Development of the lateral ventricular choroid plexus in a marsupial, Monodelphis domestica

<p>Abstract</p> <p>Background</p> <p>Choroid plexus epithelial cells are the site of blood/cerebrospinal fluid (CSF) barrier and regulate molecular transfer between the two compartments. Their mitotic activity in the adult is low. During development, the pattern of growth and timing of acquisition of functional properties of plexus epithelium are not known.</p> <p>Methods</p> <p>Numbers and size of choroid plexus epithelial cells and their nuclei were counted and measured in the lateral ventricular plexus from the first day of its appearance until adulthood. Newborn <it>Monodelphis </it>pups were injected with 5-bromo-2-deoxyuridine (BrdU) at postnatal day 3 (P3), P4 and P5. Additional animals were injected at P63, P64 and P65. BrdU-immunopositive nuclei were counted and their position mapped in the plexus structure at different ages after injections. Double-labelling immunocytochemistry with antibodies to plasma protein identified post-mitotic cells involved in protein transfer.</p> <p>Results</p> <p>Numbers of choroid plexus epithelial cells increased 10-fold between the time of birth and adulthood. In newborn pups each consecutive injection of BrdU labelled 20-40 of epithelial cells counted. After 3 injections, numbers of BrdU positive cells remained constant for at least 2 months. BrdU injections at an older age (P63, P64, P65) resulted in a smaller number of labelled plexus cells. Numbers of plexus cells immunopositive for both BrdU and plasma protein increased with age indicating that protein transferring properties are acquired post mitotically. Labelled nuclei were only detected on the dorsal arm of the plexus as it grows from the neuroependyma, moving along the structure in a 'conveyor belt' like fashion.</p> <p>Conclusions</p> <p>The present study established that lateral ventricular choroid plexus epithelial cells are born on the dorsal side of the structure only. Cells born in the first few days after choroid plexus differentiation from the neuroependyma remain present even two months later. Protein-transferring properties are acquired post-mitotically and relatively early in plexus development.</p

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).Methods/Principal Findings: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.Conclusions/Significance: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.This work received financial support from the Ministry of Science and Technology of Argentina [PICT 2011-0207 to AGS] and the National Scientific and Technical Research Council in Argentina (CONICET) [PIP 112 2011-010-0974 to AGS]. Work related to evaluation of biological samples was partially sponsored by the Pan-American Health Organization (PAHO) [Small Grants Program PAHO-TDR]; the Drugs and Neglected Diseases Initiative (DNDi, Geneva, Switzerland), Wellcome Trust (London, United Kingdom), SANOFI-AVENTIS (Buenos Aires, Argentina) and the National Council for Science and Technology in Mexico (CONACYT) [FONSEC 161405 to JMR]

Testis-specific glyceraldehyde-3-phosphate dehydrogenase: origin and evolution

<p>Abstract</p> <p>Background</p> <p>Glyceraldehyde-3-phosphate dehydrogenase (GAPD) catalyses one of the glycolytic reactions and is also involved in a number of non-glycolytic processes, such as endocytosis, DNA excision repair, and induction of apoptosis. Mammals are known to possess two homologous GAPD isoenzymes: GAPD-1, a well-studied protein found in all somatic cells, and GAPD-2, which is expressed solely in testis. GAPD-2 supplies energy required for the movement of spermatozoa and is tightly bound to the sperm tail cytoskeleton by the additional N-terminal proline-rich domain absent in GAPD-1. In this study we investigate the evolutionary history of GAPD and gain some insights into specialization of GAPD-2 as a testis-specific protein.</p> <p>Results</p> <p>A dataset of GAPD sequences was assembled from public databases and used for phylogeny reconstruction by means of the Bayesian method. Since resolution in some clades of the obtained tree was too low, syntenic analysis was carried out to define the evolutionary history of GAPD more precisely. The performed selection tests showed that selective pressure varies across lineages and isoenzymes, as well as across different regions of the same sequences.</p> <p>Conclusions</p> <p>The obtained results suggest that GAPD-1 and GAPD-2 emerged after duplication during the early evolution of chordates. GAPD-2 was subsequently lost by most lineages except lizards, mammals, as well as cartilaginous and bony fishes. In reptilians and mammals, GAPD-2 specialized to a testis-specific protein and acquired the novel N-terminal proline-rich domain anchoring the protein in the sperm tail cytoskeleton. This domain is likely to have originated by exonization of a microsatellite genomic region. Recognition of the proline-rich domain by cytoskeletal proteins seems to be unspecific. Besides testis, GAPD-2 of lizards was also found in some regenerating tissues, but it lacks the proline-rich domain due to tissue-specific alternative splicing.</p

Specific patterns of histone marks accompany X chromosome inactivation in a marsupial

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Husbandry Of Monodelphis Domestica In The Study Of Mammalian Embryogenesis

Monodelphis domestica, commonly called the laboratory opossum, is a useful laboratory animal for studying marsupial embryogenesis and mammalian development. Females breed year-round and the animals can be sustainably bred indoors. The authors draw on their own laboratory\u27s experience to supplement previously published research on laboratory opossums. They describe a breeding protocol that reliably produces timed-pregnant M. domestica. Additionally, the authors discuss general laboratory opossum husbandry techniques and describe how to collect, handle and culture embryos

Human pedigree-based quantitative-trait-locus mapping: localization of two genes influencing HDL-cholesterol metabolism.

Common disorders with genetic susceptibilities involve the action of multiple genes interacting with each other and with environmental factors, making it difficult to localize the specific genetic loci responsible. An important route to the disentangling of this complex inheritance is through the study of normal physiological variation in quantitative risk factors that may underlie liability to disease. We present an analysis of HDL-cholesterol (HDL-C), which is inversely correlated with risk of heart disease. A variety of HDL subphenotypes were analyzed, including HDL particle-size classes and the concentrations and proportions of esterified and unesterified HDL-C. Results of a complete genomic screen in large, randomly ascertained pedigrees implicated two loci, one on chromosome 8 and the other on chromosome 15, that influence a component of HDL-C-namely, unesterified HDL2a-C. Multivariate analyses of multiple HDL phenotypes and simultaneous multilocus analysis of the quantitative-trait loci identified permit further characterization of the genetic effects on HDL-C. These analyses suggest that the action of the chromosome 8 locus is specific to unesterified cholesterol levels, whereas the chromosome 15 locus appears to influence both HDL-C concentration and distribution of cholesterol among HDL particle sizes