Food availability aligns with contrasting demographics in populations of an at-risk songbird.

Golden-winged Warblers (Vermivora chrysoptera) have become rare across much of their historic breeding range and response to conservation efforts is variable. Evidence from several recent studies suggests that breeding output is a primary driver explaining responses to conservation and it is hypothesized that differences in food availability may be driving breeding output disparity between two subpopulations of the warbler's Appalachian breeding range. Herein, we studied two subpopulations: central Pennsylvania ("central subpopulation"), where breeding productivity is relatively low, and eastern Pennsylvania ("eastern subpopulation"), where breeding productivity is relatively high. To test the food-availability hypothesis in this system, we measured density of caterpillars, plasma lipid metabolites (triglycerides [TRIG; fat deposition] and glycerol [GLYC; fat breakdown]), body mass of adults males, and acquired body mass data for fledglings at 38 sites managed for nesting habitat. Consistent with our prediction, leaf-roller caterpillar density, the group upon which Golden-winged Warblers specialize, was 45× lower in the central subpopulation than the eastern subpopulation. TRIG concentrations were highest within the eastern subpopulation during breeding grounds arrival. The change in TRIG concentrations from the breeding-grounds-arrival stage to the nestling-rearing stage was subpopulation dependent: TRIG decreased in the eastern subpopulation and was constant in the central subpopulation, resulting in similar concentrations during the nestling-rearing stage. Furthermore, GLYC concentrations were higher in the eastern subpopulation, which suggests greater energy demands in this region. Despite this, adult male warblers in the eastern subpopulation maintained a higher average body mass. Finally, fledgling body mass was 16% greater in the eastern subpopulation than the central subpopulation before and after fledging. Collectively, our results suggest that poor breeding success of Golden-winged Warblers in the central subpopulation could be driven by lower availability of primary prey during the breeding season (leaf-roller caterpillars), and this, in turn, limits their response to conservation efforts.

Assessment of the added value of intravenous gadolinium for knee osteosarcoma resection planning in pediatric and young adult patients.

BACKGROUND AND OBJECTIVES: To assess the impact of Gadolinium-enhanced magnetic resonance imaging (MRI) sequences on Preoperative imaging evaluation and surgical planning parameters for osteosarcoma (OS) of the knee in pediatric and young adult patients. METHODS: Thirty MRI scans of patients with OS about the knee were reviewed by five orthopedic oncologists. Key preoperative parameters (neurovascular bundle involvement, intra-articular tumor extension, extent of intramedullary extension) and surgical plans were evaluated based on non-contrast versus Gd contrast enhanced sequences. Assessment agreement, inter-rater agreement, and intrarater agreement between pre and postcontrast images were evaluated via Kappa statistics. RESULTS: Moderate agreement was seen between non and contrast-enhanced assessment of neurovascular involvement and intra-articular tumor extension. Intrarater reproducibility was substantial for neurovascular bundle involvement (precontrast Kappa: 0.63, postcontrast Kappa: 0.69). Intrarater reproducibility was also substantial for precontrast (Kappa: 0.70) and moderate for postcontrast (Kappa: 0.50) assessment of intra-articular tumor extension. Planned resection length and choice of surgical approach were similar between sequences. The addition of Gd-enhanced sequences improved the inter-rater agreement across collected parameters. CONCLUSIONS: While some findings suggest that contrast enhanced sequences may not significantly alter the assessment of key preoperative planning parameters by orthopedic oncologists, they may help reduce variability among providers with differing experience levels.

High Complication Rate Associated With Arthroscopic Lysis of Adhesions Versus Manipulation Under Anesthesia for Arthrofibrosis After Total Knee Arthroplasty.

BACKGROUND: Arthrofibrosis after total knee arthroplasty (TKA) is often treated by arthroscopic lysis of adhesions (ALAs) or manipulation under anesthesia (MUA). This study compared the 2-year complication rates of ALA and MUA and range-of-motion (ROM) outcomes for ALA, early MUA (<3 months after TKA), and delayed MUA (>3 months after TKA). METHODS: This retrospective cohort study included 425 patients undergoing ALA or MUA after primary TKA from 2001 to 2018. Demographics, clinical variables, and complication rates were collected from clinical records and compared using Student t -tests and Kaplan-Meier log-rank tests. Multivariable logistic regressions were used for adjusted analysis. ROM data were analyzed using fixed and mixed-effects models. RESULTS: ALA patients were younger (55.2 versus 58.9 years, P < 0.001) and underwent surgery later from the index TKA (12 versus 1.9 months, P < 0.001). The Charlson Comorbidity Index was higher in the MUA group. Preoperative ROM was significantly worse in the MUA cohort, but did not differ between groups after the procedure (117°, P = 0.27) or at 2 years. Demographics and ROM outcomes were equivalent between early MUA and delayed MUA ( P = 0.75). The incidence of repeat arthrofibrosis (7.1%) and revision arthroplasty (2.4%) was similar between ALA and MUA cohorts while ALA patients had significantly more surgical site infections (3.8%) compared with MUA patients (0.47%, P = 0.017). DISCUSSION: Equivalent ROM outcomes were seen between ALA, early MUA, and delayed MUA for the treatment of arthrofibrosis after TKA. However, this study demonstrated a markedly higher complication rate, particularly surgical site infection, after ALA, suggesting that MUA may be the preferred option for treating arthrofibrosis at both early and late time points.

