Collagen-hyaluronic acid scaffolds for adipose tissue engineering.

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen-HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.This was supported by the Biotechnology and Biological Sciences Research Council

Energy density of foods and beverages in the Australian food supply: influence of macronutrients and comparison to dietary intake.

OBJECTIVES: The energy density (ED) of the diet is considered an important determinant of total energy intake and thus energy balance and weight change. We aimed to compare relationships between ED and macronutrient content in individual food and beverage items as well as population diet in a typical Western country. DESIGN: Nutrient data for 3673 food items and 247 beverage items came from the Australian Food and Nutrient database (AusNut). Food and beverage intake data came from the 1995 Australian National Nutrition Survey (a 24-h dietary recall survey in 13 858 people over the age of 2). Relationships between ED and macronutrient and water content were analysed by linear regression with 95% prediction bands. RESULTS: For both individual food items and population food intake, there was a positive relationship between ED and percent energy as fat and negative relationships between ED and percent energy as carbohydrate and percent water by weight. In all cases, there was close agreement between the slopes of the regression lines between food items and dietary intake. There were no clear relationships between ED and macronutrient content for beverage items. Carbohydrate (mostly sucrose) contributed 91, 47, and 25% of total energy for sugar-based, fat-based, and alcohol-based beverages respectively. CONCLUSIONS: The relationship between ED and fat content of foods holds true across both population diets and individual food items available in the food supply in a typical Western country such as Australia. As high-fat diets are associated with a high BMI, population measures with an overall aim of reducing the ED of diets may be effective in mediating the growing problem of overweight and obesity

An assessment of diet overlap of two mesocarnivores in the North-West Province, South Africa

We used scat analysis to study the diet of two sympatric medium-sized carnivores: brown hyaena and black-backed jackal, in the NorthWest Province of South Africa. Seven major dietary categories were identified from the scats, with mammal remains being most common for both species. Brown hyaena scats contained more large mammal remains, which together with the presence of invertebrates (in 50% of all brown hyaena scats), suggests that they mainly scavenged. Jackal scats contained a higher proportion of small mammal remains, suggesting that jackals actively hunted more often than brown hyaenas did. The diets differed significantly between the two species, even though diet overlap was fairly high (0.79). Further analysis, albeit based on small sample sizes, suggests that diet of these mesopredators differ between protected reserves with apex predators and unprotected areas without apex predators, thus confounding generalizations. Further studies are therefore required to investigate possible mesopredator release when apex predators are absent

sFlt-1 and NTproBNP independently predict mortality in a cohort of heart failure patients.

Objective: Soluble fms-like tyrosine kinase-1 (sFlt-1) is a circulating receptor for VEGF-A. Recent reports of elevated plasma levels of sFlt-1 in coronary heart disease and heart failure (HF) motivated our study aimed at investigating the utility of sFlt-1 as a prognostic biomarker in heart failure patients. Methods: ELISA assays for sFlt-1 and NTproBNP were performed in n=858 patients from a prospective multicentre, observational study (the PEOPLE study) of outcome among patients after appropriate treatment for an episode of acute decompensated HF in New Zealand. Plasma was sampled at a baseline visit and stored at -80°C. Statistical tests were adjusted for patient age at baseline visit, skewed data were log-adjusted and the endpoint for clinical outcome analysis was all-cause death. Patients were followed for a median of 3.63 (range 0.74-5.50) years. Results: Mean baseline plasma sFlt-1 was 125 +/- 2.01 pg/ml. sFlt-1 was higher in patients with HF with reduced ejection fraction (HFrEF) (130 +/- 2.62 pg/ml, n=553) compared to those with HF with preserved EF (HFpEF) (117 +/-3.59 pg/ml, n=305; p=0.005). sFlt-1 correlated with heart rate (r=0.148, p<0.001), systolic blood pressure (r=-0.139, p<0.001) and LVEF (r=-0.088, p=0.019). A Cox proportional hazards model showed sFlt-1 was a predictor of all-cause death (HR=6.30, p<0.001) in the PEOPLE cohort independent of age, NTproBNP, ischaemic aetiology, and NYHA class (n=842, 274 deaths), established predictors of mortality in the PEOPLE cohort. Conclusion: sFlt-1 levels at baseline should be investigated further as a predictor of death; complementary to established prognostic biomarkers in heart failure

