Photochemical cross-linking of plastically compressed collagen gel produces an optimal scaffold for corneal tissue engineering

The experiments were designed to use photochemically cross-linked plastically compressed collagen (PCPCC) gel to support corneal epithelial cells. A plastically compressed collagen (PCC) scaffold was photo cross-linked by UVA in the presence of riboflavin to form a biomaterial with optimal mechanical properties. The breaking force, rheology, surgical suture strength, transparency, ultrastructure, and cell-based biocompatibility were compared between PCPCC and PCC gels. The breaking force increased proportionally with an increased concentration of riboflavin. The stress required to reach breaking point of the PCPCC scaffolds was over two times higher compared to the stress necessary to break PCC scaffolds in the presence of 0.1% riboflavin. Rheology results indicated that the structural properties of PCC remain unaltered after UVA cross-linking. The PCC gels were more easily broken than PCPCC gels when sutured on to bovine corneas. The optical density values of PCPCC and PCC showed no significant differences (p > 0.05). SEM analyses showed that the collagen fibres within the PCPCC gels were similar in morphology to PCC gels. No difference in cell-based biocompatibility was seen between the PCPCC and PCC scaffolds in terms of their ability to support the ex vivo expansion of corneal epithelial cells or their subsequent differentiation evidenced by similar levels of cytokeratin 14. In conclusion, PCPCC scaffold is an optimal biomaterial for use in therapeutic tissue engineering of the cornea.

Doubled haploid ramets via embryogenesis of haploid tissue cultures

Tissue culture in the oil palm business is generally concerned with the multiplication (clonal production) of dura, pisifera and tenera palms. These are all normal diploids (2n=2x=36). Sumatra Bioscience has pioneered haploid tissue culture of oil palm (n=x=18). Haploid oil palm is the first step in producing doubled haploid palms which in turn provide parental lines for F1 hybrid production. Chromosome doubling is known to occur during embryogenesis in other haploid cultures, e.g. barley anther culture. Haploid tissue cultures in oil palm were therefore set up to investigate and exploit spontaneous chromosome doubling during embryogenesis. Flow cytometry of embryogenic tissue showed the presence of both haploid (n) and doubled haploid (2n) cells indicating spontaneous doubling. Completely doubled haploid ramets were regenerated suggesting that doubling occurred during the first mitoses of embryogenesis. This is the first report of doubled haploid production in oil palm via haploid tissue culture. The method provides a means of producing a range of doubled haploids in oil palm from the 1,000 plus haploids available at Sumatra Bioscience, in addition the method also produced doubled haploid (and haploid) clones. 1.

Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in commercial-line turkeys

The objective of this study was to determine the concentration of total selenium (Se) and proportions of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in the tissues of female turkeys offered diets containing graded additions of selenized-enriched yeast (SY), or sodium selenite (SS). Oxidative stability and tissue glutathione peroxidase (GSH-Px) activity of breast and thigh muscle were assessed at 0 and 10 days post mortem. A total of 216 female turkey poults were enrolled in the study. A total of 24 birds were euthanized at the start of the study and samples of blood, breast, thigh, heart, liver, kidney and gizzard were collected for determination of total Se. Remaining birds were blocked by live weight and randomly allocated to one of four dietary treatments(n548 birds/treatment) that differed either in Se source (SY v. SS) or dose (Con [0.2 mg/kg total Se], SY-L and SS-L [0.3mg/kg total Se as SY and SS, respectively] and SY-H [0.45mg total Se/kg]). Following 42 and 84 days of treatment 24 birds per treatment were euthanized and samples of blood, breast, thigh, heart, liver, kidney and gizzard were retained for determination of total Se and the proportion of total Se comprised as SeMet or SeCys. Whole blood GSH-Px activity was determined at each time point. Tissue GSH-Px activity and thiobarbituric acid reactive substances were determined in breast and thigh tissue at the end of the study. There were responses (P,0.001) in all tissues to the graded addition of dietary Se, although rates of accumulation were highest in birds offered SY. There were notable differences between tissue types and treatments in the distribution of SeMet and SeCys, and the activity of tissue and erythrocyte GSH-Px (P,0.05). SeCys was the predominant form of Se in visceral tissue and SeMet the predominant form in breast tissue. SeCys contents were greater in thigh when compared with breast tissue. Muscle tissue GSH-Px activities mirrored SeCys contents. Despite treatment differences in tissue GSH-Px activity, there were no effects of treatment on any meat quality parameter.

