Coexistence of a novel NDM-1-encoding MDR plasmid and an IMP-4-encoding IncN-IncU hybrid plasmid in a clinical isolate of Citrobacter freundii BC73.

Objectives: To investigate the genetic characteristics and transmission mechanism of the NDM-1-, IMP-4-, and SHV-12-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii BC73. Methods: C. freundii BC73 was isolated from a urine specimen of a urological patient diagnosed with bladder cancer at a Chinese teaching hospital. Antimicrobial susceptibility testing was carried out using DL-120E susceptibility cards and DL-96A system. Whole genome sequencing (WGS) of the isolate was performed using the Illumina and Oxford Nanopore platforms to analyze the genetic context of drug resistance genes and plasmid characteristics. The phylogenetic tree was constructed and visualized by KSNP3.0 software and iTOL5.0 online database. Results: C. freundii isolate BC73 co-carrying bla NDM-1, bla IMP-4 and bla SHV-12 were multidrug-resistant. bla NDM-1 and bla IMP-4 were located on a novel IncFIB-like plasmid, pCFBC1, and an IncN-IncU hybrid plasmid, pCFBC2, respectively. The transferability of bla NDM-1 and bla IMP-4 from C. freundii BC73 to E. coli J53 was successfully demonstrated. The genetic context of the bla NDM-1 and bla IMP-4 genes were ISCR27-groEL-∆groES-cutA-dsbD-trpF-ble MBL-bla NDM-1-∆ISAba125-IS3000 and intI1-bla IMP-4-Kl.pn.13-mobC-IS6100, respectively. Additionally, two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2) were identified and numerous antimicrobial resistance genes were discovered on it. Conclusion: To our knowledge, our study represents the first characterization of a ST22 C. freundii isolate co-harboring bla NDM-1, bla IMP-4, and bla SHV-12, obtained from a urine sample. The dissemination of this MDR isolate should be of close concern in future clinical surveillance.

Genomic Characterization of Two Escherichia fergusonii Isolates Harboring mcr-1 Gene From Farm Environment.

The prevalence and transmission of mobile colistin resistance (mcr) genes have led to a severe threat to humans and animals. Escherichia fergusonii is an emerging pathogen which is closely related to a variety of diseases. However, the report of mcr genes harboring E. fergusonii is still rare. One study in Brazil reported the E. fergusonii isolates with IncHI2-type plasmids harboring mcr-1. A Chinese study reported two strains carrying mcr-1 gene with the same plasmid type IncI2. Here, we identified two strains of E. fergusonii carrying mcr-1 gene from farm environments with IncX4-type and IncI2-type plasmids, respectively. To our best knowledge, this is the first report about mcr-1 gene located on IncX4-type plasmid in E. fergusonii. We investigate the resistance mechanism of colistin-resistant Escherichia fergusonii strains 6S41-1 and 5ZF15-2-1 and elucidate the genetic context of plasmids carrying mcr-1 genes. In addition, we also investigated chromosomal mutations mediated colistin resistance in these two strains. Species identification was performed using MALDI-TOF MS and 16S rRNA gene sequencing. The detection of mcr-1 gene was determined by PCR and Sanger sequencing. S1-pulsed-field gel electrophoresis (PFGE), Southern blotting, antimicrobial susceptibility testing, conjugation experiments, complete genome sequencing, and core genome analysis were conducted to investigate the characteristics of isolates harboring mcr-1. The mcr-1 genes on two strains were both plasmids encoded and the typical IS26-parA-mcr-1-pap2 cassette was identified in p6S41-1 while a nikA-nikB-mcr-1 locus sites on the conjugative plasmid p5ZF15-2-1. In addition, Core genome analysis reveals that E. fergusonii 6S41-1 and 5ZF15-2-1 have close genetic relationships. The mcr-1 gene is located on conjugative IncI2-type plasmid p5ZF15-2-1, which provides support for its further transmission. In addition, there's the possibility of mcr-1 spreading to humans through farm environments and thereby threatening public health. Therefore, continuous monitoring and investigations of mcr-1 among Enterobacteriaceae in farm environments are necessary to control the spread.

Emergence and Genetic Characterization of Plasmid-Encoded VIM-2-Producing Pseudomonas stutzeri with Novel Integron In1998 Isolated from Cerebrospinal Fluid.

