A Bruch’s membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a similar thickness as Bruch’s membrane (3 μm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell). Cultures established on either material developed a cobblestoned morphology with partial pigmentation within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+/K+-ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned medium collected from above and below both membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrate that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model.

Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5pl5. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

Ankylosing spondylitis is associated with the anthrax toxin receptor 2 gene (ANTXR2)

Objectives ANTXR2 variants have been associated with ankylosing spondylitis (AS) in two previous genome-wide association studies (GWAS) (p∼9×10-8). However, a genome-wide significant association (p<5×10-8) was not observed. We conducted a more comprehensive analysis of ANTXR2 in an independent UK sample to confirm and refine this association. Methods A replication study was carried out with 2978 cases and 8365 controls. Then, these were combined with non-overlapping samples from the two previous GWAS in a meta-analysis. Human leukocyte antigen (HLA)-B27 stratification was also performed to test for ANTXR2-HLA-B27 interaction. Results Out of nine single nucleotide polymorphisms (SNP) in the study, five SNPs were nominally associated (p<0.05) with AS in the replication dataset. In the meta-analysis, eight SNPs showed evidence of association, the strongest being with rs12504282 (OR=0.88, p=6.7×10-9). Seven of these SNPs showed evidence for association in the HLA-B27-positive subgroup, but none was associated with HLA-B27-negative AS. However, no statistically significant interaction was detected between HLA-B27 and ANTXR2 variants. Conclusions ANTXR2 variants are clearly associated with AS. The top SNPs from two previous GWAS (rs4333130 and rs4389526) and this study (rs12504282) are in strong linkage disequilibrium (r2≥0.76). All are located near a putative regulatory region. Further studies are required to clarify the role played by these ANTXR2 variants in AS.

High-throughput sequencing of IL23R reveals a low-frequency, nonsynonymous single-nucleotide polymorphism that is associated with ankylosing spondylitis in a Han Chinese population

Objective Ankylosing spondylitis (AS) is a highly heritable common inflammatory arthritis that targets the spine and sacroiliac joints of the pelvis, causing pain and stiffness and leading eventually to joint fusion. Although previous studies have shown a strong association of IL23R with AS in white Europeans, similar studies in East Asian populations have shown no association with common variants of IL23R, suggesting either that IL23R variants have no role or that rare genetic variants contribute. The present study was undertaken to screen IL23R to identify rare variants associated with AS in Han Chinese. Methods A 170-kb region containing IL23R and its flanking regions was sequenced in 50 patients with AS and 50 ethnically matched healthy control subjects from a Han Chinese population. In addition, the 30-kb region of peak association in white Europeans was sequenced in 650 patients with AS and 1,300 healthy controls. Validation genotyping was undertaken in 846 patients with AS and 1,308 healthy controls. Results We identified 1,047 variants, of which 729 were not found in the dbSNP genomic build 130. Several potentially functional rare variants in IL23R were identified, including one nonsynonomous single-nucleotide polymorphism (nsSNP), Gly149Arg (position 67421184 GA on chromosome 1). Validation genotyping showed that the Gly149Arg variant was associated with AS (odds ratio 0.61, P = 0.0054). Conclusion This is the first study to implicate rare IL23R variants in the pathogenesis of AS. The results identified a low-frequency nsSNP with predicted loss-of-function effects that was protectively associated with AS in Han Chinese, suggesting that decreased function of the interleukin-23 (IL-23) receptor protects against AS. These findings further support the notion that IL-23 signaling has an important role in the pathogenesis of AS.