A comparison of global mangrove maps: Assessing spatial and bioclimatic discrepancies at poleward range limits.

Mangrove distribution maps are used for a variety of applications, ranging from estimates of mangrove extent, deforestation rates, quantify carbon stocks, to modelling response to climate change. There are multiple mangrove distribution datasets, which were derived from different remote sensing data and classification methods, and so there are some discrepancies among these datasets, especially with respect to the locations of their range limits. We investigate the latitudinal discrepancies in poleward mangrove range limits represented by these datasets and how these differences translate climatologically considering factors known to control mangrove distributions. We compare four widely used global mangrove distribution maps - the World Atlas of Mangroves, the World Atlas of Mangroves 2, the Global Distribution of Mangroves, the Global Mangrove Watch. We examine differences in climate among 21 range limit positions by analysing a set of bioclimatic variables that have been commonly related to the distribution of mangroves. Global mangrove maps show important discrepancies in the position of poleward range limits. Latitudinal differences between mangrove range limits in the datasets exceed 5°, 7° and 10° in western North America, western Australia and northern West Africa, respectively. In some range limit areas, such as Japan, discrepancies in the position of mangrove range limits in different datasets correspond to differences exceeding 600 mm in annual precipitation and > 10 °C in the minimum temperature of the coldest month. We conclude that dissimilarities in mapping mangrove range limits in different parts of the world can jeopardise inferences of climatic thresholds. We expect that global mapping efforts should prioritise the position of range limits with greater accuracy, ideally combining data from field-based surveys and very high-resolution remote sensing data. An accurate representation of range limits will contribute to better predicting mangrove range dynamics and shifts in response to climate change.

Activating alternative transport modes in a multidrug resistance efflux pump to confer chemical susceptibility.

Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds. Previous biophysical studies of the E. coli SMR transporter EmrE suggest that it should also be able to perform proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several previously uncharacterized small-molecule substrates in E. coli, including harmane. In vitro electrophysiology assays demonstrate that harmane binding triggers uncoupled proton flux through EmrE. Assays in E. coli are consistent with EmrE-mediated dissipation of the transmembrane pH gradient as the mechanism underlying the in vivo phenotype of harmane susceptibility. Furthermore, checkerboard assays show this alternative EmrE transport mode can synergize with some existing antibiotics, such as kanamycin. These results demonstrate that it is possible to not just inhibit multidrug efflux, but to activate alternative transport modes detrimental to bacteria, suggesting a strategy to address antibiotic resistance.

A solid-supported membrane electrophysiology assay for efficient characterization of ion-coupled transport.

Transport stoichiometry determination can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transporter behavior under multiple experimental conditions. Solid-supported membrane electrophysiology (SSME) allows multiple recordings of liposomal or membrane samples adsorbed onto a sensor and is sensitive enough to detect transport currents from moderate-flux transporters that are inaccessible to traditional electrophysiology techniques. Here, we use SSME to develop a new method for measuring transport stoichiometry with greatly improved throughput. Using this technique, we were able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx, reproduce the 1H+:2Cl- antiport stoichiometry of CLC-ec1, and confirm loose proton:nitrate coupling for CLC-ec1. Furthermore, we were able to demonstrate quantitative exchange of internal contents of liposomes adsorbed onto SSME sensors to allow multiple experimental conditions to be tested on a single sample. Our SSME method provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

Trees outside forests are an underestimated resource in a country with low forest cover.

