Wide Dispersal and Possible Multiple Origins of Low-Copy-Number Plasmids in Rickettsia Species Associated with Blood-Feeding Arthropods

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, ""Candidatus Rickettsia amblyommii,"" R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two ""Ca. Rickettsia amblyommii"" isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of ""Ca. Rickettsia amblyommii,"" R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.

Non-homologous sex chromosomes in two species of the genus Eigenmannia (Teleostei : Gymnotiformes)

The Neotropical genus Eigenmannia is a fish group with unknown species diversity where representatives possess a broad range of chromosomal sex determining systems namely XY/XX, X1X2Y/X1X1X2X2, ZZ/ZW as well as homomorphic sex chromosomes. To test the homology of two heteromorphic XY sex chromosome systems present in two sympatric populations, reciprocal cross-species FISH experiments were performed using probes derived by microdissection of X and Y chromosomes present in analyzed specimens of Eigenmannia virescens and Eigenmannia sp.2, respectively. While X and Y paint probes hybridized to species-specific sex chromosomes, in reciprocal cross-FISH both probes hybridized exclusively to autosomes. The result suggests multiple independent origins of the XY systems in the analyzed populations. Copyright (C) 2008 S. Karger AG, Basel.

From Offshore to Onshore: Multiple Origins of Shallow-Water Corals from Deep-Sea Ancestors

Shallow-water tropical reefs and the deep sea represent the two most diverse marine environments. Understanding the origin and diversification of this biodiversity is a major quest in ecology and evolution. The most prominent and well-supported explanation, articulated since the first explorations of the deep sea, holds that benthic marine fauna originated in shallow, onshore environments, and diversified into deeper waters. In contrast, evidence that groups of marine organisms originated in the deep sea is limited, and the possibility that deep-water taxa have contributed to the formation of shallow-water communities remains untested with phylogenetic methods. Here we show that stylasterid corals (Cnidaria: Hydrozoa: Stylasteridae)-the second most diverse group of hard corals-originated and diversified extensively in the deep sea, and subsequently invaded shallow waters. Our phylogenetic results show that deep-water stylasterid corals have invaded the shallow-water tropics three times, with one additional invasion of the shallow-water temperate zone. Our results also show that anti-predatory innovations arose in the deep sea, but were not involved in the shallow-water invasions. These findings are the first robust evidence that an important group of tropical shallow-water marine animals evolved from deep-water ancestors.

Multiple origins of vagrant Subantarctic fur seals: A long journey to the Brazilian coast detected by molecular markers

In this study, we present the first data about putative source populations of the vagrant Subantarctic fur seal, Arctocephalus tropicalis, found on the Brazilian coast, through the comparison of their mitochondrial DNA control sequences to exclusive haplotypes from the main breeding colonies of the species. The results indicated that, despite the majority of the vagrant individuals are from Gough Island (the closest breeding site to the Brazilian coast), they also come from other reproductive colonies, such as Crozet Island, a distance around 16,500 km from the Brazilian coast. Furthermore, the molecular data identified three possible management units: (1) Gough, (2) Amsterdam, and (3) Marion, Macquarie and Crozet. This significant genetic subdivision must be taken into account in any future management plan for the species conservation, including rehabilitation and even reintroduction of vagrant fur seals.

Litter size, effects of maternal body size, and date of birth in South American scorpions (Arachnida: Scorpiones)

