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Tuberculosis (TB) diagnosis among sputum-scarce patients is time consuming. Thus, a nonsputum diagnostic alternative is urgently needed. The Mycobacterium tuberculosis–specific transrenal (Tr) DNA from urine is a potential target for TB diagnostics. In this study, a new urine-based Tr-DNA molecular assay was evaluated for diagnosis of pulmonary tuberculosis among 428 adults suspected of having pulmonary TB (164 HIV positive, 263 HIV negative) from Cape Town, South Africa. Tr-DNA was isolated from 4 mL of EDTA urine, and a rapid, double-stranded, primer-based PCR method was performed targeting the Mycobacterium tuberculosis–specific direct repeat region. Each Tr-DNA eluate was tested in triplicate using an automated molecular analyzer with controls included in each test. With liquid culture used as the gold standard, the Tr-DNA assay showed sensitivity of 42.9% (n = 75/175; 95% CI, 35.4%–50.5%) and specificity of 88.6% (n = 210/237; 95% CI, 83.9%–92.4%). Among HIV-infected patients with TB, sensitivity and specificity were 45.2% and 89.0%, respectively. The combination of smear microscopy and Tr-DNA increased the sensitivity to 83.8% (smear microscopy alone, 75.1%), with 96.6% specificity. This study indicates that Tr-DNA has a moderate specificity with low sensitivity for diagnosis of pulmonary TB. Despite low sensitivity, this diagnostic test may have potential in combination with smear microscopy to support TB diagnosis in HIV-endemic regions, where sputum-scarce patients are common.

Tuberculosis (TB) diagnosis among sputum-scarce patients is time consuming. Thus, a nonsputum diagnostic alternative is urgently needed. The Mycobacterium tuberculosis-specific transrenal (Tr) DNA from urine is a potential target for TB diagnostics. In this study, a new urine-based Tr-DNA molecular assay was evaluated for diagnosis of pulmonary tuberculosis among 428 adults suspected of having pulmonary TB (164 HIV positive, 263 HIV negative) from Cape Town, South Africa. Tr-DNA was isolated from 4 mL of EDTA urine, and a rapid, double-stranded, primer-based PCR method was performed targeting the Mycobacterium tuberculosis-specific direct repeat region. Each Tr-DNA eluate was tested in triplicate using an automated molecular analyzer with controls included in each test. With liquid culture used as the gold standard, the Tr-DNA assay showed sensitivity of 42.9% (n = 75/175; 95% CI, 35.4%-50.5%) and specificity of 88.6% (n = 210/237; 95% CI, 83.9%-92.4%). Among HIV-infected patients with TB, sensitivity and specificity were 45.2% and 89.0%, respectively. The combination of smear microscopy and Tr-DNA increased the sensitivity to 83.8% (smear microscopy alone, 75.1%), with 96.6% specificity. This study indicates that Tr-DNA has a moderate specificity with low sensitivity for diagnosis of pulmonary TB. Despite low sensitivity, this diagnostic test may have potential in combination with smear microscopy to support TB diagnosis in HIV-endemic regions, where sputum-scarce patients are common.

Molecular diagnostics of patients with MTB tuberculosis from urine samples. We developed a new molecular assay based on the detection of M. tuberculosis-specific transrenal DNA (trDNA) and tested it for the diagnosis of active tuberculosis at the initiation of anti-tuberculosis therapy and during treatment follow-up. The overall sensitivity of trDNA was 96 and 100% when smear-microscopy and trDNA was combined. In a subset of TB treatment naive patients (n = 11) sensitivity and specificity of trDNA was 64 and 100%, respectively. For this subset of patients the sensitivity was 91% when smear-microscopy and trDNA diagnosis were combined. After treatment initiation, trDNA showed a significant reduction in concentration over time reaching undetectable trDNA values at week 12 in 9 of 11 accessible patients (82%). Kinetics in treatment-naive patients showed low base-line trDNA levels, which increased to maximal trDNA levels within one week indicating bactericidal activity of anti-tuberculosis drugs after the initiation of effective therapy. Maximal trDNA levels correlated positively with a radiological score, suggesting that the process of DNA excretion may reflect the extent of pulmonary disease. Matched samples showed an inverse correlation between the time to positivity of solid culture with maximum trDNA levels as well as the expected positive correlation between smear grade and maximum trDNA values. The detection of M. tuberculosis trDNA from urine specimen is a promising method for the diagnosis tuberculosis. The assay may be a candidate diagnostic tool for patients with paucibacillary and extrapulmonary disease, as method to assess treatment responses and could be helpful to diagnose tuberculosis in children.

