An analysis of the main driving factors of renewable energy consumption in the European Union.

Climate change is a major global concern closely related to the strategies aimed at reducing energy consumption and increasing energy efficiency. Over the last decades, the interest in the development of renewable energy (RE) has grown exponentially. In the case of the European Union (EU), the Renewable Energy Directive sets rules to achieve a 32% of total energy consumption to be covered through RE by 2030. In order to achieve this goal, it is important to know what are the main driving factors of RE consumption (REC). This study aims to analyze the impact of economic and social factors on the share of REC in total energy consumption in the EU over the period 2001-2015. For doing so, we estimate a Panel Corrected Standard Error (PCSE) model. The results obtained show that economic factors have a negative effect on REC. In contrast, social factors like education exert a positive effect. This suggests that it is necessary to adopt a holistic approach that includes not only economic but also social aspects in order to foster REC.

ZP4 Is Present in Murine Zona Pellucida and Is Not Responsible for the Specific Gamete Interaction.

Mammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1-ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus, although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus, which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human.

Composition of marsupial zona pellucida: a molecular and phylogenetic approach.

The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett's wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species.

Economic evaluation of elective single-embryo transfer with subsequent single frozen embryo transfer in an in vitro fertilization/intracytoplasmic sperm injection program.

OBJECTIVE: To analyze the cost-effectiveness of IVF-ICSI cycles with elective single-embryo transfer (eSET), plus elective single frozen embryo transfer (eSFET) if pregnancy is not achieved, compared with double-embryo transfer (DET). DESIGN: Cost-effectiveness analysis. SETTING: Public hospital. PATIENT(S): A population of 121 women (<38 years old), undergoing their first or second IVF cycles. INTERVENTION(S): We conducted a cost-effectiveness analysis using the results of a prospective clinical trial. The women in group 1 received eSET plus eSFET, and those in group 2 received DET. A probabilistic sensitivity analysis was performed. MAIN OUTCOME MEASURE(S): Live birth delivery rate. RESULT(S): The cumulative live birth delivery rate was 38.60% in the eSET+eSFET group versus 42.19% in the DET group. The mean costs per patient were €5,614.11 in the eSET+eSFET group and €5,562.29 in the DET group. These differences were not statistically significant. The rate of multiple gestation was significantly lower in the eSET group than in the DET group (0 vs. 25.9%). CONCLUSION(S): This study does not show that eSET is superior to DET in terms of effectiveness or of costs. The lack of superiority of the results for the eSET+eSFET and the DET groups corroborates that the choice of strategy to be adopted should be determined by the context of the health care system and the individual prognosis.

Phospholipase D2 is involved in the formation of Golgi tubules and ArfGAP1 recruitment.

Lipids and lipid-modifying enzymes play a key role in the biogenesis, maintenance and fission of transport carriers in the secretory and endocytic pathways. In the present study we demonstrate that phosphatidic acid generated by phospholipase D2 (PLD2) is involved in the formation of Golgi tubules. The main evidence to support this is: 1) inhibitors of phosphatidic acid formation and PLD2 depletion inhibit the formation of tubules containing resident enzymes and regulators of intra-Golgi transport in a low temperature (15°C) model of Golgi tubulation but do not affect brefeldin A-induced tubules, 2) inhibition of PLD2 enzymatic activity and PLD2 depletion in cells cultured under physiological conditions (37°C) induce the formation of tubules specifically containing Golgi matrix proteins, and, 3) over-expression of PLD2 induces the formation of a tubular network. In addition, it was found that the generation of this lipid by the isoenzyme is necessary for ArfGAP1 recruitment to Golgi membranes. These results suggest that both proteins are involved in the molecular mechanisms which drive the formation of different types of Golgi tubules.

Direct and indirect generation of waste in the Spanish paper industry.

The paper industry has a relatively high degree of reliance on suppliers when compared to other industries. Exploring the role of the paper industry in terms of consumption of intermediate inputs from other industries may help to understand how the production of paper does not only generate waste by itself but also affects the amount of waste generated by other industries. The product Life Cycle Assessment (LCA) is a useful analytical tool to examine and assess environmental impacts over the entire life cycle of a product "from cradle to grave" but it is costly and time intensive. In contrast, Economic Input Output Life Cycle Assessment Models (IO-LCA) that combine LCA with Input-Output analysis (IO) are more accurate and less expensive, as they employ publicly available data. This paper represents one of the first Spanish studies aimed at estimating the waste generated in the production of paper by applying IO-LCA. One of the major benefits is the derivation of the contribution of direct and indirect suppliers to the paper industry. The results obtained show that there was no direct relationship between the impact on output and the impact on waste generation exerted by the paper industry. The major contributors to waste generation were the mining industry and the forestry industry.