We present new data on litter size and date of birth (month) for 21 South American scorpions species. We provide data for one katoikogenic species, the liochelid Opisthacanthus cayaporum Vellard, 1932 (offspring = 3; birth month: Jan); and for several apoikogenic species, such as the bothriurids Bothriurus araguayae Vellard, 1934 (53; Sep), B. rochensis San Martín, 1965 (22-28; Jan, Aug); the buthids Ananteris balzanii Thorell, 1891 (10-34; Jan-Mar), Physoctonus debilis (Koch, 1840) (2; Sep), Rhopalurus amazonicus Lourenço, 1986 (19; Nov), R. lacrau Lourenço & Pinto-da-Rocha, 1997 (30; Dec), R. laticauda Thorell, 1876 (41; Nov), R. rochai Borelli, 1910 (11-47; Dec-Jan, Mar-Apr), Tityus bahiensis (Perty, 1833) (4-23; Oct-Mar), T. clathratus Koch, 1844 (8-18; Nov-Jan), T. costatus (Karsch, 1879) (21-25; Jan, Apr), T. kuryi Lourenço, 1997 (4-16; Mar), T. mattogrossensis Borelli, 1901(8-9; May), T. obscurus (Gervais, 1843) (16-31; Jan-Feb, May, Jul), T. serrulatus Lutz & Mello, 1922 (8-36; Dec, Feb-Apr), T. silvestris Pocock, 1897 (5-14; Dec-Jan, Apr), T. stigmurus (Thorell, 1876) (10-18; Nov, Jan, Mar), Tityus sp. 1 (T. clathratus group - 7-12; Feb-Apr), Tityus sp. 2 (T. bahiensis group - 2; Mar); and the chactid Brotheas sp. (8-21; Jan, Apr). We observed multiple broods: R. lacrau (offspring in the 2nd brood = 27), T. kuryi (6-16), T. obscurus (2-32), T. silvestris (8), T. stigmurus (4-9), T. bahiensis (offspring in the 2nd brood = 2-18; 3rd = 1), and T. costatus (2nd brood = 18; 3rd = 4). We found statistically significant positive correlation between female size and litter size for T. bahiensis and T. silvestris, and nonsignificant correlation for T. serrulatus.

Origins of the Xylella fastidiosa Prophage-Like Regions and Their Impact in Genome Differentiation

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes.

Exposure to maternal smoking during fetal life affects food preferences in adulthood independent of the effects of intrauterine growth restriction

Experimental animal studies have shown that nicotine exposure during gestation alters the expression of fetal hypothalamic neuropeptides involved in the control of appetite. We aimed to determine whether the exposure to maternal smoking during gestation in humans is associated with an altered feeding behavior of the adult offspring. A longitudinal prospective cohort study was conducted including all births from Ribeirao Preto (Sao Paulo, Brazil) between 1978 and 1979. At 24 years of age, a representative random sample was re-evaluated and divided into groups exposed (n = 424) or not (n = 1586) to maternal smoking during gestation. Feeding behavior was analyzed using a food frequency questionnaire. Covariance analysis was used for continuous data and the chi(2) test for categorical data. Results were adjusted for birth weight ratio, body mass index, gender, physical activity and smoking, as well as maternal and subjects` schooling. Individuals exposed to maternal smoking during gestation ate more carbohydrates than proteins (as per the carbohydrate-to-protein ratio) than non-exposed individuals. There were no differences in the consumption of the macronutrients themselves. We propose that this adverse fetal life event programs the individual`s physiology and metabolism persistently, leading to an altered feeding behavior that could contribute to the development of chronic diseases in the long term.

Health surveys and use of maternal and child health care services in three municipalities within the Sao Paulo metropolitan area

Objectives. To describe the changes in the use of maternal and child health care services by residents of three municipalities-Embu, Itapecerica da Serra, and Taboao da Serra-in the Sao Paulo metropolitan area, 12 years after the implementation of the Unified Health System (SUS) in Brazil, and to analyze the potential of population-based health care surveys as sources of data to evaluate these changes. Methods. Two population-based, cross-sectional surveys were carried out in 1990 and 2002 in municipalities located within the Sao Paulo metropolitan area. For children under 1 year of age, the two periods were compared in terms of outpatient services utilization and hospital admission; for the mothers, the periods were compared in terms of prenatal care and deliveries. In both surveys, stratified and multiple-stage conglomerate sampling was employed, with standardization of interview questions. Results. The most important changes observed were regarding the location of services used for prenatal care, deliveries, and hospitalization of children less than 1 year of age. There was a significant increase in the use of services in the surrounding region or hometown, and decrease in the utilization of services in the city of Sao Paulo (in 1990, 80% of deliveries and almost all admissions for children less than 1 year versus 32% and 46%, respectively, in 2002). The use of primary care units and 24-hour walk-in clinics also increased. All these changes reflect care provided by public resources. In the private sector, there was a decrease in direct payments and payments through company-paid health insurance and an increase in payments through self-paid health insurance. Conclusions. The major changes observed in the second survey occurred simultaneous to the changes that resulted from the implementation of the SUS. Population-based health surveys are adequate for analyzing and comparing the utilization of health care services at different times.

