[Drug resistance and molecular typing of diarrheagenic escherichia coli from the diarrhea clinics in Qingpu District, Shanghai].

Objective: To analyze the drug resistance and multilocus sequence typing of five types of diarrheagenic Escherichia coli (DEC) isolated from diarrhea outpatients of diarrhea comprehensive monitoring designated hospital in Qingpu District, Shanghai from 2015 to 2019. Methods: From January 2015 to December 2019, five types of DEC, isolated and identified from diarrhea outpatient cases' anal swabs of the Qingpu branch of Zhongshan Hospital were collected to determine the minimal inhibitory concentration by using the micro broth dilution susceptibility test. The strains, resistant to the third-generation cephalosporins or carbapenems, or producing ESBLs, were selected based on the results of sensitivity tests and determined by WGS. The MLST typing of DEC was analyzed based on the WGS technology and the minimum spanning tree was constructed by BioNumerics 7.6 software to analyze the local dominant flora. Results: A total of 513 strains of DEC were detected and isolated from 4 494 anal swabs, with a detection rate of 11.42%. About 500 strains were tested for drug sensitivity to nine antibiotics in four classes, including 330 strains of enterotoxigenic E.coli (ETEC), 72 strains of enteroaggregative E.coli (EAEC), 95 strains of enteropathogenic E.coli (EPEC), 1 strain of enterohemorrhagic E.coli (EHEC), and 2 strains of enteroinvasive E.coli (EIEC). From 2015 to 2019, the resistance rate of cefotaxime-clavulanic acid was significantly different (P<0.05). The resistance rate of virulence types of DEC to nalixic acid was significantly different (P<0.05). About 71 strains of DEC were determined by WGS, and 77 drug-resistant genes were detected. Strains were classified into 32 ST subtypes, with the dominant genotypes being ST-1491 (29.6%, 21/71) and ST-10 Complex (23.9%, 17/71). All ST-1491 produced ESBLs, which were blaCTX-M gene mutant strains. The dominant type of ST-10 complex was ST-218 (35.3%, 6/17). In addition, 8 strains of EAEC, 14 strains of EPEC and 49 strains of ETEC were classified into 7, 14 and 18 ST subtypes, respectively. Conclusion: The drug resistance of DEC strains from the diarrhea outpatient case of Qingpu District is serious. The ST types of EAEC and EPEC are highly polymorphic. The dominant ST types of DEC are basically consistent with the common genotypes in southeast China.

[Drug resistance and molecular typing of diarrheagenic escherichia coli from the diarrhea clinics in Qingpu District, Shanghai City from 2015 to 2019].

Objective: To analyze the drug resistance and multilocus sequence typing of five types of diarrheagenic Escherichia coli (DEC) isolated from diarrhea outpatients of diarrhea comprehensive monitoring designated hospital in Qingpu District, Shanghai City from 2015 to 2019. Methods: From January 2015 to December 2019, five types of DEC, isolated and identified from diarrhea outpatient cases' anal swabs of the Qingpu branch of Zhongshan Hospital were collected to determine the minimal inhibitory concentration by using the micro broth dilution susceptibility test. The strains, resistant to the third-generation cephalosporins or carbapenems, or producing ESBLs, were selected based on the results of sensitivity tests and determined by WGS. The MLST typing of DEC was analyzed based on the WGS technology and the minimum spanning tree was constructed by BioNumerics 7.6 software to analyze the local dominant flora. Results: A total of 513 strains of DEC were detected and isolated from 4 494 anal swabs, with a detection rate of 11.42%. About 500 strains were tested for drug sensitivity to nine antibiotics in four classes, including 330 strains of enterotoxigenic E.coli (ETEC), 72 strains of enteroaggregative E.coli (EAEC), 95 strains of enteropathogenic E.coli (EPEC), 1 strain of enterohemorrhagic E.coli (EHEC), and 2 strains of enteroinvasive E.coli (EIEC). From 2015 to 2019, the resistance rate of cefotaxime-clavulanic acid was significantly different (P<0.05). The resistance rate of virulence types of DEC to nalixic acid was significantly different (P<0.05). About 71 strains of DEC were determined by WGS, and 77 drug-resistant genes were detected. Strains were classified into 32 ST subtypes, with the dominant genotypes being ST-1491 (29.6%, 21/71) and ST-10 Complex (23.9%, 17/71). All ST-1491 produced ESBLs, which were blaCTX-M gene mutant strains. The dominant type of ST-10 complex was ST-218 (35.3%, 6/17). In addition, 8 strains of EAEC, 14 strains of EPEC and 49 strains of ETEC were classified into 7, 14 and 18 ST subtypes, respectively. Conclusion: The drug resistance of DEC strains from the diarrhea outpatient case of Qingpu District is serious. The ST types of EAEC and EPEC are highly polymorphic. The dominant ST types of DEC are basically consistent with the common genotypes in southeast China.

