Isolation of epithelial stem cells from dermis by a three-dimensional culture system

Skin is a representative self-renewing tissue containing stem cells. Although many attempts have been made to define and isolate skin-derived stem cells, establishment of a simple and reliable isolation procedure remains a goal to be achieved. Here, we report the isolation of cells having stem cell properties from mouse embryonic skin using a simple selection method based on an assumption that stem cells may grow in an anchorage-independent manner. We inoculated single cell suspensions prepared from mouse embryonic dermis into a temperature-sensitive gel and propagated the resulting colonies in a monolayer culture. The cells named dermis-derived epithelial progenitor-1 (DEEP) showed epithelial morphology and grew rapidly to a more than 200 population doubling level over a period of 250 days. When the cells were kept confluent, they spontaneously formed spheroids and continuously grew even in spheroids. Immunostaining revealed that all of the clones were positive for the expression of cytokeratin-8, -18, -19, and E-cadherin and negative for the expression of cytokeratin-1, -5, -6, -14, -20, vimentin, nestin, a ckit. Furthermore, they expressed epithelial stem cell markers such as p63, integrin beta1, and S100A6. On exposure to TGFbeta in culture, some of DEEP-1 cells expressed alpha-smooth muscle actin. When the cells were transplanted into various organs of adult SCID mice, a part of the inoculated cell population acquired neural, hepatic, and renal cell properties. These results indicate that the cells we isolated were of epithelial stem cell origin and that our new approach is useful for isolation of multipotent stem cells from skin tissues.

A novel three-dimensional culture system for isolation and clonal propagation of neural stem cells using a thermo-reversible gelation polymer

In the present study, we examined the possible utility of a three-dimensional culture system using a thermo-reversible gelation polymer to isolate and expand neural stem cells (NSCs). The polymer is a synthetic biologically inert polymer and gelates at temperatures higher than the gel-sol transition point ( approximately 20 degrees C). When fetal mouse brain cells were inoculated into the gel, spherical colonies were formed ( approximately 1% in primary culture and approximately 9% in passage cultures). The spheroid-forming cells were positive for expression of the NSC markers nestin and Musashi. Under conditions facilitating spontaneous neural differentiation, the spheroid-forming cells expressed genes characteristic to astrocytes, oligodendrocytes, and neurons. The cells could be successively propagated at least to 80 poly-D-lysines over a period of 20 weeks in the gel culture with a growth rate higher than that observed in suspension culture. The spheroids formed by fetal mouse brain cells in the gel were shown to be of clonal origin. These results indicate that the spheroid culture system is a convenient and powerful tool for isolation and clonal expansion of NSCs in vitro.

Effect of corrosion of reinforcement on the mechanical behaviour of highly corroded RC beams

This paper describes an experimental investigation of the behaviour of corroded reinforced concrete beams. These have been stored in a chloride environment for a period of 26 years under service loading so as to be representative of real structural and environmental conditions. The configuration and the widths of the cracks in the two seriously corroded short-span beams were depicted carefully, and then the beams were tested until failure by a three-point loading system. Another two beams of the same age but without corrosion were also tested as control specimens. A short span arrangement was chosen to investigate any effect of a reduction in the area and bond strength of the reinforcement on shear capacity. The relationship of load and deflection was recorded so as to better understand the mechanical behaviour of the corroded beams, together with the slip of the tensile bars. The corrosion maps and the loss of area of the tensile bars were also described after having extracted the corroded bars from the concrete beams. Tensile tests of the main longitudinal bars were also carried out. The residual mechanical behaviour of the beams is discussed in terms of the experimental results and the cracking maps. The results show that the corrosion of the reinforcement in the beams induced by chloride has a very important effect on the mechanical behaviour of the short-span beams, as loss of cross-sectional area and bond strength have a very significant effect on the bending capacity.

Are causal and intervention judgments inextricably linked? A developmental study.

The application of the formal framework of causal Bayesian Networks to children's causal learning provides the motivation to examine the link between judgments about the causal structure of a system, and the ability to make inferences about interventions on components of the system. Three experiments examined whether children are able to make correct inferences about interventions on different causal structures. The first two experiments examined whether children's causal structure and intervention judgments were consistent with one another. In Experiment 1, children aged between 4 and 8years made causal structure judgments on a three-component causal system followed by counterfactual intervention judgments. In Experiment 2, children's causal structure judgments were followed by intervention judgments phrased as future hypotheticals. In Experiment 3, we explicitly told children what the correct causal structure was and asked them to make intervention judgments. The results of the three experiments suggest that the representations that support causal structure judgments do not easily support simple judgments about interventions in children. We discuss our findings in light of strong interventionist claims that the two types of judgments should be closely linked. © 2011 Cognitive Science Society, Inc.


