Comparison of the Effects of Methadone and Heroin on Human <Emphasis Type="Italic">ether</Emphasis>-<Emphasis Type="Italic">à</Emphasis>-<Emphasis Type="Italic">go</Emphasis>-<Emphasis Type="Italic">go</Emphasis>-Related Gene Channels

Torsades de pointes (TdP) is a life-threatening form of ventricular arrhythmia that occurs under conditions of delayed cardiac repolarization indicated by prolonged QT intervals in ECG recordings. The main mechanism of QT prolongation and TdP is block of the rapid component of the cardiac delayed rectifier K⁺ current (I_Kr), which is encoded by hERG (human ether-à-go-go-related gene). The opioid agonist methadone has previously been demonstrated to inhibit hERG currents, and there are reports of serious cardiac arrhythmias and deaths from TdP and ventricular fibrillation in patients taking methadone. The aim of the present study was to compare the effects of the opioid agonists methadone and heroin (3,6-diacetylmorphine) on hERG currents stably expressed in human embryonic kidney (HEK 293) cells using the whole-cell configuration of the patch-clamp technique. Both methadone and heroin inhibit hERG currents in a concentration-dependent manner. The following values were calculated for IC₅₀ (concentration causing half-maximal inhibition) and n (the Hill coefficient): 4.8 μM and 0.9 for methadone, 427 μM and 0.7 for heroin. In conclusion, the potency for block of hERG currents is about 100-fold lower for heroin when compared to methadone.     

Longevity-modulating effects of symbiosis: insights from <Emphasis Type="Italic">Drosophila</Emphasis>–<Emphasis Type="Italic">Wolbachia</Emphasis> interaction

Microbial communities are known to significantly affect various fitness components and survival of their insect hosts, including Drosophila. The composition of symbiotic microbiota has been shown to change with the host’s aging. It is unclear whether these changes are caused by the aging process or, vice versa, they affect the host’s aging and longevity. Recent findings indicate that fitness and lifespan of Drosophila are affected by endosymbiotic bacteria Wolbachia. These effects, however, are inconsistent and have been reported both to extend and shorten longevity. The main molecular pathways underlying the lifespan-modulating effects of Wolbachia remain unclear, however insulin/insulin-like growth factor, immune deficiency, ecdysteroid synthesis and signaling and c-Jun N-terminal kinase pathways as well as heat shock protein synthesis and autophagy have been proposed to play a role. Here we revise the current evidence that elucidates the impact of Wolbachia endosymbionts on the aging processes in Drosophila.     

Genetic epidemiology of MODY in the Czech republic: new mutations in the MODY genes <Emphasis Type="Italic">HNF-4α</Emphasis>, <Emphasis Type="Italic">GCK</Emphasis> and <Emphasis Type="Italic">HNF-1α</Emphasis>

AIMS/HYPOTHESIS: The aim of this study was to examine the prevalence and nature of mutations in HNF4alpha/MODY1, GCK/MODY2 and HNF-1alpha/MODY3 genes in Czech subjects with clinical diagnosis of MODY. METHODS: We studied 61 unrelated index probands of Czech origin (28 males, 33 females) with a clinical diagnosis of MODY and 202 family members. The mean age of probands was 22.7+/-12.0 years (range, 6-62) and the mean age at the first recognition of hyperglycaemia was 14.7+/-6.0 years (range, 1-25). The promotor and coding regions inclusive intron exon boundaries of the HNF-4alpha, GCK and HNF-1alpha genes were examined by PCR-dHPLC (HNF-1alpha and GCK) and direct sequencing. RESULTS: We identified 20 different mutations in the HNF-4alpha, GCK and HNF-1alpha in 29 families (48% of all families studied), giving a relative prevalence of 5% of MODY1, 31% of MODY2 and 11.5% of MODY3 among the Czech kindred with MODY. Three of 3, 10 of 11 and 1 of 6 of the mutations identified in HNF-4alpha, GCK and HNF-1alpha respectively, were new. CONCLUSION/INTERPRETATION: Of the families 48% carried mutations in the MODY1-3 genes and of the identified mutations 70% were new. In 52% of Czech families with clinical characteristics of MODY, no mutations were found in the analysed genes. This finding shows that the majority of MODY mutations in a central European population are local and that other MODY genes could be responsible for autosomal dominant transmission of diabetes mellitus.     

In situ validation of VEGFR-2 and <Emphasis Type="Italic">α</Emphasis><Subscript><Emphasis Type="Italic">v</Emphasis></Subscript><Emphasis Type="Italic">ß</Emphasis><Subscript>3</Subscript> integrin as targets for breast lesion characterization

Vascular endothelial growth factor receptor 2 (VEGFR-2) and α _v ß ₃ integrin are the most frequently addressed targets in molecular imaging of tumor angiogenesis. In preclinical studies, molecular imaging of angiogenesis has shown potential to detect and differentiate benign and malignant lesions of the breast. Thus, in this retrospective clinical study employing patient tissues, the diagnostic value of VEGFR-2, α _v ß ₃ integrin and vascular area fraction for the diagnosis and differentiation of breast neoplasia was evaluated. To this end, tissue sections of breast cancer (n = 40), pre-invasive ductal carcinoma in situ (DCIS; n = 8), fibroadenoma (n = 40), radial scar (n = 6) and normal breast tissue (n = 40) were used to quantify (1) endothelial VEGFR-2, (2) endothelial α _v ß ₃ integrin and (3) total α _v ß ₃ integrin expression, as well as (4) the vascular area fraction. Sensitivity and specificity to differentiate benign from malignant lesions were calculated for each marker by receiver operating characteristics (ROC) analyses. Whereas vessel density, as commonly used, did not significantly differ between benign and malignant lesions (AUROC: 0.54), VEGFR-2 and α _v ß ₃ integrin levels were gradually up-regulated in carcinoma versus fibroadenoma versus healthy tissue. The highest diagnostic accuracy for differentiating carcinoma from fibroadenoma was found for total α _v ß ₃ integrin expression (AUROC: 0.76), followed by VEGFR-2 (AUROC: 0.71) and endothelial α _v ß ₃ integrin expression (AUROC: 0.68). In conclusion, total α _v ß ₃ integrin expression is the best discriminator between breast cancer, fibroadenoma and normal breast tissue. With respect to vascular targeting and molecular imaging of angiogenesis, endothelial VEGFR-2 appeared to be slightly superior to endothelial α _v ß ₃ for differentiating benign from cancerous lesions.     

Analysis of the 3′ Variable Region of the <Emphasis Type="Italic">cagA</Emphasis> Gene of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Isolated in Koreans

CagA protein of Helicobacter pylori is injected into epithelial cells, and it undergoes tyrosine phosphorylation, resulting in inducing cytoskeletal rearrangements. A few studies have suggested that the number of CagA tyrosine phosphorylation motifs (EPIYA) and subtypes of CagA were associated with gastric cancer. This study was performed to characterize the 3' variable regions of the cagA gene of H. pylori and to investigate whether or not there is any relationship between the diversities of cagA and the disease outcome in Korea. Seventy-nine patients (chronic gastritis, 15; duodenal ulcer, 27; benign gastric ulcer, 18; gastric cancer, 19) were enrolled. Biopsy specimens were taken from the antrum for H. pylori culture, and genomic DNA was extracted. PCR and DNA sequence analysis was carried out for the 3′ variable region of the cagA gene. Seventy-eight strains (98.8%) contained three EPIYA motifs and one strain (1.2%) isolated from a patient with duodenal ulcer contained four EPIYA motifs. Seventy-six strains (96.2%) were the East Asian type. In conclusion, there was no significant difference between the number of EPIYA motifs or CagA subtypes and various gastroduodenal diseases in Korea.     

