Mapping early fate determination in Lgr5+ crypt stem cells using a novel Ki67‐RFP allele

Cycling Lgr5⁺ stem cells fuel the rapid turnover of the adult intestinal epithelium. The existence of quiescent Lgr5⁺ cells has been reported, while an alternative quiescent stem cell population is believed to reside at crypt position +4. Here, we generated a novel Ki67^RFP knock-in allele that identifies dividing cells. Using Lgr5-GFP;Ki67^RFP mice, we isolated crypt stem and progenitor cells with distinct Wnt signaling levels and cell cycle features and generated their molecular signature using microarrays. Stem cell potential of these populations was further characterized using the intestinal organoid culture. We found that Lgr5^high stem cells are continuously in cell cycle, while a fraction of Lgr5^low progenitors that reside predominantly at +4 position exit the cell cycle. Unlike fast dividing CBCs, Lgr5^low Ki67⁻ cells have lost their ability to initiate organoid cultures, are enriched in secretory differentiation factors, and resemble the Dll1 secretory precursors and the label-retaining cells of Winton and colleagues. Our findings support the cycling stem cell hypothesis and highlight the cell cycle heterogeneity of early progenitors during lineage commitment.     

Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy

In this study, the effect of glycine receptor (GlyR) α3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both α3K and α3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for α3L compared to α3K (mean radius 92 ± 4 and 56 ± 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 ± 200 μm^?2, respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ± 6 nm). These results demonstrate that RNA splicing determines GlyR α3 membrane distribution, which has consequences for neuronal GlyR physiology and function.     

The Mn4Ca photosynthetic water-oxidation catalyst studied by simultaneous X-ray spectroscopy and crystallography using an X-ray free-electron laser

The structure of photosystem II and the catalytic intermediate states of the Mn₄CaO₅ cluster involved in water oxidation have been studied intensively over the past several years. An understanding of the sequential chemistry of light absorption and the mechanism of water oxidation, however, requires a new approach beyond the conventional steady-state crystallography and X-ray spectroscopy at cryogenic temperatures. In this report, we present the preliminary progress using an X-ray free-electron laser to determine simultaneously the light-induced protein dynamics via crystallography and the local chemistry that occurs at the catalytic centre using X-ray spectroscopy under functional conditions at room temperature.     

Mechanical control of the endothelial barrier

The integrity of the endothelial barrier is controlled by the combined action of chemical and mechanical signaling systems. Permeability-regulating factors signal through small GTPases to regulate the architecture of the cytoskeleton and this has a strong impact on the morphology and stability of VE-cadherin-based cell–cell junctions. The details of how structural and mechanical properties of the actin cytoskeleton influence cell–cell adhesion and how this impacts the dynamic regulation of the endothelial barrier, are beginning to be elucidated. In this review, we discuss the physical and regulatory interactions between the VE-cadherin complex and the actomysoin cytoskeleton, as they are the main determinants of cell–cell adhesion and the mechanical architecture of the cytoskeleton. We discuss, based on recent in vitro data, how a balance between Linear Adherens Junctions, paralleled by cortical actin bundles and Focal Adherens Junctions, connected to radial action bundles, determines endothelial barrier function. We discuss how small GTPases control this balance by regulating the spatial organization and mechanics of actomyosin. We propose a hypothetical model of how biochemical and mechanical signals cooperate locally, at the actomyosin–adhesion interface to open and re-seal the barrier in a rapid and controlled manner.     

Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue).     

Ethnopharmacological practices by livestock farmers in Uganda: Survey experiences from Mpigi and Gulu districts

Background

There is continued reliance on conventional veterinary drugs including anthelmintics, to some of which resistance has developed. Loss of indigenous technical knowledge (ITK) from societies affects the opportunities for utilization of ethnopharmacological practices unless properly documented. This study was conducted to identify common traditional practices using medicinal plants against helminthosis and other livestock diseases in Mpigi and Gulu districts of Uganda.

Methods

Seven focus group discussions with ten farmers per group plus 18 key informant interviews were held in each district from August to November 2011. Ranking was used to quantify disease burdens and to identify priority livestock and breeds. Samples of each plant were submitted to Makerere University herbarium for identification and documentation. The local name, relative availability and International Union for Conservation of Nature (IUCN) status were recorded.

Results

Seventy six farmers in Mpigi and 74 in Gulu were interviewed. Theileriosis and helminthosis were the most common disease conditions in cattle and goats, respectively. Forty plant species within 34 genera from 22 botanical families were identified, with 20 of these used against helminthosis. Other plants treated wounds and ecto-parasites, theileriosis, retained placenta and bovine ephemeral fever. Non-plant practices (7) and plants cited were used in combination depending on availability. Males older than 40 years had most ethnopharmacological knowledge. Most plants (75%, n?=?40) were common, but 10 were rare. IUCN status was not evaluated for 95% of these plants. Conventional and traditional drug use in Gulu and Mpigi districts was different (χ²?=?24; p?<?0.001). The scientific, English, Luganda and Acholi names of all plants and their availability within the communities are documented herein.

Conclusion

This is the first detailed livestock-related ethnopharmacological study in Gulu district. Farmers in Uganda are still using a variety of practices to treat livestock ailments. Scientific validation and evaluation of conservation status are urgently needed to ensure future availability and knowledge about these plant resources.