Regulation of redox and DNA repair genes by arsenic : low dose protection against oxidative stress?

We have evaluated the molecular responses of human epithelial cells to low dose arsenic to ascertain how target cells may respond to physiologically relevant concentrations of arsenic. Data gathered in numerous experiments in different cell types all point to the same conclusion: low dose arsenic induces what appears to be a protective response against subsequent exposure to oxidative stress or DNA damage, whereas higher doses often provoke synergistic toxicity. In particular, exposure to low, sub-toxic doses of arsenite, As(III), causes coordinate up-regulation of multiple redox and redox-related genes including thioredoxin (Trx) and glutathione reductase (GR). Glutathione peroxidase (GPx) is down-regulated in fibroblasts, but up-regulated in keratinocytes, as is glutathione S-transferase (GST). The maximum effect on these redox genes occurs after 24 h exposure to 5–10 mM As(III). This is 10-fold higher than the maximum As(III) concentrations required for induction of DNA repair genes, but within the dose region where DNA repair genes are co-ordinately down-regulated. These changes in gene regulation are brought about in part by changes in DNA binding activity of the transcription factors activating protein-1 (AP-1), nuclear factor kappa-B, and cAMP response element binding protein (CREB). Although sub-acute exposure to micromolar As(III) up-regulates transcription factor binding, chronic exposure to submicromolar As(III) causes persistent down-regulation of this response. Similar long-term exposure to micromolar concentrations of arsenate in drinking water results in a decrease in skin tumour formation in dimethylbenzanthracene (DMBA)/phorbol 12-tetradecanoate 13-acetate (TPA) treated mice. Altered response patterns after long exposure to As(III) may play a significant role in As(III) toxicology in ways that may not be predicted by experimental protocols using short-term exposures.

Activation of the selenopritein SEPS1 gene expression by pro-inflammatory cytokines in Hep G2 cells

SEPS1 (also called selenoprotein S, SelS) plays an important role in the production of inflammatory cytokines and its expression is activated by endoplasmic reticulum (ER) stress. In this report, we have identified two binding sites for the nuclear factor kappa B in the human SEPS1 promoter. SEPS1 gene expression, protein levels and promoter activity were all increased 2–3-fold by TNF-α and IL-1β in HepG2 cells. We have also confirmed that the previously proposed ER stress response element GGATTTCTCCCCCGCCACG in the SEPS1 proximate promoter is fully functional and responsive to ER stress. However, concurrent treatment of HepG2 cells with IL-1β and ER stress produced no additive effect on SEPS1 gene expression. We conclude that SEPS1 is a new target gene of NF-κB. Together with our previous findings that SEPS1 may regulate cytokine production in macrophage cells, we propose a regulatory loop between cytokines and SEPS1 that plays a key role in control of the inflammatory response.