Trees outside forests (TOF) are an underrepresented resource in forest poor nations. As a result of their frequent omission from national forest resource assessments and a lack of readily available very-high-resolution remotely sensed imagery, TOF status and characterization has until now, been unknown. Here, we assess the capacity of openly available 10 m ESA Sentinel constellation satellite imagery for mapping TOF extent at the national level in Bangladesh. In addition, we estimate canopy height for TOF using a TanDEM-X DEM. We map 2,233,578 ha of TOF in Bangladesh with a mean canopy height of 7.3 m. We map 31 and 53% more TOF than existing estimates of TOF and forest, respectively. We find TOF in Bangladesh is nationally fragmented as a consequence of agricultural activity, yet is capable of maintaining connectedness between remaining stands. Now, TOF accounting is feasible at the national scale using readily available datasets, enabling the mainstream inclusion of TOF in national forest resource assessments for other countries.

Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers.

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.

Estimates of RelSeq, Mesh1, and SAHMex Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays.

The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3'hydroxyl of (p)ppGpp in a Mn2+ dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from Drosophila melanogaster and human MESH1 also extends to the (p)ppApp purine analogs. More importantly, we developed real time enzymatic assays, coupling ppNpp hydrolysis to NADH oxidation and pppNpp hydrolysis to NADP+ reduction, which facilitate estimation of kinetic constants. Furthermore, by using this assay technique we confirmed TLC observations and also revealed that purified small alarmone hydrolase (SAHMex) from Methylobacterium extorquens displays a strong hydrolase activity toward (p)ppApp but only negligible activity toward (p)ppGpp. In contrast, the substrate specificity of the hydrolase present in catalytically active N-terminal domain of the RSH protein from Streptococcus equisimilis (RelSeq) includes (p)ppGpp but not (p)ppApp. It is noteworthy that the RSH protein from M. extorquens (RSHMex) has been recently shown to synthesize both (p)ppApp and (p)ppGpp.

Global declines in human-driven mangrove loss.

Global mangrove loss has been attributed primarily to human activity. Anthropogenic loss hotspots across Southeast Asia and around the world have characterized the ecosystem as highly threatened, though natural processes such as erosion can also play a significant role in forest vulnerability. However, the extent of human and natural threats has not been fully quantified at the global scale. Here, using a Random Forest-based analysis of over one million Landsat images, we present the first 30 m resolution global maps of the drivers of mangrove loss from 2000 to 2016, capturing both human-driven and natural stressors. We estimate that 62% of global losses between 2000 and 2016 resulted from land-use change, primarily through conversion to aquaculture and agriculture. Up to 80% of these human-driven losses occurred within six Southeast Asian nations, reflecting the regional emphasis on enhancing aquaculture for export to support economic development. Both anthropogenic and natural losses declined between 2000 and 2016, though slower declines in natural loss caused an increase in their relative contribution to total global loss area. We attribute the decline in anthropogenic losses to the regionally dependent combination of increased emphasis on conservation efforts and a lack of remaining mangroves viable for conversion. While efforts to restore and protect mangroves appear to be effective over decadal timescales, the emergence of natural drivers of loss presents an immediate challenge for coastal adaptation. We anticipate that our results will inform decision-making within conservation and restoration initiatives by providing a locally relevant understanding of the causes of mangrove loss.

Highly coupled transport can be achieved in free-exchange transport models.

Secondary active transporters couple the transport of an ion species down its concentration gradient to the uphill transport of another substrate. Despite the importance of secondary active transport to multidrug resistance, metabolite transport, and nutrient acquisition, among other biological processes, the microscopic steps of the coupling mechanism are not well understood. Often, transport models illustrate coupling mechanisms through a limited number of "major" conformations or states, yet recent studies have indicated that at least some transporters violate these models. The small multidrug resistance transporter EmrE has been shown to couple proton influx to multidrug efflux via a mechanism that incorporates both "major" and "minor" conformational states and transitions. The resulting free exchange transport model includes multiple leak pathways and theoretically allows for both exchange and cotransport of ion and substrate. To better understand how coupled transport can be achieved in such a model, we numerically simulate a free-exchange model of transport to determine the step-by-step requirements for coupled transport. We find that only moderate biasing of rate constants for key transitions produce highly efficient net transport approaching a perfectly coupled, stoichiometric model. We show how a free-exchange model can enable complex phenotypes, including switching transport direction with changing environmental conditions or substrates. This research has broad implications for synthetic biology, as it demonstrates the utility of free-exchange transport models and the fine tuning required for perfectly coupled transport.

Development of a novel murine femur fracture and fixation model.

