Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

Purpose: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Methods: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c. 3277C>T, a nonsense mutation, and c. 3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. Results: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. Conclusions: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE) x S(SM)(EK) x S(SM)(SSRs)). Clustering analyses showed a mean of 9 +/- 12.4 isolates per cluster (3.8 +/- 8 isolates/taxon) for MLEE, 6.2 +/- 4.9 isolates per cluster (4 +/- 4.5 isolates/taxon) for SSRs, and 4.1 +/- 2.3 isolates per cluster (2.6 +/- 2.3 isolates/taxon) for EK. A total of 45 (13%), 39(11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (Si) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (C) 2010 Elsevier B.V. All rights reserved.

Exercise training reduces cardiac angiotensin II levels and prevents cardiac dysfunction in a genetic model of sympathetic hyperactivity-induced heart failure in mice

The role of exercise training (ET) on cardiac renin-angiotensin system (RAS) was investigated in 3-5 month-old mice lacking alpha(2A-) and alpha(2C-)adrenoceptors (alpha(2A)/alpha(2C)ARKO) that present heart failure (HF) and wild type control (WT). ET consisted of 8-week running sessions of 60 min, 5 days/week. In addition, exercise tolerance, cardiac structural and function analysis were made. At 3 months, fractional shortening and exercise tolerance were similar between groups. At 5 months, alpha(2A)/alpha(2C)ARKO mice displayed ventricular dysfunction and fibrosis associated with increased cardiac angiotensin (Ang) II levels (2.9-fold) and increased local angiotensin-converting enzyme activity (ACE 18%). ET decreased alpha(2A)/alpha(2C)ARKO cardiac Ang II levels and ACE activity to age-matched untrained WT mice levels while increased ACE2 expression and prevented exercise intolerance and ventricular dysfunction with little impact on cardiac remodeling. Altogether, these data provide evidence that reduced cardiac RAS explains, at least in part, the beneficial effects of ET on cardiac function in a genetic model of HF.

The role of local and systemic renin angiotensin system activation in a genetic model of sympathetic hyperactivity-induced heart failure in mice

Sympathetic hyperactivity (SH) and renin angiotensin system (RAS) activation are commonly associated with heart failure (HF), even though the relative contribution of these factors to the cardiac derangement is less understood. The role of SH on RAS components and its consequences for the HF were investigated in mice lacking alpha(2A) and alpha(2C) adrenoceptor knockout (alpha(2A)/alpha(2C) ARKO) that present SH with evidence of HF by 7 mo of age. Cardiac and systemic RAS components and plasma norepinephrine (PN) levels were evaluated in male adult mice at 3 and 7 mo of age. In addition, cardiac morphometric analysis, collagen content, exercise tolerance, and hemodynamic assessments were made. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF, while displaying elevated PN, activation of local and systemic RAS components, and increased cardiomyocyte width (16%) compared with wild-type mice (WT). In contrast, at 7 mo, alpha(2A)/alpha(2C)ARKO mice presented clear signs of HF accompanied only by cardiac activation of angiotensinogen and ANG II levels and increased collagen content (twofold). Consistent with this local activation of RAS, 8 wk of ANG II AT(1) receptor blocker treatment restored cardiac structure and function comparable to the WT. Collectively, these data provide direct evidence that cardiac RAS activation plays a major role underlying the structural and functional abnormalities associated with a genetic SH-induced HF in mice.

Multidimensional cluster stability analysis from a Brazilian Bradyrhizobium sp RFLP/PCR data set

The taxonomy of the N(2)-fixing bacteria belonging to the genus Bradyrhizobium is still poorly refined, mainly due to conflicting results obtained by the analysis of the phenotypic and genotypic properties. This paper presents an application of a method aiming at the identification of possible new clusters within a Brazilian collection of 119 Bradryrhizobium strains showing phenotypic characteristics of B. japonicum and B. elkanii. The stability was studied as a function of the number of restriction enzymes used in the RFLP-PCR analysis of three ribosomal regions with three restriction enzymes per region. The method proposed here uses Clustering algorithms with distances calculated by average-linkage clustering. Introducing perturbations using sub-sampling techniques makes the stability analysis. The method showed efficacy in the grouping of the species B. japonicum and B. elkanii. Furthermore, two new clusters were clearly defined, indicating possible new species, and sub-clusters within each detected cluster. (C) 2008 Elsevier B.V. All rights reserved.

