Evidence of Bos javanicus x Bos indicus hybridization and major QTLs for birth weight in Indonesian Peranakan Ongole cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inactivation of candida albicans by cold atmospheric pressure plasma jet

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Complex sex chromosome system in Eigenmannia sp. (Pisces, Gymnotiformes)

Chromosomes of Eigenmannia sp. (7 males and 15 females) collected from the Tietê River in Botucatu (SP, Brazil) were examined from gill, kidney and testicular cells. The diploid chromosome number in males was 2n=31 and in females, 2n=32. In both sexes the number of chromosomal arms was 40. The difference in diploid number was due to the fusion of two acrocentrics. Mitotic and meiotic studies suggested that one of the fused acrocentrics was the Y chromosome. The sex-determining mechanism in Eigenmannia sp. could therefore be XX, AA in the female and X, \-YA A in the males. One of the males presented 2n=30 chromosomes due to the occurrence of another fusion of acrocentrics. C-banding analysis of the mitotic chromosomes revealed constitutive heterochromatin in the centromeric regions of all acrocentrics. However, small metacentrics were C-band negative. The YA chromosome is C-band negative except for a small amount of heterochromatin in the centromeric region. The nucleolar organizer region as identified by Ag-staining is present in the interstitial region of chromosome pair No. 10. © 1984 Dr W. Junk Publishers.

Synaptomenal complex analysis of four breeds of Bos taurus taurus x B. taurus indicus hybrids

The synaptonemal complex (SC) was analyzed in four F1 hybrids of Bos taurus taurus and B. taurus indicus including Gyr-Simmental (G-S), Nelore Simmental (N-S), Gyr-Holstein-Friesian (G-H) and Nelore-Piemontese (N-P). We analysed the frequency of various types of SC abnormalities and the frequency of cells with SC abnormalities. The results were compared with similar observations made on purebred animals. All the animals studied possessed 29 autosomal and one sex bivalent. The frequency of cells with abnormalities in the hybrids were 28.0% in the N-P, 29.1% in the G-S, 33.3% in the N-S and 40.0% in the G-H. The frequency of cells with abnormalities in the four hybrids was 31.5%; 57.9% of these abnormalities occurred in zygotene and 42.0% occurred in pachytene. The comparisons among the hybrids and among the hybrids and their parental breeds showed that the only significant difference was between Gyr and Gyr-Holstein-Friesian animals. Some aspects of the relationship between the frequency of cells with anomalies and the fertility of hybrids are discussed.

Mutations, Clinical Findings and Survival Estimates in South American Patients with X-Linked Adrenoleukodystrophy

In this study, we analyzed the ABCD1 gene in X-linked adrenoleukodystrophy (X-ALD) patients and relatives from 38 unrelated families from South America, as well as phenotypic proportions, survival estimates, and the potential effect of geographical origin in clinical characteristics. Methods: X-ALD patients from Brazil, Argentina and Uruguay were invited to participate in molecular studies to determine their genetic status, characterize the mutations and improve the genetic counseling of their families. All samples were screened by SSCP analysis of PCR fragments, followed by automated DNA sequencing to establish the specific mutation in each family. Age at onset and at death, male phenotypes, genetic status of women, and the effect of family and of latitude of origin were also studied. Results: We identified thirty-six different mutations (twelve novel). This population had an important allelic heterogeneity, as only p. Arg518Gln was repeatedly found (three families). Four cases carried de novo mutations. Intra-familiar phenotype variability was observed in all families. Out of 87 affected males identified, 65% had the cerebral phenotype (CALD). The mean (95% CI) ages at onset and at death of the CALD were 10.9 (9.1-12.7) and 24.7 (19.8-29.6) years. No association was found between phenotypic manifestations and latitude of origin. One index-case was a girl with CALD who carried an ABCD1 mutation, and had completely skewed X inactivation. Conclusions: This study extends the spectrum of mutations in X-ALD, confirms the high rates of de novo mutations and the absence of common mutations, and suggests a possible high frequency of cerebral forms in our population.