Polysialic acid (polySia) is mainly described as a glycan modification of the neural cell adhesion molecule NCAM1. PolySia-NCAM1 has multiple functions during the development of vertebrate nervous systems including axon extension and fasciculation. Phylogenetic analyses reveal the presence of two related gene clusters, NCAM1 and NCAM2, in tetrapods and fishes. Within the ncam1 cluster, teleost fishes express ncam1a (ncam) and ncam1b (pcam) as duplicated paralogs which arose from a second round of ray-finned fish-specific genome duplication. Tetrapods, in contrast, express a single NCAM1 gene. Using the zebrafish model, we identify Ncam1b as a novel major carrier of polySia in the nervous system. PolySia-Ncam1a is expressed predominantly in rostral regions of the developing nervous system, whereas polySia-Ncam1b prevails caudally. We show that ncam1a and ncam1b have different expression domains which only partially overlap. Furthermore, Ncam1a and Ncam1b and their polySia modifications serve different functions in axon guidance. Formation of the posterior commissure at the forebrain/ midbrain junction requires polySia-Ncam1a on the axons for proper fasciculation, whereas Ncam1b, expressed by midbrain cell bodies, serves as an instructive guidance cue for the dorso-medially directed growth of axons. Spinal motor axons, on the other hand, depend on axonally expressed Ncam1b for correct growth toward their target region. Collectively, these findings suggest that the genome duplication in the teleost lineage has provided the basis for a functional diversification of polySia carriers in the nervous system.

Sequences from two mitochondrial genes (cytochrome b and NADH1) were used to produce a molecular phylogeny for 12 named and two undescribed species of the genus Oligoryzomys. All analyses placed Oligoryzomys microtis as the most basal taxon, a finding consistent with previous studies that suggested the west-central Amazon as a centre of origin for the tribe Oryzomyini to which Oligoryzomys belongs. Biogeographically, this suggests that Oligoryzomys had a South American origin, and later advanced northwards, entering Central America and Mexico more recently. Different analyses have provided consistent support for several additional clades that did not necessarily agree with the species groups hypothesized by previous studies. A molecular clock derived for these data suggests an origin for the genus of 6.67 Mya, with most speciation within the genus occurring between 3.7 and 1.5 Mya. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160, 551–566.

Enzymes of the St8Sia family, a subgroup of the glycosyltransferases, mediate the transfer of sialic acid to glycoproteins or glycolipids. Here, we describe the cloning of the zebrafish St8SiaIII gene and study its developmental activity. A conserved synteny relationship among vertebrate chromosome regions containing St8SiaIII loci underscores an ancient duplication of this gene in the teleost fish lineage and a specific secondary loss of one paralog in the zebrafish. The single zebrafish St8SiaIII enzyme, which is expected to function as an oligosialyltransferase, lacks maternal activity, is weakly expressed during nervous system development, and shows a highly dynamic expression pattern in somites and somite-derived structures. Morpholino knock-down of St8SiaIII leads to anomalous somite morphologies, including defects in segment boundary formation and myotendious-junction integrity. These phenotypes hint for a basic activity of zebrafish St8SiaIII during segmentation and somite formation, providing novel evidence for a non-neuronal function of sialyltransferases during vertebrate development.

Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.

Reggies (flotillins) are detergent-resistant microdomains involved in the scaffolding of large heteromeric complexes that signal across the plasma membrane. Based on the presence of an evolutionarily widespread motif, reggies/flotillins have been included within the SPFH (stomatin-prohibitin-flotillin-HflC/K) protein superfamily. To better understand the origin and evolution of reggie/flotillin structure and function, we searched databases for reggie/flotillin and SPFH-like proteins in organisms at the base and beyond the animal kingdom, and used the resulting dataset to compare their structural and functional domains. Our analysis shows that the SPFH grouping has little phylogenetic support, probably due to convergent evolution of its members. We also find that reggie/flotillin homologues are highly conserved among metazoans but are absent in plants, fungi and bacteria, where only proteins with "reggie-like" domains can be found. However, despite their low sequence similarities, reggie/flotillin and "reggie-like" domains appear to subserve related functions, suggesting that their basic biological role was acquired independently during evolution.

Infectious prion proteins cause neurodegenerative disease in mammals owing to the acquisition of an aberrant conformation. We cloned a Fugu rubripes gene that encodes a structurally conserved prion protein, and found rapid rates of molecular divergence among prions from different vertebrate classes, along with molecular stasis within each class. We propose that a directional trend in the evolution of prion sequence motifs associated with pathogenesis and infectivity could account for the origin of scrapie in mammals.

Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.

Background: Kcnh1 is a voltage-gated potassium channel that is primarily expressed in brain. Results: Knockdown of kcnh1 in zebrafish delays neural development and causes embryonic lethality. Conclusion: Kcnh1 is involved in cell proliferation during early zebrafish development. Significance: The finding that Kcnh1 has basic functions beyond neural signaling will help to elucidate its roles in physiology and cancer formation., The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

Prions result from the misfolding and selective accumulation of the host-encoded prion protein (PrP) in the brain. Despite intensive research on mammalian models, basic questions about the biological role of PrP and the evolutionary origin of prion disease remain unanswered. Following our previous identification of novel fish PrP homologues, here we generated new fish PrP sequences and performed genomic analysis to demonstrate the existence of two homologous PrP loci in bony fish, which display extensive molecular variation and are highly expressed in adult and developing fish brains. The fish PrP genomic regions contain PrP-related loci directly downstream of each PrP locus, suggesting an independent origin of prion-related proteins in fish and mammals. Our structural prediction analysis uncovers a conserved molecular "bauplan" for all vertebrate PrPs. The C- and N-terminal protein domains have evolved independently from one another, the former having retained its basic globular structure despite high sequence divergence and the latter having undergone differential expansion-degeneration cycles in its repetitive domains. Our evolutionary analysis redefines fundamental concepts on the functional significance of PrP domains and opens up new possibilities for the experimental analysis of prion misfolding and neurodegeneration in a non-mammalian model like the zebrafish.

Phylogeographic relationships were evaluated at the intraspecific level using nucleotide sequence data from the mitochondrial cytochrome b gene of representative specimens of ‘‘colilargo’’ (Oligoryzomys longicaudatus) from 31 localities, along its distributional range over a large part of the western Andes and southern Argentina. Based on approximately 1,000 base pairs (bp), we recognized a single species on both the Chilean and the Argentinean side as far as at least latitude 518S, rejecting the subspecific distinctiveness of longicaudatus and philippi. We thus placed the latter in full synonymy with O. longicaudatus as earlier studies proposed, and enlarged its range as far as Torres del Paine, about 518S. The occurrence of subspecies in this range is doubtful given the low sequence divergence values and the absence of significant associations between haplotypes and their geography. Additionally, we hypothesized that the entrance of this species into the Chilean side of the Andes mountains occurred through the Patagonian forests of southern Argentina, with further dispersal to the north from the south.b gene of representative specimens of ‘‘colilargo’’ (Oligoryzomys longicaudatus) from 31 localities, along its distributional range over a large part of the western Andes and southern Argentina. Based on approximately 1,000 base pairs (bp), we recognized a single species on both the Chilean and the Argentinean side as far as at least latitude 518S, rejecting the subspecific distinctiveness of longicaudatus and philippi. We thus placed the latter in full synonymy with O. longicaudatus as earlier studies proposed, and enlarged its range as far as Torres del Paine, about 518S. The occurrence of subspecies in this range is doubtful given the low sequence divergence values and the absence of significant associations between haplotypes and their geography. Additionally, we hypothesized that the entrance of this species into the Chilean side of the Andes mountains occurred through the Patagonian forests of southern Argentina, with further dispersal to the north from the south. Fil: Palma, R. Eduardo. Pontificia Universidad Católica de Chile; Chile Fil: Rivera Milla, Eric. University of Konstanz; Alemania Fil: Salazar Bravo, Jorge. Texas Tech University; Estados Unidos Fil: Torres Pérez, Fernando. Pontificia Universidad Católica de Chile; Chile Fil: Pardiñas, Ulises Francisco J.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; Argentina Fil: Marquet Iturriaga, Pablo Angel. Pontificia Universidad Católica de Chile; Chile Fil: Spotorno, Angel E.. Universidad de Chile; Chile Fil: Meynard, Andrés P.. Pontificia Universidad Católica de Chile; Chile Fil: Yates, Terry L.. University of New Mexico; Estados Unidos

