Cryptic SYT/SXX1 fusion gene in high-grade biphasic synovial sarcoma with unique complex rearrangement and extensive BCL2 overexpression

Synovial sarcomas are high-grade malignant mesenchymal tumors that account for 10% of all soft-tissue sarcomas. Almost 95% of these tumors are characterized by a nonrandom chromosomal abnormality, t(X;18)(p11.2;q11.2), that is observed in both biphasic and monophasic variants. In this article, we present the case of a 57-year-old woman diagnosed with high-grade biphasic synovial sarcoma in which conventional cytogenetic analysis revealed the constant presence of a unique t(18;22)(q12;q13), in addition to trisomy 8. The rearrangement was confirmed by fluorescence in situ hybridization. The use of the whole chromosome painting probes WCPX did not detect any rearrangements involving chromosome X, although reverse-transcriptase polymerase chain reaction (PCR) analysis demonstrated the conspicuous presence of a SYT/SXX1 fusion gene. Spectral karyotyping (SKY) was also performed and revealed an insertion of material from chromosome 18 into one of the X chromosomes at position Xp11.2. Thus, the karyotype was subsequently interpreted as 47,X,der(X)ins(X;18) (p11.2;q11.2q11.2),der(18)del(18)(q11.2q11.2)t(18;22)(q12;q13),der(22)t(18;22). Real-time PCR analysis of BCL2 expression in the tumor sample showed a 433-fold increase. This rare finding exemplifies that thorough molecular-cytogenetic analyses are required to elucidate complex and/or cryptic tumor-specific translocations. (C) 2010 Elsevier Inc. All rights reserved.

Use of chromosome microdissection in fish molecular cytogenetics

Chromosome microdissection is a technique in which whole chromosomes or chromosomal segments are dissected under an inverted microscope yielding chromosome-specific sequences. Several protocol modifications introduced during the past 15 years reduced the number of chromosomes required for most applications. This is of particular interest to fish molecular cytogenetics, since most species present highly uniform karyotypes which make impossible the collection of multiple copies of the same chromosome. Probes developed in this manner can be used to investigate chromosome homologies in closely related species. Here we describe a protocol recently used in the gymnotiform species group Eigenmannia and review the major steps involved in the generation of these markers focusing on protocol modifications aiming to reduce the number of required chromosomes.

Hybridization and introgression across different ploidy levels in the Neotropical orchids Epidendrum fulgens and E-puniceoluteum (Orchidaceae)

The hypothesis of gene flow between species with large differences in chromosome numbers has rarely been tested in the wild, mainly because species of different ploidy are commonly assumed to be reproductively isolated from each other because of instantaneous and strong postzygotic barriers. In this study, a broad-scale survey of molecular variation was carried out between two orchid species with different ploidy levels: Epidendrum fulgens (2n = 2x = 24 chromosomes) and Epidendrum puniceoluteum (2n = 4x = 52 chromosomes). To test the strength of their reproductive barriers, we investigated the distribution of genetic variation in sympatric and allopatric populations of these two species and conducted crossing experiments. Nuclear and plastid microsatellite loci were used to genotype 463 individuals from eight populations across the geographical range of both species along the Brazilian coastal plain. All six sympatric populations analysed presented hybrid zones, indicating that hybridization between E. fulgens and E. puniceoluteum is a common phenomenon. Bayesian assignment analysis detected the presence of F(1) and F(2) individuals and also signs of introgression, demonstrating a high potential for interspecific gene flow. Introgression occurs preferentially from E. fulgens to E. puniceoluteum. Pure parental individuals of both species display strong genotype-habitat associations, indicating that environment-dependent selection could be acting in all hybrid zones. This study suggests that hybridization and introgression are evolutionary processes playing a role in the diversification of Epidendrum and indicates the importance of investigations of hybrid zones in understanding reproductive barriers and speciation processes in Neotropical orchid species.

A Comparative Cytogenetic Analysis of 2 Bothriuridae Species and Overview of the Chromosome Data of Scorpiones

The order Scorpiones is one of the most cytogenetically interesting groups within Arachnida by virtue of the combination of chromosome singularities found in the 59 species analyzed so far. In this work, mitotic and meiotic chromosomes of 2 species of the family Bothriuridae were detailed. This family occupies a basal position within the superfamily Scorpionoidea. Furthermore, review of the cytogenetic data of all previously studied scorpions is presented. Light microscopy chromosome analysis showed that Bothriurus araguayae and Bothriurus rochensis possess low diploid numbers compared with those of species belonging to closely related families. Gonadal cells examined under light and in transmission electron microscopy revealed, for the first time, that the Bothriuridae species possess typical monocentric chromosomes, and male meiosis presented chromosomes with synaptic and achiasmatic behavior. Moreover, in the sample of B. araguayae studied, heterozygous translocations were verified. The use of techniques to highlight specific chromosomal regions also revealed additional differences between the 2 Bothriurus species. The results herein recorded and the overview elaborated using the available cytogenetic information of Scorpiones elucidated current understanding regarding the processes of chromosome evolution that have occurred in Bothriuridae and in Scorpiones as a whole.