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Simultaneous use of mephedrone and alcohol: A qualitative study of users' experiences

Mephedrone (4-methylmethcathinone) gained popularity across "recreational" drug scenes in the United Kingdom and Ireland during 2009. Although mephedrone was banned in both jurisdictions in 2010, the drug was subsequently sourced through street dealers. This qualitative study explores the simultaneous use of mephedrone and alcohol, among study participants who used mephedrone following legislative controls. Data were collected through semi-structured interviews with male and female respondents. The results suggest a three-tier classification system that describes users' experiences with simultaneous use of mephedrone and alcohol. Most participants engaged in "heavy" alcohol use immediately prior to consuming mephedrone, and then reduced alcohol consumption as the effects of mephedrone were experienced during the drug episode. Spontaneous use of mephedrone often was associated with larger amounts of alcohol being consumed just prior to the mephedrone episode. The findings have the potential for informing socioepidemiological surveys as well as peer interventions to reduce harm associated with
simultaneous drug use.

Temporal and statistical information in causal structure learning

Three experiments examined children’s and adults’ abilities to use statistical and temporal information to distinguish between common cause and causal chain structures. In Experiment 1, participants were provided with conditional probability information and/or temporal information and asked to infer the causal structure of a three-variable mechanical system that operated probabilistically. Participants of all ages preferentially relied on the temporal pattern of events in their inferences, even if this conflicted with statistical information. In Experiments 2 and 3, participants observed a series of interventions on the system, which in these experiments operated deterministically. In Experiment 2, participants found it easier to use temporal pattern information than statistical information provided as a result of interventions. In Experiment 3, in which no temporal pattern information was provided, children from 6-7 years, but not younger children, were able to use intervention information to make causal chain judgments, although they had difficulty when the structure was a common cause. The findings suggest that participants, and children in particular, may find it more difficult to use statistical information than temporal pattern information because of its demands on information processing resources. However, there may also be an inherent preference for temporal information.

Perceptions of collaboration between general practitioners and community pharmacists: Findings from a qualitative study based in Spain

According to the theory of reasoned action (TRA), collaboration is only possible when it is perceived as useful by the participants involved. This paper describes a qualitative study using semi-structured interviews to explore the preceived usefulness of general practitioner (GPs)-community pharmacists (CPs)' collaboration from these professionals' perspectives based in two Spanish regions. Thirty-seven interviews were conducted with GPs and CPs with and without previous experience of collaborating with the other groups of professionals. Analysis of the data indicated that the GPs and CPs considered that collaboration between practitioners and pharmacists to have different forms of usefulness, ranging from positive to negative perceptions of usefulness. Negative and neutral opinions (collaboration generates conflict and/or is not benefitial) could prevent practitioners from initiating collaboration with the other group of professionals, which is explained by the TRA. These perceptions were only found among those participants without experience in collaboration. When collaboration was perceived as advantageous, it could be beneficial on three levels: health system (i.e. provision of integrated care, increased efficiency of the system), GPs and CPs (i.e. increased job satisfaction and patient loyalty) and patients (i.e. improved patient safety). Although GPs and CPs with experience identified benefits using a range of examples, GPs and CPs who had never collaborated also believed that if collaboration was undertaken there would be benefits for the health system, patients and health professionals. These results should be considered when developing strategies to encourage and improve the implementation of collaborative working relationships between GPs and pharmacists in primary care.

Dynamics of interacting Dicke model in a coupled-cavity array

We consider the dynamics of an array of mutually interacting cavities, each containing an ensemble of N two-level atoms. By exploring the possibilities offered by ensembles of various dimensions and a range of atom-light and photon-hopping values, we investigate the generation of multisite entanglement, as well as the performance of excitation transfer across the array, resulting from the competition between on-site nonlinearities of the matter-light interaction and intersite photon hopping. In particular, for a three-cavity interacting system it is observed that the initial excitation in the first cavity completely transfers to the ensemble in the third cavity through the hopping of photons between the adjacent cavities. Probabilities of the transfer of excitation of the cavity modes and ensembles exhibit characteristics of fast and slow oscillations governed by coupling and hopping parameters, respectively. In the large-hopping case, by seeding an initial excitation in the cavity at the center of the array, a tripartite W state, as well as a bipartite maximally entangled state, is obtained, depending on the interaction time. Population of the ensemble in a cavity has a positive impact on the rate of excitation transfer between the ensembles and their local cavity modes. In particular, for ensembles of five to seven atoms, tripartite W states can be produced even when the hopping rate is comparable to the cavity-atom coupling rate. A similar behavior of the transfer of excitation is observed for a four-coupled-cavity system with two initial excitations.