GOOAL: A routine based on Genetic Algorithms to track harmonics in electric power systems

Voltage and current waveforms of a distribution or transmission power system are not pure sinusoids. There are distortions in these waveforms that can be represented as a combination of the fundamental frequency, harmonics and high frequency transients. This paper presents a novel approach to identifying harmonics in power system distorted waveforms. The proposed method is based on Genetic Algorithms, which is an optimization technique inspired by genetics and natural evolution. GOOAL, a specially designed intelligent algorithm for optimization problems, was successfully implemented and tested. Two kinds of representations concerning chromosomes are utilized: binary and real. The results show that the proposed method is more precise than the traditional Fourier Transform, especially considering the real representation of the chromosomes.

Hormonal modulation of photomorphogenesis-controlled anthocyanin accumulation in tomato (Solanum lycopersicum L. cv Micro-Tom) hypocotyls: Physiological and genetic studies

Hormones are likely to be important factors modulating the light-dependent anthocyanin accumulation. Here we analyzed anthocyanin contents in hypocotyls of near isogenic Micro-Tom (MT) tomato lines carrying hormone and phytochrome mutations, as single and double-mutant combinations. In order to recapitulate mutant phenotype, exogenous hormone applications were also performed Anthocyanin accumulation was promoted by exogenous abscisic acid (ABA) and inhibited by gibberellin (GA), in accordance to the reduced anthocyanin contents measured in ABA-deficient (notabills) and GA-constitutive response (procera) mutants. Exogenous cytokinin also enhanced anthocyanin levels in MT hypocotyls. Although auxin-insensitive chageotropica mutant exhibited higher anthocyanin contents, pharmacological approaches employing exogenous auxin and a transport inhibitor did not support a direct role of the hormone in anthocyanin accumulation Analysis of mutants exhibiting increased ethylene production (epwastic) or reduced sensitivity (Never ripe), together with pharmacological data obtained from plants treated with the hormone, indicated a limited role for ethylene in anthocyanin contents. Phytochrome-deficiency (aurea) and hormone double-mutant combinations exhibited phenotypes suggesting additive or synergistic interactions, but not fully espistatic ones, in the control of anthocyanin levels in tomato hypocotyls. Our results indicate that phytochrome-mediated anthocyanin accumulation in tomato hypocotyls is modulated by distinct hormone classes via both shared and independent pathways. (C) 2010 Elsevier Ireland Ltd. All rights reserved

Spatial genetic structure of Hymenaea stigonocarpa Mart. ex Hayne assessed with chloroplast microsatellite markers

The use of chloroplast DNA markers (cpDNA) helps to elucidate questions related to ecology, evolution and genetic structure. The knowledge of inter-and intra-population genetic structure allows to design effective conservation and management strategies for tropical tree species. With the aim to help the conservation of Hymenaea stigonocarpa of the Cerrado (Brazilian savanna) in Sao Paulo State, an analysis of the spatial genetic structure (SGS) was conducted in two populations using five universal chloroplast microsatellite loci (cpSSR). The population of 68 trees of H. stigonocarpa in the Ecological Station of Itirapina (ESI) had a single haplotype, indicating a strong founder effect. In turn, the population of 47 trees of H. stigonocarpa in a contiguous area that includes the Ecological Station of Assis and the Assis State Forest (ESA), showed six haplotypes ((n) over cap (h) = 6) with a moderate haplotype diversity ((h) over cap = 0667 + 0094), revealing that it was founded by a small number of maternal lineages. The SGS analysis for the population ESA/ASF, using Moran`s I index, indicated limited seed dispersal. Considering SGS, for ex situ conservation strategies in the population ESA/ASF, seed harvesting should require a minimum distance of 750 m among seed-trees.

