The Mitochondrial Genome of Toxocara canis

Toxocara canis (Ascaridida: Nematoda), which parasitizes (at the adult stage) the small intestine of canids, can be transmitted to a range of other mammals, including humans, and can cause the disease toxocariasis. Despite its significance as a pathogen, the genetics, epidemiology and biology of this parasite remain poorly understood. In addition, the zoonotic potential of related species of Toxocara, such as T. cati and T. malaysiensis, is not well known. Mitochondrial DNA is known to provide genetic markers for investigations in these areas, but complete mitochondrial genomic data have been lacking for T. canis and its congeners. In the present study, the mitochondrial genome of T. canis was amplified by long-range polymerase chain reaction (long PCR) and sequenced using a primer-walking strategy. This circular mitochondrial genome was 14162 bp and contained 12 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes consistent for secernentean nematodes, including Ascaris suum and Anisakis simplex (Ascaridida). The mitochondrial genome of T. canis provides genetic markers for studies into the systematics, population genetics and epidemiology of this zoonotic parasite and its congeners. Such markers can now be used in prospecting for cryptic species and for exploring host specificity and zoonotic potential, thus underpinning the prevention and control of toxocariasis in humans and other hosts

Control de la calidad del diagnóstico coproparasitológico en la provincia de Ciudad de La Habana, Cuba External quality assessment in coproparasitology in Havana City Province, Cuba

Se realizó un estudio sobre la calidad del diagnóstico coproparasitológico en 77 laboratorios de la red de salud pública de la provincia Ciudad de La Habana, Cuba. El procedimiento se basó en la entrega a cada jefe de laboratorio de un modelo de encuesta, y una bolsa de nylon conteniendo 10 viales plásticos con distintos especímenes parasitarios, preservados en formaldehído al 7%. Recogidos los resultados en las primeras 72 horas después de su entrega, se realizó la evaluación mediante una escala de puntuación establecida. La mayoría de los laboratorios aprobaron (70%); sin embargo aún existen centros, sobre todo policlínicas, con calificaciones deficientes. Los municipios con resultados más desfavorables fueron, Lisa, Marianao y Habana del Este, alcanzándose mejores resultados en los hospitales que en las policlínicas. En el análisis de Protozooarios, el mejor diagnosticado fué Giardia lamblia, con solo un centro que erró al identificarlo. Las mayores dificultades se presentaron en Blastocystis hominis con 61% de fallas, Endolimax nana, con 24,6%, y Entamoeba histolytica, con 22%. Entre los helmintos, la mayor aprobación fué en Trichuris trichiura y los errores diagnósticos predominaron con Fasciola hepatica y Taenia sp., ambos con 66,2% de fallas. Dados los resultados obtenidos, hemos organizado una intervención educativa en la red de laboratorios de la provincia.<br>An external quality assessment in coproparasitology was carried out in 77 laboratories from Havana City. A questionnaire and ten plastic vials with different intestinal parasites in a small nylon bag, duly sealed, were sent to each laboratory. Answers were collected during the 72 hours after delivery. Results were analyzed by means of a computer program. The majority of the laboratories (70%) passed the test; the municipalities with the worst scores in the province were Lisa, Marianao, and Habana del Este. Better results were obtained among technologists working only in parasitology than those who were also performing other laboratory work, and better averages were observed in hospitals than in polyclinics. The best identified intestinal protozoan was Giardia lamblia and the worst identified was Blastocystis hominis (with a 61% mistake rate), followed by Endolimax nana (24.6%), and Entamoeba histolytica (22%). Among helminths, the best identified was Trichuris trichiura (9.2% mistake rate) and the highest percentage of incorrect diagnoses was for Taenia sp. and Fasciola hepatica (both with 66.2%). Taking into account these results, we feel it is necessary to provide training in parasitology among these laboratories

Response of Osteoblasts on Amine-Based Nanocoatings Correlates with the Amino Group Density

