Hypoxia impacts human MSC response to substrate stiffness during chondrogenic differentiation

Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-β1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-β may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia's influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167 kPa) or stiff (49.6 kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-β3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia's effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage

Discarding IVF embryos: reporting on global practices

Purpose: To provide a global scale report on a representative sample of the clinical embryology community depicting the practice of discarding supernumerary IVF embryos. Methods: A web-based questionnaire titled “Anonymous questionnaire on embryo disposal practices” was designed in order to ensure anonymous participation of practicing clinical embryologists around the world. Results: During a data collection period of 8 months, 703 filled-in questionnaires from 65 countries were acquired. According to the data acquired, the majority of practitioners, dispose of embryos by placing them directly in a trash can strictly dedicated for embryo disposal for both fresh and frozen cycles (39% and 36.7% respectively). Moreover, 66.4% of practitioners discard the embryos separately—case by case—at different time points during the day. Over half of embryologists (54%) wait until day 6 to discard the surplus embryos, while 65.5% do not implement a specially allocated incubator space as a designated waiting area prior to disposal. The majority of 63.1% reported that this is a witnessed procedure. The vast majority of embryologists (93%) do not employ different protocols for different groups of patients. Nonetheless, 17.8% reported the request to perform a ceremony for these embryos. Assessing the embryologists’ perspective, 59.5% of participants stated that the embryology practice would benefit from a universally accepted and practiced protocol. Conclusion(s): This study uniquely provides insight into global embryo disposal practices and trends. Results highlight the divergence between reported practices, while indicating the significance on standardization of practice, with embryologists acknowledging the need for a universally accepted protocol implementation

An overview of reliability assessment and control for design of civil engineering structures

Random variations, whether they occur in the input signal or the system parameters, are phenomena that occur in nearly all engineering systems of interest. As a result, nondeterministic modeling techniques must somehow account for these variations to ensure validity of the solution. As might be expected, this is a difficult proposition and the focus of many current research efforts. Controlling seismically excited structures is one pertinent application of nondeterministic analysis and is the subject of the work presented herein. This overview paper is organized into two sections. First, techniques to assess system reliability, in a context familiar to civil engineers, are discussed. Second, and as a consequence of the first, active control methods that ensure good performance in this random environment are presented. It is the hope of the authors that these discussions will ignite further interest in the area of reliability assessment and design of controlled civil engineering structures

Binding Kinetics Redefine the Antagonist Pharmacology of the Corticotropin-Releasing Factor Type 1 Receptor

ABSTRACT Corticotropin-releasing factor (CRF) receptor antagonists are under preclinical and clinical investigation for stress-related disorders. In this study the impact of receptor-ligand binding kinetics on CRF 1 receptor antagonist pharmacology was investigated by measuring the association rate constant (k 1 ), dissociation rate constant (k Ϫ1 ), and kinetically derived affinity at 37°C. Three aspects of antagonist pharmacology were reevaluated: comparative binding activity of advanced compounds, in vivo efficacy, and structure-activity relationships. Twelve lead compounds, with little previously noted difference of affinity, varied substantially in their kinetic binding activity with a 510-fold range of kinetically derived affinity (k Ϫ1 /k 1 ), 170-fold range of k Ϫ1 , and 13-fold range of k 1 . The k Ϫ1 values indicated previous affinity measurements were not close to equilibrium, resulting in compression of the measured affinity range. Dissociation was exceptionally slow for three ligands (k Ϫ1 t 1/2 of 1.6 -7.2 h at 37°C). Differences of binding behavior were consistent with in vivo pharmacodynamics (suppression of adrenocorticotropin in adrenalectomized rats). Ligand concentration-effect relationships correlated with their kinetically derived affinity. Two ligands that dissociated slowly (53 and 130 min) produced prolonged suppression, whereas only transient suppression was observed with a more rapidly dissociating ligand (16 min). Investigating the structure-activity relationship indicated exceptionally low values of k 1 , approaching 100,000-fold less than the diffusion-limited rate. Retrospective interpretation of medicinal chemistry indicates optimizing specific elements of chemical structure overcame kinetic barriers in the association pathway, for example, constraint of the pendant aromatic orthogonal to the ligand core. Collectively, these findings demonstrate receptor binding kinetics provide new dimensions for understanding and potentially advancing the pharmacology of CRF 1 receptor antagonists

