Trafficking of Leishmania donovani promastigotes in non-lytic compartments in neutrophils enables the subsequent transfer of parasites to macrophages

Inoculation of Leishmania ( L.) spp. promastigotes in the dermis of mammals by blood-feeding sand flies can be accompanied by the rapid recruitment of neutrophils, inflammatory monocytes and dendritic cells. Despite the presence of these lytic leucocytes, parasitism is efficiently established. We show here that Leishmania donovani promastigotes are targeted to two different compartments in neutrophils. The compartments harbouring either damaged or non-damaged parasites were characterized at the electron microscopy (EM) level using the glucose 6-phosphatase cytochemistry and endosome–phagosome fusion assays. One involves the contribution of lysosomes leading to the formation of highly lytic compartments where parasites are rapidly degraded. The other is lysosome-independent and involves the contribution of a compartment displaying some features of the endoplasmic reticulum (ER) where parasites are protected from degradation. Using genetically modified parasites, we show that the promastigote surface lipophosphoglycan (LPG) is required to inhibit lysosome fusion and maintain parasites in neutrophil compartments displaying ER features. L. donovani -harbouring neutrophils that eventually enter apoptosis can be phagocytosed by macrophages enabling the stealth entry of parasites into their final replicative host cells. Thus, the ability of L. donovani to avoid trafficking into lysosomes-derived compartments in short-lived neutrophils constitutes a key process for the subsequent establishment of long-term parasitism.     

Expression of a chloroplast ATP/ADP transporter in E. coli membranes: behind the Mistic strategy

Eukaryotic membrane protein expression is still a major bottleneck for structural studies. Production in E. coli often leads to low expression level and/or aggregated proteins. In the last decade, strategies relying on new fusion protein expression revealed promising results. Fusion with the amphipatic Mistic protein has been described to favor expression in E. coli membranes. Although, this approach has already been reported for a few membrane proteins, little is known about the activity of the fused proteins. We used this strategy and obtained high expression levels of a chloroplast ATP/ADP transporter from A. thaliana (NTT1) and characterized its transport properties. NTT1 fused to Mistic has a very low transport activity which can be recovered after in vivo Mistic fusion cleavage. Moreover, detailed molecular characterization of purified NTT1 mature form, NTT1 fused to Mistic or NTT1 cleaved-off from this fusion highlights the correct fold of the latter one. Therefore, considering the higher quantity of purified NTT1 mature form obtained via the Mistic fusion approach, this is a valuable strategy for obtaining quantities of pure and active proteins that are adequate for structural studies.     

Assessing the assessors: Quantifying observer variation in vegetation and habitat assessment

Vegetation assessment protocols used in research, monitoring and statutory planning often rely on the consistent application of methods by multiple assessors. Consequently, a study was undertaken to evaluate the application of a vegetation condition assessment protocol by a range of assessors. The aim of the study was to quantify variation among assessors in their measurement of field‐based vegetation attributes using Queensland’s BioCondition protocol, and to determine the effect of variability on the final condition score. The study consisted of 77 assessors, ranging from nil to 25 years experience in vegetation assessment, who each undertook an assessment at one site following training. Six of the 20 attributes used to derive the final condition score were not assessed consistently, this being because of a small number of assessors who had misinterpreted the protocol and had inappropriately assessed some attributes. Despite inclusion of outliers, 82% of assessors were within 10 points of the median condition score for the site. Based on the results, the definition and measurement of problematic attributes have been either clarified or removed from the revised assessment procedure. The study demonstrated that with training prior to use, assessors were able to consistently assess condition at the study site.     

The blood vessels of the regenerating limb of the adult newt,Triturus

The vessels of the forelimb stump and regenerate were perfused with Prussian blue and studied as whole mounts and in histological sections to reveal the condition and disposition of the blood vessels in various stages of forelimb regeneration in the adult newt, Triturus viridescens. The development of the vessels in the regenerate seemed to be comparable in all its essential features to that which has been described for the normal developing limb in urodele, chick and pig embryos. The first signs of regeneration of the vessels are seen during wound healing when fine sprouts appear from the old vessels near the amputation wound. These grow and anastomose, but are limited to the transition region between old and new tissues and avoid the growing blastema during the early stages of regeneration. As the regenerate enlarges into a conical structure vessels invade the proximal part of the growth and avoid the distal regions. It is only during the stages of histogenesis and morphogenesis that vessels grow into more distal regions. The regions of most active enlargement of the early or later regenerate are those most poorly vascularized. These results are discussed against the background of the activity of certain enzymes during regeneration. In the advanced regenerate, preferential channels are consolidated until in the palette and digital stages the pattern of the blood vessels resembles that of the normal limb.