Endothelin suppresses cell migration via the JNK signaling pathway in a manner dependent upon Src kinase, Rac1, and Cdc42

AbstractCell migration is a complex phenomenon that is stimulated by chemoattractive factors such as chemokines, a family of ligands for G protein-coupled receptors (GPCRs). In contrast, factors that suppress cell migration, and the mechanism of their action, remain largely unknown. In this study, we show that endothelin, a GPCR ligand, inhibits cell motility in a manner dependent upon signaling through the c-Jun N-terminal kinase (JNK) pathway. We further demonstrate that this effect is dependent upon Src kinase and small GTPases Rac1 and Cdc42. These findings provide new insight into GPCR-mediated regulation of cell migration

Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin

Botulinum neurotoxin's nontoxic HA protein binds E-cadherin to disrupt cell–cell adhesion in a species-specific manner

PCR-Dipstick-Oriented Surveillance and Characterization of mcr-1- and Carbapenemase-Carrying Enterobacteriaceae in a Thai Hospital

Colistin is used as an alternative therapeutic for carbapenemase-producing Enterobacteriaceae (CPE) infections which are spreading at a very high rate due to the transfer of carbapenemase genes through mobile genetic elements. Due to the emergence of mcr-1, the plasmid-mediated colistin resistance gene, mcr-1-positive Enterobacteriaceae (MCRPEn) pose a high risk for the transfer of mcr-1-carrying plasmid to CPE, leading to a situation with no treatment alternatives for infections caused by Enterobacteriaceae possessing both mcr-1 and carbapenemase genes. Here, we report the application of PCR-dipstick-oriented surveillance strategy to control MCRPEn and CPE by conducting the PCR-dipstick technique for the detection of MCRPEn and CPE in a tertiary care hospital in Thailand and comparing its efficacy with conventional surveillance method. Our surveillance results showed a high MCRPEn (5.9%) and CPE (8.7%) carriage rate among the 219 rectal swab specimens examined. Three different CPE clones were determined by pulsed-field gel electrophoresis (PFGE) whereas only two MCRPEn isolates were found to be closely related as shown by single nucleotide polymorphism-based phylogenetic analysis. Whole genome sequencing (WGS) and plasmid analysis showed that MCRPEn carried mcr-1 in two plasmids types—IncX4 and IncI2 with ~99% identity to the previously reported mcr-1-carrying plasmids. The identification of both MCRPEn and CPE in the same specimen indicates the plausibility of plasmid-mediated transfer of mcr-1 genes leading to the emergence of colistin- and carbapenem-resistant Enterobacteriaceae. The rapidity (<2 h) and robust sensitivity (100%)/specificity (~99%) of PCR-dipstick show that this specimen-direct screening method could aid in implementing infection control measures at the earliest to control the dissemination of MCRPEn and CPE

Achieving LDL cholesterol target levels <1.81 mmol/L may provide extra cardiovascular protection in patients at high risk: Exploratory analysis of the Standard Versus Intensive Statin Therapy for Patients with Hypercholesterolaemia and Diabetic Retinopathy study

Aims To assess the benefits of intensive statin therapy on reducing cardiovascular (CV) events in patients with type 2 diabetes complicated with hyperlipidaemia and retinopathy in a primary prevention setting in Japan. In the intension-to-treat population, intensive therapy [targeting LDL cholesterol = 2.59 to = 100 to = 2.59 to <3.10 mmol/L in patients with hypercholesterolaemia and diabetic retinopathy

Functional dissection of the Clostridium botulinum type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites.

Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption

Crystal structure and the carbohydrate and E-cadherin binding sites of the HA/B complex.

<p>The galactose binding sites of HA1 (Asn286) are colored blue and marked with dotted blue circles. The sialic acid-binding sites of HA3 (Arg528) are colored magenta and marked with dotted magenta circles. The E-cadherin binding sites of HA3 (Leu473, M508, and Lys607) are colored red and marked with dotted red circles. These sites are structurally independent. Each of the three HA monomers is colored pale cyan, pale yellow, or pink.</p

HA/B complex binds to mucins through carbohydrate recognition by HA1/B and HA3/B.

<p>(A) Mucin binding of GST-HA1/B. Mucin binding of GST, GST-HA1/B, and GST-HA1/B harboring Asn286 to Ala mutation (N286A) was assessed as described in Materials and Methods. Binding of GST-HA1/B was measured in the presence or absence of 100 mM glucose (Glc) or galactose (Gal). HA1/B preferentially bound to PGM in a galactose-binding-dependent manner. (B) Mucin binding of GST, GST-HA3/B, and GST-HA3/B harboring Arg528 to Ala mutation (R528A). Binding of GST-HA3/B was assessed with the coated mucin pretreated with (+Neu) or without 5 mU/ml neuraminidase. HA3/B preferentially bound to BSM in a sialic-acid-dependent manner. (C) Mucin binding of HA/B complex (WT) and those harboring either HA1/B N286A or HA3/B R528A, or both (HA/B harboring HA1 N286A mutation, HA1 m; HA/B harboring HA3 R528A mutation, HA3 m; HA/B harboring HA1N286A and HA3 R528A mutations, HA1 m/HA3 m). The results show that carbohydrate binding activities of HA1 and HA3 are fully functional in the HA/B complex. Values are means ± S.E.M. from three independent experiments. *<i>p</i><0.05, unpaired <i>t</i> test.</p

Carbohydrate binding of HA/B is not required for E-cadherin binding.

