Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

Bovine DNA methylation imprints are established in an oocyte size-specific manner, which are coordinated with the expression of the DNMT3 family proteins

A subset of genes, known as imprinted genes, is present in the mammalian genome. Genomic imprinting governs the monoallelic expression of these genes, depending on whether the gene was inherited from the sperm or the egg. This parent-of-origin specific gene expression is generally dependent on the epigenetic modification, DNA methylation, and the DNA methylation status of CpG dinucleotides residing in loci known as differentially methylated regions (DMRs). The enzymatic machinery responsible for the addition of methyl (-CH(3)) groups to the cytosine residue in the CpG dinucleotides are known as DNA methyltransferases (DNMTs). Correct establishment and maintenance of methylation patterns at imprinted genes has been associated with placental function and regulation of embryonic/fetal development. Much work has been carried out on imprinted genes in mouse and human; however, little is known about the methylation dynamics in the bovine oocyte. The primary objective of the present study was to characterize the establishment of methylation at maternally imprinted genes in bovine growing oocytes and to determine if the expression of the bovine DNMTs-DNMT3A, DNMT3B, and DNMT3L-was coordinated with DNA methylation during oocyte development. To this end, a panel of maternally imprinted genes was selected (SNRPN, MEST, IGF2R, PEG10, and PLAGL1) and putative DMRs for MEST, IGF2R, PEG10, and PLAGL1 were identified within the 5' regions for each gene; the SNRPN DMR has been reported previously. Conventional bisulfite sequencing revealed that methylation marks were acquired at all five DMRs investigated in an oocyte size-dependent fashion. This was confirmed for a selection of genes using pyrosequencing analysis. Furthermore, mRNA expression and protein analysis revealed that DNMT3A, DNMT3B, and DNMT3L are also present in the bovine oocyte during its growth phase. This study demonstrates for the first time that an increase in bovine imprinted gene DMR methylation occurs during oocyte growth, as is observed in mouse.     

Large deformation shear properties of liver tissue

Characterization of the mechanical properties of soft biological tissues is important for establishing the mechanical tolerances of the tissues, and for input to computational models. In this work, the viscoelastic properties of bovine liver tissue in shear loading have been measured using relaxation and constant shear rate loading. The tissue is nonlinearly viscoelastic for strains greater than 0.2%, has a yield strain of approximately 10, and shows moderate strain-rate sensitivity. The response can be modelled using a nonlinear viscoelastic differential model previously developed for brain tissue.     

Contrasting relatedness patterns in bottlenose dolphins (Tursiops sp.) with different alliance strategies

Male bottlenose dolphins (Tursiops sp.) in Shark Bay have one of the most complex male societies outside humans. Two broad mating strategies have been identified in males. In the first strategy, there are two types of alliances: stable 'first-order' pairs and trios that herd individual females in reproductive condition, and 'second-order' teams of two first-order alliances (five or six individuals) that join forces against rivals in contests for females. In the alternative strategy, a 'super-alliance' of ca. 14 individuals, males form pairs or trios to herd females, but in contrast to the stable alliances, these pairs and trios are highly labile. Here, we show that males in stable first-order alliances and the derived second-order alliances are often strongly related, so that they may gain inclusive fitness benefits from alliance membership. By contrast, members of the super-alliance are no more closely related than expected by chance. Further, the strength of the association of alliance partners within the super-alliance, as measured by an index of joint participation in consorting a female, was not correlated with their genetic relatedness. Thus, within one population and one sex, it appears that there may be simultaneous operation of more than one mode of group formation.     

Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method

Rhodococcus equi is a facultative pathogen of foals. Infection causes an often fatal pulmonary pneumonia. The organism has also been isolated from pigs, cattle, humans and the environment. Equine virulence has a high positive correlation with the expression of a 17.4 kD polypeptide of unknown function, VapA, the product of the plasmid-encoded vapA gene. More recently an isogene of vapA, referred to as vapB and encoding an 18.2 kDa polypeptide, has been identified among pig and human isolates. The two genes share > 80% sequence identity, yet their host strains apparently exhibit different pathogenicity profiles (for example by reference to virulence in mouse model system and host specificity). In this study, a polymerase chain reaction (PCR) technique was developed that permits the selective amplification of vapA and vapB. Using this technique the distribution of the two genes among 35 randomly selected isolates of Rhodococcus equi from various animal and environmental sources was determined. Using this technique the genotype of each isolate could be unambiguously assigned as vapA+, vapB+ or vap- (i.e., scoring negative for both vapA and vapB). No isolate scored positive for both vapA and vapB. 100% of equine isolates scored vapA+, confirming the status of vapA as a reliable marker of equine virulence. All three genotypes were found among human isolates; porcine isolates scored either vapB+ or vap- and no vapA+ isolates were present in this sample. Rigorous statistical analysis using the Fisher Exact test confirmed that the high frequency of vapA+ among equine isolates is significant; however the sample size was too small to draw statistically significant conclusions regarding the distribution of genotypes among within other animal groups.     

