Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application

BACKGROUND: Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS: Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS: In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS: We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.

Inquiry of antibodies anti-Neospora caninum in dairy cattle, dogs and rural workers of the south-west region of Mato Grosso State

Considering the importance of neosporosis in the animal health and production, the frequency of antibodies to Neospora caninum was evaluated in dairy cattle of the Southwestern region of Mato Grosso State, Brazil, in addition to serum samples obtained from dogs and humans living in the farms. A total of 1036 serum samples were analyzed, from which 932 were from dairy bovine females, 37 from dogs and 67 from humans, from 24 farms and examined by the indirect fluorescent antibody test (IFAT). Reactive human scrum samples were retested by Western-blotting to confirm the results. Antibodies to N. caninum were found in 499 cattle sera (53.5 %), with at least one positive in each farm, 25 dog sera (67.6 %) and seven human sera (10.5 %). There was no significant difference in the number of positive cattle sera according to age group. The results indicate a wide dissemination of N caninum in the studied region.

Synteny of human chromosomes 14 and 15 in the platyrrhines (Primates, Platyrrhini)

In order to study the intra- and interspecific variability of the 14/15 association in Platyrrhini, we analyzed 15 species from 13 genera, including species that had not been described yet. The DNA libraries of human chromosomes 14 and 15 were hybridized to metaphases of Alouatta guariba clamitans, A. caraya, A. sara, Ateles paniscus chamek, Lagothrix lagothricha, Brachyteles arachnoides, Saguinus midas midas, Leontopithecus chrysomelas, Callimico goeldii, Callithrix sp., Cebus apella, Aotus nigriceps, Cacajao melanocephalus, Chiropotes satanas and Callicebus caligatus. The 14/15 hybridization pattern was present in 13 species, but not in Alouatta sara that showed a 14/15/14 pattern and Aotus nigriceps that showed a 15/14/15/14 pattern. In the majority of the species, the HSA 14 homologue retained synteny for the entire chromosome, whereas the HSA 15 homologue displayed fragmented segments. Within primates, the New World monkeys represent the taxon with the highest variability in chromosome number (2n = 16 to 62). The presence of the HSA 14/15 association in all species and subspecies studied herein confirms that this association is the ancestral condition for platyrrhines and that this association has been retained in most platyrrhines, despite the occurrence of extensive inter- and intrachromosomal rearrangements in this infraorder of Primates.

Interleukin-10 production by tumor infiltrating macrophages plays a role in Human Papillomavirus 16 tumor growth

Background: Human Papillomavirus, HPV, is the main etiological factor for cervical cancer. Different studies show that in women infected with HPV there is a positive correlation between lesion grade and number of infiltrating macrophages, as well as with IL-10 higher expression. Using a HPV16 associated tumor model in mice, TC-1, our laboratory has demonstrated that tumor infiltrating macrophages are M2-like, induce T cell regulatory phenotype and play an important role in tumor growth. M2 macrophages secrete several cytokines, among them IL-10, which has been shown to play a role in T cell suppression by tumor macrophages in other tumor models. In this work, we sought to establish if IL-10 is part of the mechanism by which HPV tumor associated macrophages induce T cell regulatory phenotype, inhibiting anti-tumor activity and facilitating tumor growth. Results: TC-1 tumor cells do not express or respond to IL-10, but recruit leukocytes which, within the tumor environment, produce this cytokine. Using IL-10 deficient mice or blocking IL-10 signaling with neutralizing antibodies, we observed a significant reduction in tumor growth, an increase in tumor infiltration by HPV16 E7 specific CD8 lymphocytes, including a population positive for Granzyme B and Perforin expression, and a decrease in the percentage of HPV specific regulatory T cells in the lymph nodes. Conclusions: Our data shows that in the HPV16 TC-1 tumor mouse model, IL-10 produced by tumor macrophages induce regulatory phenotype on T cells, an immune escape mechanism that facilitates tumor growth. Our results point to a possible mechanism behind the epidemiologic data that correlates higher IL-10 expression with risk of cervical cancer development in HPV infected women.