<p>(A) Caco-2 cells were grown on Transwell chambers, and treated with 10 nM HA/B (WT) or those harboring carbohydrate-binding-defective mutations (HA/B harboring HA1 N286A mutation, HA1 m; HA/B harboring HA3 R528A mutation, HA3 m; HA/B harboring HA1 N286A and HA3 R528A mutations, HA1 m/HA3 m) from the lower side of the chambers. HA1 mutation affected the barrier-disrupting activity, whereas HA3 mutation largely did not. (B) Caco-2 cell monolayers were treated with 10 nM HA/B or the indicated concentrations of HA/B carbohydrate-binding-defective mutant (HA1m/HA3m). These HAs were treated with the lower side of the chamber. Values are means ± S.E.M. of triplicate wells. The mutant showed sufficient activity to reduce the TER value completely when applied at higher concentrations (50 nM and 100 nM). (C) Caco-2 cells were treated the same as in A. After 1 h of treatment, cells were fixed and the basolateral surface of the cell was stained with the anti-B16S antibody. E-cadherin staining is also indicated. Binding to the basolateral surface of the cells was reduced in the carbohydrate-binding-defective mutant. Scale bar: 50 µm. (D) Varying concentrations of the recombinant E-cadherin ectodomain protein (300, 100, and 30 nM) was pulled-down with resin-bound HA/B complex or its carbohydrate-binding-defective mutant. The mutations did not affect E-cadherin binding.</p

Fluoroquinolone resistance determinants in carbapenem-resistant Escherichia coli isolated from urine clinical samples in Thailand

Background Escherichia coli is the most common cause of urinary tract infections and has fluoroquinolone (FQ)-resistant strains, which are a worldwide concern. Objectives To characterize FQ-resistant determinants among 103 carbapenem-resistant E. coli (CREc) urinary isolates using WGS. Methods Antimicrobial susceptibility, biofilm formation, and short-read sequencing were applied to these isolates. Complete genome sequencing of five CREcs was conducted using short- and long-read platforms. Results ST410 (50.49%) was the predominant ST, followed by ST405 (12.62%) and ST361 (11.65%). Clermont phylogroup C (54.37%) was the most frequent. The genes NDM-5 (74.76%) and CTX-M-15 (71.84%) were the most identified. Most CREcs were resistant to ciprofloxacin (97.09%) and levofloxacin (94.17%), whereas their resistance rate to nitrofurantoin was 33.98%. Frequently, the gene aac(6′)-Ib (57.28%) was found and the coexistence of aac(6′)-Ib and blaCTX-M-15 was the most widely predominant. All isolates carried the gyrA mutants of S83L and D87N. In 12.62% of the isolates, the coexistence was detected of gyrA, gyrB, parC, and parE mutations. Furthermore, the five urinary CREc-complete genomes revealed that blaNDM-5 or blaNDM-3 were located on two plasmid Inc types, comprising IncFI (60%, 3/5) and IncFI/IncQ (40%, 2/5). In addition, both plasmid types carried other resistance genes, such as blaOXA-1, blaCTX-M-15, blaTEM-1B, and aac(6′)-Ib. Notably, the IncFI plasmid in one isolate carried three copies of the blaNDM-5 gene. Conclusions This study showed FQ-resistant determinants in urinary CREc isolates that could be a warning sign to adopt efficient strategies or new control policies to prevent further spread and to help in monitoring this microorganism

Transcription Factor Homeobox D9 Drives the Malignant Phenotype of HPV18-Positive Cervical Cancer Cells via Binding to the Viral Early Promoter

Persistent infections with two types of human papillomaviruses (HPV), HPV16 and HPV18, are the most common cause of cervical cancer (CC). Two viral early genes, E6 and E7, are associated with tumor development, and expressions of E6 and E7 are primarily regulated by a single viral promoter: P97 in HPV16 and P105 in HPV18. We previously demonstrated that the homeobox D9 (HOXD9) transcription factor is responsible for the malignancy of HPV16-positive CC cell lines via binding to the P97 promoter. Here, we investigated whether HOXD9 is also involved in the regulation of the P105 promoter using two HPV18-positive CC cell lines, SKG-I and HeLa. Following the HOXD9 knockdown, cell viability was significantly reduced, and E6 expression was suppressed and was accompanied by increased protein levels of P53, while mRNA levels of TP53 did not change. E7 expression was also downregulated and, while mRNA levels of RB1 and E2F were unchanged, mRNA levels of E2F-target genes, MCM2 and PCNA, were decreased, which indicates that the HOXD9 knockdown downregulates E7 expression, thus leading to an inactivation of E2F and the cell-cycle arrest. Chromatin immunoprecipitation and promoter reporter assays confirmed that HOXD9 is directly associated with the P105 promoter. Collectively, our results reveal that HOXD9 drives the HPV18 early promoter activity to promote proliferation and immortalization of the CC cells