Experimental and numerical investigations on the impact behaviour of pristine and patch-repaired composite laminates.

The present paper investigates the impact behaviour of both pristine carbon-fibre-reinforced-plastic (CFRP) composite laminates and repaired CFRP laminates. For the patch-repaired CFRP specimen, the pristine CFRP panel specimen has been damaged by cutting out a central disc of the CFRP material and then repaired using an adhesively bonded patch of CFRP to cover the hole. Drop-weight, impact tests are performed on these two types of specimens and a numerical elastic-plastic, three-dimensional damage model is developed and employed to simulate the impact behaviour of both types of specimen. This numerical model is meso-scale in nature and assumes that cracks initiate in the CFRP at a nano-scale, in the matrix around fibres, and trigger sub-micrometre intralaminar matrix cracks during the impact event. These localized regions of intralaminar cracking then lead to interlaminar, i.e. delamination, cracking between the neighbouring plies which possess different fibre orientations. These meso-scale, intralaminar and interlaminar, damage processes are modelled using the numerical finite-element analysis model with each individual ply treated as a continuum. Good agreement is found between the results from the experimental studies and the predictions from the numerical simulations. This article is part of the theme issue 'Nanocracks in nature and industry'.

Pharmacotoxicology of clinically-relevant concentrations of obeticholic acid in an organotypic human hepatocyte system.

Nonalcoholic steatohepatitis (NASH) is an emerging health crisis with no approved therapies. Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, shows promise in NASH trials. However, the precise mechanisms mediating OCA effects and impact on cholesterol metabolism are not fully understood. We explored the pharmaco-toxicological effects of OCA on patho-physiological pathways in hepatocytes using a previously described perfused organotypic liver system that allows culture in near-physiological insulin/glucose milieus, and exhibits drug responses at clinically-relevant concentrations. Primary hepatocytes experienced 48-hour exposure to OCA at concentrations approximating therapeutic (0.5µM) and supratherapeutic (10µM) levels. Global transcriptomics by RNAseq was complimented by cellular viability (MTT), CYP activity assays, and secreted FGF19 levels in the media. Dose-dependent, transcriptional effects suggested suppression of bile acid synthesis (↓CYP7A1, ↓CYP27A1) and increased bile efflux (↑ABCB4, ↑ABCB11, ↑OSTA, ↑OSTB). Pleiotropic effects included suppression of TGFß and IL-6 signaling pathways, and signatures suggestive of HDL suppression (↑SCARB1, ↓ApoAI, ↓LCAT) and LDL elevation (↑ApoB, ↓CYP7A1). OCA exhibited direct FXR-mediated effects with increased FGF19 secretion. Transcriptomics revealed regulation of metabolic, anti-inflammatory, and anti-fibrotic pathways beneficial in NASH, and predicted cholesterol profiles consistent with clinical findings. Follow-up studies under lipotoxic/inflammatory conditions would corroborate these effects in a disease-relevant environment.

The timing of molecular and morphological changes underlying reproductive transitions in wild tomatoes (Solanum sect. Lycopersicon).

Molecular mechanisms underlying the transition from genetic self-incompatibility to self-compatibility are well documented, but the evolution of other reproductive trait changes that accompany shifts in reproductive strategy (mating system) remains comparatively under-investigated. A notable exception is the transition from exserted styles to styles with recessed positions relative to the anthers in wild tomatoes (Solanum Section Lycopersicon). This phenotypic change has been previously attributed to a specific mutation in the promoter of a gene that influences style length (style2.1); however, whether this specific regulatory mutation arose concurrently with the transition from long to short styles, and whether it is causally responsible for this phenotypic transition, has been poorly investigated across this group. To address this gap, we assessed 74 accessions (populations) from 13 species for quantitative genetic variation in floral and reproductive traits as well as the presence/absence of deletions at two different locations (StyleD1 and StyleD2) within the regulatory region upstream of style2.1. We confirmed that the putatively causal deletion variant (a 450-bp deletion at StyleD1) arose within self-compatible lineages. However, the variation and history of both StyleD1 and StyleD2 was more complex than previously inferred. In particular, although StyleD1 was statistically associated with differences in style length and stigma exsertion across all species, we found no evidence for this association within two species polymorphic for the StyleD1 mutation. We conclude that the previous association detected between phenotypic and molecular differences is most likely due to a phylogenetic association rather than a causal mechanistic relationship. Phenotypic variation in style length must therefore be due to other unexamined linked variants in the style2.1 regulatory region.