Analysis of the stimulated whole saliva in overweight and obese school children

OBJECTIVE: To determine if some stimulated whole saliva parameters are influenced by an increase of Body Mass Index. METHODS: Controlled cross-sectional study involving 90 school children of both genders between 7 and 10 years of age, from Bragança Paulista - SP. Three groups were formed: overweight, obese and control. Body Mass Index and diet intake by the Food Register method were evaluated. The salivary pH, flow rate, buffer capacity, protein, phosphate, calcium, fluoride, total and free sialic acid, and peroxidase activity were determined. RESULTS: The overweight and obese groups showed greater energy and lipid intake (P< 0.001) than the control group. There was no difference in the saliva flow rate between groups, however only the control group showed a mean value considered normal. In the overweight and obese groups a decrease in both the concentration of phosphate (P< 0.001) and peroxidase activity (P<0.001) was observed. In the obese group an increase in the concentrations of free sialic acid (P= 0.004) and protein (P= 0.003) occurred. CONCLUSION: Overweight and obese children show alterations in the concentrations of phosphate, free sialic acid and proteins, and in the peroxidase activity that are favorable conditions for dental caries.

Array-CGH testing in spontaneous abortions with normal karyotypes

In about 50% of first trimester spontaneous abortion the cause remains undetermined after standard cytogenetic investigation. We evaluated the usefulness of array-CGH in diagnosing chromosome abnormalities in products of conception from first trimester spontaneous abortions. Cell culture was carried out in short- and long-term cultures of 54 specimens and cytogenetic analysis was successful in 49 of them. Cytogenetic abnormalities (numerical and structural) were detected in 22 (44.89%) specimens. Subsequent, array-CGH based on large insert clones spaced at ~1 Mb intervals over the whole genome was used in 17 cases with normal G-banding karyotype. This revealed chromosome aneuplodies in three additional cases, giving a final total of 51% cases in which an abnormal karyotype was detected. In keeping with other recently published works, this study shows that array-CGH detects abnormalities in a further ~10% of spontaneous abortion specimens considered to be normal using standard cytogenetic methods. As such, array-CGH technique may present a suitable complementary test to cytogenetic analysis in cases with a normal karyotype.

Chorion biopsy in mongrel dogs

With the great development of the gestational studies in all of the species, we noticed the necessity of adaptations of these techniques for prenatal diagnosis in dogs. Based on this, we studied the feasibility of chorion biopsy guided by ultrasound. Our results demonstrated accuracy on the sex determination being 2 males and 12 females, as well as it would be possible to identify chromosome alteration due to the quality of samplings. Sex determination was accomplished with the identification of Y gene chromosomes in PCR technique. After the collection, fragments were prepared for light microscopy studies and revealed fetal chorion tissue, blood colloid and erythrocyte. In the whole material we found hemosiderin impregnations due to the hemolysis and to the residue of blood of the placental marginal hematomes. The submitted female dogs to this technique demonstrated normal puppy births without death.

Comparison of agglutinating and neutralizing antibodies to serovar hardjo in sows immunized with two commercial whole culture polivalent anti-leptospira bacterins

It was performed the comparison of the intensity and duration of agglutinating and neutralizing antibodies to serovar Hardjo in swines vaccinated with two commercial anti-leptospira bacterins. Sows no reactive to 24 Leptospira sp serovars in the microscopic agglutination test (MAT) were divided in three groups: Group A (n=08): received two vaccine A doses with 30 days interval, Group B (n=08) two vaccine B doses with 30 days interval and Group C (n=08): control no vaccinated against leptospirosis.Blood samples were collected each 30 days during six months following the first vaccination. The sera were tested by MAT and growth inhibition test (GIT) to serovar Hardjo in order to evaluate respectively agglutinating and neutralizing antibodies. It was found that neutralizing antibodies persisted for a longer time than the agglutinating ones and that the absence of agglutinating antibodies does not means in the absence of the neutralizing. The peaks of agglutinating antibodies was obtained at least 30 days earlier than that produced by neutralizing. The duration of both kinds of antibodies measured differed between the two bacterines tested. The period for inducing neutralizing antibodies against serovar Hardjo indicated that gilts must be immunized with two doses of whole culture anti-leptospira bacterines applied 30 days each other at least 90 days before the first mating. For the maintenance of hight levels of neutralizing antibodies the revaccinations must be performed every six months after the first vaccination.

