RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport.

Rho GTPases are key regulators of the actin-based cytoskeleton; Rab GTPases are key regulators of membrane traffic. We report here that the atypical Rho GTPase family member, RhoBTB3, binds directly to Rab9 GTPase and functions with Rab9 in protein transport from endosomes to the trans Golgi network. Gene replacement experiments show that RhoBTB3 function in cultured cells requires both RhoBTB3's N-terminal, Rho-related domain and C-terminal sequences that are important for Rab9 interaction. Biochemical analysis reveals that RhoBTB3 binds and hydrolyzes ATP rather than GTP. Rab9 binding opens the autoinhibited RhoBTB3 protein to permit maximal ATP hydrolysis. Because RhoBTB3 interacts with TIP47 on membranes, we propose that it may function to release this cargo selection protein from vesicles to permit their efficient docking and fusion at the Golgi.

Conversion of char from pyrolysis of plastic wastes into alternative activated carbons for heavy metal removal.

The valorization of post-consumer mixed plastics in pyrolysis processes represents an abundant reservoir of carbon that can be effectively converted into useful chars. This process not only holds appeal in terms of improving plastic waste concerns but also contributes to the reduction of greenhouse gas emissions, thus aligning with the principles of a circular economy paradigm. In this study, the char produced from the pyrolysis of post-consumer mixed plastic waste has been activated with Na2CO3, KOH, NaOH, and K2CO3 to improve the textural, structural, and composition characteristics, leading to improved adsorption capability. These characteristics were studied by N2 adsorption-desorption isotherms, scanning electron microscopy, elemental and immediate analysis, and X-ray photoelectron spectroscopy. The developed surface area (SBET) was 573, 939, 704 and 592 m2 g-1 for Na2CO3, KOH, NaOH and K2CO3 activated carbons, respectively. These activated chars (ACs) were tested for the adsorption of heavy metals in both synthetic waters containing Pb, Cd, and Cu and industrial wastewater collected at an agrochemical production plant. Na2CO3-AC was the best performing material. The metal uptake in synthetic waters using a batch set-up was 40, 13 and 12 mg g-1 for Pb, Cd and Cu. Experiments in a column set-up using Na2CO3-AC resulted in a saturation time of 290, 16, and 80 min for Pb, Cd, and Cu synthetic waters, respectively, and metal uptakes of 26.8, 4.1, and 7.9 mg g-1, respectively. The agrochemical effluents, containing mainly Cr, Cu, Mn, and Zn were tested in a plug-flow column. The metal uptake notably decreased compared to synthetic water due to a competition effect for active sites.

Promoting the circular economy: Valorization of a residue from industrial char to activated carbon with potential environmental applications as adsorbents.

Pyrolysis of residues enriched with carbon, such as in agroforestry or industrial activities, has been postulated as an emerging technology to promote the production of biofuels, contributing to the circular economy and minimizing waste. However, during the pyrolysis processes a solid fraction residue is generated. This work aims to study the viability of these chars to develop porous carbonaceous materials that can be used for environmental applications. Diverse chars discharged by an industrial pyrolysis factory have been activated with KOH. Concretely, the char residues came from the pyrolysis of olive stone, pine, and acacia splinters, spent residues fuel, and cellulose artificial casings. The changes in the textural, structural, and composition characteristics after the activation process were studied by N2 adsorption-desorption isotherms, scanning electron microscopy, FTIR, elemental analysis, and XPS. A great porosity was developed, SBET within 776-1186 m2 g-1 and pore volume of 0.37-0.59 cm3 g-1 with 70-90% of micropores contribution. The activated chars were used for the adsorption of CO2, leading to CO2 maximum uptakes of 90-130 mg g-1. There was a good correlation between the CO2 uptake with microporosity and oxygenated surface groups of the activated chars. Moreover, their ability to adsorption of contaminants in aqueous solution was also evaluated. Concretely, there was studied the adsorption of aqueous heavy metals, i.e., Cd, Cu, Ni, Pb, and Zn, and organic pollutants of emerging concern such as caffeine, diclofenac, and acetaminophen.

Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble.

Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.

Effect of COVID-19 pandemic on municipal solid waste generation: a case study in Granada city (Spain).

The 2020 coronavirus pandemic has involved a series of political and social measures that have been adapted to the spread of the disease. Apart from the severe effects on health sector, the most effects of the pandemic have been felt in households and day-to-day life. Consequently, COVID-19 has had a significant impact on the generation of not only medical and health care waste but also of municipal solid waste production and composition. In this context, this work studied the implications of COVID-19 for municipal solid waste generation in Granada, Spain. Granada is a city where the economy is based mainly on the services sector, tourism and the University. Therefore, the impact of COVID-19 pandemic has greatly affected the city and it can be analyzed through the municipal solid waste generation. For the study of the incidence of COVID-19 in the generation of waste a period that goes from March 2019 to February 2021 has been chosen. The results show that, in the global calculation, a decrease in the generation of waste in the city is observed in this last year, reaching - 13.8%. Regarding the organic-rest fraction, the decrease in the COVID year represents - 11.7%. However, bulky waste has shown an increase in the COVID year which may be related to higher home furnishings renovation rates than in other years. Finally, glass is the waste flow that best indicates the effect of COVID in the service sector. In leisure areas, a significant reduction in the collection of glass is observed (- 45%). Supplementary Information: The online version contains supplementary material available at 10.1007/s10163-023-01671-2.

Boosted Activity of g-C3N4/UiO-66-NH2 Heterostructures for the Photocatalytic Degradation of Contaminants in Water.

The combination of graphitic carbon nitride and the metal-organic framework UiO-66-NH2 has been developed with the aim to enhance the photocatalytic activity of pure semiconductors. Different proportions of g-C3N4 and UiO-66-NH2 were combined. Complete characterization analysis of the resulting photocatalytic materials was conducted, including N2 adsorption isotherms, XRD, FTIR, STEM-EDX microscopy, DRS-UV-visible, and photoluminescence. The photocatalytic activity was tested in an aqueous solution for the removal of acetaminophen as the target pollutant. From the obtained results, less than 50% of UiO-66-NH2 incorporated in the g-C3N4 structure enhanced the photocatalytic degradation rate of both bare semiconductors. Concretely, 75% of g-C3N4 in the final g-C3N4/UiO-66-NH2 heterostructure led to the best results, i.e., complete acetaminophen elimination initially at 5 mg·L-1 in 2 h with a pseudo-first order rate constant of ca. 2 h-1. The presence of UiO-66-NH2 in the g-C3N4 enhanced the optoelectronic properties, concretely, the separation of the photo-generated charges was improved according to photoluminescence characterization. The better photo-absorption uptake was also confirmed by the determination of the quantum efficiency values of the heterostructure if compared to either pure g-C3N4 or UiO-66-NH2. This photocatalyst with the best activity was further tested at different pH values, with the best degradation rate at a pH close to the pHpzc ~4.15 of the solid. Sequential recycling tests demonstrated that the heterostructure was stable after five cycles of use, i.e., 15 h. A high contribution of photo-generated holes in the process of the degradation of acetaminophen, followed marginally by superoxide radicals, was suggested by scavenger tests.

Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria.

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella-containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane-bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP-bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63-containing late endosomes. Nischarin is recruited to the SCV in a Rab14-dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners--Rac1, Rab14 and Rab9 GTPases--reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.

Use of transmission electron microscopy to identify nanocrystals of challenging protein targets.

The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 µm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as "hits." We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.

A general path for large-scale solubilization of cellular proteins: from membrane receptors to multiprotein complexes.

Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential "druggable" targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format.

Life-Cycle Assessment of the thermal and catalytic pyrolysis over sepiolite of face masks.