Increased life expectancy in industrialized countries is causing an increased incidence of osteoporosis and the need for bioactive bone implants. The integration of implants can be improved physically, but mainly by chemical modifications of the material surface. It was recognized that amino-group-containing coatings improved cell attachment and intracellular signaling. The aim of this study was to determine the role of the amino group density in this positive cell behavior by developing controlled amino-rich nanolayers. This work used covalent grafting of polymer-based nanocoatings with different amino group densities. Titanium coated with the positively-charged trimethoxysilylpropyl modified poly(ethyleneimine) (Ti-TMS-PEI), which mostly improved cell area after 30 min, possessed the highest amino group density with an N/C of 32%. Interestingly, changes in adhesion-related genes on Ti-TMS-PEI could be seen after 4 h. The mRNA microarray data showed a premature transition of the MG-63 cells into the beginning differentiation phase after 24 h indicating Ti-TMS-PEI as a supportive factor for osseointegration. This amino-rich nanolayer also induced higher bovine serum albumin protein adsorption and caused the cells to migrate slower on the surface after a more extended period of cell settlement as an indication of a better surface anchorage. In conclusion, the cell spreading on amine-based nanocoatings correlated well with the amino group density (N/C)

Multiplex real-time PCR for the diagnosis of malaria : correlation with microscopy

Clin Microbiol Infect Abstract Malaria is generally diagnosed by microscopy and rapid antigen testing. Molecular methods become more widely used. In the present study, the contribution of a quantitative multiplex malaria PCR was investigated. We assessed: (i) the agreement between PCR-based identification and microscopy and (ii) the correlation between the parasite load as determined by quantitative PCR and by microscopy. For 83 patients positive by microscopy for Plasmodium spp., the first EDTA-blood sample was tested by multiplex PCR to confirm smear-based species identification. Parasite load was assessed daily using both microscopy and PCR. Among the 83 patients tested, one was positive by microscopy only and 82 were positive by microscopy and PCR. Agreement between microscopy and PCR for the identification at the species level was 89% (73/82). Six of the nine discordant results corresponded to co-infections by two or three species and were attributed to inaccurate morphological identification of mixed cases. The parasite load generally decreased rapidly after treatment had been started, with similar decay curves being obtained using both microscopy and PCR. Our PCR proved especially useful for identifying mixed infections. The quantification obtained by PCR closely correlated with microscopy-based quantification and could be useful for monitoring treatment efficacy, at least in clinical trial

Ca2+ Release to Lumen from ADP-sensitive Phosphoenzyme E1PCa2 without Bound K+ of Sarcoplasmic Reticulum Ca2+-ATPase*

During Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase, the conformation change of ADP-sensitive phosphoenzyme (E1PCa2) to ADP-insensitive phosphoenzyme (E2PCa2) is followed by rapid Ca2+ release into the lumen. Here, we find that in the absence of K+, Ca2+ release occurs considerably faster than E1PCa2 to E2PCa2 conformation change. Therefore, the lumenal Ca2+ release pathway is open to some extent in the K+-free E1PCa2 structure. The Ca2+ affinity of this E1P is as high as that of the unphosphorylated ATPase (E1), indicating the Ca2+ binding sites are not disrupted. Thus, bound K+ stabilizes the E1PCa2 structure with occluded Ca2+, keeping the Ca2+ pathway to the lumen closed. We found previously (Yamasaki, K., Wang, G., Daiho, T., Danko, S., and Suzuki, H. (2008) J. Biol. Chem. 283, 29144–29155) that the K+ bound in E2P reduces the Ca2+ affinity essential for achieving the high physiological Ca2+ gradient and to fully open the lumenal Ca2+ gate for rapid Ca2+ release (E2PCa2 → E2P + 2Ca2+). These findings show that bound K+ is critical for stabilizing both E1PCa2 and E2P structures, thereby contributing to the structural changes that efficiently couple phosphoenzyme processing and Ca2+ handling