Regulation of ER-Golgi intermediate compartment tubulation and mobility by COPI coats, motor proteins and microtubules.

Little is known about the formation and regulation of endoplasmic reticulum (ER)-Golgi transport intermediates, although previous studies suggest that cargo is the main regulator of their morphology. In this study, we analyze the role of coat protein I (COPI) and cytoskeleton in the formation of tubular ER-Golgi intermediate compartment (ERGIC) and also show that partial COPI detachment by means of low temperature (15 degrees C) or brefeldin A induces the formation of transient tubular ERGIC elements. Most of them moved from the cell periphery to the perinuclear area and were 2.5x slower than vesicles. Time-lapse analysis of living cells demonstrates that the ERGIC elements are able to shift very fast from tubular to vesicular forms and vice versa, suggesting that the amount of cargo is not the determining factor for ERGIC morphology. Both the partial microtubule depolymerization and the inhibition of uncoating of the membranes result in the formation of long tubules that grow from round ERGICs and form at complex network. Interestingly, both COPI detachment and microtubule depolymerization induce a redistribution of kinesin from peripheral ERGIC elements to the Golgi area, while dynein distribution is not affected. However, both kinesin and dynein downregulation by RNA interference induced ERGIC tubulation. The tubules induced by kinesin depletion were static, whereas those resulting from dynein depletion were highly mobile. Our results strongly suggest that the interaction of motor proteins with COPI-coated membranes and microtubules is a key regulator of ERGIC morphology and mobility.

Cytochemical and biochemical evidences for a complex tridimensional structure of the hamster zona pellucida.

Zona pellucida (ZP) is an extracellular matrix that surrounds eggs and pre-implantation embryos and is required for in vivo fertility. A key event in successful fertilization is sperm binding to the surface of the ZP. It has been previously described that the hamster sperm binds mainly the outer region of the ZP which corresponds to the porous region in contact with the cumulus cells. Using ultrastructural cytochemistry approaches with an antibody developed against porcine ZP, this study shows that the pig ZP shares epitopes with some rodent species like hamster, rat and mouse. In the hamster, these epitopes are located mainly in the outer region of the ZP of preovulatory and ovulated oocytes. By means of biochemical approaches it was demonstrated that 1) the antibody is specific for the native hamster ZP3, 2) four different bands with a molecular weight of 67, 60, 48 and 38 kDa after N-linked deglycosylation suggesting that the hamster ZP is formed by four proteins, and 3) the different composition observed in the outer region of the hamster ZP could be due to a specific supramolecular structure that makes some epitopes accessible for the antibodies. In summary, this study provides evidence that the different composition observed in the different regions of the ZP is mediated by a different organization of the components of the ZP produced during the oocyte maturation. This different organization could be responsible for the different sperm binding affinity observed for sperm to the outer region versus the inner region of the ZP.

Heterotypic tubular connections at the endoplasmic reticulum-Golgi complex interface.

Electron microscopy and cryoimmunocytochemistry were used to characterize tubular connections in the secretory pathway using rat spermatids as model. Our results support the existence of a complex tubular network enriched in the Golgi matrix protein GM130 that transiently joins the cis-Golgi side and the endoplasmic reticulum. These tubules occasionally contain the endoplasmic reticulum resident protein PDI but not COPII complexes or KDEL receptor. At the lateral edges of the stacks tubules were seen to connect cisternae belonging to the same or adjacent stacks. These connections were observed in all cisternae but preferentially on the cis side. Giantin, Gos28 and Rab6 were detected in the tubules; importantly, we reported the presence of cis-trans heterotypic connections between cisternae. On the trans-Golgi side, we occasionally observed tubules highly immunoreactive for Rab6 connecting the stack with the forming acrosome. Together, our results support the existence of transient continuities throughout the secretory pathways.

Low temperature (15 degrees C) induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum in HeLa cells.

