Application of benchtop total-reflection X-ray fluorescence spectrometry and chemometrics in classification of origin and type of Croatian wines

The contents of selected metals (K, Ca, Fe, Cu, Zn, Mn, Sr, Rb, Ba, Pb, Ni, Cr and V) in 70 wine samples from Continental and Adriatic part of Croatia and different types of wine (red and white) were determined by TXRF. The aim of this study was to compare the elemental composition of wines from two different regions and to determine the discriminant ability of each variable and to indicate which variables discriminate between the four categories considered. Principal component analysis and cluster analysis showed that K, Mn, Ba and Ni can be considered as the most important characteristics to distinguish between Continental red and white wines, Rb, Ni and Ba for Continental red and Adriatic red wines while Sr is the only metal that completely distinguishes the samples of each category. Finally, linear discriminant analysis showed good recognition (100%) and prediction abilities (96.43%) using these selected elements

Bacterial Transmembrane Proteins that Lack N-Terminal Signal Sequences

Tail-anchored membrane proteins (TAMPs), a class of proteins characterized by their lack of N-terminal signal sequence and Sec-independent membrane targeting, play critical roles in apoptosis, vesicle trafficking and other vital processes in eukaryotic organisms. Until recently, this class of membrane proteins has been unknown in bacteria. Here we present the results of bioinformatic analysis revealing proteins that are superficially similar to eukaryotic TAMPs in the bacterium Streptomyces coelicolor. We demonstrate that at least four of these proteins are bona fide membrane-spanning proteins capable of targeting to the membrane in the absence of their N-terminus and the C-terminal membrane-spanning domain is sufficient for membrane targeting. Several of these proteins, including a serine/threonine kinase and the SecE component of the Sec translocon, are widely conserved in bacteria

Integrated management of ash from industrial and domestic combustion : a new sustainable approach for reducing greenhouse gas emissions from energy conversion

This work supports, for the first time, the integrated management of waste materials arising from industrial processes (fly ash from municipal solid waste incineration and coal fly ash), agriculture (rice husk ash), and domestic activities (ash from wood biomass burning in domestic stoves). The main novelty of the paper is the reuse of wood pellet ash, an underestimated environmental problem, by the application of a new technology (COSMOS-RICE) that already involves the reuse of fly ashes from industrial and agricultural origins. The reaction mechanism involves carbonation: this occurs at room temperature and promotes permanent carbon dioxide sequestration. The obtained samples were characterized using XRD and TGA (coupled with mass spectroscopy). This allowed quantification of the mass loss attributed to different calcium carbonate phases. In particular, samples stabilized using wood pellet ash show a weight loss, attributed to the decomposition of carbonates greater than 20%. In view of these results, it is possible to conclude that there are several environmental benefits from wood pellet ash reuse in this way. In particular, using this technology, it is shown that for wood pellet biomass the carbon dioxide conversion can be considered negative

PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1α-mediated unfolded protein response

Poly(ADP-ribose) polymerases (PARPs; also known as ADP-ribosyl transferase D proteins) modify acceptor proteins with ADP-ribose modifications of varying length (reviewed in refs 1, 2, 3). PARPs regulate key stress response pathways, including DNA damage repair and the cytoplasmic stress response. Here, we show that PARPs also regulate the unfolded protein response (UPR) of the endoplasmic reticulum (ER). Human PARP16 (also known as ARTD15) is a tail-anchored ER transmembrane protein required for activation of the functionally related ER stress sensors PERK and IRE1α during the UPR. The third identified ER stress sensor, ATF6, is not regulated by PARP16. As is the case for other PARPs that function during stress, the enzymatic activity of PARP16 is upregulated during ER stress when it ADP-ribosylates itself, PERK and IRE1α. ADP-ribosylation by PARP16 is sufficient for activating PERK and IRE1α in the absence of ER stress, and is required for PERK and IRE1α activation during the UPR. Modification of PERK and IRE1α by PARP16 increases their kinase activities and the endonuclease activity of IRE1α. Interestingly, the carboxy-terminal luminal tail of PARP16 is required for PARP16 function during ER stress, suggesting that it transduces stress signals to the cytoplasmic PARP catalytic domain.National Cancer Institute (U.S.) (Cancer Center Support Core Grant P30-CA14051)National Institutes of Health (U.S.) (Grant 5R01 GM087465-02)Kathy and Curt Marble Cancer Research FundJeptha H. and Emily V. Wade FundVirginia and D.K. Ludwig Fund for Cancer Researc

