Heritability and shared environment estimates for myopia and associated ocular biometric traits: the Genes in Myopia (GEM) family study

To examine the familial correlations, heritability (h ²) and common environmental components (c ²) of myopia and ocular biometric traits (all treated as continuous outcomes) in families collected through the Genes in Myopia (GEM) family study in Australia. A total of 132 pedigrees (723 participants) were recruited for this study. All individuals completed a risk factor questionnaire and underwent a detailed eye examination including spherical equivalent (SphE) and ocular biometric measurements of axial length (AL), anterior chamber depth (ACD) and corneal curvature (CC). Familial correlations were calculated and h ² and c² were estimated using a variance component model that assumes a multivariate t distribution within each pedigree. Two definitions of common environments (c ²) were considered: nuclear family (current) shared environment (Model 1) and sib-ship (childhood) shared environment (Model 2). Population ascertainment adjustment was performed using the Blue Mountains eye study dataset. The trends observed for familial correlations suggested that SphE is influenced by both environmental and genetic factors whereas AL, ACD and CC are predominantly genetically determined. This was largely confirmed by variance components modelling. Heritability estimates (adjusted for age, sex and years of education) from the best fitting ACE model (Model 2, childhood shared environment) were 0.50 ± 0.05 for SphE, 0.73 ± 0.04 for AL, 0.78 ± 0.04 for ACD and 0.16 ± 0.06 for CC. Childhood environmental effects were significant with c ² estimated to be 0.33 ± 0.04 for SphE, 0.06 ± 0.03 for AL, 0.22 ± 0.04 for ACD and 0.10 ± 0.05 for CC. Age was associated with SphE, total years of education was associated with AL and sex was associated with all traits studied. We used a novel and conservative approach to account for and estimate common environmental effects by specifying either nuclear family or sib-ship environment when estimating heritability estimates and showed that all traits examined (SphE, AL, ACD and CC) are heritable, thus reflecting a genetic component. These traits therefore all represent candidates for quantitative trait linkage analyses.     

N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution

Humans and chimpanzees share >99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors approximately 2 to 3 million years ago likely had immediate and long-term consequences for human biology.     

Amber suppression coupled with inducible surface display identifies cells with high recombinant protein productivity

Cell line development (CLD) for biotherapeutics is a time- and resource-intensive process requiring the isolation and screening of large numbers of clones to identify high producers. Novel methods aimed at enhancing cell line screening efficiency using markers predictive of productivity early in the CLD process are needed to reliably generate high-yielding cell lines. To enable efficient and selective isolation of antibody expressing Chinese hamster ovary cells by fluorescence-activated cell sorting, we developed a strategy for the expression of antibodies containing a switchable membrane-associated domain to anchor an antibody to the membrane of the expressing cell. The switchable nature of the membrane domain is governed by the function of an orthogonal aminoacyl transfer RNA synthetase/tRNApyl pair, which directs a nonnatural amino acid (nnAA) to an amber codon encoded between the antibody and the membrane anchor. The process is “switchable” in response to nnAA in the medium, enabling a rapid transition between the surface display and secretion. We demonstrate that the level of cell surface display correlates with productivity and provides a method for enriching phenotypically stable high-producer cells. The strategy provides a means for selecting high-producing cells with potential applications to multiple biotherapeutic protein formats.     

Liver Stiffness by Transient Elastography Predicts Liver-Related Complications and Mortality in Patients with Chronic Liver Disease

Background

Liver stiffness measurement (LSM) by transient elastography (TE, FibroScan) is a validated method for noninvasively staging liver fibrosis. Most hepatic complications occur in patients with advanced fibrosis. Our objective was to determine the ability of LSM by TE to predict hepatic complications and mortality in a large cohort of patients with chronic liver disease.

Methods

In consecutive adults who underwent LSM by TE between July 2008 and June 2011, we used Cox regression to determine the independent association between liver stiffness and death or hepatic complications (decompensation, hepatocellular carcinoma, and liver transplantation). The performance of LSM to predict complications was determined using the c-statistic.

