Lighting consumes about 20% to 40% of the total electricity use in large office buildings in China. Commonly in building simulations, static time schedules for typical weekdays, weekends and holidays are assumed to represent the dynamics of lighting energy use in buildings. This approach does not address the stochastic nature of lighting energy use, which can be influenced by occupant behavior in buildings. This study analyzes the main characteristics of lighting energy use over various timescales, based on the statistical analysis of measured lighting energy use data from 15 large office buildings in Beijing and Hong Kong. It was found that in these large office buildings, the 24-hourly variation in lighting energy use was mainly driven by the schedules of the building occupants. Outdoor illuminance levels had little impact on lighting energy use due to the lack of automatic daylighting controls (an effective retrofit measure to reduce lighting energy use) and the relatively small perimeter area exposed to natural daylight. A stochastic lighting energy use model for large office buildings was further developed to represent diverse occupant activities, at six different time periods throughout a day, and also the annual distribution of lighting power across these periods. The model was verified using measured lighting energy use from the 15 buildings. The developed stochastic lighting model can generate more accurate lighting schedules for use in building energy simulations, improving the simulation accuracy of lighting energy use in real buildings.

The serine/threonine protein kinase Akt integrates diverse upstream inputs to regulate cell survival, growth, metabolism, migration, and differentiation. Mounting evidence suggests that Akt activity is differentially regulated depending on its subcellular location, which can include the plasma membrane, endomembrane, and nuclear compartment. This spatial control of Akt activity is critical for achieving signaling specificity and proper physiological functions, and deregulation of compartment-specific Akt signaling is implicated in various diseases, including cancer and diabetes. Understanding the spatial coordination of the signaling network centered around this key kinase and the underlying regulatory mechanisms requires precise tracking of Akt activity at distinct subcellular compartments within its native biological contexts. To address this challenge, new molecular tools are being developed, enabling us to directly interrogate the spatiotemporal regulation of Akt in living cells. These include, for instance, the newly developed genetically encodable fluorescent-protein-based Akt kinase activity reporter (AktAR2), which serves as a substrate surrogate of Akt kinase and translates Akt-specific phosphorylation into a quantifiable change in Förster resonance energy transfer (FRET). In addition, we developed the Akt substrate tandem occupancy peptide sponge (Akt-STOPS), which allows biochemical perturbation of subcellular Akt activity. Both molecular tools can be readily targeted to distinct subcellular localizations. Here, we describe a workflow to study Akt kinase activity at different subcellular locations in living cells. We provide a protocol for using genetically targeted AktAR2 and Akt-STOPS, along with fluorescence imaging in living NIH3T3 cells, to visualize and perturb, respectively, the activity of endogenous Akt kinase at different subcellular compartments. We further describe a protocol for using chemically inducible dimerization (CID) to control the plasma membrane-specific inhibition of Akt activity in real time. Lastly, we describe a protocol for maintaining NIH3T3 cells in culture, a cell line known to exhibit robust Akt activity. In all, this approach enables interrogation of spatiotemporal regulation and functions of Akt, as well as the intricate signaling networks in which it is embedded, at specific subcellular locations. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualizing and perturbing subcellular Akt kinase activity using AktAR and Akt-STOPS Basic Protocol 2: Using chemically inducible dimerization (CID) to control inhibition of Akt at the plasma membrane Support Protocol: Maintaining NIH3T3 cells in culture.

Cell signaling networks are intricately regulated in time and space to determine the responses and fates of cells to different cues. Genetically encodable fluorescent and bioluminescent biosensors enable the direct visualization of these spatiotemporal signaling dynamics within the native biological context, and have therefore become powerful molecular tools whose unique benefits are being used to address challenging biological questions. We first review the basis of biosensor design and remark on recent technologies that are accelerating biosensor development. We then discuss a few of the latest advances in the development and application of genetically encodable fluorescent and bioluminescent biosensors that have led to scientific or technological breakthroughs.