The association between ambient temperature and children's lung function in Baotou, China

The objective of this study is to examine the association between ambient temperature and children’s lung function in Baotou, China. We recruited 315 children (8–12 years) from Baotou, China in the spring of 2004, 2005, and 2006. They performed three successive forced expiratory measurements three times daily (morning, noon, and evening) for about 5 weeks. The highest peak expiratory flow (PEF) was recorded for each session. Daily data on ambient temperature, relative humidity, and air pollution were monitored during the same period. Mixed models with a distributed lag structure were used to examine the effects of temperature on lung function while adjusting for individual characteristics and environmental factors. Low temperatures were significantly associated with decreases in PEF. The effects lasted for lag 0–2 days. For all participants, the cumulative effect estimates (lag 0–2 days) were −1.44 (−1.93, −0.94) L/min, −1.39 (−1.92, −0.86) L/min, −1.40 (−1.97, −0.82) L/min, and −1.28 (−1.69, −0.88) L/min for morning, noon, evening, and daily mean PEF, respectively, associated with 1 °C decrease in daily mean temperature. Generally, the effects of temperature were slightly stronger in boys than in girls for noon, evening, and daily mean PEF, while the effects were stronger in girls for morning PEF. PM2.5 had joint effects with temperature on children’s PEF. Higher PM2.5 increased the impacts of low temperature. Low ambient temperatures are associated with lower lung function in children in Baotou, China. Preventive health policies will be required for protecting children from the cold weather.

Spatiotemporal pattern of bacillary dysentery in China from 1990 to 2009: What is the driver behind?

BACKGROUND Little is known about the spatiotemporal pattern of bacillary dysentery (BD) in China. This study assessed the geographic distribution and seasonality of BD in China over the past two decades. METHODS Data on monthly BD cases in 31 provinces of China from January 1990 to December 2009 obtained from Chinese Center for Disease Control and Prevention, and data on demographic and geographic factors, as well as climatic factors, were compiled. The spatial distributions of BD in the four periods across different provinces were mapped, and heat maps were created to present the seasonality of BD by geography. A cosinor function combined with Poisson regression was used to quantify the seasonal parameters of BD, and a regression analysis was conducted to identify the potential drivers of morbidity and seasonality of BD. RESULTS Although most regions of China have experienced considerable declines in BD morbidity over the past two decades, Beijing and Ningxia still had high BD morbidity in 2009. BD morbidity decreased more slowly in North-west China than other regions. BD in China mainly peaked from July to September, with heterogeneity in peak time between regions. Relative humidity was associated with BD morbidity and peak time, and latitude was the major predictor of BD amplitude. CONCLUSIONS The transmission of BD was heterogeneous in China. Improved sanitation and hygiene in North-west China, and better access to clean water and food in the big floating population in some metropolises could be the focus of future preventive interventions against BD. BD control efforts should put more emphasis on those dry areas in summer.

Frequency response function based damage identification using principal component analysis and pattern recognition technique

Pattern recognition is a promising approach for the identification of structural damage using measured dynamic data. Much of the research on pattern recognition has employed artificial neural networks (ANNs) and genetic algorithms as systematic ways of matching pattern features. The selection of a damage-sensitive and noise-insensitive pattern feature is important for all structural damage identification methods. Accordingly, a neural networks-based damage detection method using frequency response function (FRF) data is presented in this paper. This method can effectively consider uncertainties of measured data from which training patterns are generated. The proposed method reduces the dimension of the initial FRF data and transforms it into new damage indices and employs an ANN method for the actual damage localization and quantification using recognized damage patterns from the algorithm. In civil engineering applications, the measurement of dynamic response under field conditions always contains noise components from environmental factors. In order to evaluate the performance of the proposed strategy with noise polluted data, noise contaminated measurements are also introduced to the proposed algorithm. ANNs with optimal architecture give minimum training and testing errors and provide precise damage detection results. In order to maximize damage detection results, the optimal architecture of ANN is identified by defining the number of hidden layers and the number of neurons per hidden layer by a trial and error method. In real testing, the number of measurement points and the measurement locations to obtain the structure response are critical for damage detection. Therefore, optimal sensor placement to improve damage identification is also investigated herein. A finite element model of a two storey framed structure is used to train the neural network. It shows accurate performance and gives low error with simulated and noise-contaminated data for single and multiple damage cases. As a result, the proposed method can be used for structural health monitoring and damage detection, particularly for cases where the measurement data is very large. Furthermore, it is suggested that an optimal ANN architecture can detect damage occurrence with good accuracy and can provide damage quantification with reasonable accuracy under varying levels of damage.