Outside-the-(cavity-prep)-box thinking.

Direct placement restorative materials must interface with tooth structures that are often compromised by caries or trauma. The material must seal the interface while providing sufficient strength and wear resistance to assure function of the tooth for, ideally, the lifetime of the patient. Needed are direct restorative materials that are less technique-sensitive than current resin-based composite systems while having improved properties. The ideal material could be successfully used in areas of the world with limited infrastructure. Advances in our understanding of the interface between the restoration adhesive system and the stages of carious dentin can be used to promote remineralization. Application of fracture mechanics to adhesion at the tooth-restoration interface can provide insights for improvement. Research in polymer systems suggests alternatives to current composite resin matrix systems to overcome technique sensitivity, while advances in nano- and mesoparticle reinforcement and alignment in composite systems can increase material strength, toughness, and wear resistance, foreshadowing dental application.

Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro.

In vitro primary hepatocyte systems typically elicit drug induction and toxicity responses at concentrations much higher than corresponding in vivo or clinical plasma C(max) levels, contributing to poor in vitro-in vivo correlations. This may be partly due to the absence of physiological parameters that maintain metabolic phenotype in vivo. We hypothesized that restoring hemodynamics and media transport would improve hepatocyte architecture and metabolic function in vitro compared with nonflow cultures. Rat hepatocytes were cultured for 2 wk either in nonflow collagen gel sandwiches with 48-h media changes or under controlled hemodynamics mimicking sinusoidal circulation within a perfused Transwell device. Phenotypic, functional, and metabolic parameters were assessed at multiple times. Hepatocytes in the devices exhibited polarized morphology, retention of differentiation markers [E-cadherin and hepatocyte nuclear factor-4α (HNF-4α)], the canalicular transporter [multidrug-resistant protein-2 (Mrp-2)], and significantly higher levels of liver function compared with nonflow cultures over 2 wk (albumin ~4-fold and urea ~5-fold). Gene expression of cytochrome P450 (CYP) enzymes was significantly higher (fold increase over nonflow: CYP1A1: 53.5 ± 10.3; CYP1A2: 64.0 ± 15.1; CYP2B1: 15.2 ± 2.9; CYP2B2: 2.7 ± 0.8; CYP3A2: 4.0 ± 1.4) and translated to significantly higher basal enzyme activity (device vs. nonflow: CYP1A: 6.26 ± 2.41 vs. 0.42 ± 0.015; CYP1B: 3.47 ± 1.66 vs. 0.4 ± 0.09; CYP3A: 11.65 ± 4.70 vs. 2.43 ± 0.56) while retaining inducibility by 3-methylcholanthrene and dexamethasone (fold increase over DMSO: CYP1A = 27.33 and CYP3A = 4.94). These responses were observed at concentrations closer to plasma levels documented in vivo in rats. The retention of in vivo-like hepatocyte phenotype and metabolic function coupled with drug response at more physiological concentrations emphasizes the importance of restoring in vivo physiological transport parameters in vitro.

Connecting genetic variation to phenotypic clines.

From early allozyme work to recent genome-wide scans, many studies have reported associations between molecular markers and latitude. These geographic patterns are tantalizing because they hint at the possibility of identifying specific mutations responsible for climatic adaptation. Unfortunately, few studies have done so because these exciting first glances often prove extremely challenging to follow up. Many difficulties can hinder connecting genetic and phenotypic variation in this context, and without such links, distinguishing the action of spatially varying selection from the other evolutionary processes capable of generating these patterns can be quite thorny. Nevertheless, two papers in this issue report excellent progress in overcoming these obstacles and provide persuasive evidence supporting the involvement of specific natural variants in clinal adaptation of Drosophila melanogaster populations (Fig. 1). In the first paper, Paaby et al. (2010) describe replicated allele frequency clines for a coding polymorphism in the Insulin-like Receptor (InR) gene on two continents, findings that strongly point to selection acting at this locus and that likely reflect life history adaptation. McKechnie et al. (2010) report compelling functional evidence that cis-regulatory variation in the Dca (drosophila cold acclimation) gene contributes to an adaptive cline in wing size. Notably, these papers employ largely alternative and complementary approaches, and together they exemplify how diverse strategies may be interwoven to draw convincing connections between genotype, phenotype, and evolutionary process.