Children's use of interventions to learn causal structure

Children aged between 5 and 8 years freely intervened on a three-variable causal system, with their task being to discover whether it was a common-cause structure or one of two causal chains. From 6-7 years, children were able to use information from their interventions to correctly disambiguate the structure of a causal chain. We used a Bayesian model to examine children’s interventions on the system; this showed that with development children became more efficient in producing the interventions needed to disambiguate the causal structure and that the quality of interventions, as measured by their informativeness, improved developmentally. The latter measure was a significant predictor of children’s correct inferences about the causal structure. A second experiment showed that levels of performance were not reduced in a task in which children did not select and carry out interventions themselves, indicating no advantage for self-directed learning. However, children’s performance was not related to intervention quality in these circumstances, suggesting that children learn in a different way when they carry out interventions themselves.

Characterization of melanoidins from a glucose-glycine model system

The properties of melanoidins prepared from glucose and glycine (GG) were investigated by a three step purification protocol consisting of dialysis, gel filtration at high ionic strength and ion metal affinity chromatography. The high molecular weight fraction obtained in the GG system is responsible for 80% of the total brown colour and its antioxidative ability was about 1/4 of that of Trolox measured by the inhibition of linoleic acid oxidation. GG melanoidins have good affinity towards Cu (II) (32% bound to the resin) while it is much lower towards Pb (II) (10%) and Fe (II) (5%). Capillary zone electrophoresis analysis suggests that GG melanoidins are positively charged, although no signal was observed analysing melanoidins by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF/MS).

Clinical and immunohistochemical assessment of vulval intraepithelial neoplasia following photodynamic therapy using a novel bioadhesive patch-type system loaded with 5-aminolevulinic acid

Background: The work in this study appraised photodynamic treatment (PDT) as a treatment method for vulval intraepithelial neoplasia (VIN) using a novel bioadhesive patch to deliver aminolevulinic acid. An analysis of changes in expression of apoptotic and cell cycle proteins (p53, p21, Mdm2, Blc-2, Bax, Ki-67) in response to PDT was evaluated. Methods: PDT was performed using non-laser light, either as a one or two-cycle treatment, with clinical and pathological assessment following after 6 weeks. Twenty-three patients with 25 VIN lesions underwent 49 cycles of PDT Patches were designed to conform to uneven vulval skin and contained 38 mg cm(-2) aminolevulinic acid. Assessment was carried out at 6 weeks post-treatment. Patient-based treatment assessment, along with clinical and pathological changes, were monitored. Immunohistochemical staining was used to elucidate a possible biomolecular basis for induced cellular changes. Results: Most patients (52%) reported a symptomatic response, with normal pathology restored in 38% of lesions. The patch was easy to apply and remove, causing minimal discomfort. Fluorescence inspection confirmed protoporphyrin accumulation. Pain during implementation of PDT was problematic, necessitating some form of local analgesia. Changes in expression of cell cycle and apoptotic-related proteins suggested involvement of apoptotic pathways. Down regulation of p21 and inverse changes in Bcl-2 and Bax were key findings. Conclusion: Treatment of VIN lesions using a novel bioadhesive patch induced changes in cell cycle and apoptotic proteins in response to PDT with possible utilisation of apoptotic pathways. The efficacy of PDT in treating VIN could be improved by a better understanding of these apoptotic mechanisms, the influence of factors, such as HPV status, and of the need for effective pain management.

Three dimensional number density mapping in the plume of a low temperature laser ablated magnesium plasma

Simultaneous optical absorption and laser-induced fluorescence measurements have been used to map the three-dimensional number densities of ground-state ions and neutrals within a low-temperature KrF laser-produced magnesium plasma expanding into vacuum. Data is reported for the symmetry plane of the plasma, which includes the laser interaction point at a delay of 1 μs after the ∼30 ns KrF laser ablation pulse and for a laser fluence of 2 J cm−2 on target. The number density distributions of ion and neutral species within this plane indicate that two distinct regions exist within the plume; one is a fast component containing ions and neutrals at maximum densities of ∼3×1013 cm−3 and ∼4×1012 cm−3, respectively and the second is a high-density region containing slow neutral species, at densities up to ∼1×1015 cm−3.