Development of three-dimensional collagen scaffolds with controlled architecture for cell migration studies using breast cancer cell lines

Cancer is characterized by cell heterogeneity and the development of 3D $\textit{in vitro }$ assays that can distinguish more invasive or migratory phenotypes could enhance diagnosis or drug discovery. 3D collagen scaffolds have been used to develop analogues of complex tissues $\textit{in vitro }$ and are suited to routine biochemical and immunological assays. We sought to increase 3D model tractability and modulate the migration rate of seeded cells using an ice-templating technique to create either directional/anisotropic or non-directional/isotropic porous architectures within cross-linked collagen scaffolds. Anisotropic scaffolds supported the enhanced migration of an invasive breast cancer cell line MDA-MB-231 with an altered spatial distribution of proliferative cells in contrast to invasive MDA-MB-468 and non-invasive MCF-7 cells lines. In addition, MDA-MB-468 showed increased migration upon epithelial-to-mesenchymal transition (EMT) in anisotropic scaffolds. The provision of controlled architecture in this system may act both to increase assay robustness and as a tuneable parameter to capture detection of a migrated population within a set time, with consequences for primary tumour migration analysis. The separation of invasive clones from a cancer biomass with in vitro platforms could enhance drug development and diagnosis testing by contributing assay metrics including migration rate, as well as modelling cell-cell and cell-matrix interaction in a system compatible with routine histopathological testing.The authors gratefully acknowledge the financial support of the ERC Advanced Grant 320598 3D-E and the Newton Trust. A.H. held a Daphne Jackson Fellowship funded by the University of Cambridge for part of the work. R.D.H. is funded through a NC3Rs studentship

Effects of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR.

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C(T)) values were established for beta-actin, beta2-microglobulin (beta2M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C(T) values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C(T) values and the linear value of 2(-C(T)), respectively. Interassay variability was 2.3% for raw C(T) values and 34% for the linear value of 2(-C(T)). We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P &lt; 0.05) muscle total creatine content above placebo levels; however, there were no changes (P &gt; 0.05) in C(T) values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas beta-actin, beta2M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for beta2M with more stable expressions for both beta-actin and CYC. We conclude that, using real-time RT-PCR, beta-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise

Soy protein ingestion results in less prolonged p70S6 kinase phosphorylation compared to whey protein after resistance exercise in older men

Background The phosphorylation of p70S6 Kinase (p70S6K) is an important step in the initiation of protein translation. p70S6K phosphorylation is enhanced with graded intakes of whey protein after resistance exercise. Soy protein ingestion results in lower muscle protein synthesis after exercise compared with whey; however, the underlying mechanisms responsible for this difference have not been reported. Findings 13 older men (60–75) completed an acute bout of lower body resistance exercise and ingested 30 g of soy protein or carbohydrate. Muscle biopsies were obtained in the rested and fasted state and 2 and 4 hours post exercise. Phosphorylation status of p70S6K was measured with western blot. Results were compared with previously reported data from the ingestion of 30 g of whey protein or placebo. p70S6K phosphorylation was increased 2, but not 4 hours post exercise with soy protein ingestion. p70S6K phosphorylation was not increased post exercise with carbohydrate ingestion. Conclusions Ingesting 30 g of either whey or soy protein resulted in equivalent p70S6K phosphorylation at 2 hours post exercise, however, unlike whey, soy protein failed to promote prolonged phosphorylation of p70S6K to 4 hours post-exercise

Active immunization with myelin-derived altered peptide ligand reduces mechanical pain hypersensitivity following peripheral nerve injury

