Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.     

Supplementary feeding during the chick-rearing period is ineffective in increasing the breeding success in the bearded vulture (Gypaetus barbatus)

One of the most widespread tools in the conservation of scavenger species is the provision of supplementary food. However, scientific studies on its effectiveness have been rarely conducted. Here, we present the first results of an experimental specific supplementary feeding programme applied from hatching to 45–60 days, aimed at increasing the breeding success of an obligate brood reducer, the threatened bearded vulture Gypaetus barbatus. We tested its effectiveness in the Spanish Pyrenees, the locale of the most important population of this species in the European Union, in which a regressive trend in breeding parameters has been observed in recent years. We compared the breeding success in nests with supplementary food to non-supplemented control nests. Supplementary food did not significantly increase global breeding success (supplemented nests, 0.793 ± 0.193 chicks per pair with eggs hatched vs non-supplemented nests, 0.771 ± 0.185) or the individual breeding performance of the territories (supplemented period, 0.712 ± 0.307 vs non-supplemented period, 0.642 ± 0.311). The similar values obtained suggest that the specific supplementary feeding programme applied during the chick-rearing period is apparently ineffective at increasing breeding success. The results suggest that, at least in the study area, factors that provoke breeding failure after hatching continue to operate independently of the supplementary feeding programme. Although more research on this subject is required, these preliminary conclusions should be taken into account by managers in order to optimise the investment of economic resources and to better prioritise the future establishment of alternative conservation actions.     

Structural Integrity of the Contralesional Hemisphere Predicts Cognitive Impairment in Ischemic Stroke at Three Months

After stroke, white matter integrity can be affected both locally and distally to the primary lesion location. It has been shown that tract disruption in mirror’s regions of the contralateral hemisphere is associated with degree of functional impairment. Fourteen patients suffering right hemispheric focal stroke (S) and eighteen healthy controls (HC) underwent Diffusion Weighted Imaging (DWI) and neuropsychological assessment. The stroke patient group was divided into poor (SP; n = 8) and good (SG; n = 6) cognitive recovery groups according to their cognitive improvement from the acute phase (72 hours after stroke) to the subacute phase (3 months post-stroke). Whole-brain DWI data analysis was performed by computing Diffusion Tensor Imaging (DTI) followed by Tract Based Spatial Statistics (TBSS). Assessment of effects was obtained computing the correlation of the projections on TBSS skeleton of Fractional Anisotropy (FA) and Radial Diffusivity (RD) with cognitive test results. Significant decrease of FA was found only in right brain anatomical areas for the S group when compared to the HC group. Analyzed separately, stroke patients with poor cognitive recovery showed additional significant FA decrease in several left hemisphere regions; whereas SG patients showed significant decrease only in the left genu of corpus callosum when compared to the HC. For the SG group, whole brain analysis revealed significant correlation between the performance in the Semantic Fluency test and the FA in the right hemisphere as well as between the performance in the Grooved Pegboard Test (GPT) and theTrail Making Test-part A and the FA in the left hemisphere. For the SP group, correlation analysis revealed significant correlation between the performance in the GPT and the FA in the right hemisphere.     

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd⁻/b⁺ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de- O -acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de- O -acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy- d - manno -octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.     

Plant–Environment Interactions in the Low Arctic Torngat Mountains of Labrador

Ecosystems - The eastern Canadian Subarctic and Arctic are experiencing significant environmental change with widespread implications for the people, plants, and animals living there. In this...     

Regulated expression of the rat medium chain hydrolase gene in transgenic rape seed

Medium chain hydrolase (MCH) is an enzyme which regulates the chain length of fatty acid synthesis specifically in the mammary gland of the rat. During lactation, MCH interacts with fatty acid synthase (FAS) to cause premature release of acyl chains, thus providing medium chain fatty acids for synthesis of milk fat. In this study we have investigated the ability of rat MCH to interact with the phylogenetically more distant FAS structure present in plant systems and to cause a perturbation of fatty acid synthesis. Inin vitro experiments, addition of purified MCH to rapeseed homogenates was found to cause a significant perturbation of fatty acid synthesis towards medium chain length products. The rat MCH gene was expressed in transgenic oilseed rape using a seed specific rape acyl carrier protein (ACP) promoter and a rape ACP plastid targeting sequence. Western analysis showed MCH protein to be present in transgenic seed and for its expression to be developmentally regulated in concert with storage lipid synthesis. The chimaeric preprotein was correctly processed and immunogold labelling studies confirmed MCH to be localized within plastid organelles. However, fatty acid analysis of oil from MCH-expressing rape seed showed no significant differences to that from control seed.     

Co-populated conformational ensembles of beta2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry

Ordered assembly of monomeric human beta(2)-microglobulin (beta(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 degrees C), wild-type beta(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, beta(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of beta(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of beta(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.     

Toward Standardization of Mesenchymal Stromal Cell‐Derived Extracellular Vesicles for Therapeutic Use: A Call for Action

Extracellular vesicles (EVs), which include a variety of nano‐sized membrane‐encapsulated particles, are released to the extracellular microenvironment by the vast majority of cells and carry lipids, proteins, mRNA, and miRNA or non‐coding RNA. Increasing evidence suggests the great versatility and potential of EV‐based applications in humans. In this issue, van Balkom et al. explore and compare the reported proteomic signature of mesenchymal stromal cell (MSC)‐derived small EVs. In particular, their paper offers a valuable approach and point of view on MSC‐EV manufacturing and therapeutic potential. Briefly, van Balkom et al. aimed to identify a common protein signature that may be useful in ensuring the homogeneity of therapeutic MSC‐EVs. In addition to excessive variability in EV‐producing cell sources and culture conditions, the harvesting time for the EV‐containing conditioned medium, and EV isolation procedure, the authors found a specific protein signature from the publicly available MSC‐EVs proteome. In light of their findings and those from the plentiful studies published in this continuously growing area of research, potential focus areas and issues are outlined for the more rational design and optimization of MSC‐EV production and potency for therapeutics.