The impact of maternal cortisol concentrations on child arterial elasticity

Epidemiological studies suggest that glucocorticoid excess in the fetus may contribute to the pathophysiology of cardiovascular diseases in adulthood. However, the impact of maternal glucocorticoid on the cardiovascular system of the offspring has not been much explored in studies involving humans, especially in childhood. The objective of this study was to assess the influence of maternal cortisol concentrations on child arterial elasticity. One hundred and thirty pregnant women followed from 1997 to 2000, and respective children 5-7 years of age followed from 2004 to 2006 were included in the study. Maternal cortisol was determined in saliva by an enzyme immunoassay utilizing the mean concentration of nine samples of saliva. Arterial elasticity was assessed by the large artery elasticity index (LAEI; the capacitive elasticity of large arteries) by recording radial artery pulse wave, utilizing the equipment HDI/PulseWave CR-2000 Cardiovascular Profiling System (R). The nutritional status of the children was determined by the body mass index (BMI). Insulin concentration was assessed by chemiluminescence, and insulin resistance by the homeostasis model assessment. Blood glucose, total cholesterol and fractions (LDL-c and HDL-c) and triglyceride concentrations were determined by automated enzymatic methods. The association between maternal cortisol and child arterial elasticity was assessed by multivariate linear regression analysis. There was a statistically significant association between maternal cortisol and LAEI (P=0.02), controlling for birth weight, age, BMI and HDL-c of the children. This study suggests that exposure to higher glucocorticoid concentrations in the prenatal period is associated to lower arterial elasticity in childhood, an earlier cardiovascular risk marker.

Independent fusions and recent origins of sex chromosomes in the evolution and diversification of glass knife fishes (Eigenmannia)

The genus Eigenmannia comprises several species groups that display a surprising variety of diploid chromosome numbers and sex-determining systems. In this study, hypotheses regarding phylogenetic relationships and karyotype evolution were investigated using a combination of molecular and cytogenetic methods. Phylogenetic relationships were analyzed for 11 cytotypes based on sequences from five mitochondrial DNA regions. Parsimony-based character mapping of sex chromosomes confirms previous suggestions of multiple origins of sex chromosomes. Molecular cytogenetic analyses involved chromosome painting using probes derived from whole sex chromosomes from two taxa that were hybridized to metaphases of their respective sister cytotypes. These analyses showed that a multiple XY system evolved recently (<7 mya) by fusion. Furthermore, one of the chromosomes that fused to form the neo-Y chromosome is fused independently to another chromosome in the sister cytotype. This may constitute an efficient post-mating barrier and might imply a direct function of sex chromosomes in the speciation processes in Eigenmannia. The other chromosomal sex-determination system investigated is shown to have differentiated by an accumulation of heterochromatin on the X chromosome. This has occurred in the past 0.6 my, and is the most recent chromosomal sex-determining system described to date. These results show that the evolution of sex-determining systems can proceed very rapidly. Heredity (2011) 106, 391-400; doi:10.1038/hdy.2010.82; published online 23 June 2010

Can physical exercise during gestation attenuate the effects of a maternal perinatal low-protein diet on oxygen consumption in rats?