PURPOSE: To investigate the genomic and plasmid characteristics of a newly discovered Pseudomonas stutzeri strain with a bla VIM-2-carrying plasmid and novel integron In1998 isolated from a cerebrospinal fluid specimen in a teaching hospital. METHODS: Species identification was performed by MALDI-TOF MS, and bla VIM-2 was identified by PCR and Sanger sequencing. Whole-genome sequencing analysis was conducted using the Illumina NovaSeq 6000 and Oxford Nanopore platforms. Integron detection was performed using INTEGRALL. The phylogenetic tree was constructed by using kSNP3.0. Plasmid characteristics were assessed by S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, conjugation experiments, and whole-genome sequencing analysis. Comparative genomics analysis of the plasmid and genetic context of bla VIM-2 were conducted by using BLAST Ring Image Generator (BRIG) and Easyfig 2.3, respectively. RESULTS: ZDHY95, an MDR strain of P. stutzeri harboring bla VIM-2, was identified. It was sensitive only to amikacin and was resistant to carbapenems, ß-lactams, aztreonam, fluoroquinolones, and aminoglycosides. Joint S1-PFGE, Southern blot, conjugation assay, and whole-genome sequencing experiments confirmed that the bla VIM-2 gene was located within class I integron In1722 of the plasmid and that the surrounding genetic environment was 5'CS-aacA4'-30-bla VIM-2-aacA4'-3'CS. The novel class I integron In1998 was detected on the chromosome of P. stutzeri ZDHY95, and the gene cassette array was 5'CS-aacA3-aadA13-cmlA8-bla OXA-246-arr3-dfrA27-3'CS. Phylogenetic analysis showed that antimicrobial resistance gene-carrying P. stutzeri isolates were divided into two clusters, mainly containing isolates from the USA and Pakistan. CONCLUSION: A novel bla VIM-2-carrying conjugative plasmid, pZDHY95-VIM-2, was reported for the first time in P. stutzeri, elucidating the genetic environment and transfer mechanism. The gene structure of the novel class I integron In1998 was also clarified. We explored the phylogenetic relationship of P. stutzeri with drug resistance genes and suggested that Pseudomonas with metallo-ß-lactamases (MBLs) in the hospital environment may cause infection in patients with long-term intubation or after interventional surgery.

HCG18 Participates in Vascular Invasion of Hepatocellular Carcinoma by Regulating Macrophages and Tumor Stem Cells.

OBJECTIVES: To identify key genes involved in vascular invasion in hepatocellular carcinoma (HCC), to describe their regulatory mechanisms, and to explore the immune microenvironment of HCC. METHODOLOGY: In this study, the genome, transcriptome, and immune microenvironment of HCC were assessed by using multi-platform data from The Cancer Genome Atlas (n = 373) and GEO data (GSE149614). The key regulatory networks, transcription factors and core genes related to vascular invasion and prognosis were explored based on the CE mechanism. Survival analysis and gene set enrichment were used to explore pathways related to vascular invasion. Combined with single-cell transcriptome data, the distribution of core gene expression in various cells was observed. Cellular communication analysis was used to identify key cells associated with vascular invasion. Pseudo-temporal locus analysis was used to explore the regulation of core genes in key cell phenotypes. The influence of core genes on current immune checkpoint therapy was evaluated and correlations with tumor stem cell scores were explored. RESULTS: We obtained a network containing 1,249 pairs of CE regulatory relationships, including 579 differential proteins, 28 non-coding RNAs, and 37 miRNAs. Three key transcription factors, ILF2, YBX1, and HMGA1, were identified, all regulated by HCG18 lncRNA. ScRNAseq showed that HCG18 co-localized with macrophages and stem cells. CIBERSORTx assessed 22 types of immune cells in HCC and found that HCG18 was positively correlated with M0 macrophages, while being negatively correlated with M1 and M2 macrophages, monocytes, and dendritic cells. Cluster analysis based on patient prognosis suggested that regulating phenotypic transformation of macrophages could be an effective intervention for treating HCC. At the same time, higher expression of HCG18, HMGA1, ILF2, and YBX1 was associated with a higher stem cell score and less tumor differentiation. Pan cancer analysis indicated that high expression of HCG18 implies high sensitivity to immune checkpoint therapy. CONCLUSION: HCG18 participates in vascular invasion of HCC by regulating macrophages and tumor stem cells through three key transcription factors, YBX1, ILF2, and HMGA1.

SOD3 deficiency induces liver fibrosis by promoting hepatic stellate cell activation and epithelial-mesenchymal transition.