Effects of heating on the secondary structure of proteins in milk powders using mid-infrared spectroscopy.

Milk powder is an important source of protein for adults and children. Protein is very sensitive to heat, which may influence people's usage of nutrients in milk powder. In this study, we describe the temperature-induced secondary structure of protein in milk powders. In this study, whole milk powder containing 24% protein and infant formula containing 11% protein were heated from 25 to 100°C. Attenuated total reflectance (ATR) spectra in the mid-infrared range 400-4,000cm-1 were used to evaluate the heat effect on the secondary structure of protein in these 2 milk powders. The spectral changes as a function of temperature were maintained by difference spectra, second-derivative spectra and Gauss curve-fitted spectra. The secondary structures of protein in the whole milk powder began to change at 70°C and in the infant formula at 50°C. The ß-sheet and ß-turn structures in the whole milk powder both decreased in the range of 70 to 85°C, whereas α-helix structures increased. The loss of ß-sheet and ß-turn may contribute to the formation of α-helix in the whole milk powder. In infant formula powder, the ß-sheet structure showed a decrease and then increase, whereas the ß-turn structure showed an increase and then decrease in the range of 50 to 75°C, and no change was found for α-helix structures. This implies that heating may induce the transformation from ß-sheet to ß-turn. Overall, whole milk powder had better temperature stability than infant formula powder, probably because of the lower content of lipid in the former than in the latter. These results help us understand the thermal stability of protein in milk powder.

Atmospheric nourishment of global ocean ecosystems.

Over the vast open ocean, vital nutrients for phytoplankton growth in the sunlit surface layer are largely provided through physical transport from deep waters, but some nutrients are also provided through atmospheric deposition of desert dust. The extent and magnitude of dust-mediated effects on surface ocean ecosystems have been difficult to estimate globally. In this work, we use global satellite ocean color products to demonstrate widespread responses to atmospheric dust deposition across a diverse continuum of phytoplankton nutritional conditions. The observed responses vary regionally, with some areas exhibiting substantial changes in phytoplankton biomass, whereas in other areas, the response reflects a change in physiological status or health. Climate-driven changes in atmospheric aerosols will alter the relative importance of this nutrient source.

Reversing adriamycin resistance of human breast cancer cells by hyperthermia combined with Interferon alpha and Verapamil.

One of the major obstacles related to chemotherapy is resistance against anticancer drugs, including Adriamycin (ADM). The purpose of the present work is to investigate the reversal effects on ADM resistance by hyperthermia (42.5 degrees C) combined with two reversal agents (Interferon alpha and Verapamil) in MCF-7/ADR (ADM-resistant MCF-7 breast cancer cell line), and its relevant molecular mechanism of action. The cell survival rate and ADM IC50 of different experiment groups were measured by MTT test. The quantitative expression of MDR1 gene in cells was detected by Real-time PCR, and the expression of P-glycoprotein (P-gp) on the cells surface and the intracellular ADM accumulation was detected by flow cytometry (FCM). The ADM IC50 of the MCF-7/ADR cells decreased 830-fold after combined with Interferon alpha (IFN-alpha) and Verapamil (VRP). Although there was no distinction in the mRNA expression of MDR1, the P-gp on the MCF-7/ADR cell membrane was significantly reduced and the cellular ADM uptake increased markedly as compared to pretreatment. Our results suggeste that hyperthermia induces a considerably reversal activity against ADM resistance synergizing other reversal agents (IFN-alpha and VRP). The reversal mechanism needs further study. However, these features of hyperthermia may be exploited in clinical cancer chemotherapy.

