Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process

Drosophila Bruce Can Potently Suppress Rpr- and Grim-Dependent but Not Hid-Dependent Cell Death

Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain (E2) 1, 2. BRUCE upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs [2]. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Here, we demonstrate, using gain-of-function and deletion alleles, that Drosophila Bruce (dBruce) can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The dBruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. dBruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1 BIR2, be intact. dBruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that dBruce can regulate cell death at a novel point

Sticky/Citron kinase maintains proper RhoA localization at the cleavage site during cytokinesis

In many organisms, the small guanosine triphosphatase RhoA controls assembly and contraction of the actomyosin ring during cytokinesis by activating different effectors. Although the role of some RhoA effectors like formins and Rho kinase is reasonably understood, the functions of another putative effector, Citron kinase (CIT-K), are still debated. In this paper, we show that, contrary to previous models, the Drosophila melanogaster CIT-K orthologue Sticky (Sti) does not require interaction with RhoA to localize to the cleavage site. Instead, RhoA fails to form a compact ring in late cytokinesis after Sti depletion, and this function requires Sti kinase activity. Moreover, we found that the Sti Citron-Nik1 homology domain interacts with RhoA regardless of its status, indicating that Sti is not a canonical RhoA effector. Finally, Sti depletion caused an increase of phosphorylated myosin regulatory light chain at the cleavage site in late cytokinesis. We propose that Sti/CIT-K maintains correct RhoA localization at the cleavage site, which is necessary for proper RhoA activity and contractile ring dynamics

In-vitro growth characteristics of commercial probiotic strains and their potential for inhibition of Clostridium difficile and Clostridium perfringens

The effect of catheter material on intravenous catheterisation complications in horses are unknown. This study evaluated the presence of bacterial colonisation on Teflon® and polyurethane short term intravenous catheters in healthy adult horses undergoing elective surgery. Horses on admission for elective surgery were randomly allocated according to catheter type. Sixteen horses received Teflon® catheters and 19 received polyurethane. Aseptic catheter placement and removal was standardised, however systemic antibiotic treatment was case dependant and at the clinician’s discretion. To simulate routine clinical practice, face masks were not worn during placement nor were the catheters bandaged. Catheters were maintained for 74 hours and assessed for clinical evidence of catheter site reaction, phlebitis or thrombosis twice daily. Bacteria were cultured from 69% of Teflon® and 89% of polyurethane catheters. Multiple isolates were found in 31% of Teflon® and 42% of polyurethane catheters The Fisher exact test showed no difference between the proportion of catheters with colonisation (P=0.28) or multiple isolates (P=0.76). The microbes cultured were predominantly gram positive, similar to other equine and human studies. Multiple-drug resistance was seen regularly, regardless of antibiotic treatment. Despite this, no clinical evidence of phlebitis or thrombosis occurred in any horse. It was concluded, that was no clear association between bacterial colonisation of Teflon® or polyurethane catheters (0.9<RR<1.87). The unexpected large proportion of bacterial isolates in the absence of clinical signs was also evaluated and suggests that the equine immune system plays a role in the development of septic phlebitis or thrombosis

Dark Ages woodland recovery and the expansion of beech : a study of land use changes and related woodland dynamics during the Roman to Medieval transition period in northern Belgium

The results from analyses of botanical remains (pollen, wood, charcoal, seeds) from several archaeological features excavated in Kluizen (northern Belgium) are presented. The region was largely uninhabited until the Iron Age and Roman period when a rural settlement was established, resulting in small-scale woodland clearance. The site was subsequently abandoned fromc.AD 270 till the High Middle Ages. The results of the archaeological and archaeobotanical analyses provide information on changes in land use and resulting dynamics of woodland cover and composition betweenc.600 BC and AD 1200, with a spatial and temporal resolution unrivalled in northern Belgium. Especially the long period of woodland regeneration following abandonment of the site around AD 270, covering the Late Roman and Early Medieval period, could be reconstructed in detail. Abandoned fields were first covered with pioneer woodland (Salix,CorylusandBetula), thenQuercus-dominated secondary forest and finally a late-successional forest withFagus sylvatica,Carpinus betulusandIlex aquifolium, an evolution that took over 300 years. The results also indicate that the observed increase ofFagusduring the Early Middle Ages, which was never an important element in the woodland vegetation in northern Belgium before, was related to climatic changes rather than anthropogenic factors

Sticky/Citron kinase maintains proper RhoA localization at the cleavage site during cytokinesis.

In many organisms, the small guanosine triphosphatase RhoA controls assembly and contraction of the actomyosin ring during cytokinesis by activating different effectors. Although the role of some RhoA effectors like formins and Rho kinase is reasonably understood, the functions of another putative effector, Citron kinase (CIT-K), are still debated. In this paper, we show that, contrary to previous models, the Drosophila melanogaster CIT-K orthologue Sticky (Sti) does not require interaction with RhoA to localize to the cleavage site. Instead, RhoA fails to form a compact ring in late cytokinesis after Sti depletion, and this function requires Sti kinase activity. Moreover, we found that the Sti Citron-Nik1 homology domain interacts with RhoA regardless of its status, indicating that Sti is not a canonical RhoA effector. Finally, Sti depletion caused an increase of phosphorylated myosin regulatory light chain at the cleavage site in late cytokinesis. We propose that Sti/CIT-K maintains correct RhoA localization at the cleavage site, which is necessary for proper RhoA activity and contractile ring dynamics

Living on the edges : Roman to Medieval settlement dynamics and related landscape transformation (ca. 100-1200 AD, Ghent, Belgium)

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