Frequency of intrinsic pulsatile gonadotropin-releasing hormone secretion is regulated by the expression of cyclic nucleotide-gated channels in GT1 cells.

Cultures of endogenous GnRH neurons and the GT1 GnRH neuronal cell line release GnRH in pulses (intrinsic pulsatile release) with an interpulse frequency similar to that seen in castrated animals. In both GT1 cells and transgenic rats, lowering cAMP levels by expression of a phosphodiesterase decreased the frequency of intrinsic GnRH pulsatility. We asked whether the cyclic nucleotide-gated cation (CNG) channels expressed in GT1 cells participated in cAMP modulation of intrinsic GnRH pulsatility. Because expression of the CNGA2 subunit is essential for formation of functional CNG channels, we developed an adenovirus (Ad) vector expressing a short interference RNA (siRNA) to the CNGA2 subunit (Ad-CNG-siRNA) or as an infection control, to the coding region of luciferase (Ad-Luc-siRNA). Infection with the Ad-CNG-siRNA of COS cells transfected with a CNGA2 expression vector significantly inhibited CNGA2 protein levels by 74% by Western blot. Infection of GT1-1 cells with Ad-CNG-siRNA resulted in a 68% decrease in the levels of CNGA2 mRNA, a 44% decrease in protein levels, and a clear decrease in immunostaining with an antibody to CNGA2. Infection of GT1-1 cells with Ad-CNG-siRNA decreased spontaneous Ca2+ oscillations compared with Ad-Luc-siRNA-infected or uninfected cells by 71%. Furthermore infection with Ad-CNG-siRNA resulted in a 2-fold increase in the interpulse interval in GnRH secretion (49.4+/-9.1 min) compared with uninfected cells (25.9+/-2.5 min) or Ad-Luc-siRNA (29.3+/-2.8 min)-infected cells. These data provide the first direct evidence that the CNG channel is a downstream signaling molecule in the regulation of the frequency of intrinsic GnRH pulsatility by cAMP.

A framework for sequencing the rice genome.

Rice is an important food crop and a model plant for other cereal genomes. The Clemson University Genomics Institute framework project, begun two years ago in anticipation of the now ongoing international effort to sequence the rice genome, is nearing completion. Two bacterial artificial chromosome (BAC) libraries have been constructed from the Oryza sativa cultivar Nipponbare. Over 100,000 BAC end sequences have been generated from these libraries and, at a current total of 28 Mbp, represent 6.5% of the total rice genome sequence. This sequence information has allowed us to draw first conclusions about unique and redundant rice genomic sequences. In addition, more than 60,000 clones (19 genome equivalents) have been successfully fingerprinted and assembled into contigs using FPC software. Many of these contigs have been anchored to the rice chromosomes using a variety of techniques. Hybridization experiments have shown these contigs to be very robust. Contig assembly and hybridization experiments have revealed some surprising insights into the organization of the rice genome, which will have significant repercussions for the sequencing effort. Integration of BAC end sequence data with anchored contig information has provided unexpected revelations on sequence organization at the chromosomal level.

Biomechanical activation of vascular endothelium as a determinant of its functional phenotype.

One of the striking features of vascular endothelium, the single-cell-thick lining of the cardiovascular system, is its phenotypic plasticity. Various pathophysiologic factors, such as cytokines, growth factors, hormones, and metabolic products, can modulate its functional phenotype in health and disease. In addition to these humoral stimuli, endothelial cells respond to their biomechanical environment, although the functional implications of this biomechanical paradigm of activation have not been fully explored. Here we describe a high-throughput genomic analysis of modulation of gene expression observed in cultured human endothelial cells exposed to two well defined biomechanical stimuli-a steady laminar shear stress and a turbulent shear stress of equivalent spatial and temporal average intensity. Comparison of the transcriptional activity of 11,397 unique genes revealed distinctive patterns of up- and down-regulation associated with each type of stimulus. Cluster analyses of transcriptional profiling data were coupled with other molecular and cell biological techniques to examine whether these global patterns of biomechanical activation are translated into distinct functional phenotypes. Confocal immunofluorescence microscopy of structural and contractile proteins revealed the formation of a complex apical cytoskeleton in response to laminar shear stress. Cell cycle analysis documented different effects of laminar and turbulent shear stresses on cell proliferation. Thus, endothelial cells have the capacity to discriminate among specific biomechanical forces and to translate these input stimuli into distinctive phenotypes. The demonstration that hemodynamically derived stimuli can be strong modulators of endothelial gene expression has important implications for our understanding of the mechanisms of vascular homeostasis and atherogenesis.

