Structural information about organized cholesterol domains from specific antibody recognition

Cholesterol-rich domains have been observed to exist in cell membranes under physiological and pathological conditions. Their compositions and the microenvironment of their formation vary over a wide range. Very little information is however available on the molecular structure and organization of these domains. The techniques available to provide such structural information are reviewed here first. The possibility of using tailor-made antibodies as reporters of molecular organization in membranes is then considered. The concept of antibodies recognizing molecular organization rather than single molecular epitopes is established, reviewing the existing works on antibody and protein recognition of crystalline molecular arrays. The information that such antibodies could provide in cells is finally examined together with a proof of application.     

Live imaging of GLUT2 glucose-dependent trafficking and its inhibition in polarized epithelial cysts

GLUT2 is a facilitative glucose transporter, expressed in polarized epithelial cells of the liver, intestine, kidney and pancreas, where it plays a critical role in glucose homeostasis. Together with SGLT1/2, it mediates glucose absorption in metabolic epithelial tissues, where it can be translocated apically upon high glucose exposure. To track the subcellular localization and dynamics of GLUT2, we created an mCherry–hGLUT2 fusion protein and expressed it in multicellular kidney cysts, a major site of glucose reabsorption. Live imaging of GLUT2 enabled us to avoid the artefactual localization of GLUT2 in fixed cells and to confirm the apical GLUT2 model. Live cell imaging showed a rapid 15 ± 3 min PKC-dependent basal-to-apical translocation of GLUT2 in response to glucose stimulation and a fourfold slower basolateral translocation under starvation. These results mark the physiological importance of responding quickly to rising glucose levels. Importantly, we show that phloretin, an apple polyphenol, inhibits GLUT2 translocation in both directions, suggesting that it exerts its effect by PKC inhibition. Subcellular localization studies demonstrated that GLUT2 is endocytosed through a caveolae-dependent mechanism, and that it is at least partly recovered in Rab11A-positive recycling endosome. Our work illuminates GLUT2 dynamics, providing a platform for drug development for diabetes and hyperglycaemia.     

Nitrite reduction in Paracoccus sp. is affected by a novel plasmid pYR1

Two relatively low-copy plasmids of 9 and 16 kb were found to comprise the extrachromosomal DNA of a Paracoccus strain. Reduction of nitrate by plasmid-cured cells resulted in a significant intermediate nitrite accumulation as compared to wild-type cells. By examining nitrate reduction by transformants containing one of the two plasmids, it was found that nitrite accumulation was influenced by the 9.0-kb plasmid, designated as pYR1. Subcloning analysis showed that a 1.8-kb fragment of this plasmid affected nitrite accumulation. Sequence analysis of this fragment revealed the presence of five open reading frames. One of the six deduced proteins showed a strong homology to ABC transporters.     

Chickens fed with biomass of the red microalga Porphyridium sp. have reduced blood cholesterol level and modified fatty acid composition in egg yolk

The biomass of the red alga Porphyridium sp.constitutes a unique combination of soluble sulfatedpolysaccharide that accounts for about 70% of thealgal dry weight, and various polyunsaturatedfatty acids (PUFA) such as arachidonic andeicosapentaenoic acid (AA, 20:4 6 and EPA,20:5 3). In view of earlier results in ourlaboratory showing a reduction in serum cholesteroland triglyceride levels in rodents fed with red algalbiomass, we set out to examine the influence of algalbiomass as a feed additive on the metabolism ofchickens, with an emphasis on blood and eggcholesterol levels. For that purpose, lyophilizedalgal biomass was fed to 12–13, 30-week-old, WhiteLeghorn chickens for 10 days at a proportion of 5% or10% of the standard chicken diet. Twelve chickensfed with unsupplemented diet served as the control. No differences in body weight, egg number, and eggweight were found between the algal-fed chickens (atboth concentrations) and the control. However,chickens fed with algal biomass consumed 10% lessfood for both groups, and their serum cholesterollevels were significantly lower (by 11% and 28% forthe groups fed with 5% and 10% supplement,respectively) as compared with the respective valuesof the control group. Egg yolk of chickens fed withalgae tended to have reduced cholesterol levels (by10%) and increased linoleic acid and arachidonic acidlevels (by 29% and 24%, respectively). In addition,the color of the egg yolk was darker as a result ofthe higher carotenoid levels (2.4 fold higher) forchickens that fed with 5% supplement. Theseresults encourage the development of an improvedchicken feed having dietary fibers and polyunsaturatedfatty acids.     

Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da. Using genome-wide linkage analyses, we discovered an association between nerve-injury-induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.     

A versatile and tunable coating strategy allows control of nanocrystal delivery to cell types in the liver

There are many liver diseases that could be treated with delivery of therapeutics such as DNA, proteins, or small molecules. Nanoparticles are often proposed as delivery vectors for such therapeutics; however, achieving nanoparticle accumulations in the therapeutically relevant hepatocytes is challenging. In order to address this issue, we have synthesized polymer coated, fluorescent iron oxide nanoparticles that bind and deliver DNA, as well as produce contrast for magnetic resonance imaging (MRI), fluorescence imaging, and transmission electron microscopy (TEM). The composition of the coating can be varied in a facile manner to increase the quantity of poly(ethylene glycol) (PEG) from 0% to 5%, 10%, or 25%, with the aim of reducing opsonization but maintaining DNA binding. We investigated the effect of the nanoparticle coating on DNA binding, cell uptake, cell transfection, and opsonization in vitro. Furthermore, we exploited MRI, fluorescence imaging, and TEM to investigate the distribution of the different formulations in the liver of mice. While MRI and fluorescence imaging showed that each formulation was heavily taken up in the liver at 24 h, the 10% PEG formulation was taken up by the therapeutically relevant hepatocytes more extensively than either the 0% PEG or the 5% PEG, indicating its potential for delivery of therapeutics to the liver.     

Where do adaptive shifts occur during invasion? A multidisciplinary approach to unravelling cold adaptation in a tropical ant species invading the Mediterranean area

Evolution may improve the invasiveness of populations, but it often remains unclear whether key adaptation events occur after introduction into the recipient habitat (i.e. post‐introduction adaptation scenario), or before introduction within the native range (i.e. prior‐adaptation scenario) or at a primary site of invasion (i.e. bridgehead scenario). We used a multidisciplinary approach to determine which of these three scenarios underlies the invasion of the tropical ant Wasmannia auropunctata in a Mediterranean region (i.e. Israel). Species distribution models (SDM), phylogeographical analyses at a broad geographical scale and laboratory experiments on appropriate native and invasive populations indicated that Israeli populations followed an invasion scenario in which adaptation to cold occurred at the southern limit of the native range before dispersal to Israel. We discuss the usefulness of combining SDM, genetic and experimental approaches for unambiguous determination of eco‐evolutionary invasion scenarios.     

View from the top: hierarchies and reverse hierarchies in the visual system

We propose that explicit vision advances in reverse hierarchical direction, as shown for perceptual learning. Processing along the feedforward hierarchy of areas, leading to increasingly complex representations, is automatic and implicit, while conscious perception begins at the hierarchy's top, gradually returning downward as needed. Thus, our initial conscious percept--vision at a glance--matches a high-level, generalized, categorical scene interpretation, identifying "forest before trees." For later vision with scrutiny, reverse hierarchy routines focus attention to specific, active, low-level units, incorporating into conscious perception detailed information available there. Reverse Hierarchy Theory dissociates between early explicit perception and implicit low-level vision, explaining a variety of phenomena. Feature search "pop-out" is attributed to high areas, where large receptive fields underlie spread attention detecting categorical differences. Search for conjunctions or fine discriminations depends on reentry to low-level specific receptive fields using serial focused attention, consistent with recently reported primary visual cortex effects.