In Vitro Photodynamic Inactivation of Cryptococcus neoformans Melanized Cells with Chloroaluminum Phthalocyanine Nanoemulsion

The selection of fungi resistant to currently used fungicides and the emergence of new pathogenic species make the development of alternative fungus-control techniques highly desirable. Photodynamic antimicrobial chemotherapy (PACT) is a promising method which combines a nontoxic photosensitizer (PS) with visible light to cause selective killing of microbial cells. The development of PACT to treat mycoses or kill fungi in the environment depends on identifying effective PS for the different pathogenic species and delivery systems able to expand and optimize their use. In the present study, the in vitro susceptibility of Cryptococcus neoformans melanized cells to the photodynamic effects of the PS agent ClAlPc in nanoemulsion (ClAlPc/NE) was examined. Cells were killed in a PS concentration- and light dose-dependent manner. Treatment with ClAlPc/NE, using PS concentrations (e.g. 4.5 mu m) and light doses (e.g. 10 J cm-2) compatible with PACT, resulted in a reduction of up to 6 logs in survival. Washing the cells to remove unbound PS before light exposure did not inhibit fungal photodynamic inactivation. Internalization of ClAlPc by C.neoformans was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on PS concentration.

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy.

Using Prior Information from the Medical Literature in GWAS of Oral Cancer Identifies Novel Susceptibility Variant on Chromosome 4-the AdAPT Method

Background: Genome-wide association studies (GWAS) require large sample sizes to obtain adequate statistical power, but it may be possible to increase the power by incorporating complementary data. In this study we investigated the feasibility of automatically retrieving information from the medical literature and leveraging this information in GWAS. Methods: We developed a method that searches through PubMed abstracts for pre-assigned keywords and key concepts, and uses this information to assign prior probabilities of association for each single nucleotide polymorphism (SNP) with the phenotype of interest - the Adjusting Association Priors with Text (AdAPT) method. Association results from a GWAS can subsequently be ranked in the context of these priors using the Bayes False Discovery Probability (BFDP) framework. We initially tested AdAPT by comparing rankings of known susceptibility alleles in a previous lung cancer GWAS, and subsequently applied it in a two-phase GWAS of oral cancer. Results: Known lung cancer susceptibility SNPs were consistently ranked higher by AdAPT BFDPs than by p-values. In the oral cancer GWAS, we sought to replicate the top five SNPs as ranked by AdAPT BFDPs, of which rs991316, located in the ADH gene region of 4q23, displayed a statistically significant association with oral cancer risk in the replication phase (per-rare-allele log additive p-value [p(trend)] = 2.5 x 10(-3)). The combined OR for having one additional rare allele was 0.83 (95% CI: 0.76-0.90), and this association was independent of previously identified susceptibility SNPs that are associated with overall UADT cancer in this gene region. We also investigated if rs991316 was associated with other cancers of the upper aerodigestive tract (UADT), but no additional association signal was found. Conclusion: This study highlights the potential utility of systematically incorporating prior knowledge from the medical literature in genome-wide analyses using the AdAPT methodology. AdAPT is available online (url: http://services.gate.ac.uk/lld/gwas/service/config).

A complex chromosomal rearrangement involving chromosomes 2, 5, and X in autism spectrum disorder

Here, we describe a female patient with autism spectrum disorder and dysmorphic features that harbors a complex genetic alteration, involving a de novo balanced translocation t(2;X)(q11;q24), a 5q11 segmental trisomy and a maternally inherited isodisomy on chromosome 5. All the possibly damaging genetic effects of such alterations are discussed. In light of recent findings on ASD genetic causes, the hypothesis that all these alterations might be acting in orchestration and contributing to the phenotype is also considered. (C) 2012 Wiley Periodicals, Inc.

Inactivation of Alicyclobacillus acidoterrestris in orange juice by saponin extracts combined with heat-treatment