Low temperature induces a transport blockade at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in cultured cells. Our previous studies support that the primary effect of low temperature is the detachment of COPI complexes from membranes. In the present study, we have used immunofluorescence and cryoimmunoelectron microscopy to investigate the effects of low temperature on both COPII and clathrin coat complexes in HeLa cells. Strikingly, COPII proteins moved from membranes to the cytosol at 15 degrees C, accumulating into electron-dense areas. In agreement with this observation, we also showed that ER exit is delayed in cells cultured at this temperature. In contrast, clathrin coat is not affected. Together, our results demonstrate that low temperature induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum.

Low-temperature-induced Golgi tubules are transient membranes enriched in molecules regulating intra-Golgi transport.

The incubation of HeLa cells at 15 degrees C induces the formation of Golgi tubules, which contain glycosylation enzymes but neither cargo nor matrix proteins. We now show by immunofluorescence and immunoelectron microscopy that these tubules are enriched in a specific set of SNARE and Rab proteins mediating intra-Golgi transport (Gos28, GS15 and Rab6) but excluded others involved in endoplasmic reticulum-Golgi trafficking (Sec22, membrin, Rab 1 and Rab2). In vivo experiments using cyan fluorescent protein-tagged galactosyltransferase showed that most of these tubules are dynamic transient membranes that grow to the cell periphery but then decrease until disappearing into the perinuclear area. Interestingly, in experiments carried out with cells cultured under physiological conditions, Golgi tubules containing Gos28, GS15, Rab6 and glycosylation enzymes and showing in vivo dynamics identical to that detected in low-temperature-cultured cells were observed. Together, our results support that low-temperature-induced tubules may be representatives of the carriers mediating intra-Golgi recycling of enzymes.

Role of sialic acid in bovine sperm-zona pellucida binding.

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.

Structure and dynamics of the Golgi complex at 15 degrees C: low temperature induces the formation of Golgi-derived tubules.

Immunofluorescence and cryoimmunoelectron microscopy were used to examine the morphologic and functional effects on the Golgi complex when protein transport is blocked at the ERGIC (endoplasmic reticulum-Golgi intermediate compartment) in HeLa cells incubated at low temperature (15 degrees C). At this temperature, the Golgi complex showed long tubules containing resident glycosylation enzymes but not matrix proteins. These Golgi-derived tubules also lacked anterograde (VSV-G) or retrograde (Shiga toxin) cargo. The formation of tubules was dependent on both energy and intact microtubule and actin cytoskeletons. Conversely, brefeldin A or cycloheximide treatments did not modify the appearance. When examined at the electron microscope, Golgi stacks were long and curved and appeared connected to tubules immunoreactive to galactosyltransferase antibodies but devoid of Golgi matrix proteins. Strikingly, COPI proteins moved from membranes to the cytosol at 15 degrees C, which could explain the formation of tubules.

Carbohydrate analysis of the zona pellucida and cortical granules of human oocytes by means of ultrastructural cytochemistry.

BACKGROUND: The zona pellucida (ZP), the mammalian oocyte coat, consists of a restricted number of highly glycosylated proteins. In vitro sperm binding studies suggest a higher binding affinity for the outer region of the ZP compared to its inner region in different species. However, the reason for this difference in binding distribution remains unresolved. Many studies suggest that the carbohydrate sequences linked to ZP glycoproteins act as ligands for sperm binding to this matrix. METHODS: Lectins and antibodies that recognize different carbohydrates were employed to perform an ultrastructural analysis of human ZP and cortical granule glycosylation. RESULTS: This study reveals variable glycosylation of the human ZP throughout its thickness, with pronounced differences between the most external and internal regions of this matrix. The binding studies also indicate that ZP glycoproteins express some carbohydrate sequences not previously detected in other species. Finally, cytochemical analysis of human cortical granules suggests similarities in glycosylation to ZP glycoproteins but not to cortical granules from other mammalian species. CONCLUSION: A heterogeneous carbohydrate composition was observed in the thickness of the human ZP that could be responsible for the different sperm binding affinity detected between the outer and inner regions of the ZP.

MAL regulates clathrin-mediated endocytosis at the apical surface of Madin-Darby canine kidney cells.

MAL is an integral protein component of the machinery for apical transport in epithelial Madin-Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.

Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP.

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner.