Morphological differentiation between S. mutans and S. sobrinus on modified SB-20 culture medium

Due to the major role of Streptococcus mutans and Streptococcus sobrinus in the etiology of dental caries, it is important to use culture media that allow for differentiating these bacterial species. The aim of this study was to evaluate the suitability of a modified SB-20 culture medium (SB-20M) for the isolation and morphological differentiation of S. mutans and S. sobrinus, compared to biochemical identification (biotyping). Saliva samples were collected using the spatula method from 145 children, seeded on plates containing the SB-20M, in which sucrose was replaced by coarse granular cane sugar, and incubated in microaerophilia at 37 degrees C during 72 h. Identification of the microorganisms was performed under stereomicroscopy based on colony morphology of 4904 colonies. The morphological identification was examined by biochemical tests of 94 randomly selected colonies with the macroscopic characteristic of S. mutans and S. sobrinus using sugar fermentation, resistance to bacitracin and production of hydrogen peroxide. There was no statistically significant difference (p> 0.05) between morphological identification in the SB-20M medium and biochemical identification (biotyping). Biotyping confirmed that S. mutans and S. sobrinus colonies were correctly characterized in the SB-20M in 95.8% and 95.5% of the cases, respectively. Of the mutans streptococci detected in the children 98% were S. mutans and 2% S. sobrinus. The SB-20M medium is reliable for detection and direct morphological identification of S. mutans and S. sobrinus. (C) 2010 Elsevier GmbH. All rights reserved.

Health in Brazil 2 Maternal and child health in Brazil: progress and challenges

In the past three decades, Brazil has undergone rapid changes in major social determinants of health and in the organisation of health services. In this report, we examine how these changes have affected indicators of maternal health, child health, and child nutrition. We use data from vital statistics, population censuses, demographic and health surveys, and published reports. In the past three decades, infant mortality rates have reduced substantially, decreasing by 5.5% a year in the 1980s and 1990s, and by 4.4% a year since 2000 to reach 20 deaths per 1000 livebirths in 2008. Neonatal deaths account for 68% of infant deaths. Stunting prevalence among children younger than 5 years decreased from 37% in 1974-75 to 7% in 2006-07. Regional differences in stunting and child mortality also decreased. Access to most maternal-health and child-health interventions increased sharply to almost universal coverage, and regional and socioeconomic inequalities in access to such interventions were notably reduced. The median duration of breastfeeding increased from 2.5 months in the 1970s to 14 months by 2006-07. Official statistics show stable maternal mortality ratios during the past 10 years, but modelled data indicate a yearly decrease of 4%, a trend which might not have been noticeable in official reports because of improvements in death registration and the increased number of investigations into deaths of women of reproductive age. The reasons behind Brazil`s progress include: socioeconomic and demographic changes (economic growth, reduction in income disparities between the poorest and wealthiest populations, urbanisation, improved education of women, and decreased fertility rates), interventions outside the health sector (a conditional cash transfer programme and improvements in water and sanitation), vertical health programmes in the 1980s (promotion of breastfeeding, oral rehydration, and immunisations), creation of a tax-funded national health service in 1988 (coverage of which expanded to reach the poorest areas of the country through the Family Health Program in the mid-1990s); and implementation of many national and state-wide programmes to improve child health and child nutrition and, to a lesser extent, to promote women`s health. Nevertheless, substantial challenges remain, including overmedicalisation of childbirth (nearly 50% of babies are delivered by caesarean section), maternal deaths caused by illegal abortions, and a high frequency of preterm deliveries.

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H-heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs. (C) 2010 Elsevier B.V. All rights reserved.

