In vitro template-change PCR to create single crossover libraries: a case study with B. thuringiensis Cry2A toxins

During evolution the creation of single crossover chimeras between duplicated paralogous genes is a known process for increasing diversity. Comparing the properties of homologously recombined chimeras with one or two crossovers is also an efficient strategy for analyzing relationships between sequence variation and function. However, no well-developed in vitro method has been established to create single-crossover libraries. Here we present an in vitro template-change polymerase change reaction that has been developed to enable the production of such libraries. We applied the method to two closely related toxin genes from B. thuringiensis and created chimeras with differing properties that can help us understand how these toxins are able to differentiate between insect species

ADAM15 mediates upregulation of Claudin-1 expression in breast cancer cells

A Disintegrin and Metalloproteinase-15 (ADAM15) is a transmembrane protein involved in protein ectodomain shedding, cell adhesion and signalling. We previously cloned and characterised alternatively spliced variants of ADAM15 that differ in their intracellular domains and demonstrated correlation of the expression of specific variants with breast cancer prognosis. In this study we have created isogenic cell panels (MDA-MB-231 and MCF-7) expressing five ADAM15 variants including wildtype and catalytically inactive forms. The expression of ADAM15 isoforms in MDA-MB-231 cells led to cell clustering to varying degree, without changes in EMT markers vimentin, slug and E-cadherin. Analysis of tight junction molecules revealed ADAM15 isoform specific, catalytic function dependent upregulation of Claudin-1. The expression of ADAM15A, and to a lesser degree of C and E isoforms led to an increase in Claudin-1 expression in MDA-MB-231 cells, while ADAM15B had no effect. In MCF-7 cells, ADAM15E was the principal variant inducing Claudin-1 expression. Sh-RNA mediated down-regulation of ADAM15 in ADAM15 over-expressing cells reduced Claudin-1 levels. Additionally, downregulation of endogenous ADAM15 expression in T47D cells by shRNA reduced endogenous Claudin-1 expression confirming a role for ADAM15 in regulating Claudin-1 expression. The PI3K/Akt/mTOR pathway was involved in regulating Claudin-1 expression downstream of ADAM15. Immunofluorescence analysis of MDA-MB-231 ADAM15A expressing cells showed Claudin-1 at cell-cell junctions, in the cytoplasm and nuclei. ADAM15 co-localised with Claudin-1 and ZO1 at cell-cell junctions. Immunoprecipitation analysis demonstrated complex formation between ADAM15 and ZO1/ZO2. These findings highlight the importance of ADAM15 Intra Cellular Domain-mediated interactions in regulating substrate selection and breast cancer cell phenotype

Haemodynamics and flow modification stents for peripheral arterial disease:a review

Endovascular stents are widely used for the treatment of peripheral arterial disease (PAD). However, the development of in-stent restenosis and downstream PAD progression remain a challenge. Stent revascularisation of PAD causes arterial trauma and introduces abnormal haemodynamics, which initiate complicated biological processes detrimental to the arterial wall. The interaction between stent struts and arterial cells in contact, and the blood flow field created in a stented region, are highly affected by stent design. Spiral flow is known as a normal physiologic characteristic of arterial circulation and is believed to prevent the development of flow disturbances. This secondary flow motion is lost in atheromatous disease and its re-introduction after endovascular treatment of PAD has been suggested as a method to induce stabilised and coherent haemodynamics. Stent designs able to generate spiral flow may support endothelial function and therefore increase patency rates. This review is focused on secondary flow phenomena in arteries and the development of flow modification stent technologies for the treatment of PAD

Hippocampal state-dependent behavioral reflex to an identical sensory input in rats.

We examined the local field potential of the hippocampus to monitor brain states during a conditional discrimination task, in order to elucidate the relationship between ongoing brain states and a conditioned motor reflex. Five 10-week-old Wistar/ST male rats underwent a serial feature positive conditional discrimination task in eyeblink conditioning using a preceding light stimulus as a conditional cue for reinforced trials. In this task, a 2-s light stimulus signaled that the following 350-ms tone (conditioned stimulus) was reinforced with a co-terminating 100-ms periorbital electrical shock. The interval between the end of conditional cue and the onset of the conditioned stimulus was 4±1 s. The conditioned stimulus was not reinforced when the light was not presented. Animals successfully utilized the light stimulus as a conditional cue to drive differential responses to the identical conditioned stimulus. We found that presentation of the conditional cue elicited hippocampal theta oscillations, which persisted during the interval of conditional cue and the conditioned stimulus. Moreover, expression of the conditioned response to the tone (conditioned stimulus) was correlated with the appearance of theta oscillations immediately before the conditioned stimulus. These data support hippocampal involvement in the network underlying a conditional discrimination task in eyeblink conditioning. They also suggest that the preceding hippocampal activity can determine information processing of the tone stimulus in the cerebellum and its associated circuits