Mutations in CIC and FUBP1 Contribute to Human Oligodendroglioma

Oligodendrogliomas are the second most common malignant brain tumor in adults and exhibit characteristic losses of chromosomes 1p and 19q. To identify the molecular genetic basis for this alteration, we performed exomic sequencing of seven tumors. Among other changes, we found that the CIC gene (homolog of the Drosophila gene capicua) on chromosome 19q was somatically mutated in six cases and that the FUBP1 gene [encoding far-upstream element (FUSE) binding protein] on chromosome 1p was somatically mutated in two tumors. Examination of 27 additional oligodendrogliomas revealed 12 and 3 more tumors with mutations of CIC and FUBP1, respectively, 58% of which were predicted to result in truncations of the encoded proteins. These results suggest a critical role for these genes in the biology and pathology of oligodendrocytes.

Afro-Derived Brazilian Populations: Male Genetic Constitution Estimated by Y-Chromosomes STRs and AluYAP Element Polymorphisms

The genetic constitution of Afro-derived Brazilian populations is barely studied. To improve that knowledge, we investigated the AluYAP element and five Y-chromosome STRs (DYS19, DYS390, DYS391, DYS392, and DYS393) to estimate ethnic male contribution in the constitution of four Brazilian quilombos remnants: Mocambo, Rio das Ras, Kalunga, and Riacho de Sacutiaba. Results indicated significant differences among communities, corroborating historical information about the Brazilian settlement. We concluded that besides African contribution, there was a great European participation in the constitution of these four populations and that observed haplotype variability could be explained by gene flow to quilombos remnants and mutational events in microsatellites (STRs). Am. J. Hum. Biol. 21:354-356, 2009. (C) 2009 Wiley-Liss, Inc.

Caries Resistance of Lased Human Root Surface with 10.6 mu m CO(2) Laser-Thermal, Morphological, and Microhardness Analysis

Although the cariostatic effects of CO(2) laser on enamel have been shown, its effects on root surface demineralization remains uncertain. The objectives of this in vitro research was to establish safe parameters for a pulsed 10.6 mu m CO(2) laser and to evaluate its effect on morphological features of the root surface, as well as on the reduction of root demineralization. Ninety-five human root surfaces were randomly divided into five groups: G1-No treatment (control); G2-2.5 J/cm(2); G3-4.0 J/cm(2); G4-5.0 J/cm(2); and G5-6.0 J/cm(2). Intrapulpal temperature was evaluated during root surface irradiation by a thermocouple and morphological changes were evaluated by SEM. After the surface treatment, the specimens were submitted to a 7-day pH-cycling model. Subsequently, the cross-sectional Knoop microhardness values were measured. For all irradiated groups, intrapulpal temperature changes were less than 1.5 degrees C. Scanning electron microscopy images indicated that fluences as low as 4.0 J/cm(2) were sufficient to induce morphological changes in the root surface. Additionally, for fluences reaching or exceeding 4.0 J/cm(2), laser-induced inhibitory effects on root surface demineralization were observed. It was concluded that laser energy density in the range of 4.0 to 6.0 J/cm(2) could be applied to a dental root to reduce demineralization of this surface without compromising pulp vitality.

Conventional and ultrastructural analyses of the chromosomes of Discocyrtus pectinifemur (Opiliones, Laniatores, Gonyleptidae)

Among the Opiliones, species of the suborders Cyphophthalmi, Eupnoi, Dyspnoi and Laniatores have shown very diverse diploid chromosome numbers. However, only certain Eupnoi species exhibit XY/XX and ZZ/ZW sex chromosome systems. Considering the scarcity of karyotypical information and the absence of structurally identifiable sex chromosomes in the suborder Laniatores, we decided to analyse the chromosomes and bivalents of Discocyrtus pectinifemur (Gonyleptidae) to identify possible sex differences. Testicular cells examined under light microscopy showed it high diploid number, 2n = 88, meta/submetacentric chromosome morphology and a nucleolar organizer region on pair 35. Prophase I microspreading observed in transmission electron microscopy exhibited 44 synaptonemal complexes with similar electron density and thickness. The total and regular synapsis between the chromosomes of the bivalents was also noted in pachytene nuclei. Male mitotic and meiotic chromosomes revealed no distinct characteristic that could be related to the occurrence of heteromorphic sex chromosomes. Evolutionary trends of chromosome differentiation in the four suborders of Opiliones are discussed here.