Deleterious effect of TRIS buffer on growth rates and pigment content of Gracilaria birdiae Plastino & EC Oliveira (Gracilariales, Rhodophyta)

This work evaluated the effects of Tris (hydroxymethyl)-aminomethane (TRIS) buffer and its interaction with nutrient concentration on the development of Gracilaria birdie, a common species on the Brazilian coast that has been exploited for agar production. Responses to different conditions were assessed through growth rates and pigment content (chlorophyll a, phycoerythrin, phycocyanin and allophycocyanin). Provasoli`s nutrient solution with and without TRIS addition was tested at concentrations of 12.5, 25 and 50%. The pH was also monitored. G. birdiae grew better in the absence of TRIS and at low nutrient concentrations, 12.5 and 25% (growth rates of 10.8-11.3%.day(-1)). Higher contents of phycoerythrin and chlorophyll a were observed without TRIS at 12.5 and 25% (Phycoerythrin, 649.6-698.0 mu g g(-1) fresh biomass; Chlorophyll a, 156.0-168.6 mu g g(-1) fresh biomass). These findings highlight the deleterious effect of TRIS on growth and phycoerythrin and chlorophyll a content. They also demonstrate the importance of appropriate nutrient concentration for laboratory cultures, depending on the intrinsic characteristics of each species.

Color inheritance and pigment characterization of red (wild-type), greenish-brown, and green strains of Gracilaria birdiae (Gracilariales, Rhodophyta)

Species of Gracilaria are some of the most useful algae in the world for the production of agar. As a consequence of its economic importance, the genus has been the subject of many studies worldwide. Color variants of Gracilaria birdiae have been found in the natural population on the Brazilian coast, and they have also been isolated from plants cultivated in laboratory. These findings raised new questions regarding intraspecific variation and the prospects of cultivating such variants for their agar production. Therefore, this work aimed to determine the mode of color inheritance for two G. birdiae strains: a greenish-brown strain (gb) found in a natural population and a green strain (gr) which had arisen as a spontaneous mutation in a red plant cultured in the laboratory. The pigment contents of these strains, as well as the red wildtype (rd), were also characterized. Crosses between female and male plants of the same color (rd, gr, or gb) and between different colors were performed. Crosses between plants of the same color showed tetrasporophytic and gametophytic descendents of the parental color. Recessive nuclear inheritance was found in the greenish-brown strain, and cytoplasmic maternal inheritance was found in the green strain; both had lower phycoerythrin and higher concentrations of allophycocyanin and phycocyanin than the wild-type. Chlorophyll a contents were similar among all strains. Taken together, our results contribute to knowledge about the variability of this important red algae. In addition, since greenish-brown and green strains showed stability of color, both could be selected and tested in experimental sea cultivation to evaluate if mutants have advantageous performance when compared with red strain.

Iridaea cordata (Gigartinales, Rhodophyta): responses to artificial UVB radiation

The effects of UVB radiation on the different developmental stages of the carrageenan-producing red alga Iridaea cordata were evaluated considering: (1) carpospore and discoid germling mortality; (2) growth rates and morphology of young tetrasporophytes; and (3) growth rates and pigment content of field-collected plant fragments. Unialgal cultures were submitted to 0.17, 0.5, or 0.83 W m(-2) of UVB radiation for 3 h per day. The general culture conditions were as follows: 12 h light/12 h dark cycles; irradiance of 55 mu mol photon. per square meter per second; temperature of 9 +/- 1 degrees C; and seawater enriched with Provasoli solution. All UVB irradiation treatments were harmful to carpospores (0.17 W m(-2) = 40.9 +/- 6.9%, 0.5 W m(-2) = 59.8 +/- 13.4%, 0.83 W m(-2) = 49 +/- 17.4% mortality in 3 days). Even though the mortality of all discoid germlings exposed to UVB radiation was unchanged when compared to the control, those germlings exposed to 0.5 and 0.83 W m(-2) treatments became paler and had smaller diameters than those cultivated under control treatment. Decreases in growth rates were observed in young tetrasporophytes, mainly in 0.5 and 0.83 W m(-2) treatments. Similar effects were only observed in fragments of adult plants cultivated at 0.83 W m(-2). Additionally, UVB radiation caused morphological changes in fragments of adult plants in the first week, while the young individuals only displayed this pattern during the third week. The verified morphological alterations in I. cordata could be interpreted as a defense against UVB by reducing the area exposed to radiation. However, a high level of radiation appears to produce irreparable damage, especially under long-term exposure. Our results suggest that the sensitivity to ultraviolet radiation decreases with increased algal age and that the various developmental stages have different responses when exposed to the same doses of UVB radiation.

