The structural organization and protein composition of lens fiber junctions.

The structural organization and protein composition of lens fiber junctions isolated from adult bovine and calf lenses were studied using combined electron microscopy, immunolocalization with monoclonal and polyclonal anti-MIP and anti-MP70 (two putative gap junction-forming proteins), and freeze-fracture and label-fracture methods. The major intrinsic protein of lens plasma membranes (MIP) was localized in single membranes and in an extensive network of junctions having flat and undulating surface topologies. In wavy junctions, polyclonal and monoclonal anti-MIPs labeled only the cytoplasmic surface of the convex membrane of the junction. Label-fracture experiments demonstrated that the convex membrane contained MIP arranged in tetragonal arrays 6-7 nm in unit cell dimension. The apposing concave membrane of the junction displayed fracture faces without intramembrane particles or pits. Therefore, wavy junctions are asymmetric structures composed of MIP crystals abutted against particle-free membranes. In thin junctions, anti-MIP labeled the cytoplasmic surfaces of both apposing membranes with varying degrees of asymmetry. In thin junctions, MIP was found organized in both small clusters and single membranes. These small clusters also abut against particle-free apposing membranes, probably in a staggered or checkerboard pattern. Thus, the structure of thin and wavy junctions differed only in the extent of crystallization of MIP, a property that can explain why this protein can produce two different antibody-labeling patterns. A conclusion of this study is that wavy and thin junctions do not contain coaxially aligned channels, and, in these junctions, MIP is unlikely to form gap junction-like channels. We suggest MIP may behave as an intercellular adhesion protein which can also act as a volume-regulating channel to collapse the lens extracellular space. Junctions constructed of MP70 have a wider overall thickness (18-20 nm) and are abundant in the cortical regions of the lens. A monoclonal antibody raised against this protein labeled these thicker junctions on the cytoplasmic surfaces of both apposing membranes. Thick junctions also contained isolated clusters of MIP inside the plaques of MP70. The role of thick junctions in lens physiology remains to be determined

OPTIMIZING THE USE OF ENERGY STORAGE AS A DEMAND RESPONSE TOOL

The renewable energies expansion over last years, due to the need to bring electricity production towards ever higher levels of green production and the increase of the demand, have brought further stability problems to the main grid. The handling of the integration of these alternative sources and the optimization of the electricity grid have given high attention on the role of demand response program as a key part for the target. The combination of battery storage units with real-time prices is part of the research effort that aims to reduce the instability of the grid and the energy costs of the users. Literature shows good potential for the control strategies as the relative wide range of technologies developed recently for the scope, even if for the residential customers usually the potential is constrained by the limited controllable loads and their significant share of consumption. However, the aspect of user comfort is not always fully considered leading to less realistic conclusions. The objective of the work described in the dissertation was then to obtain a reduction in residential energy costs through the optimal scheduling of user appliances supported by the use of battery storage, under a real-time price scheme, while limiting the discomfort for the customer. Although the first results of applying a real time pricing scheme based on the current variations in price observed in the Iberian wholesale market led only to small profits when not considering additional self-generation, they increased significantly if a small photovoltaic based production is considered, and reached significant cost savings (circa 70%) in periods of high solar generation. But, when applying a real time price following the fluctuations of the renewable energy supply, which produced much higher variations in price, the results improved considerably, reaching cost savings as high as 85%. The implemented model shows the true relevance of Demand Response and Energy Storage, producing meaningful savings if the supply costs change with the availability of renewable energy supply. With self-generation, the obtained value is even higher in the perspective of the individual customer, maximizing the cost-effectiveness of such investment

The Three-Dimensional Distribution of αA-Crystalline in Rat Lenses and Its Possible Relation to Transparency

Lens transparency depends on the accumulation of massive quantities (600–800 mg/ml) of twelve primary crystallines and two truncated crystallines in highly elongated “fiber” cells. Despite numerous studies, major unanswered questions are how this heterogeneous group of proteins becomes organized to bestow the lens with its unique optical properties and how it changes during cataract formation. Using novel methods based on conical tomography and labeling with antibody/gold conjugates, we have profiled the 3D-distribution of the αA-crystalline in rat lenses at ∼2 nm resolutions and three-dimensions. Analysis of tomograms calculated from lenses labeled with anti-αA-crystalline and gold particles (∼3 nm and ∼7 nm diameter) revealed geometric patterns shaped as lines, isosceles triangles and polyhedrons. A Gaussian distribution centered at ∼7.5 nm fitted the distances between the ∼3 nm diameter gold conjugates. A Gaussian distribution centered at ∼14 nm fitted the Euclidian distances between the smaller and the larger gold particles and another Gaussian at 21–24 nm the distances between the larger particles. Independent of their diameters, tethers of 14–17 nm in length connected files of gold particles to thin filaments or clusters to ∼15 nm diameter “beads.” We used the information gathered from tomograms of labeled lenses to determine the distribution of the αA-crystalline in unlabeled lenses. We found that αA-crystalline monomers spaced ∼7 nm or αA-crystalline dimers spaced ∼15 nm center-to-center apart decorated thin filaments of the lens cytoskeleton. It thus seems likely that lost or gain of long-range order determines the 3D-structure of the fiber cell and possible also cataract formation

Functional and Morphological Correlates of Connexin50 Expressed in Xenopus laevis Oocytes

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 ± 3 helices, (b) when their density reached 300–400/μm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1–2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in ∼0.1-μm diameter “coated” and in larger 0.2–0.5-μm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5–40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80,000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60–500 μm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in ∼24 h

Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells

Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Kitchen and Conner (2015). For the underlying methodology development, please refer to Fenton et al. (2010) and Solenov et al. (2004)

High p CO 2 promotes coral primary production

While research on ocean acidification (OA) impacts on coral reefs has focused on calcification, relatively little is known about effects on coral photosynthesis and respiration, despite these being among the most plastic metabolic processes corals may use to acclimatize to adverse conditions. Here, we present data collected between 2016 and 2018 at three natural CO2 seeps in Papua New Guinea where we measured the metabolic flexibility (i.e. in hospite photosynthesis and dark respiration) of 12 coral species. Despite some species-specific variability, metabolic rates as measured by net oxygen flux tended to be higher at high pCO(2) (ca 1200 mu atm), with increases in photosynthesis exceeding those of respiration, suggesting greater productivity of Symbiodiniaceae photosynthesis in hospite, and indicating the potential for metabolic flexibility that may enable these species to thrive in environments with high pCO(2). However, laboratory and field observations of coral mortality under high CO2 conditions associated with coral bleaching suggests that this metabolic subsidy does not result in coral higher resistance to extreme thermal stress. Therefore, the combined effects of OA and global warming may lead to a strong decrease in coral diversity despite the stimulating effect on coral productivity of OA alone