Thy-1 is a developmentally regulated, immunoglobulin superfamily member (IgSF), glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein expressed most strongly in neurons and lymphocytes. Thy-1 is expressed in all vertebrates and has been implicated in a variety of processes, including axon regeneration and transmembrane signaling, but its specific function remains elusive. A Thy-1-like molecule in teleost fish was recently identified, with evidence for its role in lipid-raft based signal transduction linked to optic nerve regeneration. For a better characterization of Thy-1, the evolutionary relationships between novel fish homologues and other vertebrate Thy-1s were analyzed. Although the sequence similarity between fish and mammals is very low, there appeared conservation of gene structure and disrupted but recognizable synteny. In addition, the detailed expression analysis of teleost Thy-1 showed nervous system Thy-1 mainly in sensory systems. Strong Thy-1 expression was detected in the youngest retinal ganglion cells and in some neurons in deeper retinal layers, probably amacrine cells. From the olfactory bulbs, Thy-1-positive cells extended axons into the telencephalon. The vagal lobe stained intensively as well as facial and glossopharyngeal lobes and nerves. Outside the CNS, skin cells, blood vessels, kidney macrophages, swim bladder, spleen, gut-associated nerve fibers and the palatal organ were labeled.

Nucleotide sequence data from the mitochondrial cytochrome b gene were used to evaluate the phylogenetic relationships among mouse opossum species of the genus Thylamys. Based on approximately 1000 bp in five of the six species of the genus and including different localities for some of the species, we concluded that T. macrura from the subtropical forests of eastern Paraguay is the most primitive taxon. Subsequent radiation of the genus is explained mainly via founder effect speciation. This evolutionary scenario would account for the speciation of T. pusilla, T. venusta, T. pallidior, and T. elegans in the Chaco, southern Bolivia and northern Argentina, the Andean Altiplano, the Coastal Desert of Chile, and coastal Peru, respectively. Calibration of a molecular clock set the Pleistocene as the period for the differentiation of Thylamys species. The molecular results confirm the strong genetic connection between populations that inhabit the “pre-cordillera” of northern Chile (T. pallidior) and the canyons that run through the Atacama Desert to the lowlands in northern Chile. Our results confirm the occurrence of two Thylamys species in Chile, T. pallidior and T. elegans, within and south to the Atacama Desert, respectively.

Six mouse opossum species are currently recognized for the genus Thylamys in South America, of which we hypothesized the evolutionary relationships for those located in the Andean Altiplano and canyons of the Region I of Chile, as well as for those located south of the Atacama Desert down to 37˚ S. To that goal we sequenced the cytochrome b mitochondrial gene and data were analyzed using different phylogenetic criteria such as parsimony, distance and likelihood available in the program PAUP 4.0. The different phylogenetic approaches agreed in recovering two well defined clades: one constituted by Thylamys pallidior that included specimens from the coast and the “pre-cordillera” of the Region I of Chile, and the other clade constituted by Thylamys elegans that included populations located south to the Atacama Desert. Therefore, we demonstrate that populations located in the canyons and andean areas of northern Chile belong to T. pallidior, while those located south to the Atacama desert are recognized as T. elegans, results that confirm the occurrence of two thylamyine species in Chile.

Polysialic acid (PSA) is a developmentally regulated carbohydrate attached to the neural cell adhesion molecule (NCAM). PSA is involved in dynamic processes like cell migration, neurite outgrowth and neuronal plasticity. In mammals, polysialylation of NCAM is catalyzed independently by two polysialyltransferases, STX (ST8Sia II) and PST (ST8Sia IV), with STX mainly acting during early development and PST at later stages and into adulthood. Here, we functionally characterize zebrafish Stx and Pst homolog genes during fish development and evaluate their catalytic affinity for NCAM in vitro . Both genes have the typical gene architecture and share conserved synteny with their mammalian homologues. Expression analysis, gene-targeted knockdown experiments and in vitro catalytic assays indicate that zebrafish Stx is the principal–if not unique–polysialyltransferase performing NCAM-PSA modifications in both developing and adult fish. The knockdown of Stx exclusively affects PSA synthesis, producing defects in axonal growth and guidance. Zebrafish Pst is in principle capable of synthesizing PSA, however, our data argue against a fundamental function of the enzyme during development. Our findings reveal an important divergence of Stx and Pst enzymes in vertebrates, which is also characterized by a differential gene loss and rapid evolution of Pst genes within the bony-fish class.