Functional analysis of SOX9 in chondrogenesis by targeted gene inactivation

Sox9 is a Sry-related HMG-domain containing transcription factor. Lines of evidence suggest that Sox9 has a potential role in skeletal development. During mouse development, Sox9 is predominantly expressed in all chondroprogenitors and differentiated chondrocytes, throughout the deposition of cartilage matrix. Mutations in one allele of SOX9 in humans result in campomelic dysplasia (CD), a skeletal dysplasia. syndrome characterized by the bowing of long bones. Moreover, Sox9 binds to and activates chondrocyte-specific enhancers in Col2a1 and Col11a2 genes. To further investigate the function of Sox9 in chondrogenesis, we analyzed chimeras derived from Sox9 heterozygous and homozygous null embryonic stem (ES) cells. In mouse chimeras, Sox9 −/− cells were excluded from all cartilages and did not express chondrocyte-specific genes. The segregation occurred during mesenchymal condensation. No cartilages developed in teratocarcinomas derived from Sox9 −/− ES cells. Mice heterozygous for the Sox9 mutation died neonatally and exhibited skeletal abnormalities resembling those of the CD patients. The Sox9 +/− mutants had a cleft palate and hypoplasia of scapula, pelvis and other skeletal structures derived by endochondral ossification. Bending of the radius, ulna and tibia cartilage was prominent at embryonic day 14.5 (E14.5). At E12.5 many pre-cartilaginous condensations were already defective. Advanced ossification was observed and the hypertrophic zone was enlarged in the growth plates, suggesting that Sox9 also regulates hypertrophic chondrocyte differentiation. Our results identify Sox9 as the first essential regulator of chondrocyte differentiation, which plays multiple roles in chondrogenesis. ^

Analysis of Manganese nodules from the Challenger Expedition

Manganese nodules from Stations 252 and 281 of the Challenger Expedition, collected in 1875, have recently been discovered in the Redpath Museum. The nodules have been found to be quite typical specimens of two areas in the Central Pacific Ocean except for dehydration and other changes that have taken place during storage. The principal resolvable manganese mineral in nodules from Station 252 proved to be 10 A manganite; there was a very thin surface coating of birnessite. Delta manganite was the only manganese mineral found in nodules from Station 281. Through electron microprobe studies, findings from chemical, optical and x-ray crystallographic work were correlated with the detailed picture of the occurrence and quantities of the different elements within the nodules. In all cases it was found that the iron and manganese had an antithetical relationship, and that nickel and copper were associated with the manganese. Special study was given to a 300-micron-square area in a nodule from Station 252 which included a segregation of 49.39% Mn, 5.31 % Ni, and 1.64% Cu. Crystallization of the manganese phases is thought to have provided a mech¨anism for formation of segregations which were further enriched through chemical scavenger action as long as ocean floor conditions permitted.

Integration versus segregation in functional brain networks

We propose a new methodology to evaluate the balance between segregation and integration in functional brain networks by using singular value decomposition techniques. By means of magnetoencephalography, we obtain the brain activity of a control group of 19 individuals during a memory task. Next, we project the node-to-node correlations into a complex network that is analyzed from the perspective of its modular structure encoded in the contribution matrix. In this way, we are able to study the role that nodes play I/O its community and to identify connector and local hubs. At the mesoscale level, the analysis of the contribution matrix allows us to measure the degree of overlapping between communities and quantify how far the functional networks are from the configuration that better balances the integrated and segregated activity

Mapping and Introgression of QTL Involved in Fruit Shape Transgressive Segregation into 'Piel de Sapo' Melon (Cucumis melo L.)

A mapping F2 population from the cross ‘Piel de Sapo’ × PI124112 was selectively genotyped to study the genetic control of morphological fruit traits by QTL (Quantitative Trait Loci) analysis. Ten QTL were identified, five for FL (Fruit Length), two for FD (Fruit Diameter) and three for FS (Fruit Shape). At least one robust QTL per character was found, flqs8.1 (LOD = 16.85, R2 = 34%), fdqs12.1 (LOD = 3.47, R2 = 11%) and fsqs8.1 (LOD = 14.85, R2 = 41%). flqs2.1 and fsqs2.1 cosegregate with gene a (andromonoecious), responsible for flower sex determination and with pleiotropic effects on FS. They display a positive additive effect (a) value, so the PI124112 allele causes an increase in FL and FS, producing more elongated fruits. Conversely, the negative a value for flqs8.1 and fsqs8.1 indicates a decrease in FL and FS, what results in rounder fruits, even if PI124112 produces very elongated melons. This is explained by a significant epistatic interaction between fsqs2.1 and fsqs8.1, where the effects of the alleles at locus a are attenuated by the additive PI124112 allele at fsqs8.1. Roundest fruits are produced by homozygous for PI124112 at fsqs8.1 that do not carry any dominant A allele at locus a (PiPiaa). A significant interaction between fsqs8.1 and fsqs12.1 was also detected, with the alleles at fsqs12.1 producing more elongated fruits. fsqs8.1 seems to be allelic to QTL discovered in other populations where the exotic alleles produce elongated fruits. This model has been validated in assays with backcross lines along 3 years and ultimately obtaining a fsqs8.1-NIL (Near Isogenic Line) in ‘Piel de Sapo’ background which yields round melons.