Levels of (1→3)-β-D-glucan, <Emphasis Type="Italic">Candida</Emphasis> mannan and <Emphasis Type="Italic">Candida</Emphasis> DNA in serum samples of pediatric cancer patients colonized with <Emphasis Type="Italic">Candida</Emphasis>species

Background  

Surveillance cultures may be helpful in identifying patients at increased risk of developing invasive candidiasis. However, only scant information exists on the effect of Candida colonization on serum levels of diagnostic biomarkers. This prospective surveillance study determined the extent of Candida colonization among pediatric cancer patients and its possible impact on serum levels of (1-3)-β-D-glucan (BDG), Candida mannan and Candida DNA.     

<Emphasis Type="Italic">Corynebacterium</Emphasis> species nasopharyngeal carriage in asymptomatic individuals aged ≥ 65 years in Germany

Purpose

The prevalence of protective anti-diphtheria toxin antibodies decreases with age. Therefore, the elderly might serve as reservoir for potentially toxigenic Corynebacterium (C.) species (C. diphtheriae, C. ulcerans, and C. pseudotuberculosis). This study aimed to examine the colonization rate of the nasopharynx with corynebacteria of individuals aged 65 years and older.

Methods

In the period from October 2012 to June 2013, nasal and throat swabs were taken from 714 asymptomatic subjects aged 65–106 years (average age 77.2) at three regions in Germany and investigated for Corynebacterium species.

Results

A total of 402 strains of Corynebacterium species were isolated from 388 out of 714 asymptomatic subjects (carriage rate 54.3%). The carriage rate was significantly higher in study participants living in retirement homes (68.4%) compared to those living autonomously at home (51.1%). Strains were isolated mostly from the nose (99%). Corynebacterium accolens was the most often isolated species (39.8%), followed by C. propinquum (24.1%), C. pseudodiphtheriticum (19.4%), and C. tuberculostearicum (10.2%). No C. diphtheriae, C. ulcerans, and C. pseudotuberculosis strains were isolated. A subsample of 74 subjects was tested serologically for anti-diphtheria antibodies. Protective anti-diphtheria toxin antibodies were found in 29.7% of the subjects; 70.3% showed no protective immunity.

Conclusions

These results suggest that carriage of potentially toxigenic corynebacteria is very rare among people aged 65 and older in Germany. However, the low prevalence of protective anti-diphtheria toxin antibodies might pose a risk for acquiring diphtheria especially for the elderly.

     

<Emphasis Type="Italic">PAX3/7</Emphasis>–<Emphasis Type="Italic">FOXO1</Emphasis> fusion status in older rhabdomyosarcoma patient population by fluorescent in situ hybridization

Purpose  

In pediatric alveolar rhabdomyosarcoma, the PAX3–FOXO1 and PAX7–FOXO1 gene fusions are prognostic indicators, while little is known concerning this disease in older patients. To determine whether PAX3/7–FOXO1 fusion gene status correlates with outcome in adolescent, young adult, and adult rhabdomyosarcoma patients, the histological, immunohistochemical, and clinical characteristics of 105 patients followed at The University of Texas MD Anderson Cancer Center from 1957 to 2001 were evaluated.     

Does Cyclophosphamide Play a Protective Role Against Neuronal Loss in Chronic <Emphasis Type="Italic">T.</Emphasis><Emphasis Type="Italic"> cruzi</Emphasis> Infection?

In this study, we verified the possible role of cyclophosphamide (CY) in protecting or not against neuronal losses in young and aged male Calomys callosus chronically infected with the MORC-1 strain of Trypanosoma cruzi through numerical quantification of neurons from the myenteric plexus of the colon and quantification of nitric-oxide concentration (NO) during the acute and chronic phase of infection. For this purpose, groups of young C. callosus were infected with the MORC-1 strain of T. cruzi. A group of infected animals received i.p. 0.2 mg/ml genuxal dissolved in distilled water treatment with CY. NO concentration in aged animals displayed reduced levels when compared to those found in young animals. No significant alterations in the number of neurons were observed in young animals, but for aged ones, a protective role of CY in reducing neuron loss was noted, in addition to enhancing the neuronal volume, area, and perimeter. These results suggest that CY administration, depending on the dose and time span, can act as a protective agent against neuronal losses.     

<Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG-itis and Cervical Lymphadenitis due to <Emphasis Type="Italic">Salmonella enteritidis</Emphasis> in a Patient with Complete Interleukin-12/-23 Receptor <Emphasis Type="Italic">β</Emphasis>1 Deficiency

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare disorder with predisposition to severe, sometimes lethal, disease caused by otherwise poorly virulent, non-tuberculous environmental mycobacteria and poorly virulent salmonellae. In patients with MSMD, mutations have been identified in five genes that encode for the proteins IL-12/IL-23p40, IL-12/ IL-23Rβ1, IFN-コR1, IFN-γR2 and STAT1. These proteins play important roles in the type-1 cytokine pathway, which is crucial for human host defence against intracellular pathogens such as mycobacteria and salmonellae. We report a girl with mild Mycobacterium bovis Bacille Calmette–Guérin (BCG) disease and Salmonella enteritidis cervical lymphadenitis. Despite treatment, she has remained a fecal carrier of S. enteritidis for the past 14 years. She was found to have complete IL-12/IL-23Rβ1 deficiency. A homozygous r.518G>C IL12RB1 mutation was identified, leading to a non-functional R173P substitution in the IL-12/IL-23Rβ1 protein. This mutation abrogated IL-12/IL-23Rβ1 cell-surface expression and resulted in complete lack of T cell responsiveness to both IL-12 and IL-23.     

Antimicrobial resistance in <Emphasis Type="Italic">Enterobacteriaceae</Emphasis> from healthy broilers in Egypt: emergence of colistin-resistant and extended-spectrum β-lactamase-producing <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

Poultry remains one of the most important reservoir for zoonotic multidrug resistant pathogens. The global rise of antimicrobial resistance in Gram-negative bacteria is of reasonable concern and demands intensified surveillance.

Methods

In 2016, 576 cloacal swabs were collected from 48 broiler farms located in five governorates in northern Egypt. Isolates of Enterobacteriaceae could be cultivated on different media and were identified by MALDI-TOF MS and PCR. Escherichia coli isolates were genotyped by DNA-microarray-based assays. The antimicrobial susceptibility to 14 antibiotics was determined and resistance-associated genes were detected. The VITEK-2 system was applied for phenotypical confirmation of extended-spectrum β-lactamase-producing isolates. The determination of colistin resistance was carried out phenotypically using E-test and genotypically using PCR for detection of the mcr-1 gene.

Results

Out of 576 samples, 72 representatives of Enterobacteriaceae were isolated and identified as 63 E. coli (87.5%), 5 Enterobacter cloacae (6.9%), 2 Klebsiella pneumoniae (2.8%) and 2 Citrobacter spp. (2.8%). Seven out of 56 cultivated E. coli (12.5%) were confirmed as ESBL-producing E. coli and one isolate (1.8%) as ESBL/carbapenemase-producing E. coli. Five out of 63 E. coli isolates (7.9%) recovered from different poultry flocks were phenotypically resistant to colistin and harboured mcr-1 gene.

Conclusions

This is the first study reporting colistin resistance and emergence of multidrug resistance in Enterobacteriaceae isolated from healthy broilers in the Nile Delta region, Egypt. Colistin-resistant E. coli in poultry is of public health significance. The global rise of ESBL- and carbapenemase-producing Gram-negative bacteria demands intensified surveillance. ESBL-producing E. coli in poultry farms in Egypt are of major concern that emphasizes the possibility of spread of such strains to humans. The results also reinforce the need to develop strategies and to implement specific control procedures to reduce the use of antibiotics.

     

Emerging causes of traveler’s diarrhea: <Emphasis Type="Italic">Cryptosporidium,Cyclospora, Isospora</Emphasis>, and <Emphasis Type="Italic">Microsporidia</Emphasis>

Travel is a risk factor for acquiring infection with a sporeforming protozoa: Cryptosopridium, Cyclospora, Microsporidia, and Isospora. Certain travel destinations have a high disease burden and intense exposure. Patients present with persistent diarrhea and a history of recent travel to a developing country in the tropics. Very mild infections may be underdiagnosed and may cause typical traveler’s diarrhea. In a patient with a history of travel and persistent diarrhea unresponsive to the usual antibiotic and antidiarrhea treatment, stool studies for all four of these protozoa infections should be performed. If immune status is normal and the disease is mild, symptomatic therapy may suffice. Effective treatment is available for Cyclospora, Microsporidia, and Isospora.     