Mobilization of intracellular calcium and stimulation of calcium fluxes by endothelin-2 in cultured mouse swiss 3T3 fibroblasts

Specific receptor-induced signal transduction mechanisms for the endothelin-2 isoform (ET-2), a potent vasoconstrictor of vascular smooth muscle, were examined in Swiss 3T3 cells. Half-maximal binding (EC₅₀) and maximal, saturable binding (B_max) were estimated from Scatchard analyses and were found to be 24.2 ± 3.3 pM and 56500 ± 1700 sites/cells, respectively. A saturating concentration of ET-2 (100 nM) increased intracellular free calcium (measured by Fura-2 fluorescence) from a resting level of 100 nM to a peak level of 600–800 nM. The initial increase in intracellular free calcium was transitory and was followed by a smaller maintained elevation (250 nM). In the absence of extracellular calcium, ET-2 induced a transitory response equal in size to the peak in the presence of extracellular calcium, but the maintained response was absent. ET-2 increased intracellular free calcium in a concentration-dependent manner with an EC₅₀ of 1 nM. In calcium free solution (2 mM EGTA), ET-2 increased the efflux of ⁴⁵Ca from cells loaded to isotopic equilibrium (3 h) with ⁴⁵Ca. The intracellular second messenger, IP₃, also increased the calcium efflux from saponin permeabilized 3T3 cells loaded with ⁴⁵Ca (pCa 6) in the presence of MgATP. In the presence of extracellular calcium, ET-2 significantly increased calcium uptake into 3T3 cells by 92 ± 36.6 pmoles/million cells/2 min (n = 8). It is suggested that ET-2 binds to specific, high affinity receptors in 3T3 cells and that this receptor interaction increases the intracellular free calcium by IP₃-induced mobilization of calcium from cellular stores and by increasing influx of extracellular calcium.     

Nonlinear viscoelastic constitutive model for bovine liver tissue

Soft tissue mechanical characterisation is important in many areas of medical research. Examples span from surgery training, device design and testing, sudden injury and disease diagnosis. The liver is of particular interest, as it is the most commonly injured organ in frontal and side motor vehicle crashes, and also assessed for inflammation and fibrosis in chronic liver diseases. Hence, an extensive rheological characterisation of liver tissue would contribute to advancements in these areas, which are dependent upon underlying biomechanical models. The aim of this paper is to define a liver constitutive equation that is able to characterise the nonlinear viscoelastic behaviour of liver tissue under a range of deformations and frequencies. The tissue response to large amplitude oscillatory shear (1–50%) under varying preloads (1–20%) and frequencies (0.5–2 Hz) is modelled using viscoelastic-adapted forms of the Mooney–Rivlin, Ogden and exponential models. These models are fit to the data using classical or modified objective norms. The results show that all three models are suitable for capturing the initial nonlinear regime, with the latter two being capable of capturing, simultaneously, the whole deformation range tested. The work presented here provides a comprehensive analysis across several material models and norms, leading to an identifiable constitutive equation that describes the nonlinear viscoelastic behaviour of the liver.

     

Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts

Abstract

Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5–2.5 mM), anti-inflammatory (0.5–5.0 mM) or antiplatelet (0.18–0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0–10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575±3319 vs. 1437±348 ng ml^?1 min^?1, mean±SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo model.     

Distance-based analysis of variance for brain connectivity

The field of neuroimaging dedicated to mapping connections in the brain is increasingly being recognized as key for understanding neurodevelopment and pathology. Networks of these connections are quantitatively represented using complex structures, including matrices, functions, and graphs, which require specialized statistical techniques for estimation and inference about developmental and disorder-related changes. Unfortunately, classical statistical testing procedures are not well suited to high-dimensional testing problems. In the context of global or regional tests for differences in neuroimaging data, traditional analysis of variance (ANOVA) is not directly applicable without first summarizing the data into univariate or low-dimensional features, a process that might mask the salient features of high-dimensional distributions. In this work, we consider a general framework for two-sample testing of complex structures by studying generalized within-group and between-group variances based on distances between complex and potentially high-dimensional observations. We derive an asymptotic approximation to the null distribution of the ANOVA test statistic, and conduct simulation studies with scalar and graph outcomes to study finite sample properties of the test. Finally, we apply our test to our motivating study of structural connectivity in autism spectrum disorder.