A frailty modeling approach for parental effects in animal breeding

Survival models involving frailties are commonly applied in studies where correlated event time data arise due to natural or artificial clustering. In this paper we present an application of such models in the animal breeding field. Specifically, a mixed survival model with a multivariate correlated frailty term is proposed for the analysis of data from over 3611 Brazilian Nellore cattle. The primary aim is to evaluate parental genetic effects on the trait length in days that their progeny need to gain a commercially specified standard weight gain. This trait is not measured directly but can be estimated from growth data. Results point to the importance of genetic effects and suggest that these models constitute a valuable data analysis tool for beef cattle breeding.

Multivariate analysis of sensory characteristics of coffee grains (Coffea arabica L.) in the region of upper Paranaiba

This study aimed to examine the sensory characteristics of the grains of 21 cultivars of Coffea arabica L. and Coffea canephora Pierre from the essays of genetic improvement of EPAMIG, located in Patrocinio Municipality, Minas Gerais State, where they were collected through cloths stripping method and washed. Subsequently to dry (11 to 12% moisture b.u.), we obtained the coffee designated as natural. The evaluated varieties were: Acaia Cerrado MG 1474; Bourbon Vermelho DATERRA; Catigua MG 1; Catigua MG 2; Catual Amarelo IAC 62; Catuai Vermelho IAC 15; H 419-3-1-4-2; H 419-6-2 -5-2; H 419-6-2-5-3; H 419-6-2-7-3 Vermelho; H 493-1-2-10; H 514-7-10-1 Vermelho; H 514-7-10-6; H 515-4-2-2; H 518-3-6-1; Icatu Amarelo IAC 3282; Mundo Novo 379-19; Mundo Novo TAO 376-4; Rubi MG 1192; Sacramento MG 1 and Topazio MG 1190, from 2005/2006 and 2006/2007 seasons. The cultivars according to the first principal component with notes above 80 points, regarded as superior drink according to attributes with the highest scores (flavor, sweetness, balance, acidity, clean drink, and aspect) were: Catigua MG2, Rubi MG 1192, 514-7-10-6 H, H 419-3-1-4-2, H 419-6-2-5-2, 493-1-2-10 H, H 514-7-10-1 Vermelho, Catigua MG1, Sacramento MG1, 419-6-2-5-3 H, H 515-9-2-2 and Catuai Amarelo IAC 62.

Genetic structure and biology of Xylella fastidiosa strains causing disease in citrus and coffee in Brazil

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.

Analysis of genomic and functional RFLP derived markers associated with sucrose content, fiber and yield QTLs in a sugarcane (Saccharum spp.) commercial cross

Expressed sequence tags derived markers have a great potential to be used in functional map construction and QTL tagging. In the present work, sugarcane genomic probes and expressed sequence tags having homology to genes, mostly involved in carbohydrate metabolism were used in RFLP assays to identify putative QTLs as well as their epistatic interactions for fiber content, cane yield, pol and tones of sugar per hectare, at two crop cycles in a progeny derived from a bi-parental cross of sugarcane elite materials. A hundred and twenty marker trait associations were found, of which 26 at both crop cycle and 32 only at first ratoon cane. A sucrose synthase derived marker was associated with a putative QTL having a high negative effect on cane yield and also with a QTL having a positive effect on Pol at both crop cycles. Fifty digenic epistatic marker interactions were identified for the four traits evaluated. Of these, only two were observed at both crop cycles.