Genome-Wide Diet-Gene Interaction Analyses for Risk of Colorectal Cancer

Dietary factors, including meat, fruits, vegetables and fiber, are associated with colorectal cancer; however, there is limited information as to whether these dietary factors interact with genetic variants to modify risk of colorectal cancer. We tested interactions between these dietary factors and approximately 2.7 million genetic variants for colorectal cancer risk among 9,287 cases and 9,117 controls from ten studies. We used logistic regression to investigate multiplicative gene-diet interactions, as well as our recently developed Cocktail method that involves a screening step based on marginal associations and gene-diet correlations and a testing step for multiplicative interactions, while correcting for multiple testing using weighted hypothesis testing. Per quartile increment in the intake of red and processed meat were associated with statistically significant increased risks of colorectal cancer and vegetable, fruit and fiber intake with lower risks. From the case-control analysis, we detected a significant interaction between rs4143094 (10p14/near GATA3) and processed meat consumption (OR = 1.17; p = 8.7E-09), which was consistently observed across studies (p heterogeneity = 0.78). The risk of colorectal cancer associated with processed meat was increased among individuals with the rs4143094-TG and -TT genotypes (OR = 1.20 and OR = 1.39, respectively) and null among those with the GG genotype (OR = 1.03). Our results identify a novel gene-diet interaction with processed meat for colorectal cancer, highlighting that diet may modify the effect of genetic variants on disease risk, which may have important implications for prevention. © 2014

The use of mesenchymal stem cells for cartilage repair and regeneration: a systematic review.

BACKGROUND: The management of articular cartilage defects presents many clinical challenges due to its avascular, aneural and alymphatic nature. Bone marrow stimulation techniques, such as microfracture, are the most frequently used method in clinical practice however the resulting mixed fibrocartilage tissue which is inferior to native hyaline cartilage. Other methods have shown promise but are far from perfect. There is an unmet need and growing interest in regenerative medicine and tissue engineering to improve the outcome for patients requiring cartilage repair. Many published reviews on cartilage repair only list human clinical trials, underestimating the wealth of basic sciences and animal studies that are precursors to future research. We therefore set out to perform a systematic review of the literature to assess the translation of stem cell therapy to explore what research had been carried out at each of the stages of translation from bench-top (in vitro), animal (pre-clinical) and human studies (clinical) and assemble an evidence-based cascade for the responsible introduction of stem cell therapy for cartilage defects. This review was conducted in accordance to PRISMA guidelines using CINHAL, MEDLINE, EMBASE, Scopus and Web of Knowledge databases from 1st January 1900 to 30th June 2015. In total, there were 2880 studies identified of which 252 studies were included for analysis (100 articles for in vitro studies, 111 studies for animal studies; and 31 studies for human studies). There was a huge variance in cell source in pre-clinical studies both of terms of animal used, location of harvest (fat, marrow, blood or synovium) and allogeneicity. The use of scaffolds, growth factors, number of cell passages and number of cells used was hugely heterogeneous. SHORT CONCLUSIONS: This review offers a comprehensive assessment of the evidence behind the translation of basic science to the clinical practice of cartilage repair. It has revealed a lack of connectivity between the in vitro, pre-clinical and human data and a patchwork quilt of synergistic evidence. Drivers for progress in this space are largely driven by patient demand, surgeon inquisition and a regulatory framework that is learning at the same pace as new developments take place

