This paper describes a practical process for a SGLT2 inhibitor dapagliflozin. The target product was synthesized from 1-chloro-2-( 4-ethoxybenzyl)-4-iodobenzene and 2, 3, 4, 6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide by iodine-magnesium exchange, and coupling and acetyl removing reactions with the total yield of 50%. This practical process highlights fewer reaction steps, less waste and mild reaction conditions.

Objective:To compare the effectiveness between three methods for purifying the immunoglobulin of egg yolk(IgY) which are polyethylene glycol (PEG) method, chloroform extraction method and chloroform / PEG method, and to provide basis for obtaining the batch of IgY.Methods:The inactivated vaccine of Vibrio parahemolyticus (V. parahemolyticus) was prepared and the hens were immunized by multi-point intramuscular injection.The eggs were collected and the IgY was purified by PEG method, chloroform extraction method and chloroform/PEG method.The protein extraction rate, the IgY titer and the purity of the antibody which purified by different methods were detected.Furthermore, the operation process, cost and safety of the three methods were analyzed.Results:The protein contents of the extraction belonging three methods from high to low in turn were chloroform extraction method, chloroform/PEG method, and PEG method.There was no significant difference in the antibody titer between three methods, and the tiler of chloroform extraction method was slightly high.The purities of purified antibody from high to low in turn were PEG method, chloroform/PEG method and chloroform method.The PEG method had better security but relatively lower extraction efficiency and higher cost.The chloroform/PEG method had high extraction efficiency and good antibody purity.Conclusion:The PEG method is suitable for a small amount of extraction in the laboratory.The chloroform/PEG method is appropriate for extracting the high quality IgY in a batch as it has high extraction efficiency and good antibody purity.

Objective To study the effect of electroacupuncture on the behavior of APP/PS1 transgenic mice of different age, and to explore the optimal intervention time of electroacupuncture in treating Alzheimer's disease (AD). Method APP/PS1 double-transgenic mice of 4 months, 6 months and 9 months old, 20 in each age group, were randomized into a model group and an electroacupuncture group, and ten C57BL/6 wild-type mice were taken as a control group; after 6-week electroacupuncture treatment, the Morris water maze was adopted for spatial memory and behavioral test, and the changes of behavior in each group were observed.Result Of the 5-month-old mice, there were no significant between-group differences (P>0.05), while the time factor (day) produced a statistical significance (P<0.01); of the 7-month-old mice, there were significant differences considering the different groups, time factor and interaction (P<0.01); of the 10-month-old mice, there were significant differences considering the different groups, time factor and interaction (P<0.05). The spatial probe test showed that there were significant between-group differences in comparing the platform crossings and swimming distance in platform quadrant in each age group (P<0.05).Conclusion Electroacupuncture can improve the learning and memory of APP/PS1 double-transgenic mice, the age of 6-7 months old is possibly the optimal intervention time of electroacupuncture for AD, but there still requires further mechanism studies.

