Assessment of natural ventilation strategy to decrease the risk of COVID 19 infection at a rural elementary school.

Natural ventilation in low-budget elementary schools is the main focus to ensure the health and comfort of its occupants, specifically when looking at the global pandemic related to SARS-COV-2. This paper presents an experimental and novel study of natural ventilation in a public elementary school (Los Zumacales), with a particularly low economic budget. The study was carried out during the winter months of the Covid 19 pandemic. The school is located in the rural area of Castilla y León (North-Western Spain) far from high traffic roads. In this study, a methodology of measuring CO2 concentration was applied in nine classrooms in a school. The experimental study shows the level of natural ventilation in each classroom, expressed in Air Changes per Hour (ACH), using the Decay CO2 concentration method. The method is proven by comparing the experimental values of the obtained ACH with those determined by the most powerful methods to achieve appropriate ventilation levels. Thus, ensuring health protection protocol in rural schools, against the COVID 19 pandemic. Harvard guide and Spanish regulations (RITE), two widely recognized methods have been used together with the experimentally obtained standard by Rey et al. Only one classroom showed a value lower than 3 indicating poor ventilation. In this study, the degree of thermal comfort in the nine classrooms were also analyzed according to the EN15251 standard. An average indoor temperature of approximately 19 °C was obtained, and the relative humidity was stable and correct according to Spanish regulations. In addition, the risk of infection in each classroom was estimated following the international method recommended by the federation of European Heating, Ventilation, and Air Conditioning Associations (REHVA). The probability of infection in all the cases studied was less than 14%. Therefore, this study provides a strong response against infections illnesses, such as Covid 19, in educational buildings where economic budgets of their facilities are low in both, maintenance and investment.

Performance analysis of a hybrid ventilation system in a near zero energy building.

In this research paper, an analysis is developed on the performance of a hybrid ventilation system that combines Earth-to-Air Heat eXchangers (EAHX), free cooling and evaporative cooling Air Handling Unit Heat eXchanger (AHU-HX), all being controlled by a Building Management System (BMS) in a net Zero Energy Building (nZEB), called LUCIA. LUCIA nZEB is the first safe-building against Covid-19 in the world, certified by the international organisation WOSHIE, and located in Valladolid, Spain. The main aim is to optimize the performance of the three systems in such a way that the Indoor Air Quality (IAQ) levels remain within the allowable limits, while maximizing the use of natural resources and minimizing energy consumption and carbon emissions. The approach to satisfy the heating and cooling demand and IAQ levels through zero emissions energy systems is developed, thus anticipating the zero-energy target, set by the European Union for 2050. Results showed that the installed hybrid ventilation system uses heat exchangers for 70% of the operational time, in order to achieve the set parameters successfully. Also, the analysis made by monitoring data, have shown that the control and optimal operation of the hybrid ventilation system allows high energy recovery values with minimum additional electricity consumption. Significant reduction of carbon emissions and operational costs have been achieved.

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.

The hallmarks of cell-cell fusion.

Cell-cell fusion is essential for fertilization and organ development. Dedicated proteins known as fusogens are responsible for mediating membrane fusion. However, until recently, these proteins either remained unidentified or were poorly understood at the mechanistic level. Here, we review how fusogens surmount multiple energy barriers to mediate cell-cell fusion. We describe how early preparatory steps bring membranes to a distance of ∼10 nm, while fusogens act in the final approach between membranes. The mechanical force exerted by cell fusogens and the accompanying lipidic rearrangements constitute the hallmarks of cell-cell fusion. Finally, we discuss the relationship between viral and eukaryotic fusogens, highlight a classification scheme regrouping a superfamily of fusogens called Fusexins, and propose new questions and avenues of enquiry.

Reconstitution of Synaptic SNAREs into Large Liposomes with Reduced Curvature Stress.