Results

Among 2,052 patients (median age 51 years, 65% with hepatitis B or C), 87 patients (4.2%) died or developed a hepatic complication during a median follow-up period of 15.6 months (interquartile range, 11.0–23.5 months). Patients with complications had higher median liver stiffness than those without complications (13.5 vs. 6.0 kPa; P<0.00005). The 2-year incidence rates of death or hepatic complications were 2.6%, 9%, 19%, and 34% in patients with liver stiffness <10, 10–19.9, 20–39.9, and ≥40 kPa, respectively (P<0.00005). After adjustment for potential confounders, liver stiffness by TE was an independent predictor of complications (hazard ratio [HR] 1.05 per kPa; 95% confidence interval [CI] 1.03–1.06). The c-statistic of liver-stiffness for predicting complications was 0.80 (95% CI 0.75–0.85). A liver stiffness below 20 kPa effectively excluded complications (specificity 93%, negative predictive value 97%); however, the positive predictive value of higher results was sub-optimal (20%).

Conclusions

Liver stiffness by TE accurately predicts the risk of death or hepatic complications in patients with chronic liver disease. TE may facilitate the estimation of prognosis and guide management of these patients.     

Learning to Eat Vegetables in Early Life: The Role of Timing,Age and Individual Eating Traits

Vegetable intake is generally low among children, who appear to be especially fussy during the pre-school years. Repeated exposure is known to enhance intake of a novel vegetable in early life but individual differences in response to familiarisation have emerged from recent studies. In order to understand the factors which predict different responses to repeated exposure, data from the same experiment conducted in three groups of children from three countries (n = 332) aged 4–38 m (18.9±9.9 m) were combined and modelled. During the intervention period each child was given between 5 and 10 exposures to a novel vegetable (artichoke puree) in one of three versions (basic, sweet or added energy). Intake of basic artichoke puree was measured both before and after the exposure period. Overall, younger children consumed more artichoke than older children. Four distinct patterns of eating behaviour during the exposure period were defined. Most children were “learners” (40%) who increased intake over time. 21% consumed more than 75% of what was offered each time and were labelled “plate-clearers”. 16% were considered “non-eaters” eating less than 10 g by the 5^th exposure and the remainder were classified as “others” (23%) since their pattern was highly variable. Age was a significant predictor of eating pattern, with older pre-school children more likely to be non-eaters. Plate-clearers had higher enjoyment of food and lower satiety responsiveness than non-eaters who scored highest on food fussiness. Children in the added energy condition showed the smallest change in intake over time, compared to those in the basic or sweetened artichoke condition. Clearly whilst repeated exposure familiarises children with a novel food, alternative strategies that focus on encouraging initial tastes of the target food might be needed for the fussier and older pre-school children.     

Dysregulation of cell polarity proteins synergize with oncogenes or the microenvironment to induce invasive behavior in epithelial cells

Changes in expression and localization of proteins that regulate cell and tissue polarity are frequently observed in carcinoma. However, the mechanisms by which changes in cell polarity proteins regulate carcinoma progression are not well understood. Here, we report that loss of polarity protein expression in epithelial cells primes them for cooperation with oncogenes or changes in tissue microenvironment to promote invasive behavior. Activation of ErbB2 in cells lacking the polarity regulators Scribble, Dlg1 or AF-6, induced invasive properties. This cooperation required the ability of ErbB2 to regulate the Par6/aPKC polarity complex. Inhibition of the ErbB2-Par6 pathway was sufficient to block ErbB2-induced invasion suggesting that two polarity hits may be needed for ErbB2 to promote invasion. Interestingly, in the absence of ErbB2 activation, either a combined loss of two polarity proteins, or exposure of cells lacking one polarity protein to cytokines IL-6 or TNFα induced invasive behavior in epithelial cells. We observed the invasive behavior only when cells were plated on a stiff matrix (Matrigel/Collagen-1) and not when plated on a soft matrix (Matrigel alone). Cells lacking two polarity proteins upregulated expression of EGFR and activated Akt. Inhibition of Akt activity blocked the invasive behavior identifying a mechanism by which loss of polarity promotes invasion of epithelial cells. Thus, we demonstrate that loss of polarity proteins confers phenotypic plasticity to epithelial cells such that they display normal behavior under normal culture conditions but display aggressive behavior in response to activation of oncogenes or exposure to cytokines.     