bjective　To analyze the drug resistance screening status and drug resistance influencing factors of high-risk groups of drug-resistant pulmonary tuberculosis in Qingdao, and to understand the inclusion of rifampicin patients in treatment, so as to provide a reference for the prevention and treatment of drug-resistant pulmonary tuberculosis. Methods　The medical records of 726 cases of drug-resistant pulmonary tuberculosis among high-risk populations registered in Qingdao from 2018 to 2022 were obtained from the National Health Insurance Information System of the China Center for Disease Control and Prevention. The drug resistance to five anti-tuberculosis drugs, namely isoniazid (INH), rifampicin (RFP), ethambutol (EMB), levofloxacin (Lfx), and amikacin (Am), in the high-risk populations of drug-resistant pulmonary tuberculosis was analyzed. Univariate and multivariate logistic regression were used toidentify factors influencing rifampicin resistance, and the detection and inclusion of treatment for rifampicin-resistant patients were evaluated. Results　Of the 726 subjects, 278 were drug-resistant, with a total drug resistance rate of 38.29%. The drug resistance for the five anti-tuberculosis drugs in descending order was: INH 25.90%(188/726), RFP 22.87%(166/726), Lfx 14.19%(103/726), EMB 11.29%(82/726), Am 2.48%(18/726). Analysis of the drug resistance spectrum showed that among those resistant to one drug, RFP was most common, accounting for 13.67% (38/278); among those resistant to two drugs, INH+RFP was predominant, accounting for 15.83% (44/278); among those resistant to three drugs, INH+RFP+Lfx was most frequent, at 7.19% (22/278); and among those resistant to four drugs, INH+RFP+EMB+Lfx was highest, at 6.12% (17/278). Multivariate logistic regression analysis of rifampicin resistance showed that compared with patients under 25 years of age, the risk of developing rifampicin resistance was lower in the groups aged 45 to under 65 and those aged 65 and above (OR=0.356, 95%CI: 0.181-0.700; OR=0.352, 95%CI: 0.170-0.729). Compared with migrant patients in other provinces, local patients from within the same county or district had a lower risk of developing rifampicin resistance (OR=0.599, 95%CI:0.383-0.962). Compared with patients who were smear-positive at the end of the second month of initial treatment, the risk of developing rifampicin resistance was higher in patients with relapse/return, failure of retreatment/chronic, and other categories of patients (OR=9.380, 95%CI:3.717-23.671;OR=25.749, 95%CI:8.037-82.490; OR=36.651, 95%CI:8.438-159.201). Conclusions　The situation of drug-resistant pulmonary tuberculosis in Qingdao cannot be ignored. Individuals under 25 years old, migrants from other provinces, and patients with relapse/return, failure of retreatment/chronic, and other categories are significant risk factors for developing rifampicin resistance in the high-risk groups of drug-resistant pulmonary tuberculosis.

OBJECTIVE： To provide reference for the coordinated development of the pharmaceutical manufacturing industry and regional economy in the central and western regions of China. METHODS： With reference to China High-tech Industry Statistical Yearbook in 2011-2017 and China Statistical Yearbook in 2011-2017， relevant data of 14 provinces （municipatities， automous regions） in the central and western regions during 2010-2016 were collected， and a comprehensive evaluation index system and coupling coordination model of pharmaceutical manufacturing industry and regional economy were constructed， and the coupling coordinated relationship between pharmaceutical manufacturing industry and regional economy in the central and western regions of China were analyzed. RESULTS & CONCLUSIONS： During 2010-2016， the comprehensive development level of the pharmaceutical manufacturing industry and the regional economy in the central and western was on the rise， but the growth rate was relatively slow； the provinces in the central region were better than the western provinces. There were obvious coupling characteristics between the pharmaceutical manufacturing system and the regional economic system， and the coupling coordination degree had an obvious upward trend in 2010-2016. Although there were fluctuations during the period， the fluctuation degree was small and the overall trend was rising. Among them， pharmaceutical manufacturing industry and regional economy of Shanxi， Guangxi， Yunnan and Xinjiang had the same degree of coupling and coordination， which showed that the coupling coordination degree of them were greatly affected by the development of pharmaceutical manufacturing industry； Inner Mongolia， Jiangxi， Henan， Hubei， Chongqing， Sichuan， Shaanxi and Gansu had same coupling coordination degree and fluctuation course of regional economic development， which showed that the coupling coordination degree of them was greatly influenced by the development of regional economy； Anhui and Hunan had same coupling coordination degree with the development of pharmaceutical manufacturing industry and regional economy. The development of their pharmaceutical manufacturing industry and regional economy were on the rise， which showed that they were both in the rising stage of development. The mutual promotion of pharmaceutical manufacturing industry and regional economy showed that their coupling coordination was also improving. It is suggested to speed up the adjustment of industrial structure， promote the industrial transfer； pay attention to talent education， improve the talent reserve mechanism； increase the investment in fixed assets， improve infrastructure construction in central and western regions； increase the per capita disposable income of residents， promote the development of regional econorny，so as to premote the coordinated development of pharmaceutical manufacturing industry and regional economy.