Hypertension Due to Loss of Clock: Novel Insight From the Molecular Analysis of <Emphasis Type="Italic">Cry1</Emphasis>/<Emphasis Type="Italic">Cry2</Emphasis>–Deleted Mice

In our consumer-oriented society, in which productivity requires around-the-clock activity and demanding shift work, the biologic system that regulates our internal rhythms is being compromised. Poor sleep patterns and hectic lifestyle are detrimental to harmonious physiological and metabolic body systems, with severe impact on public health. Over a trillion peripheral cellular clocks throughout the body, supervised by the master clock located in the hypothalamic suprachiasmatic nucleus, govern most aspects of physiology and behavior. To exemplify the importance of the biologic clock for health, we have recently demonstrated that mice that are arrhythmic because of the deletion of Cry1 and Cry2 clock genes suffer from salt-sensitive hypertension. In these mice, a novel 3β-hydroxyl-steroid dehydrogenase (3β–Hsd) gene under clock control is severely overexpressed specifically in aldosterone-producing cells in the adrenal cortex, leading to hyperaldosteronism and ultimately to salt-sensitive hypertension. The human homologue of this aldosterone-producing, cell-specific enzyme was also characterized and represents a new possibility in the pathogenesis of hypertension.     

Deletion of <Emphasis Type="Italic">Ia-2</Emphasis> and/or <Emphasis Type="Italic">Ia-2β</Emphasis> in mice decreases insulin secretion by reducing the number of dense core vesicles

Aims/hypothesis  

Islet antigen 2 (IA-2) and IA-2β are dense core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes. The present experiments were initiated to test the hypothesis that the knockout of the genes encoding these proteins impairs the secretion of insulin by reducing the number of DCV.     

Post-translational protein modifications in type 1 diabetes: a role for the repair enzyme protein-<Emphasis Type="SmallCaps">l</Emphasis>-isoaspartate (<Emphasis Type="SmallCaps">d</Emphasis>-aspartate) <Emphasis Type="Italic">O</Emphasis>-methyltransferase?

Aims/hypothesis

Post-translational modifications, such as isomerisation of native proteins, may create new antigenic epitopes and play a role in the development of the autoimmune response. Protein-l-isoaspartate (d-aspartate) O-methyltransferase (PIMT), encoded by the gene PCMT1, is an enzyme that recognises and repairs isomerised Asn and Asp residues in proteins. The aim of this study was to assess the role of PIMT in the development of type 1 diabetes.

Materials and methods

Immunohistochemical analysis of 59 normal human tissues was performed with a monoclonal PIMT antibody. CGP3466B, which induces expression of Pcmt1, was tested on MIN6 and INS1 cells, to assess its effect on Pcmt1 mRNA and PIMT levels (RT-PCR and western blot) and apoptosis. Forty-five diabetes-prone BioBreeding (BB) Ottawa Karlsburg (OK) rats were randomised to receive 0, 14 or 500 μg/kg (denoted as the control, low-dose and high-dose group, respectively) of CGP3466B from week 5 to week 20.

Results

A high level of PIMT protein was detected in beta cells. CGP3466B induced a two- to threefold increase in Pcmt1 mRNA levels and reduced apoptosis by 10% in MIN6 cells. No significant effect was seen on cytokine-induced apoptosis or PIMT protein levels in INS1 cells. The onset of diabetes in the BB/OK rats was significantly delayed (85.6?±?9.0 vs 84.3?±?6.8 vs 106.6?±?13.5 days, respectively; p?p?p?

Conclusions/interpretation

The results support a role for post-translational modifications and PIMT in the development of type 1 diabetes in the diabetes-prone BB rat, and perhaps also in humans.