Up-regulation of Twist induces angiogenesis and correlates with metastasis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly invasive tumor characterized by vigorous neovascularization. The purpose of this study is to examine the expression of Twist, a highly conserved bHLH transcription factor that is known to promote EMT, and evaluate its effect on tumor angiogenesis and metastasis of HCC. The mRNA expression of Twist, VEGF, E-cadherin, and N-cadherin was determined by Real-Time RT-PCR in 30 pairs of hepatocellular carcinomas and matched non-cancerous tissues. Immunohistochemistry was carried out to analyze the protein expression of Twist, VEGF, E-cadherin, and N-cadherin in 40 hepatocellular carcinoma cases. The staining of endothelial cells for CD34 was used to evaluate the MVD. We found that Twist mRNA and protein were both increased in HCC as compared to non-cancerous tissues. The HCC specimens showing positive Twist expression had a higher microvessel density than those without Twist expression. And up-regulated Twist protein was significantly associated with intrahepatic and extrahepatic metastasis (p=0.048 and P=0.039 respectively). In addition, patients with Twist expression had poor prognosis. We also found that the expression of Twist positively correlated with up-regulation of VEGF and N-cadherin (P=0.002 and p=0.016 respectively), but not with downregulation of E-cadherin in HCC. Our results demonstrate that Twist may play an important role in the angiogenesis and metastasis of HCC. Twist expression may become a potential novel prognostic factor for the disease survival of HCC.

Proteolysis of integrin alpha5 and beta1 subunits involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells.

Our previous report demonstrated that all-trans-retinoic acid (ATRA) induces detachment and death under serum starvation in several human tumor cell lines. In this study, we examined the influence of cell-extracellular matrix interaction on the ability of ATRA to induce apoptosis. Plating of human hepatoma Hep3B cells onto poly-hydroxyethylmethacrylate-coated plates in the absence of serum resulted in the acceleration of ATRA-induced apoptosis. In contrast, ATRA-induced apoptosis was significantly suppressed by plating cells onto Matrigel-coated plates but not suppressed by culturing onto collagen-, laminin-, vitronectin-, or fibronectin-coated plates. Exogenously added soluble collagen, laminin, fibronectin, vitronectin or Matrigel failed to suppress ATRA-induced apoptosis. Results from the adhesion assay indicated that the cell attachment to fibronectin was significantly inhibited by ATRA. Treatment with perturbing antibody against integrin alpha5 or beta1 subunits resulted in promotion of ATRA-induced apoptosis. Moreover, the proteolytic cleavage of alpha5beta1 integrin and focal adhesion kinase (FAK) proteins is linked to the early phase of the ATRA-induced apoptotic process. Furthermore, ATRA-induced detachment, death, and cleavage of alpha5beta1 integrin and FAK were drastically suppressed by plating cells onto Matrigel-coated plates. These findings provide evidence that abrogation of cell adhesion, through proteolysis of alpha5beta1 integrin and FAK, is closely linked to ATRA-induced apoptosis in Hep3B cells.

Desensitization of adrenomedullin and calcitonin gene-related peptide receptors in vascular smooth muscle cells--effects of receptor activity-modifying protein.

