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In this study we report the usefulness of nested PCR for screening of the persistent B. microti infections in rodent hosts. Female BALB/c mice were inoculated with 100 μl of donor blood infected with B. microti. Infections were detected using microscopic examination of Giemsa-stained blood smears. To determine whether B. microti DNA was present in blood and/or spleen tissue, nested PCR was performed targeting a specifi c fragment of the gene encoding the 18S rRNA. Blood was sampled every 10 days post-infection (dpi) until day 30, after which mice were sampled every 30 days until the end of experiment at 360 dpi. The most extensive parasitaemia (39% of infected erythrocytes) was observed at 10 dpi. Between 20-60 dpi, less then 1% of infected erythrocytes were detected in blood smears, and from 90 dpi onwards, infected erythrocytes were no longer observed. B. microti DNA was successfully amplifi ed from the blood of mice from 10 dpi until 180 dpi, as well as from spleens of infected mice at 10 and 20 dpi. The presented results show that nested PCR is the method of choice for monitoring infections of B. microti in the blood of rodent hosts, and could therefore be a tool for environmental monitoring of naturally infected rodents which are the predominant source of infection for tick vectors.
Cryptosporidium spp. and Giardia spp. are wide-spread pathogens of humans and many species of mammals. The ways of transmission are very complex and diffi cult to defi ne. Both parasites occur in similar environments and share a broad host range. However, in Poland there is still little known about the epidemiology of these parasites due to the paucity of data on human cases and only few studies in wildlife. The aim of our study was to determine the distribution of two intestinal protozoa in a few species of protected and game mammals in North-Eastern Poland. Additionally, we wanted to compare prevalence and abundance of these parasites between wild and farm animals, and to determine the species/genotypes of Cryptosporidium. Fecal samples collected from protected species (European beaver – 22, grey wolf – 14, European bison – 55, Polish Konik (horse) – 5) and game mammals (red deer – 52, roe deer – 22, boar – 5) were examined by IFA. We also studied a group of samples collected from farm animals: beaver – 30, red deer – 66, Polish konik – 5. Cryptosporidium oocysts were identifi ed in 5 of 7 studied animal species (prevalence from 9% in roe deer to 36% in wolves), Giardia cysts in 4 of 6 studied species (prevalence from 1.7% in red deer to 7.7% in European beaver). Sequencing analysis of COWP gene fragment revealed that 5 Cryptosporidium isolates from wolves were C. parvum genotype 2 (zoonotic). The results show the important role of examined species in maintaining the natural sources of Cryptosporidium spp. and Giardia spp. infections in the environment.
hnRNP K protein, which localizes to the nucleus, cytoplasm and mitochondria, is involved in the various cellular processes that compose gene expression. We used a SAGE-based assay to profile RNAs associated with hnRNP K protein in rat mitochondria. RNA was isolated from mitoplasts obtained from highly purified and RNase-treated mitochondria. Total RNA and RNA associated with hnRNP K protein were then used as input material for generating two SAGE libraries. Mitochondrion-derived tags isolated from the total mitoplast RNA library represented 86.3%, while those isolated from the library constructed from RNA associated with hnRNP K protein represented only 28.2% of selected tags. Thus, an unexpected number of nuclear-encoded RNAs were purified from mitochondria. Many of these transcripts were co-purified with hnRNP K protein, and high levels of nuclear-encoded RNAs co-immunoprecipitating with K protein corresponded to elevated hnRNP K protein levels of the organelle. The most abundant RNAs that were co-purified with hnRNP K protein represented transcripts originating from satellite I DNA. While satellite I RNA levels were higher in the nucleus and cytoplasm than in mitochondria, the most abundant binding of satellite I transcripts to hnRNP K protein was found in mitochondria. The role of satellite I RNA in mitochondria remains to be elucidated.
 Colon anastomosis is therapeutically challenging because multiple, usually undetectable factors influence a spectrum of repair mechanisms. We hypothesized that low molecular weight heparins, routinely administered perioperatively, may differentially affect gene expression related to colon healing. Twenty pairs of untreated and enoxaparin-treated rats underwent left-side hemicolectomy with a primary end-to-end anastomosis. Normal colon and anastomotic bowel segments were resected on day 0 and on days 1, 3, 5, and 7 after surgery, respectively. Serial anastomosis transverse cross-sections were evaluated microscopically and by microarray (Rat Genome 230 2.0, Affymetrix). Differentially expressed probe sets were annotated with Gene Ontology. We also examined the influence of enoxaparin on fibroblast proliferation and viability in vitro. Among the 5476 probe sets, we identified differential expression at each healing time point, yielding 79 subcategories. Most indicated genes were involved in wound healing, including multicellular organismal development, locomotory behavior, immune response, cell adhesion, inflammatory response, cell-cell signaling, blood vessel development, and tissue remodeling. Although we found no intensity differences in histological features of healing between enoxaparin-treated and control rats, treatment did induce significant expression changes during early healing. Of these changes, 83 probe sets exhibited at least twofold changes and represented different functional annotations, including inflammatory response, regulation of transcription, regulation of apoptosis, and angiogenesis. Fibroblast culture confirmed an anti-viability effect of enoxaparin. Enoxaparin affects colon wound-related gene expression profiles, but further studies will resolve whether heparin treatment is a risk factor after intestinal surgery, at least in some patients.
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