A protocol of physical exercise, based on maximal oxygen uptake ((V) over dot(O2max)), for female rats before and during pregnancy was developed to evaluate the impact of a low-protein diet on oxygen consumption during gestation and growth rate of the offspring. Virgin female Wistar rats were divided into four groups as follows: untrained (NT, n = 5); trained (T, n = 5); untrained with low-protein diet (NT+LP, n = 5); and trained with low-protein diet (T+LP, n = 5). Trained rats were submitted to a protocol of moderate physical training on a treadmill over a period of 4 weeks (5 days week(-1) and 60 min day(-1), at 65% of (V) over dot(O2max)). At confirmation of pregnancy, the intensity and duration of the exercise was reduced. Low-protein groups received an 8% casein diet, and their peers received a 17% casein diet. The birthweight and growth rate of the pups up to the 90th day were recorded. Oxygen consumption ((V) over dot(O2)), CO(2) production and respiratory exchange ratio (RER) were determined using an indirect open-circuit calorimeter. Exercise training increased. (V) over dot(O2max) by about 20% when compared with the initial values (45.6 +/- 1.0 ml kg(-1) min(-1)). During gestation, all groups showed a progressive reduction in the resting (V) over dot(O2) values. Dams in the NT+LP group showed lower values of resting (V) over dot(O2) than those in the NT group. The growth rate of pups from low-protein-fed mothers was around 50% lower than that of their respective controls. The T group showed an increase in body weight from the 60th day onwards, while the NT+LP group presented a reduced body weight from weaning onwards. In conclusion, physical training attenuated the impact of the low- protein

Do the origins of primary teeth affect the bond strength of a self-etching adhesive system to dentin?

The aim of this in vitro study was to evaluate the tensile bond strength of a self-etching adhesive system to three different dentinal substrates. Primary molar teeth that had been recently exfoliated (RE), with unknown time of exfoliation (UT), and extracted due to prolonged retention (PR) were used for this investigation. Ten primary molar teeth of each group were cut in the middle following the mesio-distal direction, creating a total of twenty specimens per group. The specimens were included in acrylic resin and had a flat dentin surface exposed. The self-etching adhesive system was applied to this surface and a 3-millimeter high cone with diameter of 2 mm in the adhesion area was constructed using composite resin. The specimens were stored in distilled water at 37ºC for 24 hours. Fifteen specimens of each substrate were used for the tensile bond test (n = 15) and 5 had the interface analyzed by scanning electron microscopy (SEM). The data was examined by one-way ANOVA and presented no significant differences between groups (p = 0.5787). The mean values obtained for RE, UT and PR were 18.39 ± 9.70, 19.41 ± 7.80, and 23.30 ± 9.37 MPa, respectively. Any dentinal substrates of primary teeth studied are safe for tensile bond strength tests with adhesive systems.

Análise morfológica e fisiológica dos fígados e rins de ratas prenhes e seus fetos tratados pela associação zidovudina, lamivudina e ritonavir durante toda a prenhez

