Human adipose CD34+CD90+ stem cells and collagen scaffold constructs grafted in vivo fabricate loose connective and adipose tissues

Stem cell based therapies for the repair and regeneration of various tissues are of great interest for a high number of diseases. Adult stem cells, instead, are more available, abundant and harvested with minimally invasive procedures. In particular, mesenchymal stem cells (MSCs) are multi‐potent progenitors, able to differentiate into bone, cartilage, and adipose tissues. Human adult adipose tissue seems to be the most abundant source of MSCs and, due to its easy accessibility; it is able to give a considerable amount of stem cells. In this study, we selected MSCs co‐expressing CD34 and CD90 from adipose tissue. This stem cell population displayed higher proliferative capacity than CD34^?CD90^? cells and was able to differentiate in vitro into adipocytes (PPARγ⁺ and adiponectin⁺) and endothelial cells (CD31⁺VEGF⁺Flk1⁺). In addition, in methylcellulose without VEGF, it formed a vascular network. The aim of this study was to investigate differentiation potential of human adipose CD34⁺/CD90⁺ stem cells loaded onto commercial collagen sponges already used in clinical practice (Gingistat) both in vitro and in vivo. The results of this study clearly demonstrate that human adult adipose and loose connective tissues can be obtained in vivo, highlighting that CD34⁺/CD90 ASCs are extremely useful for regenerative medicine. J. Cell. Biochem. 114: 1039–1049, 2013. © 2012 Wiley Periodicals, Inc.     

Taurocholic acid feeding prevents tumor necrosis factor-alpha-induced damage of cholangiocytes by a PI3K-mediated pathway

Cholangiopathies, such as primary biliary cirrhosis and primary sclerosis cholangitis, are characterized at the end stage by ductopenia due to increased cholangiocyte apoptosis and decreased cholangiocyte proliferation. Although cholangiocyte proliferation is associated with an increased number of intra-hepatic bile ducts and secretin-stimulated ductal secretion, ductopenia is coupled with decreased ductal mass and secretin-induced ductal secretory activity. We have shown that a single injection of actinomycin D + tumor necrosis factor-alpha (TNF-alpha ) to bile duct-ligated (BDL) rats induces cholangiocyte injury, which is characterized by loss of cholangiocyte proliferation, and secretory activity and by an increase in cholangiocyte apoptosis. We also have shown that taurocholic acid both in vivo and in vitro stimulates cholangiocyte proliferation. We hypothesize that taurocholic acid feeding protects cholangiocytes against TNF-alpha -induced apoptosis through a phosphatidylinositol-3-kinase (PI3K)-dependent pathway. Immediately after BDL, rats were fed taurocholic acid or control diet in the absence/presence of daily injections of wortmannin for 1 week. Seven days later, control-fed or taurocholic acid-fed rats were treated with a single intraperitoneal injection of actinomycin D + TNF-alpha . Twenty-four hours later we evaluated: (i) cholangiocyte apoptosis and proliferation in liver sections and (ii) basal and secretin-stimulated bile and bicarbonate secretion in bile fistula rats. Taurocholic acid feeding prevented TNF-alpha -induced increases in cholangiocyte apoptosis and decreases in growth and secretin-stimulated bile and bicarbonate secretion, changes that were blocked by PI3K inhibition. The PI3K survival pathway is important in bile acid protection against immune-mediated cholangiocyte injury in cholestatic liver diseases.     

An innovative approach to molecularly imprinted capillaries for polar templates by grafting polymerization

Molecularly imprinted polymers have been successfully used as selective stationary phases in capillary electrophoresis. Notwithstanding, this technique suffers from several drawbacks as the loss of molecular recognition properties in aqueous media and the lack of feasibility for imprinted systems directed towards highly polar templates soluble in aqueous environments only. Thus, the preparation of imprinted polymers for highly polar, water-soluble analytes, represents a challenge. In this work, we present an innovative approach to overcome these drawbacks. It is based on a surface molecular imprinting technique that uses preformed macromonomers as both functional recognition elements and cross-linking agents. A poly-2-hydroxyethyl-co-methacrylic acid linear polymer was grafted from the surface of silica capillaries. The grafted polymer was exhaustively esterified with methacrylic anhydride to obtain polyethylendimethacrylate-co-methacrylic acid linear chains. Then, as a proof of concept, an adequate amount of a very polar template like penicillin V was added in a hydro-organic mixture, and a thin layer of imprinted polymer was obtained by cross-linking the polymer linear chains. The binding behaviour of the imprinted and non-imprinted capillaries was evaluated in different separation conditions in order to assess the presence of template selectivity and molecular recognition effects. The experimental results clearly show that this innovative kind of imprinted material can be easily obtained in very polar polymerization environments and that it is characterized by enhanced molecular recognition properties in aqueous buffers and good selectivity towards the template and strictly related molecules.     

Comparative study on metal homeostasis and detoxification in two Antarctic teleosts

The main characteristic of Antarctic seawater is its low constant temperature and its high concentration of O(2), which can increase the formation rate of reactive oxygen species (ROS), together with a natural occurrence of elevated cadmium and copper levels. In the present paper, we studied the presence of cadmium, copper and zinc, metallothioneins (MTs) and glutathione (GSH), and antioxidant enzyme activities in the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi, in order to determine the influence of the peculiar physico-chemical features of the Antarctic marine environment on these physiological defence systems in two species of teleosts. In both of them, cadmium and copper accumulation occurs mostly in the liver. T. bernacchii accumulates zinc mostly in the hepatic tissue, whereas T. newnesi does not show a preferential accumulation site. In addition to the intra-specific analysis, we decided to compare the two species of the Trematomus genus in order to verify if the different feeding habits and motility of these fish affects metal accumulation. Our results show that the liver of T. bernacchii accumulates cadmium and zinc at a higher extent with respect to T. newnesi. Glutathione (GSH) and metallothioneins (MTs) are present in great quantity in the liver of both species. Moreover liver is the tissue which generally showed the highest antioxidant enzyme levels. The results provide further insights in the physiological mechanisms evolved by animals living in this extreme environment.     

A restricted spectrum of mutations in the SMAD4 tumor-suppressor gene underlies Myhre syndrome

Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor β and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth.     

Dynamics and Flexibility of Human Aromatase Probed by FTIR and Time Resolved Fluorescence Spectroscopy

Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k₁) for β-sheets from 0.22±0.06 min⁻¹ for the inhibitor-bound enzyme to 0.12±0.02 min⁻¹ for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.     

Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation

Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation.

Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets.

Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis.

Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation.     

Nonparametric confidence regions for the symmetry point-based optimal cutpoint and associated sensitivity of a continuous-scale diagnostic test

In medical research, diagnostic tests with continuous values are widely employed to attempt to distinguish between diseased and non-diseased subjects. The diagnostic accuracy of a test (or a biomarker) can be assessed by using the receiver operating characteristic (ROC) curve of the test. To summarize the ROC curve and primarily to determine an “optimal” threshold for test results to use in practice, several approaches may be considered, such as those based on the Youden index, on the so-called close-to-(0,1) point, on the concordance probability and on the symmetry point. In this paper, we focus on the symmetry point-based approach, that simultaneously controls the probabilities of the two types of correct classifications (healthy as healthy and diseased as diseased), and show how to get joint nonparametric confidence regions for the corresponding optimal cutpoint and the associated sensitivity (= specificity) value. Extensive simulation experiments are conducted to evaluate the finite sample performances of the proposed method. Real datasets are also used to illustrate its application.     

Attractiveness of year-old polyethylene Isonet sex pheromone dispensers for Lobesia botrana

In this study, we have compared the release of sex pheromone from mating disruption dispensers exposed in the field for 12 months and from calling females. The main pheromone component of the grapevine moth, Lobesia botrana (D. and S.) (Lepidoptera: Tortricidae), is (E)‐7,(Z)‐9‐dodecadienyl acetate, and a minor component is (Z)‐9‐dodecenyl acetate. Aged dispensers from two different years emitted a much higher amount of both pheromone components than calling females. However, the summer temperature during field exposure influenced the release from mating disruption dispensers the following year. In the wind tunnel, male L. botrana were equally attracted to 12‐month, field‐exposed dispensers, a standard monitoring pheromone lure, and to synthetic (E)‐7,(Z)‐9‐dodecadienyl acetate sprayed at the rate of 0.6–60 ng h^?1. Field trapping tests confirmed that aged dispensers from both years were at least as attractive to L. botrana males as a standard monitoring pheromone lure. The possible contribution of previously applied dispensers to the mating disruption efficacy during following applications is discussed.