Recent researches suggest that adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) bind to the same calcitonin receptor-like receptors (CRLR), with receptor specificity being determined by a receptor activity-modifying protein (RAMP). Our objective was to explore the significance of CRLR/RAMP hypothesis in cardiovascular tissues through experiments on the phenomenon of desensitization of both ADM and CGRP receptors using cultured rat aortic vascular smooth muscle cells (VSMCs). VSMCs were incubated for 20 min either in serum-free medium (SFM) alone or in the SFM containing vasoactive agonist [10(-8) mol/L ADM, CGRP and proadrenomedullin (PAMP)]. Cells were washed twice and incubated for another 20 min in SFM containing a repetitive agonist ADM or CGRP and 0.5 mmol/L isobutyryl methylxant (an inhibitor of phosphodiesterase). VSMCs were harvested and assayed for cAMP. Exposure of VSMCs to ADM, CGRP, or PAMP alone increased intracellular cAMP generation by 191% (P < 0.01), 385% (P < 0.01) and 67% (P < 0.05), respectively, compared with SFM group. Pre-treatment of VSMCs to ADM or CGRP decreased cAMP generation in response to subsequent stimulation with CGRP by 44% (P < 0.05) and 48% (P < 0.01), respectively. Pre-treatment of VSMCs with 100 nmol/L H-89, a protein kinase A (PKA) inhibitor, abolished the desensitization of CGRP-acting receptor, implying that this desensitization was mediated through PKA. In contrast, there was no attenuation in cAMP response to stimulation with ADM by pre-exposure to ADM or CGRP. Identical results were seen with or without PKA inhibition by H-89. Pre-exposure of VSMCs to PAMP resulted in no change in cAMP generation in response to subsequent stimulation with ADM or CGRP. These results indicate that ADM receptors do not desensitize in VSMCs in contrast to CGRP-receptors, which are desensitized by pre-exposure to ADM or CGRP. These data also suggest that the desensitization phenomenon of ADM is different from that of CGRP.

Mutation analysis of human LEFTY A and LEFTY B genes in children with Ivemark syndrome.

The search for genes responsible for the abnormal development of the left-right (L/R) asymmetry has been conducted but no definite results have been reported. Recently, two human homologus mouse lefty1 genes, LEFTY A and LEFTY B, were analyzed for mutations in patients with the L/R anomalies. However, only two mutations were found in a survey of 126 patients. We collected genomic DNA from 10 children with Ivemark syndrome, a disease with anomalies in L/R asymmetry. Mutation analysis of LEFTY A and LEFTY B genes using single strand conformation polymorphism and direct sequencing was performed, but no mutations were found. This indicates that the L/R asymmetry anomaly in Ivemark syndrome may not be caused by the mutation of LEFTY A and LEFTY B genes. Other genes responsible for the anomalies of L/R asymmetry should be further investigated.

Rapid diagnosis of common aneuploidies by quantitative fluorescent polymerase chain reaction.

The most frequent autosomal aneuploidies in newborns involve chromosomes 21, 18, and 13. The pre- and postnatal detection of chromosome abnormalities has been almost exclusively performed by cytogenetic analysis. In this paper, we assess the diagnostic value of fluorescent polymerase chain reaction (PCR) using polymorphic small tandem repeats (STR). PCR products are distinguished via both size and fluorescence intensity to confirm the trisomy by either triallelic signals with similar fluorescence intensities or diallelic pattern with double-dose response. Compared with the relatively time-consuming and laborious classic cytogenetic analysis, this technique is rapid, inexpensive, and sensitive for the detection of trisomies 21, 18 and 13, particularly when the numbers of cells obtained from the prenatal diagnosis is limited or where cell culture fails. With greater samples tested and more STR markers available, this method will become more reliable. This study investigates the detection of aneuploides involving chromosomes 21, 18, and 13 by comparing quantitative fluorescent PCR with karyotyping performed by conventional cytogenetics. The results obtained by the two techniques were concordant in all trisomy cases of this study.

Wolf-Hirschhorn (4p deletion) syndrome: report of one case.

Wolf-Hirschhorn syndrome is an uncommon chromosomal disorder caused by loss of material from the distal aspect of the short arm of chromosome 4. Its characteristic features include profound growth retardation with psychomotor delay, severe mental deficiency, facial dysmorphia, midline defects and skeletal anomalies. We herein report a case of 4p deletion syndrome and review related literature.

Association of tumour necrosis factor alpha -308 gene polymorphism with primary open-angle glaucoma in Chinese.

