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Starting in 2026 Washington State building performance standards will come into effect that require commercial buildings larger than 50,000 sf to meet site energy use intensity targets. To support a state incentive program designed to encourage building owners to start complyingearly, we characterized the building stock energy use of 11 building types, analyzed 43 energy upgrade measures, and developed seven packages of energy upgrade measures using the ComStock energy analysis tool. Each energy upgrade package included from 4 to 17 energymeasures consisting of lighting, HVAC, and envelope upgrades. Package savings were calculated for four priority building types using a sample of 35,000 buildings characterized by four building area bins, three climate zones, and two county types (urban and rural). Of the 28package and building type combinations analyzed, 17 (61%) met or exceeded program energy savings targets and two packages met targets for all four building types.

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe 'autonomous hypermutation yeast surface display' (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large.

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.

Seasonal environmental conditions shape the behavior and life history of virtually all organisms. Climate change is modifying these seasonal environmental conditions, which threatens to disrupt population dynamics. It is conceivable that climatic changes may be beneficial in one season but result in detrimental conditions in another because life-history strategies vary between these time periods. We analyzed the temporal trends in seasonal survival of yellow-bellied marmots (Marmota flaviventer) and explored the environmental drivers using a 40-y dataset from the Colorado Rocky Mountains (USA). Trends in survival revealed divergent seasonal patterns, which were similar across age-classes. Marmot survival declined during winter but generally increased during summer. Interestingly, different environmental factors appeared to drive survival trends across age-classes. Winter survival was largely driven by conditions during the preceding summer and the effect of continued climate change was likely to be mainly negative, whereas the likely outcome of continued climate change on summer survival was generally positive. This study illustrates that seasonal demographic responses need disentangling to accurately forecast the impacts of climate change on animal population dynamics.

In western North America, hummingbirds can be observed systematically visiting flowers that lack the typical reddish color, tubular morphology, and dilute nectar of "hummingbird flowers." Curious about this behavior, we asked whether these atypical flowers are energetically profitable for hummingbirds. Our field measurements of nectar content and hummingbird foraging speeds, taken over four decades at multiple localities, show that atypical flowers can be as profitable as typical ones and suggest that the profit can support 24-h metabolic requirements of the birds. Thus, atypical flowers may contribute to successful migration of hummingbirds, enhance their population densities, and allow them to occupy areas seemingly depauperate in suitable resources. These results illustrate what can be gained by attending to the unexpected.

In western North America, hummingbirds can be observed systematically visiting flowers that lack the typical reddish color, tubular morphology, and dilute nectar of "hummingbird flowers." Curious about this behavior, we asked whether these atypical flowers are energetically profitable for hummingbirds. Our field measurements of nectar content and hummingbird foraging speeds, taken over four decades at multiple localities, show that atypical flowers can be as profitable as typical ones and suggest that the profit can support 24-h metabolic requirements of the birds. Thus, atypical flowers may contribute to successful migration of hummingbirds, enhance their population densities, and allow them to occupy areas seemingly depauperate in suitable resources. These results illustrate what can be gained by attending to the unexpected.

ObjectiveIslets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function.MethodsWe have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies).ResultsBy increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 μm2 area (∼210 μm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time.ConclusionsWe have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research.

Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC90 < 3 μM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzyme A (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence.

The ability to taste phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP) is a polymorphic trait mediated by the TAS2R38 bitter taste receptor gene. It has long been hypothesized that global genetic diversity at this locus evolved under pervasive pressures from balancing natural selection. However, recent high-resolution population genetic studies of TAS2Rs suggest that demographic events have played a critical role in the evolution of these genes. We here utilized the largest TAS2R38 database yet analyzed, consisting of 5,589 individuals from 105 populations, to examine natural selection, haplotype frequencies and linkage disequilibrium to estimate the effects of both selection and demography on contemporary patterns of variation at this locus. We found signs of an ancient balancing selection acting on this gene but no post Out-Of-Africa departures from neutrality, implying that the current observed patterns of variation can be predominantly explained by demographic, rather than selective events. In addition, we found signatures of ancient selective forces acting on different African TAS2R38 haplotypes. Collectively our results provide evidence for a relaxation of recent selective forces acting on this gene and a revised hypothesis for the origins of the present-day worldwide distribution of TAS2R38 haplotypes.

Climate change is altering the timing of life history events in a wide array of species, many of which are involved in mutualistic interactions. Because many mutualisms can form only if partner species are able to locate each other in time, differential phenological shifts are likely to influence their strength, duration and outcome. At the extreme, climate change-driven shifts in phenology may result in phenological mismatch: the partial or complete loss of temporal overlap of mutualistic species. We have a growing understanding of how, when, and why phenological change can alter one type of mutualism-pollination. However, as we show here, there has been a surprising lack of attention to other types of mutualism. We generate a set of predictions about the characteristics that may predispose mutualisms in general to phenological mismatches. We focus not on the consequences of such mismatches but rather on the likelihood that mismatches will develop. We explore the influence of three key characteristics of mutualism: 1) intimacy, 2) seasonality and duration, and 3) obligacy and specificity. We predict that the following characteristics of mutualism may increase the likelihood of phenological mismatch: 1) a non-symbiotic life history in which co-dispersal is absent; 2) brief, seasonal interactions; and 3) facultative, generalized interactions. We then review the limited available data in light of our a priori predictions and point to mutualisms that are more and less likely to be at risk of becoming phenologically mismatched, emphasizing the need for research on mutualisms other than plant-pollinator interactions. Future studies should explicitly focus on mutualism characteristics to determine whether and how changing phenologies will affect mutualistic interactions.