Evaluation of algorithms used to order markers on genetic maps

When building genetic maps, it is necessary to choose from several marker ordering algorithms and criteria, and the choice is not always simple. In this study, we evaluate the efficiency of algorithms try (TRY), seriation (SER), rapid chain delineation (RCD), recombination counting and ordering (RECORD) and unidirectional growth (UG), as well as the criteria PARF (product of adjacent recombination fractions), SARF (sum of adjacent recombination fractions), SALOD (sum of adjacent LOD scores) and LHMC (likelihood through hidden Markov chains), used with the RIPPLE algorithm for error verification, in the construction of genetic linkage maps. A linkage map of a hypothetical diploid and monoecious plant species was simulated containing one linkage group and 21 markers with fixed distance of 3 cM between them. In all, 700 F(2) populations were randomly simulated with and 400 individuals with different combinations of dominant and co-dominant markers, as well as 10 and 20% of missing data. The simulations showed that, in the presence of co-dominant markers only, any combination of algorithm and criteria may be used, even for a reduced population size. In the case of a smaller proportion of dominant markers, any of the algorithms and criteria (except SALOD) investigated may be used. In the presence of high proportions of dominant markers and smaller samples (around 100), the probability of repulsion linkage increases between them and, in this case, use of the algorithms TRY and SER associated to RIPPLE with criterion LHMC would provide better results. Heredity (2009) 103, 494-502; doi:10.1038/hdy.2009.96; published online 29 July 2009

Canonical discriminant analysis applied to broiler chicken performance

The mechanisms involved in the control of growth in chickens are too complex to be explained only under univariate analysis because all related traits are biologically correlated. Therefore, we evaluated broiler chicken performance under a multivariate approach, using the canonical discriminant analysis. A total of 1920 chicks from eight treatments, defined as the combination of four broiler chicken strains (Arbor Acres, AgRoss 308, Cobb 500 and RX) from both sexes, were housed in 48 pens. Average feed intake, average live weight, feed conversion and carcass, breast and leg weights were obtained for days 1 to 42. Canonical discriminant analysis was implemented by SAS((R)) CANDISC procedure and differences between treatments were obtained by the F-test (P < 0.05) over the squared Mahalanobis` distances. Multivariate performance from all treatments could be easily visualised because one graph was obtained from two first canonical variables, which explained 96.49% of total variation, using a SAS((R)) CONELIP macro. A clear distinction between sexes was found, where males were better than females. Also between strains, Arbor Acres, AgRoss 308 and Cobb 500 (commercial) were better than RX (experimental), Evaluation of broiler chicken performance was facilitated by the fact that the six original traits were reduced to only two canonical variables. Average live weight and carcass weight (first canonical variable) were the most important traits to discriminate treatments. The contrast between average feed intake and average live weight plus feed conversion (second canonical variable) were used to classify them. We suggest analysing performance data sets using canonical discriminant analysis.

Genetic Diversity and a PCR-Based Method for Xanthomonas axonopodis Detection in Passion Fruit

Xanthomonas axonopodis pv. passiflorae causes bacterial spot in passion fruit. It attacks the purple and yellow passion fruit as well as the sweet passion fruit. The diversity of 87 isolates of pv. passiflorae collected from across 22 fruit orchards in Brazil was evaluated using molecular profiles and statistical procedures, including an unweighted pair-group method with arithmetical averages-based dendrogram, analysis of molecular variance (AMOVA), and an assigning test that provides information on genetic structure at the population level. Isolates from another eight pathovars were included in the molecular analyses and all were shown to have a distinct repetitive sequence-based polymerase chain reaction profile. Amplified fragment length polymorphism technique revealed considerable diversity among isolates of pv. passiflorae, and AMOVA showed that most of the variance (49.4%) was due to differences between localities. Cluster analysis revealed that most genotypic clusters were homogeneous and that variance was associated primarily with geographic origin. The disease adversely affects fruit production and may kill infected plants. A method for rapid diagnosis of the pathogen, even before the disease symptoms become evident, has value for producers. Here, a set of primers (Xapas) was designed by exploiting a single-nucleotide polymorphism between the sequences of the intergenic 16S-23S rRNA spacer region of the pathovars. Xapas was shown to effectively detect all pv. passiflorae isolates and is recommended for disease diagnosis in passion fruit orchards.