Since the start of the global COVID-19 pandemic, extensive quantities of face masks have been used and discarded. Most of these masks end up in landfills, causing a high environmental impact and no benefits. However, there are alternative ways to deal with this waste in a more sustainable way. For example, valorisation of face masks through pyrolysis has received special attention because it offers efficient application to produce a liquid oil that can be used as a diesel substitute and a solid char that can be used as an activated carbon substitute after activation. In this context, this study applies the Life-Cycle Assessment methodology to quantify and analyse the environmental impacts of different treatment scenarios based on the pyrolysis of surgical masks and FFP2 masks. It also compares their environmental performance with the conventional practice of landfilling. The scenarios studied include both thermal and catalytic pyrolysis by using sepiolite, a low-cost material abundant in Spain. Data on the pyrolysis process were obtained from laboratory experiments. It was found that the use of the produced oil as a diesel substitute very significantly reduces the environmental impact in all pyrolysis scenarios. Consequently, the pyrolysis of face masks can reduce the environmental impact caused by the treatment of this waste material. Furthermore, the thermal pyrolysis performs environmentally better than the catalytic pyrolysis. In all scenarios, freshwater ecotoxicity and marine ecotoxicity are the environmental impact categories that cause the highest environmental impact overall.

Coffee-waste templated CeOx/TiO2 nanostructured materials for selective photocatalytic oxidations.

An environmentally friendly solvent-free approach was tested using spent coffee as a biomass sacrificial template for the preparation of TiO2 modified with CeOx. The use of coffee as a template pursues the preparation of a nanostructured heterojunction without the need for a solvent. Two variables were optimized in the synthesis process, i.e. calcination temperature and proportion of CeOx. Firstly, bare coffee-template titania was prepared to explore the effect of the calcination temperature, within 500-650 °C. The anatase phase was obtained up to 600 °C. Higher temperatures, i.e. 650 °C, led to the appearance of rutile (10%) and efficient removal of the sacrificial agent (0.6% residue). The maximum photocatalytic activity in terms of conversion, in the oxidation of benzyl alcohol, was achieved employing the bare coffee-template TiO2 at 650 °C, and it was found comparable to the benchmarked P25. The incorporation of ceria in the solvent-free approach considerably improved photocatalytic benzaldehyde production. No changes in the XRD pattern of TiO2 were appreciated in the presence of ceria due to the low amount added, within 1.5-6.0%, confirmed by XPS as superficial Ce3+/Ce4+. The UV-visible absorption spectra were considerably redshifted in the presence of Ce, reducing the bandgap values of bare titania. An optimum amount of ceria in the structure within 3-0% was found. In this case, the selectivity towards benzaldehyde was ca. 75%, 3 times higher than the selectivity value registered for the benchmarked P25 or the bare prepared TiO2.

The human connection: First evidence of microplastics in remote high mountain lakes of Sierra Nevada, Spain.

Microplastics have become one of the most serious global threats to animal and human health. While their presence has been documented in all Earth water ecosystems, including remote mountain lakes, the observation that the abundance of microplastics is largely different across nearby lakes has rarely been examined. As part of a citizen science initiative, this study analyzed for the first time the abundance of microplastics in the surface of 35 glacial lakes of Sierra Nevada National Park in Southern Spain with the objective of determining the local factors that control their abundance. First, we described the shape, size, color and nature of microplastics. Second, we tested whether the number of microplastics differed between basins and analyzed environmental and morphometrical features of lakes affecting their abundance. We found that microplastics were common in most lakes, with a maximum abundance of 21.3 particles per liter that akin to some of the most microplastic polluted lakes worldwide. Fragments were the predominant shape (59.7%) followed by fibers (38.8%) and very scarce spheres (1.5%). Microplastics were observed for all size-fractions, but the abundance of particles <45 µm was higher, what advocates for the use of low pore-size filters to prevent underestimation of microplastics. While the mean abundance of microplastics did not differ among basins, their quantity was related to the presence of meadows surrounding the lakes. This result indicates that while atmospheric transport of microsplastics may equally reach all basins, differences in microplastics among nearby-lakes has an anthropic origin caused by mountaineers who find lakes with ample meadows much more attractive to visit relative to barren lakes. The staggering number in these remote lakes, headwaters of rivers that feed drinking reservoirs, is a major concern that warrants further investigation and the strict compliance with waste management laws to reduce the harmful impacts of microplastic contamination.