Liposomes constitute a convenient biochemical model system to investigate mechanistic aspects of the membrane fusion of synaptic vesicles. The proteins responsible for mediating fusion are the SNAREs that belong to a highly conserved family of transmembrane proteins. Reconstituting SNAREs into liposomes using detergents has become a common approach not only to understand how SNAREs work, but also how fusion is regulated by the vast array of accessory proteins present at the presynapse. However, a concern has been that the high curvature stress of the small liposomes (diameters of ~40 nm) frequently used in many studies renders them prone to spontaneous fusion, bringing into question whether the measurements obtained faithfully represent SNARE-mediated fusion. By systematically varying the detergent concentration and characterizing the SNARE-liposome size distributions by light scattering, we describe a detailed procedure to reconstitute SNAREs into large liposomes with considerably reduced curvature stress.

Variable cooperativity in SNARE-mediated membrane fusion.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23-30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.

α-SNAP interferes with the zippering of the SNARE protein membrane fusion machinery.

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane.

Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods.

We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P 2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.

Controlling synaptotagmin activity by electrostatic screening.

Exocytosis of neurosecretory vesicles is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin and SNAP-25, with synaptotagmin functioning as the major Ca(2+) sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca(2+), but only when the target liposomes contain phosphatidylinositol-4,5-bisphosphate and when polyphosphate anions, such as nucleotides or pyrophosphate, are present. Ca(2+)-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis association of synaptotagmin-1 with its own membrane without affecting trans binding. Hence, the balancing of trans- and cis-membrane interactions of synaptotagmin-1 could be a crucial element in the pathway of Ca(2+)-dependent exocytosis.

Membrane fusion intermediates via directional and full assembly of the SNARE complex.

Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction, we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.

Toward a full characterization of native starch: separation and detection by size-exclusion chromatography.

The structure of starch molecules is relevant to nutrition and industrial applications. Size-exclusion chromatography (SEC, also known as GPC) of native starch generally suffers non-satisfactory repeatability and reproducibility of the dissolution and separation. This work combines two polar organic solvents: dimethylsulfoxide for complete dissolution and dimethylacetamide to limit shear degradation. The separation is as repeatable as that of polystyrene standards performing dissolution and separation at 80 degrees C. Successful covalent-labeling on the glucose unit is claimed to be published here for the first time in non-degradative conditions and allows the use of UV detector with significantly higher sensitivity than with a refractometer.

Mechanistic investigation of a starch-branching enzyme using hydrodynamic volume SEC analysis.

Two linear alpha-(1,4)-D-glucans substrates, of degrees of polymerization DP approximately 150 and 6000, were exposed to maize starch-branching enzyme IIa (mSBEIIa) in vitro. The resulting branched alpha-glucans and their constituent chains (obtained by debranching) were analyzed by nuclear magnetic resonance (NMR) and size-exclusion chromatography (SEC). SEC data for the debranched species are presented as chain-length distributions, while those for branched species are presented as hydrodynamic volume distributions (HVDs), which is the most meaningful way to present such data (because SEC separates by size, not molar mass, and a sample of branched polymers with the same size can have a range of molar masses). A rigorous interpretation of the HVDs of the substrate and its branched product show that at least part of the branching is an interchain transfer mechanism in both the short- and long-chain substrate cases. A bimodal HVD of the in vitro branched alpha-glucan derived from the short-chain substrate was observed, and it is postulated that the divergence of the two populations is due to very small chains being unable to undergo branching. In the case of the in vitro branching of the long-chain substrate, the formation of maltohexaose during the reaction and the presence of a monomodal HVD were observed, suggesting a distinct mode of action of mSBEIIa on this substrate. Quantification of the branching level by NMR showed the branched glucans from both substrates had substantial amounts of branching (2.1-4.5%), ascribed to the intrinsic nature of the action of mSBEIIa on the two substrates. It is postulated that differences in the degrees of substrate association affect the pattern of branching catalyzed by the enzyme, and a putative active site structure is proposed based on the appearance of maltohexaose. The molar mass distribution of the constituent chains of the in vitro branched alpha-glucans obtained by isoamylase treatment reveals the transfer of chains of specific size and supports the supposition given in the literature that mSBEIIa is responsible for short-chain branching in amylopectin. It is suggested that hydrodynamic volume SEC analysis should be used as a tool for the mechanistic investigation of SBEs, allowing SEC data of in vitro branched alpha-glucans to be both comparable and quantitative.