Tubulohelical membrane arrays: From the initial observation to the elucidation of nanophysical properties and cellular function

Lipids undergo self-assembly to form ordered nonlamellar, nanoperiodic arrays both in vitro and in vivo. While engineering of such membrane arrays for technical devices is envisaged, we know little about their cellular function. Do they represent building blocks of an inherent cellular nanotechnology? Prospects for answering this question could be improved if the nanophysical properties of the membrane arrays could be studied in the context of specific cellular functions. Therefore, we draw attention to exceptional complex membrane arrays found in the renal epithelial cell line PtK2 that could provide perfect conditions for both biophysical and cell functional studies. The so-called tubulohelical membrane arrays (TUHMAs) combine nanoperiodicity of lipid membranes with that of helix-like proteinaceous core structures. Strikingly, they show several characteristics of dynamic, microtubule-associated single organelles. Our initial data indicate that TUHMA formation occurs in the depth of the cytoplasm under participation of cytoplasmic nucleoporins. Once matured, they may fuse with the nuclear membrane in polarized positions, either perpendicularly or in parallel to the nucleus. As a starting point for the initiation of functional studies we found a connection between TUHMAs and primary cilia, indicated by immunolabeling patterns of detyrosynated tubulin and cytoplasmic nucleoporins. We discuss these observations in the context of the ciliary cycle and of the specific requirement of ciliated renal epithelial cells for oriented cell division. Finally, we raise the question of whether putative nanooptical properties of TUHMAs could serve for communicating orientation between dividing cells

The Cytosolic Domain of Fis1 Binds and Reversibly Clusters Lipid Vesicles

Every lipid membrane fission event involves the association of two apposing bilayers, mediated by proteins that can promote membrane curvature, fusion and fission. We tested the hypothesis that Fis1, a tail-anchored protein involved in mitochondrial and peroxisomal fission, promotes changes in membrane structure. We found that the cytosolic domain of Fis1 alone binds lipid vesicles, which is enhanced upon protonation and increasing concentrations of anionic phospholipids. Fluorescence and circular dichroism data indicate that the cytosolic domain undergoes a membrane-induced conformational change that buries two tryptophan side chains upon membrane binding. Light scattering and electron microscopy data show that membrane binding promotes lipid vesicle clustering. Remarkably, this vesicle clustering is reversible and vesicles largely retain their original shape and size. This raises the possibility that the Fis1 cytosolic domain might act in membrane fission by promoting a reversible membrane association, a necessary step in membrane fission

HIV-1 Vpr Triggers Mitochondrial Destruction by Impairing Mfn2-Mediated ER-Mitochondria Interaction

Human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) has been shown to induce host cell death by increasing the permeability of mitochondrial outer membrane (MOM). The mechanism underlying the damage to the mitochondria by Vpr, however, is not clearly illustrated. In this study, Vpr that is introduced, via transient transfection or lentivirus infection, into the human embryonic kidney cell line HEK293, human CD4+ T lymphoblast cell line SupT1, or human primary CD4+ T cells serves as the model system to study the molecular mechanism of Vpr-mediated HIV-1 pathogenesis. The results show that Vpr injures MOM and causes a loss in membrane potential (MMP) by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2) via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation. Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1) and increases bulging in mitochondria-associated membranes (MAM), the specific regions of endoplasmic reticulum (ER) which form physical contacts with the mitochondria. Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr. Furthermore, by employing time-lapse confocal fluorescence microscopy, we identify the transport of Vpr protein from the ER, via MAM to the mitochondria. Taken together, our results suggest that Vpr-mediated cellular damage may occur on an alternative protein transport pathway from the ER, via MAM to the mitochondria, which are modulated by Mfn2 and DRP1

LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones

BACKGROUND The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones. RESULTS Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61beta but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous. CONCLUSION The lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue