Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

Living with observational data in biological anthropology

The establishment of cause and effect relationships is a fundamental objective of scientific research. Many lines of evidence can be used to make cause–effect inferences. When statistical data are involved, alternative explanations for the statistical relationship need to be ruled out. These include chance (apparent patterns due to random factors), confounding effects (a relationship between two variables because they are each associated with an unmeasured third variable), and sampling bias (effects due to preexisting properties of compared groups). The gold standard for managing these issues is a controlled randomized experiment. In disciplines such as biological anthropology, where controlled experiments are not possible for many research questions, causal inferences are made from observational data. Methods that statisticians recommend for this difficult objective have not been widely adopted in the biological anthropology literature. Issues involved in using statistics to make valid causal inferences from observational data are discussed.     

Immunolocalization of tenascin and cellular fibronectins in diverse glomerulopathies

Frozen samples of minimal change glomerulopathy (MCG), and of membranous, segmental and diffuse lupus glomerulonephritis (MGN, SGN, DLGN) were studied to assess the distribution of tenascin (Ten), and the extradomains A and B (EDA-and EDB-) and oncofetal (Onc-) isoforms of cellular fibronectin (cFn). Cryosections were immunostained by the ABC method with specific monoclonal antibodies. In MCG, mesangial Ten and EDA-cFn reactions were increased. In MGN, mesangial Ten and EDA-cFn staining was enhanced except in segmental scars; convincing reactions were seen in cases with membranous transformation; spikes stained strongly. In SGN, variably intense staining for Ten and all cFn isoforms was seen in glomerular necrosis, proliferation and crescents; parietal epithelium EDA-cFn staining was noted. In DLGN, strong and extensive mesangial Ten and EDA-cFn staining was seen as were focal EDB-and Onc-cFn reactions. Parietal cells with and without crescents stained variably with all Mabs. Obsolete glomeruli were unreactive save for rare periglomerular Ten rims. Interstitial inflammation and fibrosis in MGN, SGN and DLGN had moderate to strong Ten and EDA-cFn staining with rare traces of EDB-and Onc-cFn. We conclude that enhanced Ten and EDA-cFn is a potentially reversible response to glomerular injury whereas the expression of EDB-and Onc-cFn apparently result from necrosis and/or cellular proliferation which lead to scarring. And, while mesangial cells are the major source of these molecules, epithelial cells might also partake in their synthesis.     

Generalized estimating equations approach for spatial lattice data: A case study in adoption of improved maize varieties in Mozambique

Generalized estimating equations (GEE) are extension of generalized linear models (GLM) widely applied in longitudinal data analysis. GEE are also applied in spatial data analysis using geostatistics methods. In this paper, we advocate application of GEE for spatial lattice data by modeling the spatial working correlation matrix using the Moran's index and the spatial weight matrix. We present theoretical developments and results for simulated and actual data as well. For the former case, 1,000 samples of a random variable (response variable) defined in (0, 1) interval were generated using different values of the Moran's index. In addition, 1,000 samples of a binary and a continuous variable were also randomly generated as covariates. In each sample, three structures of spatial working correlation matrices were used while modeling: The independent, autoregressive, and the Toeplitz structure. Two measures were used to evaluate the performance of each of the spatial working correlation structures: the asymptotic relative efficiency and the working correlation selection criterions. The results showed that both measures indicated that the autoregressive spatial working correlation matrix proposed in this paper presents the best performance in general. For the actual data case, the proportion of small farmers who used improved maize varieties was considered as the response variable and a set of nine variables were used as covariates. Two structures of spatial working correlation matrices were used and the results showed consistence with those obtained in the simulation study.     

Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q < 0.05; enrichment range 1.40–9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting β-cells and neurons and underline the existence of trans?nosology pathways in diabetes and its co-morbidities.     

Disentangling plastic and genetic changes in body mass of Siberian jays

Spatial and temporal phenotypic differentiation in mean body size is of commonplace occurrence, but the underlying causes remain often unclear: both genetic differentiation in response to selection (or drift) and environmentally induced plasticity can create similar phenotypic patterns. Studying changes in body mass in Siberian jays (Perisoreus infaustus) over three decades, we discovered that mean body mass declined drastically (ca. 10%) over the first two decades, but increased markedly thereafter back to almost the initial level. Quantitative genetic analyses revealed that although body mass was heritable (h² = 0.46), the pronounced temporal decrease in body mass was mainly a product of phenotypic plasticity. However, a concomitant and statistically significant decrease in predicted breeding values suggests a genetic component to this change. The subsequent increase in mean body mass was indicated to be entirely due to plasticity. Selection on body mass was estimated to be too weak to fully account for the observed genetic decline in body mass, but bias in selection differential estimates due to environmental covariance between body mass and fitness is possible. Hence, the observed body mass changes appear to be driven mainly by phenotypic plasticity. Although we were not able to identify the ecological driver of the observed plastic changes, the results highlight the utility of quantitative genetic approaches in disentangling genetic and phenotypic changes in natural populations.     

Genetic structure of Glycine canescens,a perennial relative of soybean

Summary Allozyme variation as detected by starch gel electrophoresis was used to assess the extent and spatial organization of genetic variation across the entire range of Glycine canescens sensu lato. Eleven enzyme systems were assayed in 116 accessions of this taxon and 102 alleles were detected at a total of 31 loci. Eighty-one percent of loci were polymorphic. Most of this variation occurred between and very little within accessions. Three major groupings were detected. These groupings (groups 1, 2, and 3) also differed with respect to mean seed size and their geographic distribution. A further ten accessions stood out from these distinct groups. These accessions were most closely related to group 3 but were variable among themselves. In general, they were collected from highly dissected terrain, often in the remote interior of the continent. A final group of 18 problematic accessions (group X), originally tentatively identified as G. canescens on morphological grounds, was shown to be isozymically distinct from this species and was reclassified as one form of the polytypic species G. clandestina.     

Optimization of nutritional and environmental conditions for pyocyanin production by urine isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a highly pathogenic bacteria involved in numerous diseases among which, are urinary tract infections (UTIs). The pyocyanin secreted as a virulence factor by this bacterium has many beneficial applications but its high cost remains an obstacle for its widespread use. In this study, a total of fifty urine isolates were identified as P. aeruginosa. All strains produced pyocyanin pigment with a range of 1.3–31 µg/ml. The highest producer clinical strain P21 and the standard strain PA14 were used in optimization of pyocyanin production. Among tested media, king’s A fluid medium resulted in the highest yield of pyocyanin pigment followed by nutrient broth. Growth at 37 °C was superior in pyocyanin production than growth at 30 °C. Both shaking and longer incubation periods (3–4 days) improved pyocyanin production. The pyocyanin yield was indifferent upon growth of P21 at both pH 7 and pH 8. In conclusion, the optimum conditions for pyocyanin production are to use King’s A fluid medium of pH 7 and incubate the inoculated medium at 37 °C with shaking at 200 rpm for a period of three to four days.     

Etude enzymatique comparée de champignons entomopathogènes des genres Beauveria et Metarhizium

Résumé Les extraits antigéniques de trois champignons entomopathogènes (Beauveria bassiana, Beauveria brongniartii et Metarhizium anisopliae), soumis à l'analyse éléctrophorétique en gel d'agar révèlent au total 23 activités enzymatiques différentes (5 oxydo-réductases et 18 hydrolases), dont 18 ont été retrouvées à l'analyse immunoélectrophorétique. La carte enzymatique de ces germes complète leur analyse immunoélectrophorétique; la comparaison des isoenzymes des divers isolats permet de mettre en évidence des différences interspécifiques et intraspécifiques.Chaque souche cryptogamique se caractérise par des profils enzymatiques particuliers. Ces résultats montrent donc que l'étude des enzymogrammes est un complément utile à l'analyse immunoélectrophorétique appliquée à la caractérisation des hyphomycètes entomopathogènes. Enfin, les souches n 44 et 51 de M. anisopliae paraissent être suffisamment apparentées pour être réunies au sein d'un même biotype.