Mitochondrial DNA control region diversity in a population from Espirito Santo state,Brazil

Mitochondrial DNA (mtDNA) analysis has proved to be useful for forensic identification, especially in cases which nuclear DNA markers fail, as in degraded samples or in cases where the biological material has few traces or no nuclear DNA. Moreover, it can be applied in population genetics, inferring the origin of a population. In this work, the entire mtDNA control region of 97 individuals from the state of Espirito Santo, Brazil, was analyzed. We have found 94 different haplotypes yielding a high haplotype diversity of 0.9994 ± 0.0016. The probability of a random match calculated was 1.09. Haplogroup distribution analysis confirmed a highly admixed Latin American population: African lineages (43.3 %), European lineages (32.0 %), Native American lineages (23.7 %) and Asian lineages (1.0 %). We have concluded that this type of tool can be used both in forensic genetics to the study of different human populations, such as highly admixed populations, and in the study of migration’s history and colonization of different states and countries of the world.     

Long Distance Linkage Disequilibrium and Limited Hybridization Suggest Cryptic Speciation in Atlantic Cod

Hybrid zones provide unprecedented opportunity for the study of the evolution of reproductive isolation, and the extent of hybridization across individuals and genomes can illuminate the degree of isolation. We examine patterns of interchromosomal linkage disequilibrium (ILD) and the presence of hybridization in Atlantic cod, Gadus morhua, in previously identified hybrid zones in the North Atlantic. Here, previously identified clinal loci were mapped to the cod genome with most (∼70%) occurring in or associated with (<5 kb) coding regions representing a diverse array of possible functions and pathways. Despite the observation that clinal loci were distributed across three linkage groups, elevated ILD was observed among all groups of clinal loci and strongest in comparisons involving a region of low recombination along linkage group 7. Evidence of ILD supports a hypothesis of divergence hitchhiking transitioning to genome hitchhiking consistent with reproductive isolation. This hypothesis is supported by Bayesian characterization of hybrid classes present and we find evidence of common F1 hybrids in several regions consistent with frequent interbreeding, yet little evidence of F2 or backcrossed individuals. This work suggests that significant barriers to hybridization and introgression exist among these co-occurring groups of cod either through strong selection against hybrid individuals, or genetic incompatibility and intrinsic barriers to hybridization. In either case, the presence of strong clinal trends, and little gene flow despite extensive hybridization supports a hypothesis of reproductive isolation and cryptic speciation in Atlantic cod. Further work is required to test the degree and nature of reproductive isolation in this species.     

Mutations in CSPP1 Lead to Classical Joubert Syndrome

Joubert syndrome and related disorders (JSRDs) are genetically heterogeneous and characterized by a distinctive mid-hindbrain malformation. Causative mutations lead to primary cilia dysfunction, which often results in variable involvement of other organs such as the liver, retina, and kidney. We identified predicted null mutations in CSPP1 in six individuals affected by classical JSRDs. CSPP1 encodes a protein localized to centrosomes and spindle poles, as well as to the primary cilium. Despite the known interaction between CSPP1 and nephronophthisis-associated proteins, none of the affected individuals in our cohort presented with kidney disease, and further, screening of a large cohort of individuals with nephronophthisis demonstrated no mutations. CSPP1 is broadly expressed in neural tissue, and its encoded protein localizes to the primary cilium in an in vitro model of human neurogenesis. Here, we show abrogated protein levels and ciliogenesis in affected fibroblasts. Our data thus suggest that CSPP1 is involved in neural-specific functions of primary cilia.     

Corynebacterium ulcerans isolates from humans and dogs: fibrinogen, fibronectin and collagen-binding, antimicrobial and PFGE profiles

Corynebacterium ulcerans has been increasingly isolated as an emerging zoonotic agent of diphtheria and other infections from companion animals. Since pets are able to act as symptomless carriers, it is also essential to identify virulence potential for humans of these isolates. In this work the ability of C. ulcerans to bind to fibrinogen (Fbg), fibronectin (Fn) and Type I collagen as well the genetic relationship among strains isolated from human and asymptomatic dogs in Rio de Janeiro (Brazil) were analyzed. Five pulsed-field gel electrophoresis (PFGE) profiles were demonstrated (I, II, III, IV and V). In addition, the IV and V profiles exhibiting ≥85 % similarity were expressed by the BR-AD41 and BR-AD61 strains from companion dogs living in the same neighborhood. Independent of the PFGE-types, human and dog isolates showed affinity to Fbg, Fn and collagen. Heterogeneity of PFGE profiles indicated endemicity of C. ulcerans in the Rio de Janeiro metropolitan area. Differences in the expression of adhesins to the human extracellular matrix may contribute to variations in the virulence and zoonotic potential of C. ulcerans strains.     