Alicyclobacillus acidoterrestris is a spoilage-causing bacterium in fruit juices. The inactivation of this bacterium by commercial saponin and saponin purified extract from Sapindus saponaria fruits combined with heat-treatment is described. We investigated heat treatment (87, 90, 95, and 99 degrees C) with incubation time ranging from 0 to 50 min, in both concentrated and reconstituted juice. juices were inoculated with 1.0 x 10(4) CFU/mL of A. acidoterrestris spores for the evaluation of the best temperature for inactivation. For the temperatures of 87, 90, and 95 degrees C counts of cell viability decreased rapidly within the first 10 to 20 min of incubation in both concentrated and reconstituted juices; inactivation at 99 degrees C ensued within 1 and 2 min. Combination of commercial saponin (100 mg/L) with a very short incubation time (1 min) at 99 degrees C showed a reduction of 234 log cycle for concentrated juice A. acidoterrestris spores (1.0 x 10(4) CFU/mL) in the first 24 h of incubation after treatments. The most efficient treatment was reached with 300, 400 or 500 mg/L of purified extract of saponins from S. saponaria after 5 days of incubation in concentrated juice, and after 5 days with 300 and 400 mg/L or 72 h with 500 mg/L in reconstituted juice. Commercial saponin and purified extracts from S. saponaria had similar inactivation power on A. acidoterrestris spores, without significant differences (P>0.05). Therefore, purified extract of saponins can be an alternative for the control of A acidoterrestris in fruit juices. (C) 2012 Elsevier B.V. All rights reserved.

Mechanisms of ring chromosome formation, ring instability and clinical consequences

Background The breakpoints and mechanisms of ring chromosome formation were studied and mapped in 14 patients. Methods Several techniques were performed such as genome-wide array, MLPA (Multiplex Ligation-Dependent Probe Amplification) and FISH (Fluorescent in situ Hybridization). Results The ring chromosomes of patients I to XIV were determined to be, respectively: r(3)(p26.1q29), r(4)(p16.3q35.2), r(10)(p15.3q26.2), r(10)(p15.3q26.13), r(13)(p13q31.1), r(13)(p13q34), r(14)(p13q32.33), r(15)(p13q26.2), r(18)(p11.32q22.2), r(18)(p11.32q21.33), r(18)(p11.21q23), r(22)(p13q13.33), r(22)(p13q13.2), and r(22)(p13q13.2). These rings were found to have been formed by different mechanisms, such as: breaks in both chromosome arms followed by end-to-end reunion (patients IV, VIII, IX, XI, XIII and XIV); a break in one chromosome arm followed by fusion with the subtelomeric region of the other (patients I and II); a break in one chromosome arm followed by fusion with the opposite telomeric region (patients III and X); fusion of two subtelomeric regions (patient VII); and telomere-telomere fusion (patient XII). Thus, the r(14) and one r(22) can be considered complete rings, since there was no loss of relevant genetic material. Two patients (V and VI) with r(13) showed duplication along with terminal deletion of 13q, one of them proved to be inverted, a mechanism known as inv-dup-del. Ring instability was detected by ring loss and secondary aberrations in all but three patients, who presented stable ring chromosomes (II, XIII and XIV). Conclusions We concluded that the clinical phenotype of patients with ring chromosomes may be related with different factors, including gene haploinsufficiency, gene duplications and ring instability. Epigenetic factors due to the circular architecture of ring chromosomes must also be considered, since even complete ring chromosomes can result in phenotypic alterations, as observed in our patients with complete r(14) and r(22).

Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

Molekulare Analyse des Kolumnar-Gens beim Apfel (Malus x domestica)