Effects of dietary polyunsaturated fatty acid supplementation on fatty acid composition in muscle and subcutaneous adipose tissue of lambs

Lambs (n = 48) were used in a 2 × 2 factorial arrangement of treatments to evaluate effects of inclusion of oil containing PUFA in high-concentrate diets (with or without) and duration of oil supplementation (pre- vs. postweaning) on CLA concentration of muscle and adipose tissue. Lambs were fed preweaning creep diets (with or without oil) corresponding to the dietary lactation treatment diet (with or without oil) of the dam. Dams blocked by lambing date and rearing type were randomly assigned to 1 of 2 lactation dietary treatments with or without oil supplementation. Creep diets contained approximately 70% concentrate and 30% roughage and were provided to lambs for ad libitum intake. At weaning (58.7 ± 2.5 d of age), lambs (n = 48) were randomly assigned within preweaning treatment groups to 1 of 2 postweaning dietary treatments (with or without oil) and 16 pens in a randomized block design, blocked by sex and BW. Postweaning diets were formulated to contain approximately 80% concentrate and 20% roughage and were fed once daily for ad libitum intake. Soybean and linseed oil (2:1, respectively) replaced ground corn and provided 3% additional fat in pre- and postweaning diets. Lambs were slaughtered at 60.3 ± 4.2 kg of BW. A subcutaneous fat (SQ) sample was obtained within 1 h postmortem and a LM sample at the 12th rib was obtained 24 h postmortem, and both were analyzed for fatty acid profile. Feedlot performance and carcass measurements were not affected (P ≥ 0.26) by oil supplementation. Total CLA content of LM and SQ was not affected (P ≥ 0.08) by oil supplementation pre- or postweaning, but trans-10, cis-12 CLA was greater (P = 0.02) in SQ from lambs supplemented with oil postweaning. Total PUFA content in LM was greater (P = 0.02) in lambs supplemented with oil pre- or postweaning as a result of increased concentrations of 18:2cis-9, cis-12 and longer chain PUFA. Conversely, pre- and postweaning oil supplementation resulted in less (P = 0.04) MUFA content in LM. Only postweaning oil supplementation increased (P = 0.001) SQ PUFA content. Feeding oils containing PUFA to lambs pre- and postweaning did not increase CLA content of muscle, whereas postweaning oil supplementation minimally increased CLA concentration of SQ fat. Inclusion of soybean and linseed oil in pre- and postweaning diets increased total PUFA content of SQ fat and muscle tissue without adversely affecting growth performance or carcass characteristics.