Objective: Recent studies have highlighted the active and potential role of perivascular adipose tissue (PVAT) in atherosclerosis and aneurysm progression, respectively. This study explored the link between PVAT attenuation and abdominal aortic aneurysm (AAA) progression using computed tomography angiography (CTA). Materials and Methods: This multicenter retrospective study analyzed patients with AAA who underwent CTA at baseline and follow-up between March 2015 and July 2022. The following parameters were obtained: maximum diameter and total volume of the AAA, presence or absence of intraluminal thrombus (ILT), maximum diameter and volume of the ILT, and PVAT attenuation of the aortic aneurysm at baseline CTA. PVAT attenuation was divided into high (> -73.4 Hounsfield units [HU]) and low (≤ -73.4 HU). Patients who had or did not have AAA progression during the follow-up, defined as an increase in the aneurysm volume > 10 mL from baseline, were identified. Kaplan–Meier and multivariable Cox regression analyses were used to investigate the association between PVAT attenuation and AAA progression. Results: Our study included 167 participants (148 males; median age: 70.0 years; interquartile range: 63.0–76.0 years), of which 145 (86.8%) were diagnosed with AAA accompanied by ILT. Over a median period of 11.3 months (range: 6.0–85.0 months), AAA progression was observed in 67 patients (40.1%). Multivariable Cox regression analysis indicated that high baseline PVAT attenuation (adjusted hazard ratio [aHR] = 2.23; 95% confidence interval [CI], 1.16–4.32; P = 0.017) was independently associated with AAA progression. This association was demonstrated within the patients of AAA with ILT subcohort, where a high baseline PVAT attenuation (aHR = 2.23; 95% CI, 1.08–4.60; P = 0.030) was consistently independently associated with AAA progression. Conclusion Elevated PVAT attenuation is independently associated with AAA progression, including patients of AAA with ILT, suggesting the potential of PVAT attenuation as a predictive imaging marker for AAA expansion.

Objective:To investigate the clinicopathological characteristics, immunophenotypes, molecular features, and differential diagnosis of BAP1 mutated clear cell renal cell carcinoma (CCRCC) for better understanding this entity.Methods:Clinical data, histological morphology, immunophenotypes and molecular characteristics of 18 BAP1 mutated CCRCC cases diagnosed at the Department of Pathology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China from January 2020 to December 2022 were analyzed. The patients were followed up.Results:There were 17 males and 1 female patients, aged from 39 to 72 years, with an average age of 56.3 years. Sixteen patients with primary CCRCC were followed up for an average of 24 months, 7 patients had metastases occurred from 4 to 22 months postoperatively. Thirteen of the 16 patients were alive at the time of the last follow-up while 3 patients died 12, 15, and 20 months after the surgery, respectively. One patient underwent retroperitoneal mass resection, but had lung metastasis 32 months after surgery. One case received cervical tumor resection and died at 22 months after the surgery. Characteristic CCRCC regions were identified in 11 of the 18 cases. The tumor cells were arranged in papillary, alveolar, and large nest patterns. Abundant lymphoid tissue, necrosis, and psammoma bodies were seen. Tumor cells showed abundant eosinophilic cytoplasm, and sometimes exhibited rhabdoid differentiation. Round eosinophilic globules were located in the cytoplasm and extracellular matrix. There were 9 cases with WHO/International Society of Urological Pathology grade 3, and 9 cases with grade 4. PAX8 (18/18), carbonic anhydrase 9 (CA9, 16/18), CD10 (18/18), and vimentin (18/18) were positive in the vast majority of tumors.TFE3 was expressed in 5 cases, with strong expression in only 1 case. Eighteen cases were all positive for P504s. Twelve cases harbored a BAP1 mutation combined with von Hippel-Lindau (VHL) mutation, and 2 cases had mutations in BAP1, VHL and PBRM1 simultaneously. SETD2 mutation was not found in any of the cases.Conclusions:BAP1 mutated CCRCC contained papillary, alveolar, and large nest patterns, eosinophilic cytoplasm, high-grade nucleoli, and collagen globules, with P504s positivity. In practical work, when encountering CCRCC containing these features, pathologists should consider the possibility of BAP1 mutations and conduct related molecular tests.