Climate change is altering the timing of life history events in a wide array of species, many of which are involved in mutualistic interactions. Because many mutualisms can form only if partner species are able to locate each other in time, differential phenological shifts are likely to influence their strength, duration and outcome. At the extreme, climate change-driven shifts in phenology may result in phenological mismatch: the partial or complete loss of temporal overlap of mutualistic species. We have a growing understanding of how, when, and why phenological change can alter one type of mutualism-pollination. However, as we show here, there has been a surprising lack of attention to other types of mutualism. We generate a set of predictions about the characteristics that may predispose mutualisms in general to phenological mismatches. We focus not on the consequences of such mismatches but rather on the likelihood that mismatches will develop. We explore the influence of three key characteristics of mutualism: 1) intimacy, 2) seasonality and duration, and 3) obligacy and specificity. We predict that the following characteristics of mutualism may increase the likelihood of phenological mismatch: 1) a non-symbiotic life history in which co-dispersal is absent; 2) brief, seasonal interactions; and 3) facultative, generalized interactions. We then review the limited available data in light of our a priori predictions and point to mutualisms that are more and less likely to be at risk of becoming phenologically mismatched, emphasizing the need for research on mutualisms other than plant-pollinator interactions. Future studies should explicitly focus on mutualism characteristics to determine whether and how changing phenologies will affect mutualistic interactions.

Concern regarding the biological effects of climate change has led to a recent surge in research to understand the consequences of phenological change for species interactions. This rapidly expanding research program is centered on three lines of inquiry: (1) how the phenological overlap of interacting species is changing, (2) why the phenological overlap of interacting species is changing, and (3) how the phenological overlap of interacting species will change under future climate scenarios. We synthesize the widely disparate approaches currently being used to investigate these questions: (1) interpretation of long-term phenological data, (2) field observations, (3) experimental manipulations, (4) simulations and nonmechanistic models, and (5) mechanistic models. We present a conceptual framework for selecting approaches that are best matched to the question of interest. We weigh the merits and limitations of each approach, survey the recent literature from diverse systems to quantify their use, and characterize the types of interactions being studied by each of them. We highlight the value of combining approaches and the importance of long-term data for establishing a baseline of phenological synchrony. Future work that scales up from pairwise species interactions to communities and ecosystems, emphasizing the use of predictive approaches, will be particularly valuable for reaching a broader understanding of the complex effects of climate change on the phenological overlap of interacting species. It will also be important to study a broader range of interactions: to date, most of the research on climate-induced phenological shifts has focused on terrestrial pairwise resource-consumer interactions, especially those between plants and insects.

Concern regarding the biological effects of climate change has led to a recent surge in research to understand the consequences of phenological change for species interactions. This rapidly expanding research program is centered on three lines of inquiry: (1) how the phenological overlap of interacting species is changing, (2) why the phenological overlap of interacting species is changing, and (3) how the phenological overlap of interacting species will change under future climate scenarios. We synthesize the widely disparate approaches currently being used to investigate these questions: (1) interpretation of long-term phenological data, (2) field observations, (3) experimental manipulations, (4) simulations and nonmechanistic models, and (5) mechanistic models. We present a conceptual framework for selecting approaches that are best matched to the question of interest. We weigh the merits and limitations of each approach, survey the recent literature from diverse systems to quantify their use, and characterize the types of interactions being studied by each of them. We highlight the value of combining approaches and the importance of long-term data for establishing a baseline of phenological synchrony. Future work that scales up from pairwise species interactions to communities and ecosystems, emphasizing the use of predictive approaches, will be particularly valuable for reaching a broader understanding of the complex effects of climate change on the phenological overlap of interacting species. It will also be important to study a broader range of interactions: to date, most of the research on climate-induced phenological shifts has focused on terrestrial pairwise resource-consumer interactions, especially those between plants and insects.

Mammalian cell invasion by the protozoan parasite Trypanosoma cruzi involves host cell microtubule dynamics. Microtubules support kinesin-dependent anterograde trafficking of host lysosomes to the cell periphery where targeted lysosome exocytosis elicits remodelling of the plasma membrane and parasite invasion. Here, a novel role for microtubule plus-end tracking proteins (+TIPs) in the co-ordination of T. cruzi trypomastigote internalization and post-entry events is reported. Acute silencing of CLASP1, a +TIP that participates in microtubule stabilization at the cell periphery, impairs trypomastigote internalization without diminishing the capacity for calcium-regulated lysosome exocytosis. Subsequent fusion of the T. cruzi vacuole with host lysosomes and its juxtanuclear positioning are also delayed in CLASP1-depleted cells. These post-entry phenotypes correlate with a generalized impairment of minus-end directed transport of lysosomes in CLASP1 knock-down cells and mimic the effects of dynactin disruption. Consistent with GSK3β acting as a negative regulator of CLASP function, inhibition of GSK3β activity enhances T. cruzi entry in a CLASP1-dependent manner and expression of constitutively active GSK3β dampens infection. This study provides novel molecular insights into the T. cruzi infection process, emphasizing functional links between parasite-elicited signalling, host microtubule plus-end tracking proteins and dynein-based retrograde transport. Highlighted in this work is a previously unrecognized role for CLASPs in dynamic lysosome positioning, an important aspect of the nutrient sensing response in mammalian cells.

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.