Production and dissolution rates of earthworm-secreted calcium carbonate

Earthworms secrete granules of calcium carbonate. These are potentially important in soil biogeochemical cycles and are routinely recorded in archaeological studies of Quaternary soils. Production rates of calcium carbonate granules by the earthworm Lumbricus terrestris L. were determined over 27 days in a range of soils with differing chemical properties (pH, organic matter content, water holding capacity, bulk composition, cation exchange capacity and exchangeable cations). Production rate varied between soils, lay in the range 0–0.043 mmolCaCO3 (0–4.3 mg) earthworm−1 d−1 with an average rate of 8 × 10−3 mmolCaCO3 (0.8 mg) earthworm−1 d−1 and was significantly correlated (r = 0.68, P ≤ 0.01) with soil pH. In a second experiment lasting 315 days earthworms repeatedly (over periods of 39–57 days) produced comparable masses of granules. Converting individual earthworm granule production rates into fluxes expressed on per hectare of land per year basis depends heavily on estimates of earthworm numbers. Using values of 10–20 L. terrestris m−2 suggests a rate of 18– 3139 molCaCO3 ha−1 yr−1. Data obtained from flow-through dissolution experiments suggest that at near neutral pH, granule geometric surface areanormalised dissolution rates are similar to those for other biogenic and inorganic calcium carbonate. Fits of the data to the dissolution relationship r = k(1 − ˝)n where r = dissolution rate, k = a rate constant, ˝ = relative saturation and n = the reaction order gave values of k = 1.72 × 10−10 mol cm−2 s−1 and n = 1.8 for the geometric surface area-normalised rates and k = 3.51 × 10−13 mol cm−2 s−1 and n = 1.8 for the BET surface area-normalised rates. In 196 day leaching column experiments trends in granule dissolution rate referenced to soil chemistry corresponded to predictions made by the SLIM model for dissolution of limestone in soil. If soil solution approaches saturation with respect to calcium carbonate granule dissolution will slow or even stop and granules be preserved indefinitely. Granules have the potential to be a small but significant component of the biogeochemical cycling of C and Ca in soil.

Tissue engineering a fetal membrane

The aim of this study was to construct an artificial fetal membrane (FM) by combination of human amniotic epithelial stem cells (hAESCs) and a mechanically enhanced collagen scaffold containing encapsulated human amniotic stromal fibroblasts (hASFs). Such a tissue-engineered FM may have the potential to plug structural defects in the amniotic sac after antenatal interventions, or to prevent preterm premature rupture of the FM. The hAESCs and hASFs were isolated from human fetal amniotic membrane (AM). Magnetic cell sorting was used to enrich the hAESCs by positive ATP-binding cassette G2 selection. We investigated the use of a laminin/fibronectin (1:1)-coated compressed collagen gel as a novel scaffold to support the growth of hAESCs. A type I collagen gel was dehydrated to form a material mimicking the mechanical properties and ultra-structure of human AM. hAESCs successfully adhered to and formed a monolayer upon the biomimetic collagen scaffold. The resulting artificial membrane shared a high degree of similarity in cell morphology, protein expression profiles, and structure to normal fetal AM. This study provides the first line of evidence that a compacted collagen gel containing hASFs could adequately support hAESCs adhesion and differentiation to a degree that is comparable to the normal human fetal AM in terms of structure and maintenance of cell phenotype.

Gut microbiota modulate the metabolism of brown adipose tissue in mice

A two by two experimental study has been designed to determine the effect of gut microbiota on energy metabolism in mouse models. The metabolic phenotype of germ-free (GF, n = 20) and conventional (n = 20) mice was characterized using a NMR spectroscopy-based metabolic profiling approach, with a focus on sexual dimorphism (20 males, 20 females) and energy metabolism in urine, plasma, liver, and brown adipose tissue (BAT). Physiological data of age-matched GF and conventional mice showed that male animals had a higher weight than females in both groups. In addition, conventional males had a significantly higher total body fat content (TBFC) compared to conventional females, whereas this sexual dimorphism disappeared in GF animals (i.e., male GF mice had a TBFC similar to those of conventional and GF females). Profiling of BAT hydrophilic extracts revealed that sexual dimorphism in normal mice was absent in GF animals, which also displayed lower BAT lactate levels and higher levels of (D)-3-hydroxybutyrate in liver, plasma, and BAT, together with lower circulating levels of VLDL. These data indicate that the gut microbiota modulate the lipid metabolism in BAT, as the absence of gut microbiota stimulated both hepatic and BAT lipolysis while inhibiting lipogenesis. We also demonstrated that (1)H NMR metabolic profiles of BAT were excellent predictors of BW and TBFC, indicating the potential of BAT to fight against obesity.