Microplastics and fibers from three areas under different anthropogenic pressures in Douro river.

Sources contributing to specific concentration of microplastics and fibers are still not completely understood. This study aimed at assessing the concentrations of microplastics (2-5000 µm) and fibers (18-5667 µm) in three areas of distinct influences in the Douro river, Porto, Portugal: (i) a countryside area; (ii) a wastewater treatment effluent release zone; and (iii) an area in proximity to a boat dock and maintenance station. Nile Red staining coupled with microscopy allowed the identification of small microplastics (≥2 µm) with a median concentration of the three areas of 231 MP L-1. Most were fragments (69%). Sizes <40 µm were the most abundant (84%). Highest concentrations of microplastics were found near the boat dock/maintenance and lowest in the countryside area. Fibers were mostly natural (non-synthetic, 63%). Highest concentrations of fibers were found in the area influenced by the wastewater effluent, especially of synthetic fibers, and lowest in the countryside area. Concentration of all fibers and synthetic fibers was 46 F L-1 and 6 F L-1, respectively. High concentrations of microplastics and fiber contamination suggest that the wastewater treatment plant effluent and boat dock/maintenance are the likely sources originating hotspot areas.

Effects of distance to the sea and geomorphological characteristics on the quantity and distribution of microplastics in beach sediments of Granada (Spain).

Microplastics became an unprecedented challenge and mapping their contamination all over the world is needed in order to establish baseline levels and identify the polymers in order to enhance adequate legislation and policy. The main objective of this study is to demonstrate the existence of microplastic pollution on three beaches on the coast of Granada (Spain), namely La Herradura, Motril Beach and La Rábita, characterizing the particles and the relationships in their distribution. This may contribute supporting the studies carried out at a national level in accordance with the Directive on Marine Strategy (2008/56/EC). The results showed a greater median concentration of particles/kg of dry sediment in La Herradura (45.0 ±â€¯24.7) than in Motril (31.5 ±â€¯21.5) and La Rábita (22.0 ±â€¯23.2). These data revealed a higher contamination by microplastics in an enclosed bay-type beach (La Herradura) in comparison with open delta-type beaches. The predominant morphologies were microspheres and fragments, with maximum median concentrations of 38.0 ±â€¯23.7 and 6.0 ±â€¯0.7 particles/kg, respectively. The distribution and size of the particles is affected by the geomorphological and sedimentary characteristics of these beaches, which are different from any other in Spain and in the Mediterranean in general. The beaches of Granada showed more microplastic contamination than Greek or Slovenian beaches, but less than other Spanish beaches. In this area of the Mediterranean, the presence of microplastics can be affected by the wind, sea currents or methodological aspects such as the pore size of the filters used. All of these factors were analysed when comparing the beaches of Granada with other Mediterranean beaches. This study shows that there is contamination by microplastics on the beaches of Granada, which have been little explored until now due to the difficult geological and granulometric characteristics, and gives support to other national studies.

Bioinformatic and comparative localization of Rab proteins reveals functional insights into the uncharacterized GTPases Ypt10p and Ypt11p.

A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance.

Dual prenylation is required for Rab protein localization and function.

The majority of Rab proteins are posttranslationally modified with two geranylgeranyl lipid moieties that enable their stable association with membranes. In this study, we present evidence to demonstrate that there is a specific lipid requirement for Rab protein localization and function. Substitution of different prenyl anchors on Rab GTPases does not lead to correct function. In the case of YPT1 and SEC4, two essential Rab genes in Saccharomyces cerevisiae, alternative lipid tails cannot support life when present as the sole source of YPT1 and SEC4. Furthermore, our data suggest that double geranyl-geranyl groups are required for Rab proteins to correctly localize to their characteristic organelle membrane. We have identified a factor, Yip1p that specifically binds the di-geranylgeranylated Rab and does not interact with mono-prenylated Rab proteins. This is the first demonstration that the double prenylation modification of Rab proteins is an important feature in the function of this small GTPase family and adds specific prenylation to the already known determinants of Rab localization.