PURPOSE: Genetic factors are known to play a role in the aetiology of glaucoma, and in particular the role of the immune system is highly suspected. In this study, we evaluated the association between tumour necrosis factor alpha -308 (TNF alpha -308) and primary open-angle glaucoma (POAG). METHODS: A total of sixty POAG patients and 103 healthy volunteers as control group were enrolled in this case-controlled study. Furthermore, we used polymerase chain reaction based analysis to resolve the TNF alpha -308 polymorphism. Statistical analysis for the relative risk of TNF alpha -308 polymorphism was compared by the chi(2) test. RESULTS: There were significant differences in the distribution of the polymorphism between the POAG patients and the control subjects (P = 0.00016; P < 0.05) and it was found that the A(-308) allele occurred more frequently in POAG patients (odds ratio: 2.72; 95% confidence interval: 1.66-4.45). CONCLUSION: The results of our study concluded that the distribution of TNF alpha -308 was significantly higher in the POAG patients than in the control group. Therefore, the A(-308) allele appears to be associated with POAG and, therefore, could be used as a genetic marker for disease mapping. POAG is a complex disease, and a single gene could not be responsible. Understanding the role of genetic polymorphisms, like TNF alpha, could be a prediction of the disease and useful for developing new treatments for POAG.

Polymorphism of angiotensin-1 converting enzyme gene and Kawasaki disease.

Kawasaki disease is an acute febrile illness typically elicited by vasculitis and occurring in young children. We investigated the polymorphism of the angiotensin-1 converting enzyme (ACE) gene in children with Kawasaki disease and also in age-matched controls. A total of 107 children, with a mean age at diagnosis of 1.71 +/- 1.48 years, who suffered from Kawasaki disease and who were treated with aspirin as well as intravenous immunoglobulin were enrolled in this study. Control subjects consisted of 107 children, with a mean age of 1.84 +/- 1.20 years. The polymorphisms of the ACE gene, including I/D, A-240T, and G2350A, were examined using a polymerase chain reaction method for Kawasaki disease patients and also for control subjects. We noted a significant difference in the distribution of the ACE gene I/D genotype between Kawasaki disease and control groups. The ACE gene G2350A polymorphism and associated allelic frequencies demonstrated an association with Kawasaki disease. Our results revealed no evidence of any association between the ACE gene polymorphism and the frequency of coronary artery aneurysm associated with Kawasaki disease, although our results do support a role for the I/D and G2350A polymorphism of the ACE gene in determining the risk of Kawasaki disease in the population of Taiwan.

Polymorphisms for interleukin-1 beta (IL-1 beta)-511 promoter, IL-1 beta exon 5, and IL-1 receptor antagonist: nonassociation with endometriosis.

PURPOSE: We aimed to investigate if interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1Ra) gene polymorphism could be used as markers of susceptibility in endometriosis. MATERIALS AND METHODS: Women were divided into two groups: 1) endometriosis (n = 120); 2) nonendometriosis groups (n = 103). Polymorphisms for IL-1 beta-511 promoter, IL-1 beta exon 5, and IL-1Ra were detected by polymerase chain reaction. Genotypes and allelic frequencies for these polymorphisms in both groups were compared. RESULTS: Proportions of different IL-1 and IL-1Ra polymorphisms in both groups were nonsignificantly different. Proportions of C homozygote/heterozygote/T homozygote for IL-1 beta-511 promoter in both groups were 1) 21.6/59.1/19.1% and 2) 26.2/50.5/23.3%. Proportions of E1 homozygote/heterozygote/E2 homozygote for IL-1 beta exon 5 in both groups were 1) 91.6/5/3.3% and 2) 95.15/4.85/0%. Allele I/II/IV/V for IL-1Ra in both groups were 1) 92.5/5.4/1.6/0.4% and 2) 95.1/3.9/1/0%. CONCLUSIONS: Association of endometriosis with IL-1 beta-511 promoter, IL-1 beta exon 5, and IL-1 receptor antagonist gene polymorphisms doesn't exist. These polymorphisms are not useful markers for prediction of endometriosis susceptibility.