Cell Behaviour in Chick Blastoderm Expansion

The behaviour of the leading edge cells during the early stages of epiboly (stage 3 to 16, Hamburger and Hamilton, 1951) has been investigated in this study using light, transmission and scanning electron microscopy. The results showed a consistency in the distribution of junctions between these cells throughout these stages. However, there is a significant increase in both the width of the leading edge and the number of its cells around the second day of incubation (stage 11-13). The significance of this increase is discussed in relation to the reported increase in the rate of expansion of the blastoderm at around that time (Downie, 1976). As the leading edge migrates centrifugally over the vitelline membrane, it requires the addition of new cells to cover an increasingly wider area. This study provides evidence that cells recruited into the edge are the deep layer cells which are found on the basal lamina of the peripheral part of the blastoderm and not the ectodermal cells as is generally thought. The opposite task faces the leading edge as it passes the equator of the egg and occupies a smaller area of vitelline membrane, that is how to accommodate the large number of edge cells into a smaller space? The current study provides the first account of the unique strategy that the chick blastoderm uses to achieve this task. Some of the leading edge cells become stationary at points around the circumference of the edge. This allows just enough cells to continue their movement to cover the rest of the vitelline membrane and in turn the whole yolk. As more cells are added to the original stationary cells and become stationary themselves, long stationary streaks develop, giving the blastoderm edge a crescent-shaped appearance. The difference in the end of epiboly between the chick blastoderm and other embryos with similar systems is discussed in this study. The study also reports for the first time that cell death occurs in the leading edge cells during the late stages of development. This occurs in the oldest stationary cells then progresses centrifugally to the younger ones. As the stationary edge cells die, they are replaced by the nearby ectodermal cells which attach strongly to the vitelline membrane - an unusual behaviour for ectodermal cells which never attach to this membrane during the earlier stages of epiboly. The time and position of appearance of stationary points was found to be unrelated to the stage of development. The length of the stationary streaks and their complexity was found also to be different from one blastoderm to another even in blastoderms which have been incubated for the same length of time. Another peculiar finding of this study is that the extent of blastoderm expansion and the time it seals the vitelline membrane is also different from one embryo to another at any particular time of development. Indirect immunofluorescent staining of pieces of blastoderm edges cultured on glass confirmed previous reports that the shape of the epithelial cells does not depend on the integrity of an intact microtubule system and that microfilaments are the essential elements in maintaining the shape of the leading edge cells. Immunofluorescent staining for fibronectin distribution in the blastoderm edge showed it to be present in the basal lamina, and within a few attached edge cells during the first 2 days of expansion, but not between edge cells, or at the edge cell-vitelline membrane interface. However, extracellular fibronectin was detected in the stationary streaks after 3 days. These results differ from previous reports, and reasons for these differences are discussed. Experiments to study the behaviour of blastoderm cells lacking an edge on the inner surface of the vitelline membrane revealed that the blastoderm has the ability to form a regenerated edge which moves rapidly on the substratum. Cultures of blastoderm pieces with or without the leading edge on the outer surface of the vitelline membrane showed that these cells were unable to expand on this substratum. The reasons behind this behaviour are discussed

Synthèse d'époxyphosphonates alpha-chlorés, intermédiaires potentiels dans la préparation de l'analogue phosphoré du KDO, cible privilégiée dans la recherche de nouveaux antibiotiques

Les époxyphosphonatesex-chlorés ont été préparés par action de l'acide métachloroperbenzoïque (mCPBA) sur les vinylphosphonates ex- chlorés. L'isomérisation des époxyphosphonates est en cours d'étude. Les résultats de ces travaux seront appliqués au mannose pour la préparation du KDO phosphoré

Prodrug converting enzyme gene delivery by L. monocytogenes

<p>Abstract</p> <p>Background</p> <p><it>Listeria monocytogenes </it>is a highly versatile bacterial carrier system for introducing protein, DNA and RNA into mammalian cells. The delivery of tumor antigens with the help of this carrier into tumor-bearing animals has been successfully carried out previously and it was recently reported that <it>L. monocytogenes </it>is able to colonize and replicate within solid tumors after local or even systemic injection.</p> <p>Methods</p> <p>Here we report on the delivery of two prodrug converting enzymes, purine-deoxynucleoside phosphorylase (PNP) and a fusion protein consisting of yeast cytosine deaminase and uracil phosphoribosyl transferase (FCU1) into cancer cells in culture by <it>L. monocytogenes</it>. Transfer of the prodrug converting enzymes was achieved by bacterium mediated transfer of eukaryotic expression plasmids or by secretion of the proteins directly into the host cell cytosol by the infecting bacteria.</p> <p>Results</p> <p>The results indicate that conversion of appropriate prodrugs to toxic drugs in the cancer cells occured after both procedures although <it>L. monocytogenes</it>-mediated bactofection proved to be more efficient than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging the constitutively P_CMV-promoter with the melanoma specific P_4xTETP-promoter resulted in melanoma cell-specific expression of the prodrug converting enzymes but reduced the efficiencies.</p> <p>Conclusion</p> <p>These experiments open the way for bacterium mediated tumor specific activation of prodrugs in live animals with tumors.</p

Effects of fluoxetine on functional outcomes after acute stroke (FOCUS): a pragmatic, double-blind, randomised, controlled trial

Background Results of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects. Methods FOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762. Findings Between Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months. Interpretation Fluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function. Funding UK Stroke Association and NIHR Health Technology Assessment Programme