Variations on a theme: diversification of cuticular hydrocarbons in a clade of cactophilic Drosophila

Background: We characterized variation and chemical composition of epicuticular hydrocarbons (CHCs) in the seven species of the Drosophila buzzatii cluster with gas chromatography/mass spectrometry. Despite the critical role of CHCs in providing resistance to desiccation and involvement in communication, such as courtship behavior, mating, and aggregation, few studies have investigated how CHC profiles evolve within and between species in a phylogenetic context. We analyzed quantitative differences in CHC profiles in populations of the D. buzzatii species cluster in order to assess the concordance of CHC differentiation with species divergence. Results: Thirty-six CHC components were scored in single fly extracts with carbon chain lengths ranging from C(29) to C(39), including methyl-branched alkanes, n alkenes, and alkadienes. Multivariate analysis of variance revealed that CHC amounts were significantly different among all species and canonical discriminant function (CDF) analysis resolved all species into distinct, non-overlapping groups. Significant intraspecific variation was found in different populations of D. serido suggesting that this taxon is comprised of at least two species. We summarized CHC variation using CDF analysis and mapped the first five CHC canonical variates (CVs) onto an independently derived period (per) gene + chromosome inversion + mtDNA COI gene for each sex. We found that the COI sequences were not phylogenetically informative due to introgression between some species, so only per + inversion data were used. Positive phylogenetic signal was observed mainly for CV1 when parsimony methods and the test for serial independence (TFSI) were used. These results changed when no outgroup species were included in the analysis and phylogenetic signal was then observed for female CV3 and/or CV4 and male CV4 and CV5. Finally, removal of divergent populations of D. serido significantly increased the amount of phylogenetic signal as up to four out of five CVs then displayed positive phylogenetic signal. Conclusions: CHCs were conserved among species while quantitative differences in CHC profiles between populations and species were statistically significant. Most CHCs were species-, population-, and sex-specific. Mapping CHCs onto an independently derived phylogeny revealed that a significant portion of CHC variation was explained by species' systematic affinities indicating phylogenetic conservatism in the evolution of these hydrocarbon arrays, presumptive waterproofing compounds and courtship signals as in many other drosophilid species.

Ring chromosome instability evaluation in six patients with autosomal rings

Ring chromosomes are often associated with abnormal phenotypes due to loss of genomic material and also because of ring instability at mitosis after sister chromatid exchange events. We investigated ring chromosome instability in six patients with ring chromosomes 4, 14, 15, and 18 by examining 48- and 72-h lymphocyte cultures at the first, second and subsequent cell divisions after bromodeoxyuridine incorporation. Although most cells from all patients showed only one monocentric ring chromosome, ring chromosome loss and secondary aberrations were observed both in 48-and 72-h lymphocyte cultures and in metaphase cells of the different cell generations. We found no clear-cut correlation between ring size and ring instability; we also did not find differences between apparently complete rings and rings with genetic material loss. The cytogenetic findings revealed secondary aberrations in all ring chromosome patients. We concluded that cells with ring chromosome instability can multiply and survive in vivo, and that they can influence the patient's phenotype.

An assessment of population structure in eight breeds of cattle using a whole genome SNP panel

Background: Analyses of population structure and breed diversity have provided insight into the origin and evolution of cattle. Previously, these studies have used a low density of microsatellite markers, however, with the large number of single nucleotide polymorphism markers that are now available, it is possible to perform genome wide population genetic analyses in cattle. In this study, we used a high-density panel of SNP markers to examine population structure and diversity among eight cattle breeds sampled from Bos indicus and Bos taurus. Results: Two thousand six hundred and forty one single nucleotide polymorphisms ( SNPs) spanning all of the bovine autosomal genome were genotyped in Angus, Brahman, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black, Limousin and Nelore cattle. Population structure was examined using the linkage model in the program STRUCTURE and Fst estimates were used to construct a neighbor-joining tree to represent the phylogenetic relationship among these breeds. Conclusion: The whole-genome SNP panel identified several levels of population substructure in the set of examined cattle breeds. The greatest level of genetic differentiation was detected between the Bos taurus and Bos indicus breeds. When the Bos indicus breeds were excluded from the analysis, genetic differences among beef versus dairy and European versus Asian breeds were detected among the Bos taurus breeds. Exploration of the number of SNP loci required to differentiate between breeds showed that for 100 SNP loci, individuals could only be correctly clustered into breeds 50% of the time, thus a large number of SNP markers are required to replace the 30 microsatellite markers that are currently commonly used in genetic diversity studies.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis

The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome

Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Total carbon measurement in whole tropical soil sample

Soils are an important component in the biogeochemical cycle of carbon, storing about four times more carbon than biomass plants and nearly three times more than the atmosphere. Moreover, the carbon content is directly related on the capacity of water retention, fertility. among other properties. Thus, soil carbon quantification in field conditions is an important challenge related to carbon cycle and global climatic changes. Nowadays. Laser Induced Breakdown Spectroscopy (LIBS) can be used for qualitative elemental analyses without previous treatment of samples and the results are obtained quickly. New optical technologies made possible the portable LIBS systems and now, the great expectation is the development of methods that make possible quantitative measurements with LIBS. The goal of this work is to calibrate a portable LIBS system to carry out quantitative measures of carbon in whole tropical soil sample. For this, six samples from the Brazilian Cerrado region (Argisoil) were used. Tropical soils have large amounts of iron in their compositions, so the carbon line at 247.86 nm presents strong interference of this element (iron lines at 247.86 and 247.95). For this reason, in this work the carbon line at 193.03 nm was used. Using methods of statistical analysis as a simple linear regression, multivariate linear regression and cross-validation were possible to obtain correlation coefficients higher than 0.91. These results show the great potential of using portable LIBS systems for quantitative carbon measurements in tropical soils. (C) 2008 Elsevier B.V. All rights reserved.