Measurement of paraben concentrations in human breast tissue at serial locations across the breast from axilla to sternum

The concentrations of five esters of p-hydroxybenzoic acid (parabens) were measured using HPLC-MS/MS at four serial locations across the human breast from axilla to sternum using human breast tissue collected from 40 mastectomies for primary breast cancer in England between 2005 and 2008. One or more paraben esters were quantifiable in 158/160 (99%) of the tissue samples and in 96/160 (60%) all five esters were measured. Variation was notable with respect to individual paraben esters, location within one breast and similar locations in different breasts. Overall median values in nanograms per gram tissue for the 160 tissue samples were highest for n-propylparaben [16.8 (range 0–2052.7)] and methylparaben [16.6 (range 0–5102.9)]; levels were lower for n-butylparaben [5.8 (range 0–95.4)], ethylparaben [3.4 (range 0–499.7)] and isobutylparaben 2.1 (range 0–802.9). The overall median value for total paraben was 85.5 ng g−1 tissue (range 0–5134.5). The source of the paraben cannot be identified, but paraben was measured in the 7/40 patients who reported never having used underarm cosmetics in their lifetime. No correlations were found between paraben concentrations and age of patient (37–91 years), length of breast feeding (0–23 months), tumour location or tumour oestrogen receptor content. In view of the disproportionate incidence of breast cancer in the upper outer quadrant, paraben concentrations were compared across the four regions of the breast: n-propylparaben was found at significantly higher levels in the axilla than mid (P = 0.004 Wilcoxon matched pairs) or medial (P = 0.021 Wilcoxon matched pairs) regions (P = 0.010 Friedman ANOVA).

Supranutritional selenium induces alterations in molecular targets related to energy metabolism in skeletal muscle and visceral adipose tissue of pigs

While selenium (Se) is an essential micronutrient for humans, epidemiological studies have raised concern that supranutritional Se intake may increase the risk to develop Type 2 diabetes mellitus (T2DM). We aimed to determine the impact of Se at a dose and source frequently ingested by humans on markers of insulin sensitivity and signalling. Male pigs were fed either a Se-adequate (0.17 mg Se/kg) or a Se-supranutritional (0.50 mg Se/kg; high-Se) diet. After 16 weeks of intervention, fasting plasma insulin and cholesterol levels were non-significantly increased in the high-Se pigs, whereas fasting glucose concentrations did not differ between the two groups. In skeletal muscle of high-Se pigs, glutathione peroxidase activity was increased, gene expression of forkhead box O1 transcription factor and peroxisomal proliferator-activated receptor- coactivator 1 were increased and gene expression of the glycolytic enzyme pyruvate kinase was decreased. In visceral adipose tissue of high-Se pigs, mRNA levels of sterol regulatory element-binding transcription factor 1 were increased, and the phosphorylation of Akt, AMP-activated kinase and mitogen-activated protein kinases was affected. In conclusion, dietary Se oversupply may affect expression and activity of proteins involved in energy metabolism in major insulin target tissues, though this is probably not sufficient to induce diabetes.

Characterisation of connective tissue from the hypertrophic skeletal muscle of myostatin null mice

Myostatin is a potent inhibitor of muscle development. Genetic deletion of myostatin in mice results in muscle mass increase, with muscles often weighing three times their normal values. Contracting muscle transfers tension to skeletal elements through an elaborate connective tissue network. Therefore, the connective tissue of skeletal muscle is an integral component of the contractile apparatus. Here we examine the connective tissue architecture in myostatin null muscle. We show that the hypertrophic muscle has decreased connective tissue content compared with wild-type muscle. Secondly, we show that the hypertrophic muscle fails to show the normal increase in muscle connective tissue content during ageing. Therefore, genetic deletion of myostatin results in an increase in contractile elements but a decrease in connective tissue content. We propose a model based on the contractile profile of muscle fibres that reconciles this apparent incompatible tissue composition phenotype.

Development of an ex vivo human skin model for intradermal vaccination : tissue viability and Langerhans cell behaviour

The presence of resident Langerhans cells (LCs) in the epidermis makes the skin an attractive target for DNA vaccination. However, reliable animal models for cutaneous vaccination studies are limited. We demonstrate an ex vivo human skin model for cutaneous DNA vaccination which can potentially bridge the gap between pre-clinical in vivo animal models and clinical studies. Cutaneous transgene expression was utilised to demonstrate epidermal tissue viability in culture. LC response to the culture environment was monitored by immunohistochemistry. Full-thickness and split-thickness skin remained genetically viable in culture for at least 72 h in both phosphate-buffered saline (PBS) and full organ culture medium (OCM). The epidermis of explants cultured in OCM remained morphologically intact throughout the culture duration. LCs in full-thickness skin exhibited a delayed response (reduction in cell number and increase in cell size) to the culture conditions compared with split-thickness skin, whose response was immediate. In conclusion, excised human skin can be cultured for a minimum of 72 h for analysis of gene expression and immune cell activation. However, the use of split-thickness skin for vaccine formulation studies may not be appropriate because of the nature of the activation. Full-thickness skin explants are a more suitable model to assess cutaneous vaccination ex vivo.

Characterisation of Escherichia fergusonii isolates from farm animals using an Escherichia coli virulence gene array and tissue culture adherence assays

Escherichia fergusonii has been associated with a wide variety of intestinal and extra-intestinal infections in both humans and animals but, despite strong circumstantial evidence, the degree to which the organism is responsible for the pathologies identified remains uncertain. Thirty isolates of E fergusonii collected between 2003 and 2004 were screened using an Escherichia coli virulence gene array to test for the presence of homologous virulence genes in E. fergusonii. The iss (increased serum survival) gene was present in 13/30 (43%) of the test strains and the prfB (P-related fimbriae regulatory) and ireA (siderophore receptor IreA) genes were also detected jointly in 3/30 (10%) strains. No known virulence genes were detected in 14/30 (47%) of strains. Following confirmatory PCR and sequence analysis, the E. fergusonii prfB, iss and ireA genes shared a high degree of sequence similarity to their counterparts in E. coli, and a particular resemblance was noted with the E. coli strain APEC O1 pathogenicity island. In tissue culture adherence assays, nine E. fergusonii isolates associated with HEp-2 cells with a 'localised adherence' or 'diffuse adherence' phenotype, and they proved to be moderately invasive. The E fergusonii isolates in this study possess both some phenotypic and genotypic features linked to known pathotypes of E coli, and support existing evidence that strains of E fergusonii may act as an opportunistic pathogens, although their specific virulence factors may need to be explored. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.

The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue

A LightCycler(R) real-time PCR hybridization probe-based assay that detects a conserved region of the 16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n = 180) and aborted pig foetuses (n = 24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n = 7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n = 30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.

Cryopreservation of winter-dormant apple buds: II - tissue water status after desiccation at -4°c and before further cooling

Abstract The established protocol for the cryopreservation of winter-dormant Malus buds requires that stem explants, containing a single, dormant bud are desiccated at -4°C, for up to 14 days, to reduce their water content to 25-30% of fresh weight. Using three apple cultivars, with known differences in response to cryopreservation, the pattern of evaporative water loss has been characterised, including early freezing events in the bud and cortical tissues that allow further desiccation by water migration to extracellular ice. There were no significant differences between cultivars in this respect or in the proportions of tissue water lost during the desiccation process. Differential Scanning Calorimetry (to -90°C) of intact buds indicated that bud tissues of the cultivar with the poorest response to cryopreservation had the highest residual water content at the end of the desiccation process and froze at the highest temperature Keywords: Malus, cryopreservation, dormant bud, dehydration