BACKGROUND: T cells have been implicated in neuropathic pain that is caused by peripheral nerve injury. Immunogenic myelin basic protein (MBP) peptides have been shown to initiate mechanical allodynia in a T cell-dependent manner. Antagonistic altered peptide ligands (APLs) are peptides with substitutions in amino acid residues at T cell receptor contact sites and can inhibit T cell function and modulate inflammatory responses. In the present study, we studied the effects of immunization with MBP-derived APL on pain behavior and neuroinflammation in an animal model of peripheral nerve injury. METHODS: Lewis rats were immunized subcutaneously at the base of the tail with either a weakly encephalitogenic peptide of MBP (cyclo-MBP(87-99)) or APL (cyclo-(87-99)[A(91),A(96)]MBP(87-99)) in complete Freund’s adjuvant (CFA) or CFA only (control), following chronic constriction injury (CCI) of the left sciatic nerve. Pain hypersensitivity was tested by measurements of paw withdrawal threshold to mechanical stimuli, regulatory T cells in spleen and lymph nodes were analyzed by flow cytometry, and immune cell infiltration into the nervous system was assessed by immunohistochemistry (days 10 and 30 post-CCI). Cytokines were measured in serum and nervous tissue of nerve-injured rats (day 10 post-CCI). RESULTS: Rats immunized with the APL cyclo-(87-99)[A(91),A(96)]MBP(87-99) had significantly reduced mechanical pain hypersensitivity in the ipsilateral hindpaw compared to cyclo-MBP(87-99)-treated and control rats. This was associated with significantly decreased infiltration of T cells and ED1+ macrophages in the injured nerve of APL-treated animals. The percentage of anti-inflammatory (M2) macrophages was significantly upregulated in the APL-treated rats on day 30 post-CCI. Compared to the control rats, microglial activation in the ipsilateral lumbar spinal cord was significantly increased in the MBP-treated rats, but was not altered in the rats immunized with the MBP-derived APL. In addition, immunization with the APL significantly increased splenic regulatory T cells. Several cytokines were significantly altered after CCI, but no significant difference was observed between the APL-treated and control rats. CONCLUSIONS: These results suggest that immune deviation by active immunization with a non-encephalitogenic MBP-derived APL mediates an analgesic effect in animals with peripheral nerve injury. Thus, T cell immunomodulation warrants further investigation as a possible therapeutic strategy for the treatment of peripheral neuropathic pain

Dose-dependent increases in p70S6K phosphorylation and intramuscular branched-chain amino acids in older men following resistance exercise and protein intake.

Resistance exercise and whey protein supplementation are effective strategies to activate muscle cell anabolic signaling and ultimately promote increases in muscle mass and strength. In the current study, 46 healthy older men aged 60&ndash;75 (69.0 &plusmn; 0.55 years, 85.9 &plusmn; 1.8 kg, 176.8 &plusmn; 1.0 cm) performed a single bout of unaccustomed lower body resistance exercise immediately followed by ingestion of a noncaloric placebo beverage or supplement containing 10, 20, 30, or 40 g of whey protein concentrate (WPC). Intramuscular amino acid levels in muscle biopsy samples were measured by Gas Chromatography&ndash;Mass Spectrometry (GC-MS) at baseline (before exercise and WPC supplementation) plus at 2 h and 4 h post exercise. Additionally, the extent of p70S6K phosphorylation at Thr389 in muscle biopsy homogenates was assessed by western blot. Resistance exercise alone reduced intramuscular branch chain amino acid (BCAA; leucine, isoleucine, and valine) content. Supplementation with increasing doses of whey protein prevented this fall in muscle BCAAs during postexercise recovery and larger doses (30 g and 40 g) significantly augmented postexercise muscle BCAA content above that observed following placebo ingestion. Additionally, the fold change in the phosphorylation of p70S6K (Thr389) at 2 h post exercise was correlated with the dose of whey protein consumed (r = 0.51, P &lt; 001) and was found to be significantly correlated with intramuscular leucine content (r = 0.32, P = 0.026). Intramuscular BCAAs, and leucine in particular, appear to be important regulators of anabolic signaling in aged human muscle during postexercise recovery via reversal of exercise-induced declines in intramuscular BCAAs