the end, ethyl-salicylate successfully phosphorylated by modification of the classical Atherton-Todd procedure through dropping the DEPH and tetrachloromethane into the mixed solution of ethyl-salicylate, triethylamine and dioxane with good yields. The structures were elucidated by NMR and ESI/MS/MS data.

Andreev Bound States at the Onset of Phase Coherence in Bi(2)Sr(2)CaCu(2)O(8).

A new technique of planar tunneling spectroscopy has been developed to access the in-plane density of states of optimally doped Bi(2)Sr(2)CaCu(2)O(8) single crystals. The low energy spectrum is observed to depend on crystallographic orientation. When tunnel current is injected nominally along the Cu-Cu bond direction, a zero-bias conductance peak is observed to appear simultaneously with the onset of bulk superconductivity. These data demonstrate the existence of surface-induced states in this system and confirm the d-wave symmetry of the superconducting order parameter.

Quasiparticle liquid in the highly overdoped Bi(2)Sr(2)CaCu(2)O(8+delta).

Results from the study of a highly overdoped (OD) Bi(2)Sr(2)CaCu(2)O(8+delta) with a T(c) = 51 K using angle-resolved photoemission spectroscopy are presented. We observe a sharp peak in the spectra near ( pi,0) that persists well above T(c), a nodal self-energy which approaches that seen for the Mo(110) surface state, and a more k-independent line shape at the Fermi surface than the lower-doped cuprates. This allows for a realistic comparison of the lifetime values to the experimental resistivity measurements. These observations point to the validity of the quasiparticle picture for the OD even in the normal state.