Hepatic stellate cell (HSC) activation plays an important role in the pathogenesis of liver fibrosis, and epithelial-mesenchymal transition (EMT) is suggested to potentially promote HSC activation. Superoxide dismutase 3 (SOD3) is an extracellular antioxidant defense against oxidative damage. Here, we found downregulation of SOD3 in a mouse model of liver fibrosis induced by carbon tetrachloride (CCl4 ). SOD3 deficiency induced spontaneous liver injury and fibrosis with increased collagen deposition, and further aggravated CCl4 -induced liver injury in mice. Depletion of SOD3 enhanced HSC activation marked by increased α-smooth muscle actin and subsequent collagen synthesis primarily collagen type I in vivo, and promoted transforming growth factor-ß1 (TGF-ß1)-induced HSC activation in vitro. SOD3 deficiency accelerated EMT process in the liver and TGF-ß1-induced EMT of AML12 hepatocytes, as evidenced by loss of E-cadherin and gain of N-cadherin and vimentin. Notably, SOD3 expression and its pro-fibrogenic effect were positively associated with sirtuin 1 (SIRT1) expression. SOD3 deficiency inhibited adenosine monophosphate-activated protein kinase (AMPK) signaling to downregulate SIRT1 expression and thus involving in liver fibrosis. Enforced expression of SIRT1 inhibited SOD3 deficiency-induced HSC activation and EMT, whereas depletion of SIRT1 counteracted the inhibitory effect of SOD3 in vitro. These findings demonstrate that SOD3 deficiency contributes to liver fibrogenesis by promoting HSC activation and EMT process, and suggest a possibility that SOD3 may function through modulating SIRT1 via the AMPK pathway in liver fibrosis.

GSK3ß and MCL-1 mediate cardiomyocyte apoptosis in response to high glucose.

Gestational diabetes mellitus is a risk factor for congenital heart defects. Our previous results indicated that a decrease in myocardial cells and an increase in apoptotic cells leads to heart defects under hyperglycemia, but much work remains to elucidate this important mechanism of myocardial cell apoptosis induced by high glucose (HG). In this study, we found that a decrease in GSK3ß phosphorylation on Ser9 occurred concomitantly with HG-induced cardiomyocyte apoptosis and in the heart tissues of the offspring of diabetic rats in vitro and in vivo. Decreases in GSK3ß (Ser9) phosphorylation in response to HG were remarkably restored after treatment with SC79, an activator of the Akt signaling pathway. SB216763, an effective inhibitor of the GSK3ß signaling pathway, suppressed HG-induced apoptosis in cardiomyocytes. Further studies showed a decrease in the expression of the anti-apoptotic protein MCL-1 was associated with GSK3ß-mediated apoptosis. MCL-1 overexpression partly inhibits HG-induced apoptosis in cardiomyocytes. Herein, this study revealed the roles of GSK3ß and MCL-1 in modulating HG-induced cardiomyocyte apoptosis and maternal diabetes-induced abnormalities.

[Types and risk factors of arrhythmia on young patients with acute coronary syndrome in Henan province].

OBJECTIVE: The types and risk factors of arrhythmia were analyzed on acute coronary syndrome (ACS) patients under the age of 44 years who were hospitalized in Henan province between September 2009 to June 2012. METHODS: Medical records of eligible patients were obtained from the information system of the First Affiliated Hospital of Zhengzhou University teleconsultation information center. Middle aged and elderly ACS patients who were hospitalized at the same period served as controls. Data on arrhythmia types, blood pressure, thyroid disease, respiratory sleep apnea syndrome, smoking history, history of alcohol consumption, eating habits, family history of early-onset arrhythmia, laboratory tests were analyzed. RESULTS: (1) Arrhythmia was detected in 110 out of young ACS patients (55%), which was significantly lower than that in the elderly ACS patients (71.05%, P < 0.01). (2) The top three arrhythmias in young ACS patients were: sinus tachycardia (30.50%), the premature ventricular contractions (19.00%), atrial flutter/atrial fibrillation (16.50%). Incidence of sinus tachycardia, atrial flutter/atrial fibrillation were significantly higher while incidence of ventricular tachycardia, ventricular fibrillation, paroxysmal supraventricular tachycardia were significantly lower in young ACS patients than in middle-aged ACS patients (all P < 0.05). The incidence of sinus tachycardia was higher while incidence of ventricular premature accelerated ventricular spontaneous cardiac rhythm, ventricular tachycardia, ventricular fibrillation, non-paroxysmal supraventricular tachycardia, atrial flutter/atrial fibrillation, paroxysmal supraventricular tachycardia, sinus bradycardia, nodal escape, atrioventricular block were significantly lower in young ACS patients than in elderly ACS patients (all P < 0.05). (3) Body mass index, incidence of smoking, coronary three-vessel disease, drinking, eating salty foods, thyroid dysfunction, sleep apnea were significantly higher in youth ACS patients with arrhythmia than in young ACS patients without arrhythmia (all P < 0. 05). (4) Logistic regression analysis found that number of diseased coronary vessels (OR = 24.293), smoking (OR = 1.112) and alcohol consumption (OR = 1.039) were independent risk factor for developing arrhythmia in young ACS patients from Henan province. CONCLUSIONS: The main types of arrhythmia are sinus tachycardia, premature ventricular contractions, atrial flutter/atrial fibrillation and the major risk factors related to the arrhythmia are number of diseased coronary vessels, smoking and alcohol consumption in young ACS patients from Henan province.