A review of the cytomorphology of Epstein-Barr virus-associated malignancies

The Epstein-Barr virus (EBV) is a member of the herpes family of viruses and is very common in humans. EBV is most often associated with infectious mononucleosis. However, it is estimated that 1% of tumors including lymphoproliferative, epithelial and mesenchymal are linked to EBV infection. EBV has a tropism for certain epithelial cells, lymphocytes and myocytes. Like other herpesviruses, EBV has both lytic and latent phases of infection. In the latent form, EBV-encoded genes ensure the survival of the viral genome, allowing it to circumvent the host's immune surveillance by limited expression of viral proteins and carries with it the risk of neoplastic transformation. Cytologists are likely to encounter EBV-associated malignancies in cytology material but unlike other herpesviruses, EBV does not evoke a viral cytopathic effect. The manifestation of EBV-related tumors is also often variable depending upon the patient's immune status. Therefore, knowledge of the patient's EBV status and immune competence (e.g. HIV-infection or transplant-related immunosuppression) combined with the cytomorphology and results of ancillary studies are often all required to make a diagnosis of EBV-associated malignancy. This review discusses the unique cytomorphology and ancillary studies required to diagnose EBV-related neoplasms.     

Differential expression of the putative Kex2 processed and secreted aspartic proteinase gene family of Cryphonectria parasitica

Kex2-silenced strains of Cryphonectria parasitica, the ascomycete causal agent of chestnut blight, show a significant reduction in virulence, reduced sexual and asexual sporulation and reductions in mating and fertility. Due to this and the known involvement of Kex2 in the processing of important proproteins in other systems, we searched the whole C. parasitica genome for putative Kex2 substrates. Out of 1299 open reading frames (ORFs) predicted to be secreted, 222 ORFs were identified as potential Kex2 substrates by this screen. Within the putative substrates we identified cell wall modifying proteins, putative proteinases, lipases, esterases, and oxidoreductases. This in silico screen also uncovered a family of nine secreted aspartic proteinases (SAPs) of C. parasitica. Northern blot analyses of this gene family showed differential expression when exposed to chestnut wood and Cryphonectria hypovirus 1 (CHV1). Due to the reduction in fungal virulence known to be caused upon hypoviral infection of C. parasitica, the differential gene expression observed, and the known involvement of SAPs in virulence in other systems, we conducted deletion analyses of four of these proteinases, representing different expression patterns. Deletion of each of the four SAPs did not affect growth rates, sporulation or virulence, suggesting that none of the considered SAPs is essential for the full development or virulence of C. parasitica under the conditions tested.     

Multiple mechanisms contribute to Schizosaccharomyces pombe origin recognition complex-DNA interactions

Eukaryotic DNA replication requires the assembly of multiprotein pre-replication complexes (pre-RCs) at chromosomal origins of DNA replication. Here we describe the interactions of highly purified Schizosaccharomyces pombe pre-RC components, SpORC, SpCdc18, and SpCdt1, with each other and with ars1 origin DNA. We show that SpORC binds DNA in at least two steps. The first step likely involves electrostatic interactions between the AT-hook motifs of SpOrc4 and AT tracts in ars1 DNA and results in the formation of a salt-sensitive complex. In the second step, the salt-sensitive complex is slowly converted to a salt-stable complex that involves additional interactions between SpORC and DNA. Binding of SpORC to ars1 DNA is facilitated by negative supercoiling and is accompanied by changes in DNA topology, suggesting that SpORC-DNA complexes contain underwound or negatively writhed DNA. Purified human origin recognition complex (ORC) induces similar topological changes in origin DNA, indicating that this property of ORC is conserved in eukaryotic evolution and plays an important role in ORC function. We also show that SpCdc18 and SpCdt1 form a binary complex that has greater affinity for DNA than either protein alone. In addition, both proteins contribute significantly to the stability of the initial SpORC-DNA complex and enhance the SpORC-dependent topology changes in origin DNA. Thus, the formation of stable protein-DNA complexes at S. pombe origins of replication involves binary interactions among all three proteins, as well as interactions of both SpORC and SpCdt1-SpCdc18 with origin DNA. These findings demonstrate that SpORC is not the sole determinant of origin recognition.