Mechanosensitive endothelial gene expression profiles: scripts for the role of hemodynamics in atherogenesis?

The possibility that hemodynamic forces can act as a "local risk factor" for endothelial dysfunction provides a conceptual framework for the longstanding observation that the earliest lesions of atherosclerosis develop in a nonrandom pattern, the geometries of which correlate with branch points and other regions of altered blood flow. This has led us to hypothesize that hemodynamic forces, in particular wall shear stresses generated by complex patterns of blood flow, can function as both positive and negative stimuli in atherogenesis via effects on endothelial cell gene expression. To understand how endothelial cells in different regions of the arterial tree acquire both functional and dysfunctional phenotypes due to regional hemodynamics, it was important to begin to delineate, in a comprehensive fashion, the mechanoresponsiveness of endothelial cells. To address this fundamental question, we undertook high-throughput transcriptional profiling to assess the global patterns of gene expression in cultured endothelial cells exposed to two defined biomechanical stimuli. Analyses of the transcriptional activity of thousands of genes have revealed unique patterns of gene expression associated with certain types of stimuli. These unique gene expression programs and their associated functional phenotypes constitute the strongest evidence to date that vascular endothelial cells can discriminate among different types of biomechanical stimuli. The results of these studies and the working hypotheses inspired by detailed molecular analyses of biomechanically activated vascular endothelium promise to provide new insights into the role of hemodynamics in the pathogenesis of atherosclerosis.

Selective modulation of endothelial cell [Ca2+]i response to flow by the onset rate of shear stress.

The response of endothelial cells (ECs) to their hemodynamic environment strongly influences normal vascular physiology and the pathogenesis of atherosclerosis. Unique responses to the complex flow patterns in lesion-prone regions imply that the temporal and spatial features of the mechanical stimuli modulate the cellular response to flow. We report the first systematic study of the effects of temporal gradients of shear stress on ECs. Flow was applied to cultured ECs using a novel cone-and-plate device allowing precise and independent control of the shear stress magnitude and the onset rate. Intracellular free calcium concentration ([Ca2+]i) increased rapidly following the onset of flow, and the characteristics of the transient were modulated by both the shear stress magnitude and onset rate. ECs were most sensitive to shear stress applied at physiological onset rates. Furthermore, the relative contribution of extracellular calcium and IP3-mediated release were dependent upon the specific flow regime.

In vitro cell shearing device to investigate the dynamic response of cells in a controlled hydrodynamic environment.

Mechanical stresses and strains play important roles in the normal growth and development of biological tissues, yet the cellular mechanisms of mechanotransduction have not been identified. A variety of in vitro systems for applying mechanical loads to cell populations have been developed to gain insight into these mechanisms. However, limitations in the ability to control precisely relevant aspects of the mechanical stimuli have obscured the physical relationships between mechanical loading and the biochemical signals that mediate the cellular response. We present a novel in vitro cell shearing device based on the principles of a cone and plate viscometer that utilizes microstepper motor technology to control independently the dynamic and steady components of a hydrodynamic shear-stress environment. Physical measurements of the cone velocity demonstrated faithful reproduction of user-defined input wave forms. Computational modeling of the fluid environment for the unsteady startup confirmed small inertial contributions and negligible secondary flows. Finally, we present experimental results demonstrating the onset rate dependence of functional and structural responses of endothelial cell cultures to dynamically applied shear stress. The controlled cell shearing device is a novel tool for elucidating mechanisms by which mechanical forces give rise to the biological signals that modulate cellular morphology and metabolism.