Folding protein models with a simple hydrophobic energy function: The fundamental importance of monomer inside/outside segregation

The present study explores a “hydrophobic” energy function for folding simulations of the protein lattice model. The contribution of each monomer to conformational energy is the product of its “hydrophobicity” and the number of contacts it makes, i.e., E(h⃗, c⃗) = −Σi=1N cihi = −(h⃗.c⃗) is the negative scalar product between two vectors in N-dimensional cartesian space: h⃗ = (h1, … , hN), which represents monomer hydrophobicities and is sequence-dependent; and c⃗ = (c1, … , cN), which represents the number of contacts made by each monomer and is conformation-dependent. A simple theoretical analysis shows that restrictions are imposed concomitantly on both sequences and native structures if the stability criterion for protein-like behavior is to be satisfied. Given a conformation with vector c⃗, the best sequence is a vector h⃗ on the direction upon which the projection of c⃗ − c̄⃗ is maximal, where c̄⃗ is the diagonal vector with components equal to c̄, the average number of contacts per monomer in the unfolded state. Best native conformations are suggested to be not maximally compact, as assumed in many studies, but the ones with largest variance of contacts among its monomers, i.e., with monomers tending to occupy completely buried or completely exposed positions. This inside/outside segregation is reflected on an apolar/polar distribution on the corresponding sequence. Monte Carlo simulations in two dimensions corroborate this general scheme. Sequences targeted to conformations with large contact variances folded cooperatively with thermodynamics of a two-state transition. Sequences targeted to maximally compact conformations, which have lower contact variance, were either found to have degenerate ground state or to fold with much lower cooperativity.

Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1.

We recently isolated human cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by Pseudomonas exotoxin, Pseudomonas exotoxin-derived immunotoxins, diphtheria toxin, and tumor necrosis factor. We report here that one of these fragments is an antisense fragment of a gene homologous to the essential yeast chromosome segregation gene CSE1. Cloning and analysis of the full-length cDNA of the human CSE1 homologue, which we name CAS for cellular apoptosis susceptibility gene, reveals a protein coding region with similar length (971 amino acids for CAS, 960 amino acids for CSE1) and 59% overall protein homology to the yeast CSE1 protein. The conservation of this gene indicates it has an important function in human cells consistent with the essential role of CSE1 in yeast. CAS is highly expressed in human tumor cell lines and in human testis and fetal liver, tissues that contain actively dividing cells. Furthermore, CAS expression increases when resting human fibroblasts are induced to proliferate and decreases when they are growth-arrested. Thus, CAS appears to play an important role in both toxin and tumor necrosis factor-mediated cell death, as well as in cell proliferation.

Single cell analysis of cytokine gene coexpression during CD4+ T-cell phenotype development.

CD4+ T cells from alpha beta-T-cell receptor transgenic mice were analyzed for coexpression of cytokine mRNAs during phenotype development using a double-label in situ hybridization technique. T cells that produced cytokines in the primary response were a fraction of the activated population, and only a minority of the cytokine-positive cells coexpressed two cytokines. In secondary responses, frequencies of double-positive cells increased, although they remained a minority of the total. Of the cytokine pairs examined, interleukin (IL)-4 and IL-5 were the most frequently coexpressed. IL-4 and interferon gamma showed the greatest tendency toward segregation of expression, being rarely coexpressed after the primary stimulation. These data indicate that there is significant heterogeneity of cytokine gene expression by individual CD4+ T cells during early antigenic responses. Coexpression of any pairs of cytokines, much less Th1 and Th2 cytokines, is generally the exception. The Th0 phenotype is a population phenotype rather than an individual cell phenotype.

Analysis of Plasminogen Genetic Variants in Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117), despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93-1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility.

Analysis of Melanoma Onset: Assessing Familial Aggregation by Using Estimating Equations and Fitting Variance Components via Bayesian Random Effects Models

We investigate whether relative contributions of genetic and shared environmental factors are associated with an increased risk in melanoma. Data from the Queensland Familial Melanoma Project comprising 15,907 subjects arising from 1912 families were analyzed to estimate the additive genetic, common and unique environmental contributions to variation in the age at onset of melanoma. Two complementary approaches for analyzing correlated time-to-onset family data were considered: the generalized estimating equations (GEE) method in which one can estimate relationship-specific dependence simultaneously with regression coefficients that describe the average population response to changing covariates; and a subject-specific Bayesian mixed model in which heterogeneity in regression parameters is explicitly modeled and the different components of variation may be estimated directly. The proportional hazards and Weibull models were utilized, as both produce natural frameworks for estimating relative risks while adjusting for simultaneous effects of other covariates. A simple Markov Chain Monte Carlo method for covariate imputation of missing data was used and the actual implementation of the Bayesian model was based on Gibbs sampling using the free ware package BUGS. In addition, we also used a Bayesian model to investigate the relative contribution of genetic and environmental effects on the expression of naevi and freckles, which are known risk factors for melanoma.