The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development

The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16–18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

Autosomal dominant spondylocostal dysostosis is caused by mutation in TBX6

In humans, congenital spinal defects occur with an incidence of 0.5-1 per 1000 live births. One of the most severe syndromes with such defects is spondylocostal dysostosis (SCD). Over the past decade, the genetic basis of several forms of autosomal recessive SCD cases has been solved with the identification of four causative genes (DLL3, MESP2, LFNG and HES7). Autosomal dominant forms of SCD have also been reported, but to date no genetic etiology has been described for these. Here, we have used exome capture and next-generation sequencing to identify a stoploss mutation in TBX6 that segregates with disease in two generations of one family. We show that this mutation has a deleterious effect on the transcriptional activation activity of the TBX6 protein, likely due to haploinsufficiency. In mouse, Tbx6 is essential for the patterning of the vertebral precursor tissues, somites; thus, mutation of TBX6 is likely to be causative of SCD in this family. This is the first identification of the genetic cause of an autosomal dominant form of SCD, and also demonstrates the potential of exome sequencing to identify genetic causes of dominant diseases even in small families with few affected individuals.

In situ investigation of explosive crystallization in a-Ge: Experimental determination of the interface response function using dynamic transmission electron microscopy

The crystallization of amorphous semiconductors is a strongly exothermic process. Once initiated the release of latent heat can be sufficient to drive a self-sustaining crystallization front through the material in a manner that has been described as explosive. Here, we perform a quantitative in situ study of explosive crystallization in amorphous germanium using dynamic transmission electron microscopy. Direct observations of the speed of the explosive crystallization front as it evolves along a laser-imprinted temperature gradient are used to experimentally determine the complete interface response function (i.e., the temperature-dependent front propagation speed) for this process, which reaches a peak of 16 m/s. Fitting to the Frenkel-Wilson kinetic law demonstrates that the diffusivity of the material locally/immediately in advance of the explosive crystallization front is inconsistent with those of a liquid phase. This result suggests a modification to the liquid-mediated mechanism commonly used to describe this process that replaces the phase change at the leading amorphous-liquid interface with a change in bonding character (from covalent to metallic) occurring in the hot amorphous material.

Fast decay of the velocity autocorrelation function in dense shear flow of inelastic hard spheres

We find in complementary experiments and event-driven simulations of sheared inelastic hard spheres that the velocity autocorrelation function psi(t) decays much faster than t(-3/2) obtained for a fluid of elastic spheres at equilibrium. Particle displacements are measured in experiments inside a gravity-driven flow sheared by a rough wall. The average packing fraction obtained in the experiments is 0.59, and the packing fraction in the simulations is varied between 0.5 and 0.59. The motion is observed to be diffusive over long times except in experiments where there is layering of particles parallel to boundaries, and diffusion is inhibited between layers. Regardless, a rapid decay of psi(t) is observed, indicating that this is a feature of the sheared dissipative fluid, and is independent of the details of the relative particle arrangements. An important implication of our study is that the non-analytic contribution to the shear stress may not be present in a sheared inelastic fluid, leading to a wider range of applicability of kinetic theory approaches to dense granular matter.

Genome-Wide Gene Expression Analysis Reveals a Dynamic Interplay between Luteotropic and Luteolytic Factors in the Regulation of Corpus Luteum Function in the Bonnet Monkey (Macaca radiata)