A distinct spectrum of copy number aberrations in pediatric high-grade gliomas

As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors. Purpose: As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors. Experimental Design: We carried out copy number profiling by array comparative genomic hybridization using a 32K bacterial artificial chromosome platform on 63 formalin-fixed paraffin-embedded cases of high-grade glioma arising in children and young people (<23 years). Results: The genomic profiles of these tumors could be subclassified into four categories: those with stable genomes, which were associated with a better prognosis; those with aneuploid and those with highly rearranged genomes; and those with an amplifier genotype, which had a significantly worse clinical outcome. Independent of this was a clear segregation of cases with 1q gain (more common in children) from those with concurrent 7 gain/10q loss (a defining feature of adults). Detailed mapping of all the amplification and deletion events revealed numerous low-frequency amplifications, including IGF1R, PDGFRB, PIK3CA, CDK6, CCND1, and CCNE1, and novel homozygous deletions encompassing unknown genes, including those at 5q35, 10q25, and 22q13. Despite this, aberrations targeting the “core signaling pathways” in adult glioblastomas are significantly underrepresented in the pediatric setting. Conclusions: These data highlight that although there are overlaps in the genomic events driving gliomagenesis of all ages, the pediatric disease harbors a distinct spectrum of copy number aberrations compared with adults.National Health Service funding to the NIHR Biomedical Research Centre. This work was supported by The Royal Marsden Children's Department Fund, Fundação para a Ciência e Tecnologia, Portugal, and Breakthrough Breast Cance

A Greek validation study of the Multiple Sclerosis Work Difficulties Questionnaire-23

The Multiple Sclerosis Work Difficulties Questionnaire-23 (MSWDQ-23) is a self-reportinstrument developed to assess barriers faced by People with Multiple Sclerosis (PwMS) in theworkplace. The aim of this study was to explore the psychometric properties of the Greek versionof the MSWDQ-23. The study sample consisted of 196 PwMS, all currently working in part- orfull-time jobs. Participants underwent clinical examination and cognitive screening with the BriefInternational Cognitive Assessment for Multiple Sclerosis (BICAMS) and completed self-reportmeasures of fatigue, psychological functioning, and quality of life, along with the MSWDQ-23questionnaire. Confirmatory Factor Analysis (CFA) was performed, and goodness-of-fit measureswere used to evaluate construct validity. Convergent validity was checked by correlating MSWDQ-23scores with study measures. Cronbach’s alpha value was produced to assess internal consistency.CFA yielded a model with a fair fit confirming the three-factor structure of the instrument. Higherwork difficulties were associated with higher Expanded Disability Status Scale (EDSS) scores, poorercognitive function, more fatigue, stress, anxiety, and depression, and poorer health status, supportingthe convergent validity of MSWDQ-23. Internal consistency (Cronbach’s alpha = 0.94) and test–retest reliability (ICC = 0.996, 95%, CI = 0.990–0.998) were excellent. The Greek MSWDQ-23 can beconsidered a valid patient-reported outcome measure and can be used in interventions aiming toimprove the vocational status of PwMS

Output feedback robust H∞ control with D-stability and variance constraints: A parametrization approach

This is the post print version of the article. The official published version can be obtained from the link below - Copyright 2005 Springer Ltd.In this paper, we study the problem of robust H∞ controller design for uncertain continuous-time systems with variance and D-stability constraints. The parameter uncertainties are allowed to be unstructured but norm-bounded. The aim of this problem is the design of an output feedback controller such that, for all admissible uncertainties, the closed-loop poles be placed within a specified disk, the H∞ norm bound constraint on the disturbance rejection attenuation be guaranteed, and the steady-state variance for each state of the closed-loop system be no more than the prescribed individual upper bound, simultaneously. A parametric design method is exploited to solve the problem addressed. Sufficient conditions for the existence of the desired controllers are derived by using the generalized inverse theory. The analytical expression of the set of desired controllers is also presented. It is shown that the obtained results can be readily extended to the dynamic output feedback case and the discrete-time case

Allele-Specific Up-Regulation of FGFR2 Increases Susceptibility to Breast Cancer

The recent whole-genome scan for breast cancer has revealed the FGFR2 (fibroblast growth factor receptor 2) gene as a locus associated with a small, but highly significant, increase in the risk of developing breast cancer. Using fine-scale genetic mapping of the region, it has been possible to narrow the causative locus to a haplotype of eight strongly linked single nucleotide polymorphisms (SNPs) spanning a region of 7.5 kilobases (kb) in the second intron of the FGFR2 gene. Here we describe a functional analysis to define the causative SNP, and we propose a model for a disease mechanism. Using gene expression microarray data, we observed a trend of increased FGFR2 expression in the rare homozygotes. This trend was confirmed using real-time (RT) PCR, with the difference between the rare and the common homozygotes yielding a Wilcox p-value of 0.028. To elucidate which SNPs might be responsible for this difference, we examined protein–DNA interactions for the eight most strongly disease-associated SNPs in different breast cell lines. We identify two cis-regulatory SNPs that alter binding affinity for transcription factors Oct-1/Runx2 and C/EBPβ, and we demonstrate that both sites are occupied in vivo. In transient transfection experiments, the two SNPs can synergize giving rise to increased FGFR2 expression. We propose a model in which the Oct-1/Runx2 and C/EBPβ binding sites in the disease-associated allele are able to lead to an increase in FGFR2 gene expression, thereby increasing the propensity for tumour formation