Chiari Malformation Type I: A Case-Control Association Study of 58 Developmental Genes

Chiari malformation type I (CMI) is a disorder characterized by hindbrain overcrowding into an underdeveloped posterior cranial fossa (PCF), often causing progressive neurological symptoms. The etiology of CMI remains unclear and is most likely multifactorial. A putative genetic contribution to CMI is suggested by familial aggregation and twin studies. Experimental models and human morphometric studies have suggested an underlying paraxial mesoderm insufficiency. We performed a case-control association study of 303 tag single nucleotide polymorphisms (SNP) across 58 candidate genes involved in early paraxial mesoderm development in a sample of 415 CMI patients and 524 sex-matched controls. A subgroup of patients diagnosed with classical, small-PCF CMI by means of MRI-based PCF morphometry (n = 186), underwent additional analysis. The genes selected are involved in signalling gradients occurring during segmental patterning of the occipital somites (FGF8, Wnt, and retinoic acid pathways and from bone morphogenetic proteins or BMP, Notch, Cdx and Hox pathways) or in placental angiogenesis, sclerotome development or CMI-associated syndromes. Single-marker analysis identified nominal associations with 18 SNPs in 14 genes (CDX1, FLT1, RARG, NKD2, MSGN1, RBPJ1, FGFR1, RDH10, NOG, RARA, LFNG, KDR, ALDH1A2, BMPR1A) considering the whole CMI sample. None of these overcame corrections for multiple comparisons, in contrast with four SNPs in CDX1, FLT1 and ALDH1A2 in the classical CMI group. Multiple marker analysis identified a risk haplotype for classical CMI in ALDH1A2 and CDX1. Furthermore, we analyzed the possible contributions of the most significantly associated SNPs to different PCF morphometric traits. These findings suggest that common variants in genes involved in somitogenesis and fetal vascular development may confer susceptibility to CMI.     

Oak Bark Allometry and Fire Survival Strategies in the Chihuahuan Desert Sky Islands,Texas, USA

Trees may survive fire through persistence of above or below ground structures. Investment in bark aids in above-ground survival while investment in carbohydrate storage aids in recovery through resprouting and is especially important following above-ground tissue loss. We investigated bark allocation and carbohydrate investment in eight common oak (Quercus) species of Sky Island mountain ranges in west Texas. We hypothesized that relative investment in bark and carbohydrates changes with tree age and with fire regime: We predicted delayed investment in bark (positive allometry) and early investment in carbohydrates (negative allometry) under lower frequency, high severity fire regimes found in wetter microclimates. Common oaks of the Texas Trans-Pecos region (Quercus emoryi, Q. gambelii, Q. gravesii, Q. grisea, Q. hypoleucoides, Q. muehlenbergii, and Q. pungens) were sampled in three mountain ranges with historically mixed fire regimes: the Chisos Mountains, the Davis Mountains and the Guadalupe Mountains. Bark thickness was measured on individuals representing the full span of sizes found. Carbohydrate concentration in taproots was measured after initial leaf flush. Bark thickness was compared to bole diameter and allometries were analyzed using major axis regression on log-transformed measurements. We found that bark allocation strategies varied among species that can co-occur but have different habitat preferences. Investment patterns in bark were related to soil moisture preference and drought tolerance and, by proxy, to expected fire regime. Dry site species had shallower allometries with allometric coefficients ranging from less than one (negative allometry) to near one (isometric investment). Wet site species, on the other hand, had larger allometric coefficients, indicating delayed investment to defense. Contrary to our expectation, root carbohydrate concentrations were similar across all species and sizes, suggesting that any differences in below ground storage are likely to be in total volume of storage tissue rather than in carbohydrate concentration.     

Adaptation of Cotton Shoot Apex Culture to Agrobacterium-Mediated Transformation

A protocol is presented for rapid genotype-independent transformation and regeneration of cotton (Gossypium spp.) from shoots isolated from germinating seedlings. Isolated shoots are inoculated with a super-virulent strain of Agrobacterium tumefaciens, subjected to a mild antibiotic selection, and directly regenerated as shoots in vitro. Shoots do not dedifferentiate and mutation rates are low. Rooted shoots can be obtained within 6–10 weeks of isolation and inoculation depending on the cotton cultivar.     