Das Kolumnarwachstum beim Apfel (Malus x domestica) geht auf eine in den frühen 1960er Jahren entdeckte Zufallsmutation zurück. Die daraus resultierende Sprossmutante ist von großem wirtschaftlichem Interesse, da diese sehr kompakte Wuchsform unter anderem zu einer enormen Ertragssteigerung durch eine hohe Pflanzdichte der Bäume führt. Das Ziel der Arbeit ist die Entschlüsselung der molekularen Ursache dieser Mutation, die bisher weitgehend ungeklärt ist. Die Analyse wurde durch die Erstellung einer Referenzsequenz der Co-Zielregion einer kolumnaren Apfelsorte sowie durch die Konstruktion eng gekoppelter molekularer Marker realisiert. Durch die Konstruktion von genomischen Apfel-BAC-Bibliotheken mit mehrfacher Genomabdeckung und die Erstellung geeigneter Sonden wurde die Co-Region kloniert und deren Sequenz bestimmt. In Kombination zu dieser klassischen positionellen Klonierungsstrategie wurden genomische Illumina „mate pair“-Bibliotheken erstellt, sequenziert und bioinformatisch analysiert, um die genomische Region vollständig zu annotieren. Somit wurde eine vollständige genomische Referenz der Co-Region einer kolumnaren Apfelsorte erstellt, die die Grundlage für weitere Analysen bildet. Auf Basis dieser Referenz konnte die Co-Mutation in Form der Integration des LTR-Retrotransposons Gypsy-44 im kolumnaren Chromosom an Position 18,79 Mbp auf Chromosom 10 lokalisiert werden. Darüber hinaus konnten Transposon-basierende molekulare Marker erstellt werden, die eine verlässliche Genotypisierung von Apfelbäumen in Bezug auf das Kolumnarwachstum ermöglichen und dies unabhängig von der verwendeten Apfelsorte. Der genaue Wirkmechanismus von Gypsy-44, der zur Ausprägung dieses extremen Phänotyps führt, ist bislang unklar. Zusammenfassend lässt sich sagen, dass die molekulare Ursache für das kolumnare Wachstum aufgeklärt werden konnte und zudem die ersten molekularen Marker erstellt wurden, die eine sortenunabhängige Differenzierung zwischen kolumnaren und nicht kolumnaren Apfelbäumen ermöglichen.

Molecular genetic causes of columnar growth in apple (Malus x domestica)

The columnar growth habit of apple is interesting from an economic point of view as the pillar-like trees require little space and labor. Genetic engineering could be used to speed up breeding for columnar trees with high fruit quality and disease resistance. For this purpose, this study dealt with the molecular causes of this interesting phenotype. The original bud sport mutation that led to the columnar growth habit was found to be a novel nested insertion of a Gypsy-44 LTR retrotransposon on chromosome 10 at 18.79 Mb. This subsequently causes tissue-specific differential expression of nearby downstream genes, particularly of a gene encoding a 2OG-Fe(II) oxygenase of unknown function (dmr6-like) that is strongly upregulated in developing aerial tissues of columnar trees. The tissue-specificity of the differential expression suggests involvement of cis-regulatory regions and/or tissue-specific epigenetic markers whose influence on gene expression is altered due to the retrotransposon insertion. This eventually leads to changes in genes associated with stress and defense reactions, cell wall and cell membrane metabolism as well as phytohormone biosynthesis and signaling, which act together to cause the typical phenotype characteristics of columnar trees such as short internodes and the absence of long lateral branches. In future, transformation experiments introducing Gypsy-44 into non-columnar varieties or excising Gypsy-44 from columnar varieties would provide proof for our hypotheses. However, since site-specific transformation of a nested retrotransposon is a (too) ambitious objective, silencing of the Gypsy-44 transcripts or the nearby genes would also provide helpful clues.

Inactivation of the hypermethylated in cancer 1 tumour suppressor--not just a question of promoter hypermethylation?

The chromosomal region 17p13.3 is frequently deleted or epigenetically silenced in a variety of human cancers. It includes the hypermethylated in cancer 1 (HIC1) gene placed telomerically to the p53 tumour suppressor gene. HIC1 encodes a transcriptional repressor, and its targets identified to date are genes involved in proliferation, tumour growth and angiogenesis. In addition, HIC1 functionally cooperates with p53 to suppress cancer development. Frequent allelic loss at position 17p13.1 in human cancers often points to mutations of the tumour suppressor p53. However, in a variety of cancer types, allelic loss of the short arm of chromosome 17 may hit regions distal to p53 and, interestingly, without leading to p53 mutations. Furthermore, the neighbouring region 17p13.3 often shows loss of heterozygosity or DNA hypermethylation in various types of solid tumours and leukaemias. In line with this concept, Wales et al. described a new potential tumour suppressor in this region and named it hypermethylated in cancer 1 (HIC1). Further, it was shown that in the majority of cases hypermethylation of this chromosomal region leads to epigenetic inactivation of HIC1. A role for HIC1 in tumour development is further supported by a mouse model, since various spontaneous, age- and gender-specific malignant tumours occur in heterozygous Hic1+/- knockout mice. Furthermore, exogenously delivered HIC1 leads to a significant decrease in clonogenic survival in cancer cell lines. This review highlights the role of HIC1 inactivation in solid tumours and particularly in leukaemia development.