A surveillance of human, porcine and bovine rotaviruses was carried out in Slovenia in 2004 and 2005. Stool samples were collected from a total of 406 pigs (373 from asymptomatic animals), 132 cattle (126 from asymptomatic animals) and 241 humans (all with diarrhoea), tested for group A rotaviruses using RT-PCR and analysed by sequencing. The aims of the study were to determine the incidence of asymptomatic rotavirus infection in animals, to look for evidence of zoonotic transmission and to detect reassortment among rotaviruses. The rates of asymptomatic shedding of rotaviruses in pigs and cattle were 18.0 % (67/373) and 4.0 % (5/126), respectively. Evidence for zoonotic transmission was detected in one human rotavirus strain, SI-MB6, with the G3P[6] genotype combination, as the nucleotide and predicted amino acid sequences of the VP6, VP7, VP8* and NSP4 genes of strain SI-MB6 and of porcine strains showed high nucleotide and amino acid sequence identity. Two porcine rotavirus strains carried VP7 of probable human origin, suggesting an interspecies reassortment event in the past.

In the study of 50 matched pairs of breast carcinoma and normal breast tissue, the activities of cysteine proteinases (CPs), cathepsin (Cat) B and Cat L in tumors were increased on average by 18.5-fold and 52.5-fold respectively. The differences in activity of cysteine proteinase inhibitors (CPIs) between tumor and control breast tissues was also observed: in approximately two thirds of carcinomas, lowered CPI activity was measured (group-I patients), while similar or higher tumor CPI activity was measured in the remaining samples (group-II patients). Relative increases in specific activity of Cat B and Cat L in group I were significantly higher than in group II. In group I more patients with histopathological tumor grade III and negative estrogen (ER) and progesterone receptor (PR) levels were found, but the metastatic involvement of regional lymph nodes was similar in both groups. A 2-year follow-up study showed a significant inverse correlation between disease-free survival and increased Cat L activity, but the differences in group I and group II patients were not significant in this short time interval. In 20 matched pairs of breast carcinoma and normal breast tissue, the mean activity of Cat D was 5.8-fold higher in tumors compared with controls. The hypothesis that elevated Cat D activity increased CP activity and/or lowered tumor CPI activity due to post-translational proteolytic modification appeared less likely, since no correlations between corresponding activities were observed. We suggested that lowered CPI might rather reflect changes in transcription of intracellular CPIs, the stefins. Immunoassay and Northern blot analysis showed that the average value of stefin A protein and mRNA content respectively in the majority of investigated breast carcinoma samples were lowered, suggesting the possible value of stefin A in diagnosis and/or prognosis of the disease.

Mediterranean plant Helichrysum italicum represents a rich source of versatile bioactive compounds with potential benefits for human health. Despite extensive research on the plant’s active constituents, little attention has yet been paid to characterizing the relationship between its intra-specific genetic diversity and metabolite profile. The study aimed to determine metabolic profile of H. italicum ssp. italicum (HII) and ssp. tyrrhenicum (HIT) cultivated on the experimental plantation in Slovenia and to compare the chemical composition of extracts regarding the solvent extraction process. Extracts were prepared upon conventional extract preparation procedures: maceration with 50% methanol or ethanol and cold or hot water infusion and analyzed using High Performance Liquid Chromatography-Diode Array Detection-Electrospray Ionization-Quadrupole Time-of-Flight-Mass Spectrometry (HPLC-DAD-ESI-QTOF-MS). One hundred compounds were identified in the samples, among them several isomers and derivatives were reported for the first time, while caffeoylquinic acids and pyrones were the most abundant. Semi-quantitative comparison revealed that the extraction procedure had a greater impact on the chemical profile than genetic variability. All HIT extracts showed a higher total phenolic content compared to HII, while the antioxidant potential evaluated by 1,1-diphenyl-2-picrylhydrazil test was not proportionally higher. In addition, hot water extracts proved to be comparably active as alcoholic ones, confirming high commercial potential of Helichrysum italicum as herbal functional beverages.

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Diagnosis of prosthetic joint infection with culture technique can be problematic since the causative agent(s) are not possible to cultivate in all cases. Molecular methods had been evaluated in many studies but their inclusion in routine diagnostics is still controversial. The purpose of our prospective study was to compare the diagnostic accuracy of broad-range (BR)-PCR and culture technique. Intraoperative samples of periprosthetic tissue were retrieved in 67 patients undergoing revision arthroplasty. Samples were analyzed with culture technique, immunohistochemistry and BR 16S rRNA gene PCR. Bacteria in PCR-positive samples were identified using two different methods: direct sequencing of PCR products and specific TaqMan assays. In 63 cases, full concordance was found between BR-PCR and culture technique. Specific TaqMan assays failed to identify bacteria in four culture- and BR-PCR-positive cases and therefore had a lower sensitivity in comparison with BR-PCR. Molecular methods detected bacteria with the same accuracy as culture; however, identification of bacteria was inferior to culture. Further development of species-recognition techniques is required to improve identification of causative microorganisms.

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Rotaviral RNA was detected in the stool sample of an asymptomatic fattening pig at a Slovenian pig farm. To characterize the rotavirus, RT-PCR was used, employing primers specific for the VP7, VP4 and NSP4 genes. Specific products were purified and the sequencing reaction was performed for the molecular analysis of amplified genes. Nucleotide and amino acid sequences of the VP7 gene were found highly identical (85.3-88.1% and 90.7-91.6%) to G1 genotype strains. Phylogenetic and molecular analyses of the VP7 antigen regions revealed the sample to be from a new lineage of G1 genotype. In the molecular analysis of the VP4 gene, only 70.9% nucleotide (76.2% amino acid) identity was found with the most related rotavirus VP4 gene from GenBank. Following this, the NSP4 gene was also analyzed. After the phylogenetic analysis, it clustered with the NSP4 B genotype, but also seemed to represent a new lineage of this genotype. This new rotavirus strain, named P21-5, differed greatly from all rotaviruses characterized so far in all three genes analyzed. The virulence of this strain is not clear yet and has to be investigated.

Cloning and characterization of three cDNAs and one gene coding for the aspartic proteinase inhibitor homologues, detection of four protein isoforms and analysis of the gene copy number indicate that in potato aspartic proteinase inhibitors are encoded by a multigene family.