First <Emphasis Type=Italic>in vitro</Emphasis> isolation of <Emphasis Type=Italic>Neospora caninum</Emphasis> from a naturally infected adult dairy cow in Slovakia

The impact of in vitro isolation and molecular characterisation of Neospora caninum as well as sequence analyses was studied. The brain homogenate of a naturally Neospora-infected dairy cow (positive in ELISA and Western blot) was intraperitoneally inoculated into Mongolian gerbils (Meriones unguiculatus). The brain of gerbils on day 60 post-inoculation was homogenized, and, after trypsin-digestion, cultured on Vero cells. Neospora-like tachyzoites were first observed after 77 days of cultivation. The parasite was confirmed by polymerase chain reaction (PCR) using Neospora-specific primers Np21 and Np6. The PCR product of the first Slovak isolate (NC-SKB1) was subsequently sequenced and published in GenBank under accession number GU300774. Sequencing and BLAST search identified the isolate as N. caninum.     

Differentiating the growing nosocomial infectious threats <Emphasis Type=Italic>Ralstonia pickettii</Emphasis> and <Emphasis Type=Italic>Ralstonia insidiosa</Emphasis>

Differentiation of the growing nosocomial infectious threats, Ralstonia pickettii and Ralstonia insidiosa, based on nitrate reduction, desferrioxamine susceptibility, arabinose, N-acetyl-glucosamine and phenylacetate assimilation is described. These tests can be used for preliminary identification of Ralstonia pickettii and Ralstonia insidiosa resulting in more accurate identification of these species.     

Antioxidative and <Emphasis Type=Italic>in vitro</Emphasis> antiproliferative activity of <Emphasis Type=Italic>Arctium lappa</Emphasis> root extracts

Background  

Arctium lappa, known as burdock, is widely used in popular medicine for hypertension, gout, hepatitis and other inflammatory disorders. Pharmacological studies indicated that burdock roots have hepatoprotective, anti-inflammatory, free radical scavenging and antiproliferative activities. The aim of this study was to evaluate total phenolic content, radical scavenging activity by DPPH and in vitro antiproliferative activity of different A. lappa root extracts.     

Characterization of the first Polish isolate of <Emphasis Type="Italic">Neospora caninum</Emphasis> from cattle

Neospora caninum was isolated from the brain of an apparently healthy calf born to a seropositive cow. The calf was killed 12 hrs after birth and homogenate of its brain was inoculated into Vero cells. Neospora-like tachyzoites were detected 66 days later. The identity of the parasite was confirmed by polymerase chain reaction amplification of N. canium-specific fragments using Np21 and Np6 primers. This first Polish isolate of N. caninum was designated NC-PolB1.     

Effects of <Emphasis Type=Italic>Sophora japonica</Emphasis> flowers (<Emphasis Type=Italic>Huaihua</Emphasis>) on cerebral infarction

The dried flowers and buds of Sophora japonica are used as a medicinal herb in China, Japan and Korea to treat bleeding hemorrhoids and hematemesis. This article presents an overview of the effects of Sophora japonica on cerebral infarction based on literature searched from Medline, PubMed, Cochrane Library and the China National Knowledge Infrastructure (CNKI). Sophora japonica contains both anti-hemorrhagic and anti-hemostatic substances. Sophora japonica reduces cerebral infarction partly as a result of its anti-oxidative and anti-inflammatory activities. Previous studies found that Sophora japonica reduced the size of cerebral infarction and neurological deficits and reduced microglial activation, interleukin-1β release and number of apoptotic cells in ischemia-reperfusion injured Sprague-Dawley rats. Further study is required to determine the relationship between Sophora japonica-mediated reduction in cerebral infarction size and the effects of Sophora japonica on platelet aggregation and cardiovascular function.     

The differential protein expression profiles and immunogenicity of tachyzoites and bradyzoites of in vitro cultured <Emphasis Type="Italic">Neospora caninum</Emphasis>

We report a study on the variations in the protein expression profiles of tachyzoites and bradyzoites of Neospora caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by continuous treatment of infected cultures with 70 muM sodium nitroprusside (SNP) for up to 9 days. The stage conversion indicated by the expression of the bradyzoite-specific antigen BAG1 was analyzed by immunofluoresence assay. Morphological changes between tachyzoites and bradyzoites and localization of nuclei were demonstrated by transmission electron microscopy. Notably, we showed the differential protein expression profiles of tachyzoites and bradyzoites of N. caninum upon treatment with SNP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated different protein patterns between tachyzoites and bradyzoites. Furthermore, Western blotting using rabbit polyclonal antibodies directed against tachyzoites revealed several reactive bands, one of which represented a tachyzoite-specific antigen of approximately 40 kDa remarkably expressed in the tachyzoite stage, but was absent from bradyzoites. Moreover, rabbit polyclonal serum raised against bradyzoites recognized a significant increased expression of an antigen with a MW of approximately 25 kDa in bradyzoites by Western blotting, suggesting that this protein is specifically expressed at the bradyzoite stage. Taken together, our data showed that differential protein expression profiling is a useful tool for discriminating between the two stages during tachyzoite-bradyzoite interconversion in N. caninum infections.     