Chromosomal Evolution in the Brazilian Geckos of the Genus Gymnodactylus (Squamata, Phyllodactylidae) from the Biomes of Cerrado, Caatinga and Atlantic Rain Forest: Evidence of Robertsonian Fusion Events and Supernumerary Chromosomes

Chromosomes of the South American geckos Gymnodactylus amarali and G. geckoides from open and dry areas of the Cerrado and Caatinga biomes in Brazil, respectively, were studied for the first time, after conventional and AgNOR staining, CBG- and RBG-banding, and FISH with telomeric sequences. Comparative analyses between the karyotypes of open areas and the previously studied Atlantic forest species G. darwinii were also performed. The chromosomal polymorphisms detected in populations of G. amarali from the states of Goias and Tocantins is the result of centric fusions (2n = 38, 39 and 40), suggesting a differentiation from a 2n = 40 ancestral karyotype and the presence of supernumerary chromosomes. The CBG- and RBG-banding patterns of the Bs are described. G. geckoides has 40 chromosomes with gradually decreasing sizes, but it is distinct from the 2n = 40 karyotypes of G. amarali and G. darwinii due to occurrence of pericentric inversions or centromere repositioning. NOR location seems to be a marker for Gymnodactylus, as G. amarali and G. geckoides share a medium-sized subtelocentric NOR-bearing pair, while G. darwinii has NORs at the secondary constriction of the long arm of pair 1. The comparative analyses indicate a non-random nature of the Robertsonian rearrangements in the genus Gymnodactylus. Copyright (C) 2010 S. Karger AG, Basel

Comparative genomics of the neglected human malaria parasite Plasmodium vivax

The human malaria parasite Plasmodium vivax is responsible for 25 - 40% of the similar to 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non- human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Quality evaluation of DNA obtained from stored human saliva and its applicability to identification in Forensic Dentistry

PURPOSE: This study evaluated the quality of DNA obtained from stored human saliva and its applicability to human identification. METHODS: The saliva samples of 20 subjects, collected in the form of saliva in natura and from mouth swabs and stored at -20ºC, were analyzed. After 7 days, the DNA was extracted from the 40 saliva samples and subjected to PCR and electrophoresis. After 180 days, the technique was repeated with the 20 swab samples. RESULTS: The first-stage results indicated that DNA was successfully extracted in 97.5% of reactions, 95% of saliva in natura and 100% of swab saliva samples, with no statistically significant difference between the forms of saliva. In the second phase, the result was positive for all 20 analyzed samples (100%). Subsequently, in order to analyze the quality of the DNA obtained from human saliva, the SIX3-2 gene was tested on the 20 mouth swab samples, and the PCR products were digested using the MbO1 restriction enzyme to evaluate polymorphisms in the ADRA-2 gene, with positive results for most samples. CONCLUSION: It was concluded that the quantity and quality of DNA from saliva and the techniques employed are adequate for forensic analysis of DNA.

Pulpotomies with portland cement in human primary molars

Two clinical cases in which Portland cement (PC) was applied as a medicament after pulpotomy of mandibular primary molars in children are presented. Pulpotomy using PC was carried out in two mandibular first molars and one mandibular second molar, which were further followed-up. At the 3, 6 and 12-month follow-up appointments, clinical and radiographic examinations of the pulpotomized teeth and their periradicular area revealed that the treatments were successful in maintaining the teeth asymptomatic and preserving pulpal vitality. Additionally, the formation of a dentin bridge immediately below the PC could be observed in the three molars treated. PC may be considered as an effective alternative for primary molar pulpotomies, at least in a short-term period. Randomized clinical trials with human teeth are required in order to determine the suitability of PC before unlimited clinical use can be recommended.

