Molecular detection of Babesia canis in Dermacentor reticulatus ticks collected in Slovakia

Dermacentor reticulatus ticks are recognized as the most important vectors of Babesia canis, the aetiological agent of canine babesiosis occurring throughout Europe. Vector competence of D. reticulatus for B. canis is well described and experimentally determined; however, by using molecular analysis it was proven so by one recent study in Russia. Herein, the additional molecular evidence of B. canis infection in D. reticulatus ticks collected in Slovakia is provided. Using PCR followed by sequencing of distinctive amplicons we determined the presence of Babesia canis canis in one of 100 tested adult ticks. Two zoonotic pathogens, Francisella tularensis and Coxiella burnetii, were previously isolated from D. reticulatus ticks in Slovakia. In our samples, we detected only the presence of F. tularensis.     

Individuation of monoclonal anti-HPV16 E7 antibody linear peptide epitope by computational biology

We applied computational biology to identify the linear amino acid sequence recognized by a mouse monoclonal antibody raised against the full length HPV16 E7 oncoprotein. Computer-assisted search for the epitopic peptide used two parameters: the capability of E7 peptides to bind to MHC class II molecules, and the similarity level of the oncoprotein sequence to the mouse proteome. We report that anti-E7 mAb recognized the peptide having both high binding potential to MHC II molecules and low level of molecular mimicry to mouse proteome. Peptide ability to bind to MHC II molecules appears a necessary but not sufficient condition to determine peptide immunodominance, by needing to be supported by a low degree of peptide similarity to the host’s proteome.     

Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.     

Soluble RAGE, diabetic nephropathy and genetic variability in the AGER gene

Diabetes mellitus, especially when complicated with decline of renal function due to diabetic nephropathy (DN), is associated with accumulation of advanced glycation end products (AGEs) exerting their adverse effects via receptor of AGE (RAGE). Soluble RAGE (sRAGE) is a truncated form of RAGE functioning as an inhibitor of AGE-mediated signalling. We studied relationships between sRAGE, renal function and genetic variability in the AGER gene in diabetic subjects. Study comprised a total of 265 diabetics (type 1 or 2 or LADA) with normoalbuminuria (n = 94) or DN (n = 171). sRAGE (assessed by ELISA) was significantly higher in DN than normoalbuminuria subjects (P = 0.007) and positively correlated with age, S-urea, S-creatinine and albuminuria and AGEs (determined spectrofluorimetrically), negatively with GFR (all P < 0.05); however, multivariate regression revealed that GFR was the only independent variable associated with sRAGE (P = 0.047). sRAGE did not correspond with carrier state of risk-haplotype copies (RAGE2) (P > 0.05). In conclusion, GFR is a principal determinant of sRAGE concentration and gradual sRAGE increase in subjects with advancing impairment of renal function is paralleled by AGEs.     

Chemical carcinogenesis. II. Imidazole-ring-opened derivatives from 7-ethyl- and 1,7-diethylguanosine

The UV-spectral and chromatographic analyses of the derivatives of the two synthetic standards 7-ethylguanosine and 1,7-diethylguanosine are here reported. The derivatives obtained from the dialkyl compound exhibit a striking similarity to those found in the "pyrimidine-nucleotide-like" fraction of rat liver tRNA ethylated in vivo by ethionine. The finding of imidazole-ring-opened products in tRNA ethylation by ethionine could be significant from the point of view of chemical carcinogenesis: in fact, imidazole-ring-opening of 1,7-dialkylguanosines directly at level of RNA with consequent formation of substituted pyrimidines is a transversion, i.e. a mutagenic event which would cause a change in the expression of genetic information since a purine has been transformed into a pyrimidine.     

Estimating seed dispersal distance: A comparison of methods using animal movement and plant genetic data on two primate‐dispersed Neotropical plant species

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