Life history, morphological variability and growth rates of the life phases of Gracilaria tenuistipitata (Rhodophyta: Gracilariales) in vitro

Gracilaria tenuistipitata, a species of commercial interest, is becoming a model organism for studies on red algal physiology and molecular biology as it can be grown easily in vitro under a broad range of conditions. Most of the experiments carried out around the world have been based on a tetrasporophytic clone isolated in our laboratory from a specimen collected in China. Here we describe the life history of this species, give anatomic details of the reproductive structures, illustrate the morphological variability of tetraspore progeny and compare the growth rate of gametophytic and sporophytic thalli. Tetrasporophytic branches showed higher growth rates than gametophytic branches.

Effects of kinetin and nitrogen on growth rates, pigment and protein contents in wild and phycoerythrin-deficient strains of Hypnea musciformis (Rhodophyta)

Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. is the main source for carrageenan production in Brazil and strains with selected characteristics could improve the production of raw material. The effects of kinetin on growth rates, morphology, protein content, and concentrations of pigments (chlorophyll a, phycoerythrin, phycocyanin, and allophycocyanin) were assessed in the wild strain (brown phenotype) and in the phycoerythrin-deficient strain (green phenotype) of H. musciformis. Concentrations of kinetin ranging from 0 to 50 mu M were tested in ASP 12-NTA synthetic medium with 10 mu M nitrate (N-limited) and 100 mu M nitrate (N-saturated). In N-limited condition, kinetin stimulated growth rates of the phycoerythrin-deficient strain and formation of lateral branches in both colour strains. Kinetin stimulated protein biosynthesis in both strains. However, differences between both nitrogen conditions were significant only in the phycoerythrin-deficient strain. In the wild strain, effects of kinetin on concentrations of phycobiliproteins were not significant in both nitrogen conditions, except for chlorophyll content. However, the phycoerythrin-deficient strain showed an opposite response, and kinetin stimulated the phycobiliprotein biosynthesis, with the highest concentrations of phycoerythrin in N-saturated medium, while the highest concentrations of allophycocyanin and phycocyanin were observed in N-limited medium. These results indicate that the effects of kinetin on growth, morphology, protein and phycobiliprotein contents are influenced by nitrogen availability, and the main nitrogen storage pools in phycoerythrin-deficient strain of H. musciformis submitted to N-limited conditions were phycocyanin and allophycocianin, the biosynthesis of which was enhanced by kinetin.

Obesity induced by high-fat diet promotes insulin resistance in the ovary

Besides the effects on peripheral energy homeostasis, insulin also has an important role in ovarian function. Obesity has a negative effect on fertility, and may play a role in the development of the polycystic ovary syndrome in susceptible women. Since insulin resistance in the ovary could contribute to the impairment of reproductive function in obese women, we evaluated insulin signaling in the ovary of high-fat diet-induced obese rats. Female Wistar rats were submitted to a high-fat diet for 120 or 180 days, and the insulin signaling pathway in the ovary was evaluated by immunoprecipitation and immunoblotting. At the end of the diet period, we observed insulin resistance, hyperinsulinemia, an increase in progesterone serum levels, an extended estrus cycle, and altered ovarian morphology in obese female rats. Moreover, in female obese rats treated for 120 days with the high-fat diet, the increase in progesterone levels occurred together with enhancement of LH levels. The ovary from high-fat-fed female rats showed a reduction in the insulin receptor substrate/phosphatidylinositol 3-kinase/AKT intracellular pathway, associated with an increase in FOXO3a, IL1B, and TNF alpha protein expression. These changes in the insulin signaling pathway may have a role in the infertile state associated with obesity. Journal of Endocrinology (2010) 206, 65-74