Scanning electron microscopic study of the in situ effect of salivary stimulation on erosion and abrasion in human and bovine enamel

This in situ study investigated, using scanning electron microscopy, the effect of stimulated saliva on the enamel surface of bovine and human substrates submitted to erosion followed by brushing abrasion immediately or after one hour. During 2 experimental 7-day crossover phases, 9 previously selected volunteers wore intraoral palatal devices, with 12 enamel specimens (6 human and 6 bovine). In the first phase, the volunteers immersed the device for 5 minutes in 150 ml of a cola drink, 4 times a day (8h00, 12h00, 16h00 and 20h00). Immediately after the immersions, no treatment was performed in 4 specimens (ERO), 4 other specimens were immediately brushed (0 min) using a fluoride dentifrice and the device was replaced into the mouth. After 60 min, the other 4 specimens were brushed. In the second phase, the procedures were repeated but, after the immersions, the volunteers stimulated the salivary flow rate by chewing a sugar-free gum for 30 min. Enamel superficial alterations of all specimens were then evaluated using a scanning electron microscope. Enamel prism core dissolution was seen on the surfaces submitted to erosion, while on those submitted to erosion and to abrasion (both at 0 and 60 min) a more homogeneous enamel surface was observed, probably due to the removal of the altered superficial prism layer. For all the other variables - enamel substrate and salivary stimulation -, the microscopic pattern of the enamel specimens was similar.

Digital subtraction radiographic analysis of the combination of bioabsorbable membrane and bovine morphogenetic protein pool in human periodontal infrabony defects

OBJECTIVES: This study assessed the bone density gain and its relationship with the periodontal clinical parameters in a case series of a regenerative therapy procedure. MATERIAL AND METHODS: Using a split-mouth study design, 10 pairs of infrabony defects from 15 patients were treated with a pool of bovine bone morphogenetic proteins associated with collagen membrane (test sites) or collagen membrane only (control sites). The periodontal healing was clinically and radiographically monitored for six months. Standardized pre-surgical and 6-month postoperative radiographs were digitized for digital subtraction analysis, which showed relative bone density gain in both groups of 0.034 ± 0.423 and 0.105 ± 0.423 in the test and control group, respectively (p>0.05). RESULTS: As regards the area size of bone density change, the influence of the therapy was detected in 2.5 mm² in the test group and 2 mm² in the control group (p>0.05). Additionally, no correlation was observed between the favorable clinical results and the bone density gain measured by digital subtraction radiography (p>0.05). CONCLUSIONS: The findings of this study suggest that the clinical benefit of the regenerative therapy observed did not come with significant bone density gains. Long-term evaluation may lead to a different conclusions.

Evaluation of in vitro human gingival fibroblast seeding on acellular dermal matrix

The acellular dermal matrix (ADM) was introduced in periodontology as a substitute for the autogenous grafts, which became restricted because of the limited source of donor's tissue. The aim of this study was to investigate, in vitro, the distribution, proliferation and viability of human gingival fibroblasts seeded onto ADM. ADM was seeded with human gingival fibroblasts for up to 21 days. The following parameters were evaluated: cell distribution, proliferation and viability. Results revealed that, at day 7, fibroblasts were adherent and spread on ADM surface, and were unevenly distributed, forming a discontinuous single cell layer; at day 14, a confluent fibroblastic monolayer lining ADM surface was noticed. At day 21, the cell monolayer exhibited a reduction in cell density. At 7 days, about to 90% of adherent cells on ADM surface were cycling while at 14 and 21 days this proportion was significantly reduced. A high proportion of viable cell was detected on AMD surface both on 14 and 21 days. The results suggest that fibroblast seeding onto ADM for 14 days can allow good conditions for cell adhesion and spreading on the matrix; however, migration inside the matrix was limited.