CRF type 1 receptors of the medial amygdala modulate inhibitory avoidance responses in the elevated T-maze

Corticotropin-releasing factor (CRF) plays a critical role in the mediation of physiological and behavioral responses to stressors. In the present study, we investigated the role played by the CRF system within the medial amygdala (MeA) in the modulation of anxiety and fear-related responses. Male Wistar rats were bilaterally administered into the MeA with CRF (125 and 250 ng/0.2 μl, experiment 1) or with the CRFR1 antagonist antalarmin (25 ng/0.2 μl, experiment 2) and 10 min later tested in the elevated T-maze (ETM) for inhibitory avoidance and escape measurements. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. To further verify if the anxiogenic effects of CRF were mediated by CRFR1 activation, we also investigated the effects of the combined treatment with CRF (250 ng/0.2 μl) and antalarmin (25 ng/0.2 μl) (experiment 3). All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. Results showed that CRF, in the two doses administered, facilitated ETM avoidance, an anxiogenic response. Antalarmin significantly decreased avoidance latencies, an anxiolytic effect, and was able to counteract the anxiogenic effects of CRF. None of the compounds administered altered escape responses or locomotor activity measurements. These results suggest that CRF in the MeA exerts anxiogenic effects by activating type 1 receptors, which might be of relevance to the physiopathology of generalized anxiety disorder.     

Bidirectional Modulation of Thermal and Chemical Sensitivity of TRPM8 Channels by the Initial Region of the N-terminal Domain

TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40–60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel.     

Flavonoids Modulate the Proliferation of Neospora caninum in Glial Cell Primary Cultures

Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3'',4''-dihydroxyflavone, 3'',4'',5,7-tetrahydroxyflavone (luteolin), and 3,3'',4'',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation.     

Karyoplast exchange between strontium- and 6-DMAP-parthenogenetically activated zygotes of cattle

Ooplasmic factors drive nuclear organization after fertilization and are also important for re-programming in nuclear transfer procedures, in which artificial activation is essential for reconstructed embryos to progress in development. The present research evaluated the effect of pronuclear transfer (PT) between zygotes parthenogenetically activated with ionomycin followed by strontium (S) or 6-DMAP (D) on early embryonic development. PT was performed in the same zygote to obtain embryos in control groups (S-PT and D-PT) and between zygotes activated with S and D to achieve embryos with differentially activated cytoplasm (C) and nucleus (N) (SCDN and DCSN). PT procedure did not affect cleavage and blastocyst rates, respectively, in PT control groups compared to non-manipulated control (S-PT: 73.6% and 7.3% compared with S-Control: 77.9% and 7.8%; and D-PT: 73.3% and 31.7% compared with D-Control: 83.1% and 41.5%). Cleavage, eight-cell, and blastocyst rates, respectively, were similar between SCDN (76.5%, 36.4%, and 6.8%) and DCSN (69.5%, 25.0%, and 4.9%) embryos. Developmental rates in SCDN were similar to S-PT, but inferior to D-PT. Developmental arrest up to eight-cell stage was greater in SCDN and DCSN than in S-PT and D-PT. In conclusion, karyoplast exchange between parthenogenetic zygotes activated with strontium and 6-DMAP can lead to nuclear–cytoplasmic incompatibilities and affect embryonic development to the eight-cell and blastocyst stages.     

Effect of eight benthic diatoms as feed on the growth of red abalone (Haliotis rufescens) postlarvae

The growth rate of abalone post larvae of Haliotis rufescens fed ad libitum with a benthic monoalgal diatom culture maintained as monocultures on a semi-commercial scale, was evaluated and correlated with the biochemical composition of the diatoms. The cell size (7.0 × 4.0 μm to 21.0 × 7.5 μm), protein percentage (7.42% to 13.66%), and ash content (49.03% to 59.61%) were different among diatom strains; lipid percentage, nitrogen free extract, and energy content (Kcal g⁻¹) were similar among diatom strains. The values of essential and non-essential amino and fatty acids composition differed among diatom strains. Differences in the abalone shell length and orthogonal analyses revealed postlarval growth was dependent on the quality of the food source. Postlarvae abalone displaying the longest shell lengths were fed Nitzschia thermalis var. minor and Amphiprora paludosa var. hyalina (1,712.0 ± 61 μm and 1,709 ± 67 μm, respectively), followed by Navicula incerta (1,413.3 ± 43 μm). The fatty acid content of benthic diatoms and abalone growth rate were not correlated.     

Oxidative stress induction by (+)-cordiaquinone J triggers both mitochondria-dependent apoptosis and necrosis in leukemia cells

(+)-Cordiaquinone J is a 1,4-naphthoquinone isolated from the roots of Cordia leucocephala that has antifungal and larvicidal effects. However, the cytotoxic effects of (+)-cordiaquinone J have never being explored. In the present study, the effect of (+)-cordiaquinone J on tumor cells viability was investigated, showing IC₅₀ values in the range of 2.7–6.6 μM in HL-60 and SF-295 cells, respectively. Studies performed in HL-60 leukemia cells indicated that (+)-cordiaquinone J (1.5 and 3.0 μM) reduces cell viability and 5-bromo-2-deoxyuridine incorporation after 24 h of incubation. (+)-Cordiaquinone J showed rapid induction of apoptosis, as indicated by phosphatidylserine externalization, caspase activation, DNA fragmentation, morphologic changes, and rapid induction of necrosis, as indicated by the loss of membrane integrity and morphologic changes. (+)-Cordiaquinone J altered the redox potential of cells by inducing the depletion of reduced GSH intracellular content, the generation of reactive oxygen species and the loss of mitochondrial membrane potential. However, pre-treatment of cells with N-acetyl-l-cysteine abolished most of the observed effects related to (+)-cordiaquinone J treatment, including those involving apoptosis and necrosis induction.