281 research outputs found

    The role of dissolved oxygen levels on human Mesenchymal Stem Cell culture success, regulatory compliance and therapeutic potential

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    Most cells in the human body, including human mesenchymal stem cells (hMSCs), have evolved to survive and function in a low, physiological, oxygen (O2) environment. Investigators have become increasingly aware of the effects of O2 levels on hMSC biology and culture and are mimicking the natural niche of these cells in vitro to improve cell culture yields. This presents many challenges in relation to hMSC identity and function and in the maintenance of a controlled O2 environment for cell culture. The aim of this review is to discuss a “hMSC checklist” as a guide to establishing which identity and potency assays to implement when studying hMSCs. The checklist includes markers, differentiation potential, proliferation & growth, attachment & migration, genomic stability and paracrine activity. Evidence drawn from the current literature demonstrates that low O2 environments could improve most “hMSC checklist” attributes. However, there are substantial inconsistencies around both the terminology and the equipment used in low O2 studies. Therefore, “hypoxia” as a term and as a culture condition are discussed. The biology of short (acute) vs long-term (chronic) hypoxia is considered and a nascent hypothesis to explain the behaviour of hMSCs in long-term hypoxia is presented. It is hoped that by establishing an ongoing discourse, and driving towards a regulatory recognisable “hMSC checklist”, we may be better able to provide the patient population with safe and efficacious regenerative treatments

    Genotypic variation in morphology and freezing resistance of Eucalyptus globulus seedlings subjected to drought hardening in nursery

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    Eucalyptus globulus Labill is one of the most planted species in Chile, because of its fast growth and superior pulp qualities. Nevertheless, the incidence of drought and frost damage immediately after planting is frequent. The purpose of this work was to study the effect of drought hardening on frost resistance and on variations in morphological traits that may increase drought resistance at nursery phase in four genotypes of E. globulus Labill. Drought hardening treatments consisted in induced water stress by watering restriction, until pre-dawn stem xylem water potentials (\u3a8pd) reached -0.2, -1.8 and -2.6 MPa. Two water stress-rewatering cycles were applied during 54 days of hardening. Plant and root biomasses were affected by the interaction of drought hardening and genotypes. The rest of morphological and alometrical traits were affected independently by drought or genotype. Plant height, leaf area, specific leaf area (SLA), stem, and leaf biomasses decreased with drought hardening, while collar diameter was not affected. Genotypes responded differentially to drought hardening in plant height, leaf area, SLA, and stem, and leaf biomasses. Ice nucleation temperature (INT), and freezing temperatures (FRT), and 50% freezing damage index of leaves (LT50) were affected by the interaction between drought hardening and genotypes. EG-13, EG-23 and EG-22 genotypes became freezing tolerant with drought hardening (-2.6 MPa). Additionally, EG-14 genotype increased its freezing resistance at -1.8 MPa. Therefore, freezing resistance levels and mechanism depend on genotype and drought hardening treatment. The success in tree breeding by genetic selection should be facilitated by improved understanding of the physiology of stress resistance development and survival during water supply limitations. The knowledge of morphological and freezing resistance dependency on the interaction between genotype and drought hardening may be useful nursery management information to improve plantation success

    Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor

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    The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues

    Changes in morpho-physiological attributes of Eucalyptus globulus plants in response to different drought hardening treatments

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    Morpho-physiological attributes exhibited in response to drought hardening at the end of the growing season of Eucalyptus globulus Labill under nursery conditions were studied to evaluate the effect of three drought hardening treatments in morpho-physiological traits used as suitable indicators of drought hardiness, such as, plant growth, root growth potential, plant water relationships and survival. Freezing resistance of drought hardened plants was also studied in order to evaluate cross hardening effects in cuttings of Eucalyptus globulus Labill. Drought hardening consisted in induced water stress by watering restriction, until plant stem xylem water potentials (\u3a8pd) reached to-0.2, -1.3 and -2.4 MPa. Two water stress-rewatering cycles were applied during 54 days of treatment. The hardening treatments caused a significant reduction in plant height, leaf area, specific leaf area, plant, leaf, stem and root biomass. However, stem diameter was not affected. Root growth potential increased with the exposure to moderate water stress (-1.3 MPa). Drought hardening treatments have not effect on water relationship parameters such as saturation osmotic potential (\u3a8\u3c0sat), volumetric module of elasticity (e), relative water content (RWCtlp) and osmotic potential (\u3a8\u3c0tlp) at the turgor loss point. Only 1.7% and 6% of dehydrated dead plants were observed on treatments at -1.3 and -2.4 MPa respectively. Finally, the freezing damage index of leaves (LT50) was not significantly affected by drought hardening treatments. Furthermore, a reduction of 1.1\ubaC of supercooling capacity was observed at -2.4 MPa. As a conclusion, drought hardening is an important step of plants production programs during the final phase of nursery, because changes in morphological attributes caused by exposure to moderate drought, enable the plants to maintain the balance between transpiration and absorption areas and increase the capacity of plants to generate new roots

    Using the incidence and impact of health conditions in guide dogs to investigate healthy ageing in working dogs

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    This study aimed to use retirement data from working guide dogs to investigate healthy ageing in dogs and the demographic factors that influence ageing. Using a dataset of 7686 dogs spanning 20 years, dogs withdrawn for health reasons before they reached retirement were identified. Cases of retirement for old age, rather than for health reasons, were also recorded, as was the length of working life for all dogs. Specific health reasons were grouped into 14 different health categories. The influence of purebred or crossbreed, breed, and sex on the incidence of these health categories and the length of working life within each health category was considered. The majority (n = 6465/7686; 84%) of working guide dogs were able to function as guide dogs until they had worked for 8.5 years, when they retired. This working life might constitute a reference for the different breeds considered, with the exception of the German shepherd dog, which had a shorter working life. The most common reason for health withdrawals was musculoskeletal conditions (n = 387/1362; 28%), mostly arthritis. Skin conditions (mostly comprised of cases of atopic dermatitis) reduced working life most commonly (mean, approximately 5 years). Nervous sensory conditions (35% of which were cases of epilepsy) reduced working life by 3 years