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families: functional analysis in heterozygous individuals

A protein-truncating variant of CHEK2, 1100delC, is associated with a moderate increase in breast cancer risk. We have determined the prevalence of this allele in index cases from 300 Australian multiple-case breast cancer families, 95% of which had been found to be negative for mutations in BRCA1 and BRCA2. Only two (0.6%) index cases heterozygous for the CHEK2 mutation were identified. All available relatives in these two families were genotyped, but there was no evidence of co-segregation between the CHEK2 variant and breast cancer. Lymphoblastoid cell lines established from a heterozygous carrier contained approximately 20% of the CHEK2 1100delC mRNA relative to wild-type CHEK2 transcript. However, no truncated CHK2 protein was detectable. Analyses of expression and phosphorylation of wild-type CHK2 suggest that the variant is likely to act by haploinsufficiency. Analysis of CDC25A degradation, a downstream target of CHK2, suggests that some compensation occurs to allow normal degradation of CDC25A. Such compensation of the 1100delC defect in CHEK2 might explain the rather low breast cancer risk associated with the CHEK2 variant, compared to that associated with truncating mutations in BRCA1 or BRCA2.

Atom probe field ion microscope measurements of carbon segregation at an a:a grain boundary and service failures by intergranular stress corrosion cracking

This work reports on a critical measurement to understand the intergranular stress corrosion cracking (IGSCC) of pipeline steels: the atom probe field ion microscope (APFIM) measurement of the carbon concentration at a grain boundary (GB). The APFIM measurement was related to the microstructure and to IGSCC observations. The APFIM indicated that the GB carbon concentration of X70 was similar to 10 at% or less, which correlated with a high resistance to IGSCC for X70. (C) 2005 Elsevier Ltd. All rights reserved.

Gendered dichotomies and segregation patterns in computing jobs in Australia

The uneven distribution of women and men in IT employment is often depicted as reflecting adistinction between 'hard' and 'soft' tasks, skills and attributes. This article uses detailed occupational data on professional computing jobs in Australia to examine the extent to which patterns of gender segregation are consistent with such dichotomies. Additionally, we draw on qualitative interview data from aset oforganisational case studies for insights into the ways in which segregation patterns are reproduced and/or reshaped at"the workplace level. While perceptions ofgendered dichotomies were evident among many of our interviewees, overall our analysis shows considerably more complexity, with segregation patterns not necessarily aligned with clear-cut dichotomies and career directions often directly influenced by the organisation ofworking time in particular occupational streams.

Effect of solute segregation on the strength of nanocrystalline alloys:Inverse Hall-Petch relation

We have used a high-energy ball mill to prepare single-phased nanocrystalline Fe, Fe90Ni10, Fe85Al4Si11, Ni99Fe1 and Ni90Fe10 powders. We then increased their grain sizes by annealing. We found that a low-temperature anneal (T < 0.4 Tm) softens the elemental nanocrystalline Fe but hardens both the body-centered cubic iron- and face-centered cubic nickel-based solid solutions, leading in these alloys to an inverse Hall–Petch relationship. We explain this abnormal Hall–Petch effect in terms of solute segregation to the grain boundaries of the nanocrystalline alloys. Our analysis can also explain the inverse Hall–Petch relationship found in previous studies during the thermal anneal of ball-milled nanocrystalline Fe (containing ∼1.5 at.% impurities) and electrodeposited nanocrystalline Ni (containing ∼1.0 at.% impurities).

Historical development of residential segregation and black housing quality in Dade County

This paper examines the relationship between the historical development of residential segregation in Black areas of Dade County and the level of housing quality in those areas. Previous literature studies the effect of hypersegregation on housing quality. Instead, this paper analyzes the nature of each Black community and the social process by which they became segregated in contrast with only hypersegregation being considered. Data were drawn from the 1990 Census of Housing and Population at the block group level for Dade County. Two indicators for housing quality were considered: crowding and rent. Six categories for Black areas in Dade County and one residual category were developed for the analysis. Regression's results show that the effect of each community on housing quality varies. For example, overcrowding goes down in first-ghetto areas when compared to second-ghetto areas, although the percentage of Blacks in both communities is about the same. ^

Responses to racial segregation in a black Miami community

The present study examines the extent to which blacks are segregated in the suburban community of Coconut Grove, Florida. Hypersegregation, or the general tendency for blacks and whites to live apart, was examined in terms of four distinct dimensions: evenness, exposure, clustering, and concentration. Together, these dimensions define the geographic traits of the target area. Alone these indices can not capture the multi-dimensional levels of segregation and, therefore, by themselves underestimate the severity of segregation and isolation in this community. This study takes a contemporary view of segregation in a Dade County community to see if segregation is the catalyst to the sometime cited violent response of blacks. This study yields results that support the information in the literature review and the thesis research questions sections namely, that the blacks within the Grove do respond violently to the negative effects that racial segregation causes. This thesis is unique in two ways. It examines segregation in a suburban environment rather than an urban inner city, and it presents a responsive analysis of the individuals studied, rather than relying only on demographic and statistical data. ^