In this paper, we establish a min-max theory for constructing minimal disks with free boundary in any closed Riemannian manifold. The main result is an effective version of the partial Morse theory for minimal disks with free boundary established by Fraser. Our theory also includes as a special case the min-max theory for Plateau problem of minimal disks, which can be used to generalize the famous work by Morse-Thompkins and Shiffman on minimal surfaces in $\mathbf{R}^n$ to the Riemannian setting. More precisely, we generalize the min-max construction of minimal surfaces using harmonic replacement introduced by Colding and Minicozzi to the free boundary setting. As a key ingredient to this construction, we show an energy convexity for weakly harmonic maps with mixed Dirichlet and free boundaries from the half unit $2$-disk in $\mathbf{R}^2$ into any closed Riemannian manifold, which in particular yields the uniqueness of such weakly harmonic maps. This is a free boundary analogue of the energy convexity and uniqueness for weakly harmonic maps with Dirichlet boundary on the unit $2$-disk proved by Colding and Minicozzi.

Nucleorhabdoviruses such as Sonchus yellow net virus (SYNV) replicate in the nuclei and undergo morphogenesis at the inner nuclear membrane (IM) in plant cells. Mature particles are presumed to form by budding of the Matrix (M) protein-nucleocapsid complexes through host IMs to acquire host phospholipids and the surface glycoproteins (G). To address mechanisms underlying nucleorhabdovirus budding, we generated recombinant SYNV G mutants containing a truncated amino-terminal (NT) or carboxyl-terminal (CT) domain. Electron microscopy and sucrose gradient centrifugation analyses showed that the CT domain is essential for virion morphogenesis whereas the NT domain is also required for efficient budding. SYNV infection induces IM invaginations that are thought to provide membrane sites for virus budding. We found that in the context of viral infections, interactions of the M protein with the CT domain of the membrane-anchored G protein mediate M protein translocation and IM invagination. Interestingly, tethering the M protein to endomembranes, either by co-expression with a transmembrane G protein CT domain or by artificial fusion with the G protein membrane targeting sequence, induces IM invagination in uninfected cells. Further evidence to support functions of G-M interactions in virus budding came from dominant negative effects on SYNV-induced IM invagination and viral infections that were elicited by expression of a soluble version of the G protein CT domain. Based on these data, we propose that cooperative G-M interactions promote efficient SYNV budding.

The mechanistic target of rapamycin complex 1 (mTORC1) senses diverse signals to regulate cell growth and metabolism. It has become increasingly clear that mTORC1 activity is regulated in time and space inside the cell, but direct interrogation of such spatiotemporal regulation is challenging. Here, we describe a genetically encoded mTORC1 activity reporter (TORCAR) that exhibits a change in FRET in response to phosphorylation by mTORC1. Co-imaging mTORC1 activity and calcium dynamics revealed that a growth-factor-induced calcium transient contributes to mTORC1 activity. Dynamic activity maps generated with the use of subcellularly targeted TORCAR uncovered mTORC1 activity not only in cytosol and at the lysosome but also in the nucleus and at the plasma membrane. Furthermore, a wide distribution of activities was observed upon growth factor stimulation, whereas leucine ester, an amino acid surrogate, induces more compartmentalized activities at the lysosome and in the nucleus. Thus, mTORC1 activities are spatiotemporally regulated in a signal-specific manner.

Administration of intravenous iron to supplement erythropoiesis stimulating agents (ESAs) has become a common practice in the management of anemia in patients with end-stage renal disease. Randomized clinical trials of anemia correction in this population have shown more adverse outcomes in CKD and ESRD patients assigned to the higher hemoglobin targets. Retrospective analysis of these trials suggests that morbidity is higher in subjects who fail to achieve the designated hemoglobin target and are typically exposed to higher doses of ESAs and iron than those that easily achieve the intended targets. Intravenous iron administration circumvents the natural biologic mechanisms for handling and utilization of iron. There is in vitro and in vivo evidence that intravenous iron preparations can cause oxidative stress, endothelial dysfunction, inflammation, impaired immunity, and renal injury. Since iron overload is known to promote endothelial dysfunction, cardiovascular disease, and immune dysfunction which are the leading causes of premature mortality in CKD and ESRD patients, it is imperative to exercise caution with the use of IV iron preparations in this population. The present review is intended to provide a brief overview of the potential adverse effects of the overzealous use of these agents.