    Mixing theory for culture and harvest in bioreactors of human mesenchymal stem cells on microcarriers

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    The use of human mesenchymal stem cells (hMSCs) in regenerative medicine is a potential major advance for the treatment of many medical conditions, especially with the use of allogeneic therapies where the cells from a single donor can be used to treat ailments in many patients. Such cells must be grown attached to surfaces and for large scale production, it is shown that stirred bioreactors containing ~200 μm particles (microcarriers) can provide such a surface. It is also shown that the just suspended condition, agitator speed NJS, provides a satisfactory condition for cell growth by minimizing the specific energy dissipation rate, εT, in the bioreactor whilst still meeting the oxygen demand of the cells. For the cells to be used for therapeutic purposes, they must be detached from the microcarriers before being cryopreserved. A strategy based on a short period (~7 min) of very high εT, based on theories of secondary nucleation, is effective at removing >99% cells. Once removed, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. This approach is shown to be successful for culture and detachment in 4 types of stirred bioreactors from 15 mL to 5 L

    Labrador retrievers under primary veterinary care in the UK: demography, mortality and disorders

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    Abstract Background Labrador retrievers are reportedly predisposed to many disorders but accurate prevalence information relating to the general population are lacking. This study aimed to describe demography, mortality and commonly recorded diseases in Labrador retrievers under UK veterinary care. Methods The VetCompass™ programme collects electronic patient record data on dogs attending UK primary-care veterinary practices. Demographic analysis covered all33,320 Labrador retrievers in the VetCompass™ database under veterinary care during 2013 while disorder and mortality data were extracted from a random sample of 2074 (6.2%) of these dogs. Results Of the Labrador retrievers with information available, 15,427 (46.4%) were female and 15,252 (53.6%) were male. Females were more likely to be neutered than males (59.7% versus 54.8%, P <  0.001). The overall mean adult bodyweight was 33.0 kg (SD 6.1). Adult males were heavier (35.2 kg, SD 5.9 kg) than adult females (30.4 kg, SD 5.2 kg) (P <  0.001). The median longevity of Labrador retrievers overall was 12.0 years (IQR 9.9–13.8, range 0.0–16.0). The most common recorded colours were black (44.6%), yellow (27.8%) and liver/chocolate (reported from hereon as chocolate) (23.8%). The median longevity of non-chocolate coloured dogs (n = 139, 12.1 years, IQR 10.2–13.9, range 0.0–16.0) was longer than for chocolate coloured animals (n = 34, 10.7 years, IQR 9.0–12.4, range 3.8–15.5) (P = 0.028). Of a random sample of 2074 (6.2%) Labrador retrievers under care in 2013 that had full disorder data extracted, 1277 (61.6%) had at least one disorder recorded. The total number of dogs who died at any date during the study was 176. The most prevalent disorders recorded were otitis externa (n = 215, prevalence 10.4%, 95% CI: 9.1–11.8), overweight/obesity (183, 8.8%, 95% CI: 7.6–10.1) and degenerative joint disease (115, 5.5%, 95% CI: 4.6–6.6). Overweight/obesity was not statistically significantly associated with neutering in females (8.3% of entire versus 12.5% of neutered, P = 0.065) but was associated with neutering in males (4.1% of entire versus 11.4% of neutered, P < 0.001). The prevalence of otitis externa in black dogs was 12.8%, in yellow dogs it was 17.0% but, in chocolate dogs, it rose to 23.4% (P < 0.001). Similarly, the prevalence of pyo-traumatic dermatitis in black dogs was 1.1%, in yellow dogs it was 1.6% but in chocolate dogs it rose to 4.0% (P = 0.011). Conclusions The current study assists prioritisation of health issues within Labrador retrievers. The most common disorders were overweight/obesity, otitis externa and degenerative joint disease. Males were significantly heavier females. These results can alert prospective owners to potential health issues and inform breed-specific wellness checks

    Gene-environment and protein degradation signatures characterize genomic and phenotypic diversity in wild Caenorhabditis elegans populations

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    Background: Analyzing and understanding the relationship between genotypes and phenotypes is at the heart of genetics. Research on the nematode Caenorhabditis elegans has been instrumental for unraveling genotype-phenotype relations, and has important implications for understanding the biology of mammals, but almost all studies, including forward and reverse genetic screens, are limited by investigations in only one canonical genotype. This hampers the detection and functional analysis of allelic variants, which play a key role in controlling many complex traits. It is therefore essential to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map in wild C. elegans populations. Results: We used multiple wild C. elegans populations freshly isolated from local sites to investigate gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness, and behavioral traits. The genotype, transcriptome, and a number of fitness traits showed a direct link with the original site of the strains. The separation between the isolation sites was prevalent on all chromosomes, but chromosome V was the largest contributor to this variation. These results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. Comparing polymorphic genes between the populations with a set of genes extracted from 19 different studies on gene expression in C. elegans exposed to biotic and abiotic factors, such as bacteria, osmotic pressure, and temperature, revealed a significant enrichment for genes involved in gene-environment interactions and protein degradation. Conclusions: We found that wild C. elegans populations are characterized by gene-environment signatures, and we have unlocked a wealth of genotype-phenotype relations for the first time. Studying natural isolates provides a treasure trove of evidence compared with that unearthed by the current research in C. elegans, which covers only a diminutive part of the myriad of genotype-phenotype relations that are present in the wild
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