Although LH is essential for survival and function of the corpus luteum (CL) in higher primates, luteolysis occurs during nonfertile cycles without a discernible decrease in circulating LH levels. Using genome-wide expression analysis, several experiments were performed to examine the processes of luteolysis and rescue of luteal function in monkeys. Induced luteolysis with GnRH receptor antagonist (Cetrorelix) resulted in differential regulation of 3949 genes, whereas replacement with exogenous LH (Cetrorelix plus LH) led to regulation of 4434 genes (1563 down-regulation and 2871 up-regulation). A model system for prostaglandin (PG) F-2 alpha-induced luteolysis in the monkey was standardized and demonstrated that PGF(2 alpha) regulated expression of 2290 genes in the CL. Analysis of the LH-regulated luteal transcriptome revealed that 120 genes were regulated in an antagonistic fashion by PGF(2 alpha). Based on the microarray data, 25 genes were selected for validation by real-time RT-PCR analysis, and expression of these genes was also examined in the CL throughout the luteal phase and from monkeys treated with human chorionic gonadotropin (hCG) to mimic early pregnancy. The results indicated changes in expression of genes favorable to PGF(2 alpha) action during the late to very late luteal phase, and expressions of many of these genes were regulated in an opposite manner by exogenous hCG treatment. Collectively, the findings suggest that curtailment of expression of downstream LH-target genes possibly through PGF(2 alpha) action on the CL is among the mechanisms underlying cross talk between the luteotropic and luteolytic signaling pathways that result in the cessation of luteal function, but hCG is likely to abrogate the PGF(2 alpha)-responsive gene expression changes resulting in luteal rescue crucial for the maintenance of early pregnancy. (Endocrinology 150: 1473-1484, 2009)

Immunization of male bonnet monkeys (M-radiata) with a recombinant FSH receptor preparation affects testicular function and fertility

Immunization of proven fertile adult male monkeys (n = 3) with a recombinant FSH receptor protein preparation (oFSHR-P) (representing amino acids 1-134 of the extracellular domain of the receptor Mr similar to 15KDa) resulted in production of receptor blocking antibodies. The ability of the antibody to bind a particulate FSH receptor preparation and receptors in intact granulosa cells was markedly (by 30-80%) inhibited by FSH. Serum T levels and LH receptor function following immunization remained unchanged. The immunized monkeys showed a 50% reduction (p<0.001) in transformation of spermatogonia(2C) to primary spermatocytes (4C) as determined by flow cytometry and the 4C:2C ratio showed a correlative change (R 0.81, p<0.0007) with reduction in fertility index (sperm counts X motility score). Breeding studies indicated that monkeys became infertile between 242-368 days of immunization when the fertility index was in the range of 123+/-76 to 354+/-42 (compared to a value of 1602+/-384 on day 0). As the effects observed ate near identical to that seen following immunization with FSH it is suggestive that oFSHR-P can substitute for FSH in the development of a contraceptive vaccine.

Study of texture evolution in metastable beta-Ti alloy as a function of strain path and its effect on alpha transformation texture

Texture evolution in a low cost beta titanium alloy was studied for different modes of rolling and heat treatments. The alloy was cold rolled by unidirectional and multi-step cross rolling. The cold rolled material was either aged directly or recrystallized and then aged. The evolution of texture in alpha and beta phases were studied. The rolling texture of beta phase that is characterized by the gamma fiber is stronger for MSCR than UDR; while the trend is reversed on recrystallization. The mode of rolling affects alpha transformation texture on aging with smaller alpha lath size and stronger alpha texture in UDR than in MSCR. The defect structure in beta phase influences the evolution of a texture on aging. A stronger defect structure in beta phase leads to variant selection with the rolled samples showing fewer variants than the recrystallized samples.

Conformational Changes Triggered by Mg2+ Mediate Transactivator Function

Transactivator protein C of bacteriophage mu is essential for the transition from middle to late gene expression during the phage life cycle. The unusual, multistep activation of mom promoter (Pmom) by C protein involves activator-mediated promoter unwinding to recruit RNA polymerase and subsequent enhanced promoter clearance of the enzyme. To achieve this, C binds its site overlapping the -35 region of the mom promoter with a very high affinity, in Mg2+-dependent fashion. Mg2+-mediated conformational transition in C is necessary for its DNA binding and transactivation. We have determined the residues in C which coordinate Mg2+, to induce allosteric transition in the protein, required for the specific interaction with DNA. Residues E26 and D40 in the putative metal binding motif (E26X10D37X2D40) present toward the N-terminus of the protein are found to be important for Mg2+ ion binding. Mutations in these residues lead to altered Mg2+-induced conformation, compromised DNA binding, and reduced levels of transcription activation. Although Mg2+ is widely used in various DNA transaction reactions, this report provides the first insights on the importance of the metal ion-induced allosteric transitions in regulating transcription factor function.