Novel genes in the liver of diabetic Psammomys obesus

Type 2 diabetes mellitus is a metabolic disease characterised by defects in insulin secretion and insulin action and disturbances in carbohydrate, fat and protein metabolism. Hepatic insulin resistance contributes to hyperglycemia and also leads to disturbances in fat metabolism in type 2 diabetes. Psammomys obesus is a unique poly genie animal model of type 2 diabetes and obesity, ideally suited for studies examining physiological and genetic aspects of these diseases. To identify metabolic abnormalities potentially contributing to the obesity and diabetes phenotype in P. obesus, indirect calorimetry was used to characterise whole body energy expenditure and substrate utilisation. Lean-NGT, obese-IGT and obese-diabetic animals were examined in fed and fasted states and following 14 days of dietary energy restriction. Energy expenditure and fat oxidation were elevated in the obese-IGT and obese-diabetic groups in proportion to body weight. Glucose oxidation was not different between groups. Obese-diabetic P. obesus displayed elevated nocturnal blood glucose levels and fat oxidation. Following 14 days of dietary energy restriction, body weight was reduced and plasma insulin and blood glucose levels were normalised in all groups. Glucose oxidation was reduced and fat oxidation was increased. After 24 hours of fasting, plasma insulin and blood glucose levels were normalised in all groups. Energy expenditure and glucose oxidation were greatly reduced and fat oxidation was increased. Following either dietary energy restriction or fasting, energy expenditure, glucose oxidation and fat oxidation were not different between groups of P. obesus. Energy expenditure and whole body substrate utilisation in P. obesus was similar to that seen in humans. P. obesus responded normally to short term fasting and dietary energy restriction. Elevated nocturnal fat oxidation rates and plasma glucose levels in obese-diabetic P. obesus may be an important factor in the pathogenesis of obesity and type 2 diabetes in these animals. These studies have further validated P. obesus as an ideal animal model of type 2 diabetes and obesity. It was hypothesised that many genes in the liver of P. obesus involved in glucose and fat metabolism would be differentially expressed between lean-NGT and obese-diabetic animals. These genes may represent significant factors in the pathophysiology of type 2 diabetes. Two gene discovery experiments were conducted using suppression subtractive hybridisation (SSH) to enrich a cDNA library for differentially expressed genes. Experiment 1 used cDNA dot blots to screen 576 clones with cDNA derived from lean-NGT and obese-diabetic animals. 6 clones were identified as overexpressed in lean-NGT animals and 6 were overexpressed in obese-diabetic animals. These 12 clones were sequenced and SYBR-Green PCR was used to confirm differential gene expression. 4 genes were overexpressed (≥1.5 fold) in lean-NGT animals and 4 genes were overexpressed (≥1.5 fold) in obese-diabetic animals. To explore the physiological role of these genes, hepatic gene expression was examined in several physiological conditions. One gene, encoding thyroxine binding globulin (TBG), was confirmed as overexpressed in lean-NGT P. obesus with ad libitum access to food, relative to both obese-IGT and obese-diabetic animals. TBG expression decreased with fasting in all animals. Fasting TBG expression remained greater in lean-NGT animals than obese-IGT and obese-diabetic animals. TBG expression was not significantly affected by dietary energy restriction. TBG is involved in thyroid metabolism and is potentially involved in the regulation of energy expenditure. Fasting increased hepatic site 1 protease (SIP) expression in lean-NGT animals but was not significantly affected in obese-IGT and obese-diabetic animals. SIP expression was not significantly affected by dietary energy restriction. SIP is involved in the proteolytic processing of steroid response element binding proteins (SREBP). SREBPs are insulin responsive and are known to be involved in lipid metabolism. Gene expression studies found TBG and SIP were associated with obesity and diabetes. Future research will determine whether TBG and SIP are important in the pathogenesis of these diseases. Experiment 2 used SSH and cDNA microarray to screen 8064 clones. 223 clones were identified as overexpressed in lean-NGT P. obesus and 274 clones were overexpressed in obese-diabetic P. obesus (p ≤0.05). The 9 most significantly differentially expressed clones identified from the microarray screen were sequenced (p ≤0.01). 7 novel genes were identified as well as; sulfotransferase related protein and albumin. These 2 genes have not previously been associated with either type 2 diabetes or obesity. It is unclear why hepatic expression of these genes may differ between lean-NGT and obese-diabetic groups of P. obesus. Subsequent studies will explore the potential role of these novel and known genes in the pathophysiology of type 2 diabetes.

Differentiated mTOR but not AMPK signaling after strength vs endurance exercise in training-accustomed individuals

The influence of adenosine mono phosphate (AMP)-activated protein kinase (AMPK) vs Akt-mammalian target of rapamycin C1 (mTORC1) protein signaling mechanisms on converting differentiated exercise into training specific adaptations is not well-established. To investigate this, human subjects were divided into endurance, strength, and non-exercise control groups. Data were obtained before and during post-exercise recovery from single-bout exercise, conducted with an exercise mode to which the exercise subjects were accustomed through 10 weeks of prior training. Blood and muscle samples were analyzed for plasma substrates and hormones and for muscle markers of AMPK and Akt-mTORC1 protein signaling. Increases in plasma glucose, insulin, growth hormone (GH), and insulin-like growth factor (IGF)-1, and in phosphorylated muscle phospho-Akt substrate (PAS) of 160 kDa, mTOR, 70 kDa ribosomal protein S6 kinase, eukaryotic initiation factor 4E, and glycogen synthase kinase 3α were observed after strength exercise. Increased phosphorylation of AMPK, histone deacetylase5 (HDAC5), cAMP response element-binding protein, and acetyl-CoA carboxylase (ACC) was observed after endurance exercise, but not differently from after strength exercise. No changes in protein phosphorylation were observed in non-exercise controls. Endurance training produced an increase in maximal oxygen uptake and a decrease in submaximal exercise heart rate, while strength training produced increases in muscle cross-sectional area and strength. No changes in basal levels of signaling proteins were observed in response to training. The results support that in training-accustomed individuals, mTORC1 signaling is preferentially activated after hypertrophy-inducing exercise, while AMPK signaling is less specific for differentiated exercise.