Localization of eukaryotic initiation factor 2 in neuron primary cultures and established cell lines

Eukaryotic initiation factor 2 (eIF-2) is a heterotrimeric protein with subunits α, β and γ that forms a ternary complex with Met-tRNA and GTP. It promotes the binding of Met-tRNA to ribosomes and controls translational rates via phosphorylation/dephosphorylation mechanisms. By means of immunofluorescence and post-embedding immunocytochemistry of intact cells and quantitative immunoblotting of cell extracts, the cellular distribution of the initiation factor has been examined in primary neuronal cultures as well as in two established cell lines: PC12 phaeochromocytoma cells and rat pituitary GH4C1 cells. Our results indicated that the initiation factor is located not only in the cytoplasm but also in the nuclei of the cultured neurons and cell lines. In the cytoplasm, immunocytochemical studies reveal that the factor is present mainly in those areas that are rich in ribosomes. In the nucleus, the immunolabelling of eukaryotic initiation factor 2 verified the presence of gold particles in both nucleolar and extranucleolar areas. The specific distribution of this factor on both sides of the nuclear envelope suggests that it might have some nuclear-related function(s) besides its already known role in the control of translation     

Divergent Effects of Acute Depolarization on Somatostatin Release and Protein Synthesis in Cultured Fetal and Neonatal Rat Brain Cells

The influence of membrane depolarization on somatostatin secretion and protein synthesis by fetal and neonatal cerebrocortical neurons was studied. Cortical cells obtained by mechanical dispersion were maintained as monolayer cultures for 8 days. The ability of fetal cerebrocortical and hypothalamic cells to release immunoreactive somatostatin (IR-SRIF) was confirmed. Total protein synthesis was determined by the incorporation of [3H]phenylalanine into trichloroacetic acid-precipitable proteins. To study the effect of acute depolarization on protein synthesis, cells were incubated for 30 min with [3H]phenylalanine or [3H]leucine and the depolarizing agent. In fetal cerebrocortical cells, potassium (30 and 56 mM) decreased protein synthesis and RNA levels and increased IR-SRIF release. Depolarization by veratridine, a sodium channel activator, induced a similar effect. The effect of veratridine on IR-SRIF and protein synthesis was reversed by tetrodotoxin, a sodium channel blocker, or verapamil, a calcium channel blocker. These findings suggest that protein synthesis by cerebrocortical cells is decreased in fetal brain cells by membrane depolarization and is dependent on Na+ and Ca2+ entry into cells. In postnatal (day 7) cerebrocortical cells, depolarization induced by high potassium concentrations led to a concomitant increase in protein synthesis, RNA content, and somatostatin release. These findings indicate that depolarization of the cellular membrane is coupled to an increase in protein synthesis in neonatal, but not in fetal, dispersed brain cells.     

Assessing Habitat Use by Snapper (Chrysophrys auratus) from Baited Underwater Video Data in a Coastal Marine Park

Baited Underwater Video (BUV) systems have become increasingly popular for assessing marine biodiversity. These systems provide video footage from which biologists can identify the individual fish species present. Here we explore the relevance of spatial dependence and marine park boundaries while estimating the distribution and habitat associations of the commercially and recreationally important snapper species Chrysophrys auratus in Moreton Bay Marine Park during a period when new Marine National Parks zoned as no-take or “green” areas (i.e., areas with no legal fishing) were introduced. BUV studies typically enforce a minimum distance among BUV sites, and then assume that observations from different sites are independent conditional on the measured covariates. In this study, we additionally incorporated the spatial dependence among BUV sites into the modelling framework. This modelling approach allowed us to test whether or not the incorporation of highly correlated environmental covariates or the geographic placement of BUV sites produced spatial dependence, which if unaccounted for could lead to model bias. We fitted Bayesian logistic models with and without spatial random effects to determine if the Marine National Park boundaries and available environmental covariates had an effect on snapper presence and habitat preference. Adding the spatial dependence component had little effect on the resulting model parameter estimates that emphasized positive association for particular coastal habitat types by snapper. Strong positive relationships between the presence of snapper and rock habitat, particularly rocky substrate composed of indurated freshwater sediments known as coffee rock, and kelp habitat reinforce the consideration of habitat availability in marine reserve design and the design of any associated monitoring programs.     

Toxoplasma gondii Chitinase Induces Macrophage Activation

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50°C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.     

Decision-making during NHEJ: a network of interactions in human Polμ implicated in substrate recognition and end-bridging

Human Polμ is a DNA polymerase belonging to the X family that has been implicated in the non-homologous end-joining (NHEJ) pathway during repair of double-strand breaks in DNA. Loop1 is a flexible piece of Polμ which has a critical role during terminal transferase and end-joining activities: it acts as a pseudo-template when the template strand is discontinuous or unavailable, whilst diffusing away if present to avoid steric clashes. Mutational analysis and inspection of the 3D structures available allowed us to identify a network of residues in charge of sensing the presence or absence of discontinuities in the template strand, which will in turn determine the final position adopted by Loop1. This network is formed by the previously uncharacterized thumb mini-loop (NSH motif) and the positively charged helix N, which contribute to the correct positioning of Loop1 and to juxtapose the discontinuous template strand during NHEJ of incompatible ends. Accordingly, single mutation of specific conserved residues in these motifs, whilst irrelevant in most of the cases for gap filling, largely affected terminal transferase and end-joining activities. Other point mutations in the ‘hinges’ of Loop1, such as residues Phe³⁸⁵ or Phe³⁸⁹, corroborated the flexibility requirements of this motif.     

Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity

Jorge Lobo’s disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.