Effect of torrefaction conditions on greenhouse crop residue: Optimization of conditions to upgrade solid characteristics.

This work investigated the possibility of using a greenhouse crop waste as a fuel, since it is an abundant residue in the Mediterranean area of Spain. The residue is mainly composed by biomass with a little quantity of plastic. The physical and chemical characteristics of the biomass were determined by elemental analysis, proximate analysis, FT-IR, FE-SEM and thermogravimetry. Additionally, a torrefaction process was carried out as a pre-treatment to improve the energy properties of the biomass material. The optimal conditions (time and temperature) of torrefaction were found to be 263°C and 15min using the gain and loss method. Further studies were carried out with the sample prepared with the nearest conditions to the optimal in order to determine the effect of the plastic fraction in the characteristics and torrefaction process of the waste studied.

Kinetics of thermal decomposition of some biomasses in an inert environment. An investigation of the effect of lead loaded by biosorption.

The thermal behavior of some types of raw and lead-polluted biomasses typical in south Spain was studied by non-isothermal thermogravimetry. Experiments were carried out in nitrogen atmosphere at three heating rates (5, 10 and 20°C/min). The results of thermogravimetric tests carried out proved that the presence of lead did not change the main degradation pathways of selected biomass (almond shell (AS) and olive pomace (OP)). However, from a point of view of mass loss, lead-polluted samples showed higher decomposition temperatures and decomposition at higher rate. The determination of activation energies was performed by isoconversional methods of Flynn-Wall-Ozawa (FWO), Kissinger-Akahira-Sunose (KAS) and Friedman (FR). In general, lead-polluted samples showed lower activation energies than raw ones. Then, Coast-Redfern method was applied to determine kinetic function. The kinetic function that seems to determine the mechanism of thermal degradation of main components of all samples was nth order reaction. Finally, a model based on three parallel reactions (for three pseudocomponents) that fit to nth order reactions was evaluated. This model was appropriate to predict the pyrolysis behavior of the raw and lead-polluted samples in all pyrolysis conditions studied.

Genetic analysis of yeast Yip1p function reveals a requirement for Golgi-localized rab proteins and rab-Guanine nucleotide dissociation inhibitor.

Yip1p is the first identified Rab-interacting membrane protein and the founder member of the YIP1 family, with both orthologs and paralogs found in all eukaryotic genomes. The exact role of Yip1p is unclear; YIP1 is an essential gene and defective alleles severely disrupt membrane transport and inhibit ER vesicle budding. Yip1p has the ability to physically interact with Rab proteins and the nature of this interaction has led to suggestions that Yip1p may function in the process by which Rab proteins translocate between cytosol and membranes. In this study we have investigated the physiological requirements for Yip1p action. Yip1p function requires Rab-GDI and Rab proteins, and several mutations that abrogate Yip1p function lack Rab-interacting capability. We have previously shown that Yip1p in detergent extracts has the capability to physically interact with Rab proteins in a promiscuous manner; however, a genetic analysis that covers every yeast Rab reveals that the Rab requirement in vivo is exclusively confined to a subset of Rab proteins that are localized to the Golgi apparatus.

Identification of the novel proteins Yip4p and Yip5p as Rab GTPase interacting factors.

The Rab GTPases are key regulators of membrane traffic. Yip1p is a membrane protein of unknown function that has been reported to interact with the Rabs Ypt1p and Ypt31p. In this study we identify Yif1p, and two unknown open reading frames, Ygl198p and Ygl161p, which we term Yip4p and Yip5p, as Yip1p-related sequences. We demonstrate that the Yip1p-related proteins possess several features: (i) they have a common overall domain topology, (ii) they are capable of biochemical interaction with a variety of Rab proteins in a manner dependent on C-terminal prenylation, and (iii) they share an ability to physically associate with other members of the YIP1 family.