PGC-1α and PGC-1β increase CrT expression and creatine uptake in myotubes via ERRα

Intramuscular creatine plays a crucial role in maintaining skeletal muscle energy homeostasis, and its entry into the cell is dependent upon the sodium chloride dependent Creatine Transporter (CrT; Slc6a8). CrT activity is regulated by a number of factors including extra- and intracellular creatine concentrations, hormones, changes in sodium concentration, and kinase activity, however very little is known about the regulation of CrT gene expression. The present study aimed to investigate how Creatine Transporter (CrT) gene expression is regulated in skeletal muscle. Within the first intron of the CrT gene, we identified a conserved sequence that includes the motif recognized by the Estrogen-related receptor α (ERRα), also known as an Estrogen-related receptor response element (ERRE). Additional ERREs confirming to the known consensus sequence were also identified in the region upstream of the promoter. When partnered with peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α) or beta (PGC-1β), ERRα induces the expression of many genes important for cellular bioenergetics. We therefore hypothesized that PGC-1 and ERRα could also regulate CrT gene expression and creatine uptake in skeletal muscle. Here we show that adenoviral overexpression of PGC-1α or PGC-1β in L6 myotubes increased CrT mRNA (2.1 and 1.7-fold, P<0.0125) and creatine uptake (1.8 and 1.6-fold, P<0.0125), and this effect was inhibited with co-expression of shRNA for ERRα. Overexpression of a constitutively active ERRα (VP16-ERRα) increased CrT mRNA approximately 8-fold (P<0.05), resulting in a 2.2-fold (P<0.05) increase in creatine uptake. Lastly, chromatin immunoprecipitation assays revealed that PGC-1α and ERRα directly interact with the CrT gene and increase CrT gene expression.

An experimental study of low velocity impact response in 2/2 twill weave composite laminates manufactured by a novel fabrication process

A novel fabrication process for advanced composite components—the QuicktepTM process was described. 2/2 twill weave MTM56/CF0300 carbon epoxy composite laminates were manufactured by the Quickstep and the autoclave processes. The response of these laminates to drop-weight low velocity impact at energy levels ranging from 5 to 30 J was investigated. It was found that the laminates fabricated by the Quickstep had better impact damage tolerance than those fabricated by the autoclave. Optical microscopy revealed extensive matrix fracture in the center of the backside of the autoclave laminates indicating the more brittle property of the epoxy matrix cured by the autoclave process. Interfacial shear strength (IFSS) for two composite systems were measured by micro–debond experiments. The MTM56/CF0300 material cured by the Quickstep showed stronger fibre matrix adhesion. Since the thickness and density of the impact targets produced by two processes were different, finite element analysis (FEA) was performed to study the effect of these factors on the impact response. The simulation results showed that the difference in thickness and density affects the stress distribution under impact loading. Higher thickness and lower density caused by processing lead to less endurance to drop weight impact loading. Therefore the better performance of Quickstep laminates under impact loading was not due to the thickness and density change, but resulted from stronger mechanical properties.

A case study of ground response due to diaphragm wall installation

The purpose of this research is to investigate the ground response due to diaphragm wall construction using three-dimensional numerical modelling. In this study, the commercial finite difference method software, FLAC3D, and the finite element upper and lower bound limit analysis methods are employed. In addition, a range of factors are investigated. They include the dimensions of the single panel, overconsolidation ration (OCR), soil stiffness (E/su), and the height of the bentonite slurry. The solutions from the numerical upper bound limit analysis method are used for comparison purposes. The results obtained indicate that the above factors do have influence on ground response in terms of its stability and displacements. The discussion in the paper can be utilised as the reference for practical designs.