<Emphasis Type=Italic>In situ</Emphasis> and <Emphasis Type=Italic>ex vivo</Emphasis> evaluation of a wireless magnetoelastic biliary stent monitoring system

This paper presents the in situ and ex vivo evaluation of a system that wirelessly monitors the accumulation of intimal tissue and sludge in a biliary stent. The sensing element, located within the stent, is a magnetoelastic resonator that is queried by a wireless radio frequency signal. The in situ testing uses a commercially-available self-expanding biliary stent enhanced with a 1 mm × 25 mm magnetoelastic ribbon sensor (formed from Metglas™ 2605SA1). The stent has a conformal magnetic layer (consisting of strontium ferrite particles suspended in polydimethylsiloxane) that biases the sensor. The external interrogation module is able to acquire a signal from the sensor from a distance of at least 5 cm while the sensor is implanted in a porcine carcass and loaded with biological fluids. The ex vivo testing uses bile harvested from the porcine carcass. The response of a 1 mm × 25 mm magnetoelastic ribbon sensor is first calibrated with fluids of known density and viscosity, and the calibrated sensor is used to estimate that the viscosity of the harvested bile is 2.7–3.7 cP. The test results presented in this paper illustrate the fundamental usability of the system when the sensor is implanted, loaded by biological fluids, and interrogated in a surgical setup.     

Characterization of the siderophore transport protein AsbJ in <Emphasis Type=Italic>Aeromonas salmonicida</Emphasis> subsp. <Emphasis Type=Italic>salmonicida</Emphasis>

Iron is an important nutrient for all living systems. Bacteria have developed multiple mechanisms to obtain iron from the host. Aeromonas salmonicida subsp. salmonicida produces catechol-type siderophore under iron starvation conditions. This study attempted to characterize and ascertain the role of a siderophore ABC transporter protein AsbJ in siderophore transporter system in this bacterial pathogen. Prediction of protein domains was carried out, and deleted asbJ gene mutant strain was constructed. Results showed that this protein contains special domain related to permease and ATP-binding protein, and it is essential for the siderophore transport process in this bacterial fish pathogen.     

<Emphasis Type=Italic>In vitro</Emphasis> antiproliferative activity of partially purified <Emphasis Type=Italic>Trigona laeviceps</Emphasis> propolis from Thailand on human cancer cell lines

Background  

Cancers are some of the leading causes of human deaths worldwide and their relative importance continues to increase. Since an increasing proportion of cancer patients are acquiring resistance to traditional chemotherapeutic agents, it is necessary to search for new compounds that provide suitable specific antiproliferative affects that can be developed as anticancer agents. Propolis from the stingless bee, Trigona laeviceps, is one potential interesting source that is widely available and cultivatable (as bee hives) in Thailand.     

Release of ActA into a medium via membrane anchor cleavage is required for <Emphasis Type=Italic>Listeria monocytogenes</Emphasis> host cell invasion

The ActA protein, which is involved in intra- and intercellular Listeria monocytogenes movement, is anchored in the bacterial membrane by a hydrophobic C-tail, and it is a membrane-bound form of ActA that is indispensable for intracellular movement. However, soluble ActA is detected in the culture supernatant of L. monocytogenes as a major protein. In this study, the mechanisms of ActA release into the culture supernatant and the role that soluble ActA plays in intracellular virulence were investigated. Our results indicated that the cleavage of the C-terminal membrane anchor is required for ActA release. The MALDI-TOF MS analysis of trypsin-cut soluble ActA suggested that the cleavage occurred between His582 and Thr583. The proposed cleavage site was confirmed by introduction of the His582Pro substitution preventing ActA release, although this did not disturb its surface presentation. The virulence-associated metalloprotease Mpl, which is responsible for pH-dependent ActA degradation, is not involved in membrane anchor cleavage, which requires another still unidentified protease. The mutant strain had a lower invasive effectiveness into HeLa epithelial cells, whereas it did not differ from the wild strain in intracellular proliferation and the ability to form plaques of lysis in the HeLa confluent monolayer. The obtained results suggested that ActA release is important for the invasion of L. monocytogenes into epithelial cells but is not required for intracellular motility and cell-to-cell spread.     

Assessment of <Emphasis Type=Italic>Akodon cursor</Emphasis> (Rodentia,Sigmodontinae) as permissive host to <Emphasis Type=Italic>Schistosoma mansoni</Emphasis> infection: morphology of adult worms

The natural infection with parasitic helminths is common in wild rodent populations. Once such interactions are better understood in the laboratory, it will be more feasible to extend the findings to infected hosts in nature. The flukes recovered from laboratory-infected Akodon cursor at 63 days post-infection were stained with hydrochloric carmine and individually mounted on glass slide as whole-mounts. Light and laser scanning confocal microscopy studies of adult male and female Schistosoma mansoni are reported. The parasites were examined morphologically and biometrically, which was obtained in a digital system for image analysis. Parameters used were: tegument thickness, digestive, excretory and reproductive systems. The overall conclusion of this experiment is that the morphological features of adult worm were similar to laboratory mice. It has been confirmed that the grass mouse is a permissive host to S. mansoni infection.     

Pathological and immunological findings in placentas from pregnant BALB/c mice infected with <Emphasis Type=Italic>Neospora caninum</Emphasis> at early and late stages of gestation

Neospora caninum is transmitted from a cow to its foetus by vertical transmission and the timing of infection in gestation is an important factor in determining the disease outcome. Few studies have explored the role of the placenta in the outcome of N. caninum infection during pregnancy. Here, we described the N. caninum presence, parasite load, local immune response, and histopathological lesions at the materno-foetal interface after infection of BALB/c mice at early and late stages of gestation. In mice infected at early gestation, N. caninum DNA was detected in foetoplacentary units 7 days post-infection (PI) and in the placenta, but not in viable foetuses on day 14 PI, indicating that the parasite was multiplying primarily in the placental tissues without reaching the foetus. Moreover, parasite DNA was detected in resorptions, suggesting that foetal death could be a consequence of infection. An increase in IFN-γ, TNF-α and IL-10 expression was observed in N. caninum PCR-positive placentas, which could favour N. caninum foetal transmission and be harmful to both the placenta and the foetus. Histopathological analysis revealed necrosis affecting both the maternal and foetal sides of the placenta. At late gestation, transmission occurred rapidly following infection (day 3 PI), but parasite were rarely found. In addition, an increase in cytokine expression was observed in spleen and placental tissues from infected animals, while a downregulation in IL-4 expression was only observed in the spleen. Finally, necrosis in the placenta was limited to the maternal side, suggesting that the parasite is mainly multiplying in the placental tissue at this stage. Thus, the results of the present study indicate that the placenta may be actively involved in N. caninum pathogenesis.     

Molecular cytogenetic mapping of <Emphasis Type=Italic>Cucumis sativus</Emphasis> and <Emphasis Type=Italic>C. melo</Emphasis> using highly repetitive DNA sequences

Chromosomes often serve as one of the most important molecular aspects of studying the evolution of species. Indeed, most of the crucial mutations that led to differentiation of species during the evolution have occurred at the chromosomal level. Furthermore, the analysis of pachytene chromosomes appears to be an invaluable tool for the study of evolution due to its effectiveness in chromosome identification and precise physical gene mapping. By applying fluorescence in situ hybridization of 45S rDNA and CsCent1 probes to cucumber pachytene chromosomes, here, we demonstrate that cucumber chromosomes 1 and 2 may have evolved from fusions of ancestral karyotype with chromosome number n = 12. This conclusion is further supported by the centromeric sequence similarity between cucumber and melon, which suggests that these sequences evolved from a common ancestor. It may be after or during speciation that these sequences were specifically amplified, after which they diverged and specific sequence variants were homogenized. Additionally, a structural change on the centromeric region of cucumber chromosome 4 was revealed by fiber-FISH using the mitochondrial-related repetitive sequences, BAC-E38 and CsCent1. These showed the former sequences being integrated into the latter in multiple regions. The data presented here are useful resources for comparative genomics and cytogenetics of Cucumis and, in particular, the ongoing genome sequencing project of cucumber.     

Epithelioid Hemangioendothelioma: a Rare Primary Thyroid Tumor with Confirmation of <Emphasis Type=Italic>WWTR1</Emphasis> and <Emphasis Type=Italic>CAMTA1</Emphasis> Rearrangements

We report a rare case of epithelioid hemangioendothelioma as a primary thyroid tumor. To our knowledge, there are only two prior unequivocal cases of primary thyroid epithelioid hemangioendothelioma reported in the English literature. This is the first case in the thyroid with molecular confirmation.     

Antibodies to <Emphasis Type="Italic">Neospora caninum</Emphasis> and <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in domestic cats from Brazil

The occurrence of antibodies to Neospora caninum and Toxoplasma gondii was determined in 400 domestic cats (Siamese, Persian, and undetermined breeds) from the Municipality of Araçatuba, Sao Paulo, Brazil, through the indirect fluorescence antibody test (IFAT). Of the 400 cats, 100 were seropositive to T. gondii (25%, titer ≥64) and 98 to N. caninum (24.5%, titer ≥16). The rate of seropositive cats for T. gondii was correlated with age (χ ²=35.7; p<0.001), with a higher number of infected animals at older ages. Of the 219 cats younger than 1-year-old, 13.2% were seropositive for T. gondii, while 39.2% were positive in the 181 older animals. The presence of N. caninum was also correlated with age (÷²=8.8; p<0.01), with 18.7% (41/219) and 31.5% (57/181) of positive animals at ages below and above 12-month, respectively. An association between the occurrences of both protozoa in the felines was also observed (χ ²=19.6; p<0.001).