School Effects on the Wellbeing of Children and Adolescents

Well-being is a multidimensional construct, with psychological, physical and social components. As theoretical basis to help understand this concept and how it relates to school, we propose the Self-Determination Theory, which contends that self-determined motivation and personality integration, growth and well-being are dependent on a healthy balance of three innate psychological needs of autonomy, relatedness and competence. Thus, current indicators involve school effects on children’s well-being, in many diverse modalities which have been explored. Some are described in this chapter, mainly: the importance of peer relationships; the benefits of friendship; the effects of schools in conjunction with some forms of family influence; the school climate in terms of safety and physical ecology; the relevance of the teacher input; the school goal structure and the implementation of cooperative learning. All these parameters have an influence in promoting optimal functioning among children and increasing their well-being by meeting the above mentioned needs. The empirical support for the importance of schools indicates significant small effects, which often translate into important real-life effects as it is admitted at present. The conclusion is that schools do make a difference in children’s peer relationships and well-being

Frequency-stabilization to 6x10^-16 via spectral-hole burning

We demonstrate two-stage laser stabilization based on a combination of Fabry- Perot and spectral-hole burning techniques. The laser is first pre-stabilized by the Fabry-Perot cavity to a fractional-frequency stability of sigma_y(tau) < 10^-13. A pattern of spectral holes written in the absorption spectrum of Eu3+:Y2SiO5 serves to further stabilize the laser to sigma_y(tau) = 6x10^-16 for 2 s < tau < 8 s. Measurements characterizing the frequency sensitivity of Eu3+:Y2SiO5 spectral holes to environmental perturbations suggest that they can be more frequency stable than Fabry-Perot cavities

Incidence of osteochondrosis (dissecans) in Dutch warmblood horses presented for pre-purchase examination

Data are lacking in the literature regarding the incidence of osteochondrosis (dissecans) [OC(D)] in relation to lameness evaluation in Dutch Warmblood horses. The objective of this retrospective study was to assess the incidence of radiological abnormalities consistent with osteochondrosis or osteochondrosis dissecans in 1,231 sound Dutch Warmblood (DW) horses presented for pre-purchase examination. Standardised (Dutch) pre-purchase examination protocols were evaluated. The pre-purchase examination included a clinical, lameness and radiological evaluation, performed at a private equine clinic in the Netherlands. Radiographical examination included views of the distal (DIP) and proximal (PIP) interphalangeal, metacarpo- and metatarsophalangeal (MCP/MTP), tarsocrural (TC) and femoropatellar (FP) joints. Radiographical evidence of OC(D) was found in 44.3% of clinically sound DW horses. In this study, 443 horses (36%, n = 1,231) had evidence of OCD and 102 horses (8.3%, n = 1,231) had evidence of OC on pre-purchase radiographs. The results also indicated that the TC joints were significantly more likely to be affected. A considerable number of horses did not demonstrate any lameness, although radiographs revealed OC(D)

Faecal pharmacokinetics of orally administered vancomycin in patients with suspected Clostridium difficile infection

<p>Abstract</p> <p>Background</p> <p>Oral vancomycin (125 mg qid) is recommended as treatment of severe <it>Clostridium difficile </it>infection (CDI). Higher doses (250 or 500 mg qid) are sometimes recommended for patients with very severe CDI, without supporting clinical evidence. We wished to determine to what extent faecal levels of vancomycin vary according to diarrhoea severity and dosage, and whether it is rational to administer high-dose vancomycin to selected patients.</p> <p>Methods</p> <p>We recruited hospitalized adults suspected to have CDI for whom oral vancomycin (125, 250 or 500 mg qid) had been initiated. Faeces were collected up to 3 times/day and levels were measured with the AxSYM fluorescence polarization immunoassay.</p> <p>Results</p> <p>Fifteen patients (9 with confirmed CDI) were treated with oral vancomycin. Patients with ≥4 stools daily presented lower faecal vancomycin levels than those with a lower frequency. Higher doses of oral vancomycin (250 mg or 500 mg qid) led to consistently higher faecal levels (> 2000 mg/L), which were 3 orders of magnitude higher than the MIC₉₀of vancomycin against <it>C. difficile</it>. One patient receiving 125 mg qid had levels below 50 mg/L during the first day of treatment.</p> <p>Conclusions</p> <p>Faecal levels of vancomycin are proportional to the dosage administered and, even in patients with increased stool frequency, much higher than the MIC₉₀. Patients given the standard 125 mg qid dosage might have low faecal levels during the first day of treatment. A loading dose of 250 mg or 500 mg qid during the first 24-48 hours followed by the standard dosage should be evaluated in larger studies, since it might be less disruptive to the colonic flora and save unnecessary costs.</p