Mosquito-only flaviviruses,isolated from <Emphasis Type="Italic">Aedes albopictus</Emphasis> in Slovenia: results of a pilot mosquito monitoring program

Autochthonous transmissions of mosquito-borne diseases were described in close proximity to Slovenia where the knowledge of mosquito species and the viruses they transmit remains very scarce. Therefore we aimed to assess the burden mosquitoes bring to the local community and to test mosquitoes for the presence of the selected zoonotic arboviruses, i. e. Dengue (DENV), West Nile (WNV) and Chikungunya (CHIKV). A pilot mosquito monitoring was conducted in 2012 within two municipalities based on adult mosquito trapping. Altogether 2007 adult mosquitoes were caught. Females of Aedes albopictus and Culex pipiens represented the majority of the total mosquito catch. In sites with more households and nearby greenery, catches of Ae. albopictus were more abundant. Highest numbers of Cx. pipiens females were caught close to a nature reserve, where many bird species stop during their migrations. Trapping results are consistent with both species’ feeding behaviour. Female mosquitoes were grouped into 58 pools according to species, location and date of catch. Real time RT-PCRs for WNV, DENV and CHIKV were performed, but no arboviral genomes were detected in any of the tested mosquito pools. However, amplicons were obtained in three pools when using generic RT-PCR markers for flaviviruses. The viruses detected were propagated and isolated in mosquito cell line C3/36 and identified with sequencing as mosquito-only flaviviruses (MOFs). The important contributions of this study are a successful first isolation of MOFs and confirmation of the presence of the invasive mosquito Ae. albopictus in Slovenia.     

Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear

Introduction

Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated.

Materials and Methods

Murine NIH 3T3 fibroblasts—genetically modified to produce BDNF—were labelled with MB.

Results

Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release.

Discussion

Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest.     

High Prevalence of Multidrug Resistance and Random Distribution of Mobile Genetic Elements Among Uropathogenic Escherichia coli (UPEC) of the Four Major Phylogenetic Groups

One hundred and ten UTI Escherichia coli strains, from Ljubljana, Slovenia, were analyzed for antibiotic resistances, mobile DNA elements, serotype, and phylogenetic origin. A high prevalence of drug resistance and multidrug resistance was found. Twenty-six percent of the isolates harbored a class 1 integron, while a majority of the strains (56%) harbored rep sequences characteristic of F-like plasmids. int as well as rep sequences were found to be distributed in a random manner among strains of the four major phylogenetic groups indicating that all groups have a similar tendency to acquire and maintain mobile genetic elements frequently associated with resistance determinants.     

Modeling the bound conformation of Pemphigus Vulgaris-associated peptides to MHC Class II DR and DQ Alleles

Background

Pemphigus vulgaris (PV) is a severe autoimmune blistering disorder characterized by the presence of pathogenic autoantibodies directed against desmoglein-3 (Dsg3), involving specific DR4 and DR6 alleles in Caucasians and DQ5 allele in Asians. The development of sequence-based predictive algorithms to identify potential Dsg3 epitopes has encountered limited success due to the paucity of PV-associated allele-specific peptides as training data.

Results

In this work we constructed atomic models of ten PV associated, non-associated and protective alleles. Nine previously identified stimulatory Dsg3 peptides, Dsg3 96–112, Dsg3 191–205, Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 380–394, Dsg3 763–777, Dsg3 810–824 and Dsg3 963–977, were docked into the binding groove of each model to analyze the structural aspects of allele-specific binding.

Conclusion

Our docking simulations are entirely consistent with functional data obtained from in vitro competitive binding assays and T cell proliferation studies in DR4 and DR6 PV patients. Our findings ascertain that DRB1*0402 plays a crucial role in the selection of specific self-peptides in DR4 PV. DRB1*0402 and DQB1*0503 do not necessarily share the same core residues, indicating that both alleles may have different binding specificities. In addition, our results lend credence to the hypothesis that the alleles DQB1*0201 and *0202 play a protective role by binding Dsg3 peptides with greater affinity than the susceptible alleles, allowing for efficient deletion of autoreactive T cells.     

Optimization of the DGGE band identification method

Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucleotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis.     

Fatal Myelotoxicity After Azathioprine Treatment

Azathioprine and 6-mercaptopurine have been used for many years in the treatment of inflammatory bowel disease. Approximately 0.3% of the population are homozygous for variant alleles associated with extremely low thiopurine S-methyltransferase enzyme activity. We describe the case of a young patient with ulcerative colitis, homozygous for TPMT?3A alleles, who suffered fatal azathioprine-induced myelotoxicity after standard dosing with azathioprine. Screening for decreased activity of TPMT in patients prior to azathioprine treatment is advised to minimize the risk of drug-induced toxicity.     

Fractionation of Rat Liver tRNA by Reversed-Phase High Performance Liquid Chromatography: Isolation of ISO-tRNAsPro

Abstract

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual ³H-labeled aminoacids. Here is reported the case of tRNA^Pro, which gave three well separated radioactive peaks, most likely corresponding to tRNA^Pro isoacceptor species.     

The use of a food supplementation with D-phenylalanine, L-glutamine and L-5-hydroxytriptophan in the alleviation of alcohol withdrawal symptoms

We described the use of a food supplementation with D-phenylalanine, L-glutamine and L-5-hydroxytriptophan in the alleviation of alcohol withdrawal symptoms in patients starting a detoxification therapy. Since abstinence from ethanol causes a hypodopaminergic and a hypoopioidergic environment in the reword system circuits, manifesting with withdrawal symptoms, food supplements that contains D-phenylalanine a peptidase inhibitor (of opioide inactivation) and L-amino-acids (for dopamine synthesis) were used to replenish a lack in neurotransmitters and alleviate the symptoms of alcohol withdrawal. 20 patients suffering from alcohol addictions starting a detoxification therapy have been included in a prospective, randomized, double blind study. The patients have been randomly devided in two groups. One group recieved for a period of 40 days a food supplement containing D-phenylalanine, L-glutamine and L-5-hydroxytriptophan (investigation group), and the control (placebo) group. On the first day of hospitalization the patients performed a SCL-90-R test, and blood samples were taken for measuring liver enzymes, total bilirubin, unbound cortisol and lymphocyte populations. The same was done on the 40th day of hospitalization. During the therapy a significant decrease in SCL-90-R psychiatric symptoms scores and a significant increase in CD4 lymphocyte count was observed in the investigation group. The cortisol values were significantly, but equally decreased in both groups, the same was with the liver enzymes and the total bilirubin values. We conclude that abstinence causes a major stress for the patients. The use of food supplement containing D-phenylalanine, L-glutamine and L-5-hydroxytriptophan alleviates the withdrawal symptoms and causes a rise in CD4 lymphocyte population, but it dose not affect the serum cortisol levels, which are probably more affected by liver inflammation and the liver restitution.