[Effect of the smoking cessation services in the out-patient department for patients with coronary heart disease].

OBJECTIVE: To evaluate the effects and clinical prognosis of out-patient department-based smoking cessation services for coronary heart disease (CHD) patients. METHODS: A total of 140 smoking patients diagnosed with coronary heart disease in our cardiovascular department were randomly divided into the intensive smoking cessation clinic follow-up group (intervention group, patients were informed on the importance and methods to quit smoking at the first visit and reminded for that at months interval for 6 months, n = 70) and the conventional treatment group (control group, n = 70). After 6 months, the smoking status, cardiovascular event rates, drug usage, out-patient medical costs and quality of life were compared between the two groups. RESULTS: Age, gender, concomitant diseases, drug usage were similar between the two groups at baseline (all P > 0.05). After 6 months, smoking quit rate [34.2% (24/70) vs. 5.7% (4/70), P < 0.01], drug use rates: lipid-lowering drugs [95.3% (67/70) vs. 80.4% (56/70)], ß blockers [82.4% (57/70) vs. 41.3% (28/70)], and ACEI/ARB [61.4% (43/70) vs. 34.4% (24/70)] were significantly higher in the intervention group than in the control group, while total cardiovascular event rates [21.4% (15/70) vs. 47.1% (33/70), P < 0.01] and out-patient medical costs (3789.3 RMB vs. 4984.2 RMB, P < 0.01) were significantly lower in the intervention group than in the control group. The quality of life scores derived from MYO health survey questionnaire was significantly higher in the intervention group than in the control group (P < 0.01). The top three reasons responsible for continuous smoking for all patients failed to quit smoking were: (1) others smoked more than me and still alive and healthy [90.3% (56/62)]; (2) smoking helped me to keep relaxed and reduce trouble in daily work and life [70.9% (44/62)]; (3) smoking was essential while chatting and drinking with friends [66.1% (41/62)]. The overall satisfactory rate to this smoking cessation program was 42.8% and the satisfactory rate was up to 50.0% by patients. CONCLUSIONS: Intensive outpatient smoking cessation follow-up program can significantly improve the smoking cessation rates, the guideline drug use rate and the quality of life while reduce medical costs for coronary heart disease patients.

Protective effects of N-acetylcysteine on brain-dead rat liver.

BACKGROUND: Brain-dead donors have been the main sources in organ transplantation. But many studies show that brain-death affects the organ's function after transplantation. This study was undertaken to investigate liver injury after brain-death in rats and the protective effects of N-acetyleysteine (NAC) on liver injury. METHODS: A total of 30 Wistar rats were randomized into 3 groups: normal control group (C), brain-dead group (B), and NAC pretreatment group (N). At 4 hours after the establishment of a brain-dead model, serum was collected to determine the levels of ALT, AST, TNF-alpha and hyaluronic acid (HA). Hepatic tissue was obtained for electron microscopic examination. RESULTS: At 4 hours, the levels of ALT, AST, TNF-alpha, and HA in group N were significantly higher than those in group C, but these parameters were significantly lower than those in group B. Electron microscopy showed activated Kupffer cells, denuded sinusoidal endothelial cells (SECs), and widened fenestration in group B, but eliminated activation of Kupffer cells and intact SECs in group N. CONCLUSION: Brain death can cause liver injury, and N-acetyleysteine can protect the liver from the injury.