OBJETIVO: avaliar os efeitos da administração da associação zidovudina-lamivudina-ritonavir nos fígados e rins de ratas prenhes e seus conceptos do ponto de vista morfológico e fisiológico. MÉTODOS: 40 ratas albinas prenhes foram aleatoriamente divididas em 4 grupos: 1 controle (Ctrl: controle de veículo) e 3 experimentais (Exp1x, Exp3x e Exp9x). Estes últimos foram tratados por solução oral de zidovudina/lamivudina/ritonavir (Exp1x: 10/5/20 mg/kg; Exp3x: 30/15/60 mg/kg; Exp9x: 90/45/180 mg/kg). As drogas e o veículo foram administrados por gavagem, desde o 1º até o 20º dia de prenhez. No último dia do experimento, todos os animais foram anestesiados e sangue foi retirado da cavidade cardíaca para avaliação sérica das enzimas aspartato aminotransferase (AST) e alanina aminotransferase (ALT), por método calorimétrico, bem como da ureia, determinada por método cinético-enzimático, e creatinina, por método cinético-colorimétrico. Em seguida, fragmentos dos fígados e rins maternos e fetais foram coletados, fixados em formol a 10% e processados segundo os métodos histológicos para inclusão em parafina. Cortes com 5 µm de espessura foram corados pela hematoxilina-eosina (HE) e analisados por microscopia de luz. Na leitura das lâminas, considerou-se o padrão de normalidade para fígado e rins, tais como: hepatócitos, espaço porta íntegros e veias hepáticas bem definidas. Nos rins, a presença de corpúsculos renais, túbulos contorcidos e alças de Henle típicos. Nos fígados fetais considerou-se, ainda, a morfologia das células da linhagem eritrocitária nas diferentes fases do desenvolvimento, bem como os megacariócitos. Quando houve alteração da coloração padrão estabelecida para as estruturas hepáticas e renais, alteração na morfologia de núcleos, rompimento de limites de alguma organela citoplasmática, presença de congestão vascular, tudo isso foi entendido como provavelmente provocado pelas drogas em sua(s) dose(s) de aplicação. A avaliação estatística foi realizada por análise de variância (ANOVA), completada pelo teste de Tukey-Kramer (p<0,05). RESULTADOS: os fígados maternos dos grupos Ctrl, Exp1x e Exp3x mostraram hepatócitos típicos, espaço porta íntegros e veias hepáticas com aspecto normal. No fígado materno do grupo Exp9x, foram encontrados hepatócitos com sinais de atrofia e apoptose (eosinofilia citoplasmática e núcleos picnóticos). Além disso, identificou-se vasodilatação dos capilares sinusoides (congestão). Os rins maternos dos grupos Ctrl e Exp1x apresentaram-se normais, com corpúsculos renais, túbulos contorcidos e alças de Henle típicos. Já nos grupos Exp3x e Exp9x, foram encontrados congestão vascular, glomérulos pequenos ricos em células contendo núcleos hipercromáticos, sendo mais intensos no Exp9x. Com relação aos fígados e rins fetais, não foram observadas alterações morfológicas ou fisiológicas nos grupos estudados. Encontrou-se aumento significante nos níveis da AST (305,70±55,80; p<0,05) e da creatinina (0,50±0,09; p<0,05) no grupo Exp9x. CONCLUSÕES: nossos resultados evidenciam que a administração da associação zidovudina/lamivudina/ritonavir a ratas prenhes em altas doses causa alterações morfológicas e funcionais nos fígados e rins maternos. Não houve alterações nem morfológicas nem fisiológicas nos fígados e rins fetais.

Categorization skills and recall in brain damaged children: a multiple case study

During development, children become capable of categorically associating stimuli and of using these relationships for memory recall. Brain damage in childhood can interfere with this development. This study investigated categorical association of stimuli and recall in four children with brain damages. The etiology, topography and timing of the lesions were diverse. Tasks included naming and immediate recall of 30 perceptually and semantically related figures, free sorting, delayed recall, and cued recall of the same material. Traditional neuropsychological tests were also employed. Two children with brain damage sustained in middle childhood relied on perceptual rather than on categorical associations in making associations between figures and showed deficits in delayed or cued recall, in contrast to those with perinatal lesions. One child exhibited normal performance in recall despite categorical association deficits. The present results suggest that brain damaged children show deficits in categorization and recall that are not usually identified in traditional neuropsychological tests.

Morphine infusions into the rostrolateral periaqueductal gray affect maternal behaviors

It is well established that morphine inhibits maternal behaviors. Previous studies by our group have shown activation of the rostrolateral periaqueductal gray (rlPAG) upon inhibition-intended subcutaneous injections of morphine. In this context, we demonstrated that a single naloxone infusion into the rlPAG, following this opioid-induced inhibition, reactivated maternal behaviors. Since these data were obtained by using peripheral morphine injections, the present study was designed to test whether morphine injected directly into the rlPAG would affect maternal behaviors. Our hypothesis that morphine acting through the rlPAG would disrupt maternal behaviors was confirmed with a local infusion of morphine. The mothers showed shorter latency for locomotor behavior to explore the home cage (P = 0.049). Inhibition was especially evident regarding retrieving (P = 0.002), nest building (P = 0.05) and full maternal behavior (P = 0.023). These results support the view that opioidergic transmission plays a behaviorally meaningful inhibitory role in the rostrolateral PAG.