Trophoblast phagocytic program: roles in different placental systems

Although not belonging to the class of professional phagocytes, in many species trophoblast cells exhibit intense phagocytic activity. The complete range of physiological functions of trophoblast phagocytosis has not yet been fully characterized. Close association between the trophoblast and nutrition was determined many years ago. Hubrecht (1889) when proposing for the first time the name trophoblast to the external layer of the blastocyst, directly established the nutritive significance of this embryonic layer. Indeed, histotrophic phagocytosis, i.e. the internalization of maternal cells and secreted materials, is considered an important function of the trophoblast before the completion of the placenta. Recently, however, unexpected characteristics of the trophoblast have significantly enhanced our understanding of this process. Roles in acquisition of space for embryo development, in tissue remodeling during implantation and placentation and in defense mechanisms are highlighting how this cellular activity may be relevant for the maternal-fetal relationship beyond its nutritional function.

Tissue distribution of quiescin Q6/sulfhydryl oxidase (QSOX) in developing mouse

Quiescin Q6/sulfhydryl oxidases (QSOX) are revisited thiol oxidases considered to be involved in the oxidative protein folding, cell cycle control and extracellular matrix remodeling. They contain thioredoxin domains and introduce disulfide bonds into proteins and peptides, with the concomitant hydrogen peroxide formation, likely altering the redox environment. Since it is known that several developmental processes are regulated by the redox state, here we assessed if QSOX could have a role during mouse fetal development. For this purpose, an anti-recombinant mouse QSOX antibody was produced and characterized. In E-13.5, E-16.5 fetal tissues, QSOX immunostaining was confined to mesoderm- and ectoderm-derived tissues, while in P1 neonatal tissues it was slightly extended to some endoderm-derived tissues. QSOX expression, particularly by epithelial tissues, seemed to be developmentally-regulated, increasing with tissue maturation. QSOX was observed in loose connective tissues in all stages analyzed, intra and possibly extracellularly, in agreement with its putative role in oxidative folding and extracellular matrix remodeling. In conclusion, QSOX is expressed in several tissues during mouse development, but preferentially in those derived from mesoderm and ectoderm, suggesting it could be of relevance during developmental processes.

Metastatic melanoma positively influences pregnancy outcome in a mouse model: could a deadly tumor support embryo life?

The incidence of melanoma is increasing worldwide. It is one of the leading cancers in pregnancy and the most common malignancy to metastasize to placenta and fetus. There are no publications about experimental models of melanoma and pregnancy. We propose a new experimental murine model to study the effects of melanoma on pregnancy and its metastatic process. We tested several doses of melanoma cells until we arrived at the optimal dose, which produced tumor growth and allowed animal survival to the end of pregnancy. Two control groups were used: control (C) and stress control (SC) and three different routes of inoculation: intravenous (IV), intraperitoneal (IP) and subcutaneous (SC). All the fetuses and placentas were examined macroscopically and microscopically. The results suggest that melanoma is a risk factor for intrauterine growth restriction but does not affect placental weight. When inoculated by the SC route, the tumor grew only in the site of implantation. The IP route produced peritoneal tumoral growth and also ovarian and uterine metastases in 60% of the cases. The IV route produced pulmonary tumors. No placental or fetal metastases were obtained, regardless of the inoculation route. The injection of melanoma cells by any route did not increase the rate of fetal resorptions. Surprisingly, animals in the IV groups had no resorptions and a significantly higher number of fetuses. This finding may indicate that tumoral factors released in the host organism to favor tumor survival may also have a pro-gestational action and consequently improve the reproductive performance of these animals.

Macrophage migration inhibitory factor is up-regulated in human first-trimester placenta stimulated by soluble antigen of Toxoplasma gondii, resulting in increased monocyte adhesion on villous explants

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAB, interferon-gamma (IFN-gamma), or STAB plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAB (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAB or STAB plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.