Genotypic and Phenotypic Characterization of IncX3 Plasmid Carrying blaNDM-7 in Escherichia coli Sequence Type 167 Isolated From a Patient With Urinary Tract Infection

Infections due to New Delhi metallo-beta lactamase (NDM)-7-producing Escherichia coli are infrequent and sporadic. In this study, we report one case of recurrent urinary tract infection caused by blaNDM-7-producing E. coli belonging to phylogenetic group A, sequence type (ST) 167. In this study, we aimed to describe the genotype and phenotype of blaNDM-7-producing E. coli in China. The isolate exhibited resistance to β-lactam antimicrobials, trimethoprim-sulfamethoxazole, quinolones, and aminoglycosides. blaNDM-7 is located on a conjugative plasmid designated pJN05NDM-7 belonging to type IncX3. pJN05NDM-7 was fully sequenced and compared with all publicly available blaNDM-7-harboring plasmids. pJN05NDM-7 is almost identical to pKpN01-NDM7 and pKW53T, although the plasmids are geographically unrelated. The comparison of IncX3 plasmids harboring blaNDM in China showed high similarity, with genetic differences within insertion fragments. Notably, the differences in plasmids of animal and human origin were insignificant, because only one plasmid showed deletion inside the ISAba125 region compared with pJN05NDM7. Our study demonstrates that E. coli carrying IncX3 plasmids play an important role as a reservoir and in the spread of blaNDM. Further studies should be performed to control the dissemination of blaNDM among food animals

Epigenetics and Vascular Senescence–Potential New Therapeutic Targets?

© Copyright © 2020 Ding, Shao, Rose and Zhu. Epigenetics is defined as the heritable alterations of gene expression without changes to the coding sequence of DNA. These alterations are mediated by processes including DNA methylation, histone modifications, and non-coding RNAs mechanisms. Vascular aging consists of both structural and functional changes in the vasculature including pathological processes that drive progression such as vascular cell senescence, inflammation, oxidation stress, and calcification. As humans age, these pathological conditions gradually accumulate, driven by epigenetic alterations, and are linked to various aging-related diseases. The development of drugs targeting a spectrum of epigenetic processes therefore offers novel treatment strategies for the targeting of age-related diseases. In our previous studies, we identified HDAC4, JMJD3, Fra-1, and GATA4 as potential pharmacological targets for regulating vascular inflammation, injury, and senescence

Genotypic and Phenotypic Characterization of Clinical Escherichia coli Sequence Type 405 Carrying IncN2 Plasmid Harboring blaNDM-1

We report a blaNDM-carrying ST405 Escherichia coli recovered from the abdominal fluid of a patient in Shandong, China. This strain belonged to the high-risk phylogenetic group D and carried the virulence genes, papG II, papG III, papC, and iroN. In addition to blaNDM-1, this isolate carried the quinolone resistance gene acc(6′)-Ib and extended-spectrum β-lactamase (ESBL) genes blaCTX-M-15 and blaCTX-M-14. blaNDM-1 was located on a 41 Kb IncN2 self-transmissible plasmid. The IncN2 plasmid named as pJN24NDM1 was fully sequenced and analyzed. Genome comparative analysis showed that IncN2 plasmids harboring carbapenem-resistance genes possessed conserved backbones and variable accessory regions. Phylogenetic analysis of 87 IncN plasmids based on orthologous genes indicated that 9 IncN2 plasmids fell into one phylogenetic clade, while 4 IncN3 plasmids were in two phylogenetic clades of the 74 IncN1 plasmids. The presence of IncN2 plasmids harboring blaNDM in the high-risk clone ST405 E. coli raises serious concerns for its potential of dissemination

Rapid Determination of Antibiotic Resistance in Klebsiella pneumoniae by a Novel Antibiotic Susceptibility Testing Method Using SYBR Green I and Propidium Iodide Double Staining

Due to the broad-spectrum antibiotic usage and empirical treatments, the pathogenic bacterium, Klebsiella pneumoniae, has shown extremely high detection rates at hospitals with an increasing antibiotic resistance. Therefore, rapid detection of the antibiotic resistance is urgently required and essential for effective treatments. In this study, we evaluated the performance of a newly developed method for ultra-rapid detection of antibiotic resistance in 30–60 min in K. pneumoniae by using the SYBR Green I and propidium iodide (PI) staining. A total of 100 clinical isolates were tested for antibiotic resistance using four different antibiotics (ceftriaxone, cefepime, meropenem, and ciprofloxacin). The results showed that the SYBR Green I/PI rapid antibiotic susceptibility test (AST) could reliably detect antibiotic resistance to the four drugs in 60 min, and the results were highly concordant with the conventional AST (i.e., Kirby-Bauer method and broth microdilution method) for detection of ceftriaxone, cefepime, meropenem, and ciprofloxacin resistance with a high accuracy of 99, 96, 96, and 93%, respectively. Therefore, the rapid AST established in our study helps to enable targeted therapy to save lives and reduce the empirical use of antibiotics and ultimately the health and economic burdens of antibiotic resistance

Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants

Canine parvovirus (CPV-2) is an enteric virus causing morbidity and mortality in dogs worldwide. Since CPV-2 emerged as canine pathogen, the original CPV-2 strain has constantly evolved, and its primary variants (CPV-2a, CPV-2b, and CPV-2c) co-circulate to varying extents in canine populations worldwide. Thus, rapid and accurate laboratory diagnoses of CPV-2 variants are crucial to monitor CPV-2 evolution. Conventional methods for CPV-2 genotyping are laborious, time consuming, and determining the genotype of a CPV-2 variant often requires two or more reaction tubes. The present study developed a probe-based fluorescence melting curve analysis (FMCA) for genotyping six different CPV-2 variants (original CPV-2, CPV-2a, CPV-2b, CPV-2c, and vaccine strains of CPVpf and CPVint) in a single reaction tube using only two TaqMan probes. One of the TaqMan probes (FAM labeled) was designed to perfectly match with the target sequence of CPV-2a, this probe allows a 1-bp mismatched hybridization with the CPV-2b VP2 gene region (A4062G), and a 2-bp mismatched hybridization for CPV-2c (A4062G and T4064A); Another TaqMan probe (HEX labeled) was produced to perfectly match with the target sequence of original CPV-2, this probe enables 1-bp mismatched hybridization with the other CPV-2 variants (A3045T). Using the two TaqMan probes, all six CPV-2 variants were readily distinguished by their respective melting temperature values in a single reaction tube. The detection limits of this assay were 1–10 copies per reaction for six CPV-2 construction plasmids and no cross reactions were observed with several other common canine viruses. In this assay, co-infected samples were also directly identified via probe-based FMCA without using a mixing control; only a pure control is required. The clinical evaluation of this assay was demonstrated by analyzing 83 clinical fecal samples, among which 41 (49.39%), 8 (9.63%), and 14 (16.87%) samples were found to be positive for CPV-2a, CPV-2b, and CPV-2c, respectively. The concordance rate between probe-based FMCA and Sanger sequencing was 100%. Thus, the duplex FMCA is effective, rapid, simple, high-throughput, and straightforward for genotyping CPV-2 variants, and is useful to effectively diagnose and monitor CPV-2 epidemiology

Reduced Energy Metabolism Impairs T Cell-Dependent B Cell Responses in Patients With Advanced HBV-Related Cirrhosis

Background and AimsPatients with decompensated HBV-related liver cirrhosis (HBV D-LC) showed compromised immune responses, which manifested as a proneness to develop infections and hyporesponsiveness to vaccines, resulting in accelerated disease progression. The alterations in T cell-dependent B cell responses in this pathophysiological process were not well understood. This study aimed to investigate T cell-dependent B cell responses in this process and discuss the mechanism from the perspective of metabolism.MethodsChanges in phenotypes and subsets of peripheral B cells between HBV D-LC patients and healthy controls (HCs) were compared by flow cytometry. Isolated B cells were activated by coculture with circulating T follicular (cTfh) cells. After coculture, the frequencies of plasmablasts and plasma cells and immunoglobin levels were analyzed. Oxidative phosphorylation (OXPHOS) and glycolysis were analyzed by a Seahorse analyzer. Mitochondrial function and the AKT/mTOR pathway were analyzed by flow cytometry.ResultsThe proliferation and differentiation capacities of B cells after T cell stimulation were impaired in D-LC. Furthermore, we found that B cells from D-LC patients showed reductions in OXPHOS and glycolysis after activation, which may result from reduced glucose uptake, mitochondrial dysfunction and attenuated activation of the AKT/mTOR pathway.ConclusionsB cells from HBV D-LC patients showed dysfunctional energy metabolism after T cell-dependent activation. Understanding the regulations of B cell metabolic pathway and their changes may provide a new direction to rescue B cell hyporesponsiveness in patients with HBV D-LC, preventing these patients be infected and improving sensitivity to vaccines

An open science resource for establishing reliability and reproducibility in functional connectomics

Efforts to identify meaningful functional imaging-based biomarkers are limited by the ability to reliably characterize inter-individual differences in human brain function. Although a growing number of connectomics-based measures are reported to have moderate to high test-retest reliability, the variability in data acquisition, experimental designs, and analytic methods precludes the ability to generalize results. The Consortium for Reliability and Reproducibility (CoRR) is working to address this challenge and establish test-retest reliability as a minimum standard for methods development in functional connectomics. Specifically, CoRR has aggregated 1,629 typical individuals’ resting state fMRI (rfMRI) data (5,093 rfMRI scans) from 18 international sites, and is openly sharing them via the International Data-sharing Neuroimaging Initiative (INDI). To allow researchers to generate various estimates of reliability and reproducibility, a variety of data acquisition procedures and experimental designs are included. Similarly, to enable users to assess the impact of commonly encountered artifacts (for example, motion) on characterizations of inter-individual variation, datasets of varying quality are included