Objective:To investigate the serotypes and antimicrobial resistance of seven invasive non-typhoidal Salmonella (iNTS) isolates. Methods:For 7 iNTS strains collected, serotype identification, antimicrobial susceptibility testing and whole genome sequencing were performed. We identified, annotated and analyzed the serotypes, MLST types, and antimicrobial resistance genes.Results:Among the 7 tested iNTS isolates, we found one Salmonella Typhimurium strain and two Salmonella Ⅰ 4, [5], 12: i:- strains whose MLST types were ST34, two Salmonella Enteritidis strains, one Salmonella Corvallis strain and one strain of unknown serotype with the antigenic formulae of Ⅰ 4, [5], 12: d:- (ST279 type). Six of seven strains were monophasic and the deletion or pseudogenization of Salmonella Flagellum gene might contribute to the enhancement of Salmonella invasiveness. None was found to be resistant to tigarcycline, aztreonam, amikacin, cephalosporins and carbapenem and one Salmonella Typhimurium strain was found to be co-resistant to eight classes of antimicrobials at the same time. Resistance genes were generally in accord with relative resistant phenotypes. Conclusion:The iNTS strains could show high level multi-drug resistance, indicating that close attention should be paid to the resistance of iNTS though the overall resistance might be relatively not high.

Objective:To construct a recombinant bioluminescent bacteriophage (HT7) targeting Escherichia coli, and evaluate its ability to identify Escherichia coli. Methods:Initially, pCRISPR-sg (1-10) and PFN-1000 plasmid strains were constructed by genetic engineering, and the most efficient small guild RNA (sgRNA) were screened by bilayer plate. By the gene editing technique, which comprised homologous recombination and clustered regularly interspaced short palin dromic repeats (CRISPR)-Cas system, the Nanoluc luciferase gene was integrated into the downstream non-coding region of 10A gene of T7 phage, to constructe the bioluminescent phage HT7 successfully. The difference of biological characteristics between HT7 phage and T7 phage was evaluated by plaque assay and liquid amplification assay. In addition, 51 strains of Escherichia coli, 20 strains of Klebsiella pneumoniae, 14 strains of Staphylococcus aureus, 6 strains of Enterococcus faecium, 5 strains of Enterococcus faecalis, 3 strains of Acinetobacter baumannii and 1 strain of Pseudomonas aeruginosa were collected and isolated to evaluate the limit of detection and specificity of HT7 phage. Results:Among the 10 CRISPR-targeted cleavage systems constructed, sgRNA8 exhibited the highest cleavage efficiency, with a cleavage rate of 0.18. After three rounds of recombination screening using the pCas9/pCRISPR/PFN-1000 triple-plasmid system, PCR validation yielded recombinant phage bands at 2 798 bp, indicating the successful construction of the HT7 phage. The recombinant phage showed significant differences in biological characteristics in terms of lysis efficiency ( P<0.001), one-step growth curve ( P=0.001), and infection multiplicity ( P=0.031). Both lysis burst time and log growth node were extended by 10 min, with the optimal infection multiplicity being 0.1. Clinical sample testing identified lysis of 6 strains of Escherichia coli within 4.5 h, while other strains remained unaffected, with detection of pathogenic bacteria below 10 CFU/ml. Conclusions:The developed pCas9/pCRISPR/PFN-1000 triple-plasmid editing system efficiently edits the bacteriophage genome. The constructed HT7 fluorescent bacteriophage enables the detection of Escherichia coli below 10 CFU/ml within 4.5 hours, demonstrating low detection limits and high detection specificity.