Hybrid neural network—finite element river flow model

Results obtained from a hybrid neural network—finite element model are reported in this paper. The hybrid model incorporates artificial neural network (ANN) nodes into a numerical scheme, which solves the two-dimensional shallow water equations using finite elements (FE). First, numerical computations are carried out on the entire numerical model, using a larger mesh. The results from this computation are then used to train several preselected ANN nodes. The ANN nodes model the response for a part of the entire numerical model by transferring the system reaction to the location where both models are connected in real time. This allows a smaller mesh to be used in the hybrid ANN-FE model, resulting in savings in computation time. The hybrid model was developed for a river application, using the computational nodes located at the open boundaries to be the ANN nodes for the ANN-FE hybrid model. Real-time coupling between the ANN and FE models was achieved, and a reduction is CPU time of more than 25% was obtained.

Variation of yield loci in finite element analysis by considering texture evolution for AA5042 aluminum sheets

 Yield function has various material parameters that describe how materials respond plastically in given conditions. However, a significant number of mechanical tests are required to identify the many material parameters for yield function. In this study, an effective method using crystal plasticity through a virtual experiment is introduced to develop the anisotropic yield function for AA5042. The crystal plasticity approach was used to predict the anisotropic response of the material in order to consider a number of stress or strain modes that would not otherwise be evident through mechanical testing. A rate-independent crystal plasticity model based on a smooth single crystal yield surface, which removes the innate ambiguity problem within the rate-independent model and Taylor model for polycrystalline deformation behavior were employed to predict the material’s response in the balanced biaxial stress, pure shear, and plane strain states to identify the parameters for the anisotropic yield function of AA5042.

Application of the embedded element technique to predict interlaminar failure

In this study a modelling technique, namely, the embedded element method is assessed to evaluate its ability to predict Mode I interlaminar failure. The embedded element technique takes advantage of the embedded constraint in ABAQUS and allows the two constituents, fibre and matrix, to be meshed independently. Since the two constituents can be meshed independently a contiguous mesh is not required and the time taken to create an acceptable mesh is significantly reduced. The embedded element technique has been used to model fibre-reinforced composite structures, however, to date no studies have been conducted which combine the embedded element technique with an interlaminar damage model. The work described herein evaluates the ability of the embedded element technique to predict mode I interlaminar failure. DCB specimens were modelled using the embedded element method and a traditional 3D solid FE modelling approach with the predictions compared against experimental data. Both modelling approaches provided good agreement with experimental results. The good agreement demonstrates that the embedded element technique is capable of providing a response that is equivalent to a traditional 3D solid FE models and is particularly suited to modelling thick composite structures with complex geometry.

Multi-scale simulation using a hybrid mass-spring system and finite element model

Ply-scale finite element (FE) models are widely used to predict the performance of a composite structure based on material properties of individual plies. When simulating damage, these models neglect microscopic fracture processes which may have a significant effect on how a crack progresses within and between plies of a multidirectional laminate. To overcome this resolution limitation a multi-scale modelling technique is employed to simulate the effect micro-scale damage events have on the macro-scale response of a structure. The current paper discusses the development and validation of a hybrid mass-spring system and finite element modelling technique for multi-scale analysis. The model developed here is limited to elastic deformations; however, it is the first key step towards an efficient multi-scale damage model well suited to simulation of fracture in fibre reinforced composite materials. Various load cases have been simulated using the model developed here which show excellent accuracy compared to analytical and FE results. Future work is discussed, including extension of the model to incorporate damage modelling.

Prediction of mode I delamination resistance of z-pinned laminates using the embedded finite element technique

A new finite modelling approach is presented to analyse the mode I delamination fracture toughness of z-pinned laminates using the computationally efficient embedded element technique. In the FE model,each z-pin is represented by a single one-dimensional truss element that is embedded within the laminate. Each truss is given the material, geometric and spatial properties associated with the global crackbridging traction response of a z-pin in the laminate; this simplification provides a computationally efficient and flexible model where pin elements are independent of the underlying structural mesh for thelaminate. The accuracy of the FE modelling approach is assessed using mode I interlaminar fracture toughness data for a carbon-epoxy laminate reinforced with z-pins made of copper, titanium or stainless steel. The model is able to predict with good accuracy the crack growth resistance curves and fracture toughness properties for the different types of z-pinned laminate.