Angiotensin type 2 receptor in pancreatic islets of adult rats: a novel insulinotropic mediator Downloaded from

In the present study, we evaluated the relative abundance of angiotensin type 2 receptor (AT2R) protein in various tissues of adult rats. We found that pancreatic islets expressed the highest AT2R protein compared with all other tissues. Accordingly, we then determined the functional significance of AT2R in the endocrine pancreas in in vivo and in vitro experiments by using angiotensin II (ANG II) alone, losartan (Los; AT1R antagonist), compound 21 (C21; AT2R agonist), and PD-123319 (PD; AT2R antagonist). Experiments carried out in rats indicated that, 1) ANG II treatment significantly increased plasma insulin concentration (1.51 Ϯ 0.20 vs. 0.82 Ϯ 0.14 ng/ml, n ϭ 7, P Ͻ 0.05) in the fed state. This insulinotropic effect was further augmented by combined treatment with ANG II ϩ Los (2.31 Ϯ 0.25 ng/ml, n ϭ 7, P Ͻ 0.01). C21 also elevated insulin levels (2.13 Ϯ 0.20 ng/ml, n ϭ 7, P Ͻ 0.01), which was completely abolished by PD. 2) ANG II impaired glucose tolerance, whereas ANG II ϩ Los or C21 improved this function. 3) All treated rats displayed an enhanced insulin secretory response to a glucose challenge. 4) All treated rats displayed upregulated proinsulin 2 mRNA and insulin protein expression in the pancreas. In in vitro experiments using INS-1E cells and isolated rat islets, we found that AT2R activation significantly improved insulin biosynthesis and secretion. These results suggest that the AT2R functions as an insulinotropic mediator. AT2R and its downstream signaling pathways may be potential therapeutic targets for diabetes. compound 21; insulin production; INS-1E cells AS ONE OF THE PRIMARY ANGIOTENSIN II (ANG II) receptor subtypes, the angiotensin type 2 receptor (AT 2 R) was pharmacologically identified (10, 41) and molecularly cloned Another reason that AT 2 R actions are not well appreciated comes from the prevailing concept concerning its ontogeny (13). The AT 2 R has long been considered to express at high levels in the fetus and then dramatically decline within 24 h after birth. As a consequence, the AT 2 R in the adult animal has been believed to be a retrogressive receptor and play a minor regulatory role (5). Recent data from our laboratory, however, documented that this dogma may not be true (42). We found that adult rat (43) and mouse (15) expressed significantly higher AT 2 R protein compared with the fetus and neonate. We believe that these results suggest an important function for the AT 2 R in adulthood ANG II has been recently assumed to play a role in the regulation of pancreatic endocrine function (40). Chu and Leung (11) demonstrated that the activated AT 1 R in pancreatic islet contributes to the progressive ␤-cell failure via oxidative mechanism in the type 2 diabetic model, the db/db mouse. Given that ANG II stimulated both AT 1 R and AT 2 R, the influence of selective AT 2 R activation in the pancreas has not been previously investigated. In the current study, we first compared the relative abundance of AT 2 R in various tissues of adult rats. We found that the pancreas uniquely expressed the highest AT 2 R protein compared with all other tissues, with equal distribution in both islet and acinar components. Accordingly, we then evaluated the functional significance of AT 2 R in the endocrine pancreas. We postulated that the AT 2 R might represent a novel signaling pathway within ␤-cells to regulate insulin production and secretion. We believe that an identification of a novel pathway like this will provide an insight into the islet biology as well as a new therapeutic option to diabetes. In this experiment, the influence of AT 2 R activation or blockade on insulin biosynthesis or release was examined in vivo by using male adult rats and in vitro by using INS-1E and dissociated islets from neonatal rats. RESEARCH DESIGN AND METHODS Animals Seventy one adult male (320 -360 g) and 5 pregnant female (17-18th day gestation) Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were used in the current experiments. The adult male rats were group housed, and pregnant female rats were housed individually with standard rat food and tap water ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center and were carried out under the guidelines of the American Physiological Society and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats for tissue distribution and ontogeny of AT 2R. Eight adult male and 5 pregnant female rats were used in this experiment. The adult male rats were killed by CO 2. The tissues were removed and immediately frozen on dry ice, and then stored at Ϫ80°C. The pregnant female rats were allowed to give birth to and care for pups until the pups' brain, pancreas, and testicles were collected at the different stages of development: at 1 day, 3 days, and 1-6 wk after birth. Rats for functional experiments. Sixty three adult male rats were used in this experiment, which were assigned to five groups to receive the following treatments: vehicle control (saline), ANG II (Sigma), ANG II ϩ losartan (Los; Merck), compound 21 (C21; Vicor