BACKGROUND: Animal models have been used for decades to simulate human fractures in the laboratory setting. Fracture models in mice are attractive because they offer a high volume, relatively low-cost method of investigating fracture healing characteristics. We report on the development of a novel murine femur fracture model that is rapid, reproducible and inexpensive. METHODS: As part of a pilot study to investigate the effects of smoking on fracture healing, fifteen 35-43 g twelve-week old female CD-1 mice underwent a novel surgical protocol using direct visualization of femur fracture creation and fixation. Following surgery, mice were sacrificed at 14 days, 28 days and 42 days. After sacrifice, the femora were analyzed using MicroCT and histology to evaluate progression of healing. RESULTS: Of the 14 mice that survived the surgical procedure (one succumbed to a complication of anesthesia), two lost reduction and did not heal. Histology demonstrated at 14 days 44.1% (SD±2.9%) of callus composed of cartilage. At 28 days there was 19.0% (SD±3.4%) of callus composed of cartilage. At 42 days there was 8.4% (SD±2.6%) callus composed of cartilage (p < 0.005). MicroCT demonstrated that from 14 to 42 days the average callus volume decreased from 101.6 mm3 to 68.2 mm3 while the relative bone volume of callus increased from 14 to 42 days (15%-31%) (p = 0.068). CONCLUSIONS: Our novel fracture and fixation model is an effective, rapid, reproducible and inexpensive method to simulate a fracture in a laboratory setting. Additionally, our model reliably creates a reproducible progression of radiographic and histological bone healing.

Identification of an Alternating-Access Dynamics Mutant of EmrE with Impaired Transport.

Proteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other, resulting in the transport of bound substrates across the membrane. To better understand this process, we sought to identify mutants of the small multidrug resistance transporter EmrE with reduced rates of alternating access. We performed extensive scanning mutagenesis by changing every amino acid residue to Val, Ala, or Gly, and then screening the drug resistance phenotypes of the resulting mutants. We identified EmrE mutants that had impaired transport activity but retained the ability to bind substrate and further tested their alternating access rates using NMR. Ultimately, we were able to identify a single mutation, S64V, which significantly reduced the rate of alternating access but did not impair substrate binding. Six other transport-impaired mutants did not have reduced alternating access rates, highlighting the importance of other aspects of the transport cycle to achieve drug resistance activity in vivo. To better understand the transport cycle of EmrE, efforts are now underway to determine a high-resolution structure using the S64V mutant identified here.

Electronic Modulation of Near-Field Radiative Transfer in Graphene Field Effect Heterostructures.

Manipulating heat flow in a controllable and reversible manner is a topic of fundamental and practical interest. Numerous approaches to perform thermal switching have been reported, but they typically suffer from various limitations, for instance requiring mechanical modulation of a submicron gap spacing or only operating in a narrow temperature window. Here, we report the experimental modulation of radiative heat flow by electronic gating of a graphene field effect heterostructure without any moving elements. We measure a maximum heat flux modulation of 4 ± 3% and an absolute modulation depth of 24 ± 7 mW m-2 V-1 in samples with vacuum gap distances ranging from 1 to 3 µm. The active area in the samples through which heat is transferred is ∼1 cm2, indicating the scalable nature of these structures. A clear experimental path exists to realize switching ratios as large as 100%, laying the foundation for electronic control of near-field thermal radiation using 2D materials.

The C terminus of the bacterial multidrug transporter EmrE couples drug binding to proton release.

Ion-coupled transporters must regulate access of ions and substrates into and out of the binding site to actively transport substrates and minimize dissipative leak of ions. Within the single-site alternating access model, competitive substrate binding forms the foundation of ion-coupled antiport. Strict competition between substrates leads to stoichiometric antiport without slippage. However, recent NMR studies of the bacterial multidrug transporter EmrE have demonstrated that this multidrug transporter can simultaneously bind drug and proton, which will affect the transport stoichiometry and efficiency of coupled antiport. Here, we investigated the nature of substrate competition in EmrE using multiple methods to measure proton release upon the addition of saturating concentrations of drug as a function of pH. The resulting proton-release profile confirmed simultaneous binding of drug and proton, but suggested that a residue outside EmrE's Glu-14 binding site may release protons upon drug binding. Using NMR-monitored pH titrations, we trace this drug-induced deprotonation event to His-110, EmrE's C-terminal residue. Further NMR experiments disclosed that the C-terminal tail is strongly coupled to EmrE's drug-binding domain. Consideration of our results alongside those from previous studies of EmrE suggests that this conserved tail participates in secondary gating of EmrE-mediated proton/drug transport, occluding the binding pocket of fully protonated EmrE in the absence of drug to prevent dissipative proton transport.

Active Radiative Thermal Switching with Graphene Plasmon Resonators.

We theoretically demonstrate a near-field radiative thermal switch based on thermally excited surface plasmons in graphene resonators. The high tunability of graphene enables substantial modulation of near-field radiative heat transfer, which, when combined with the use of resonant structures, overcomes the intrinsically broadband nature of thermal radiation. In canonical geometries, we use nonlinear optimization to show that stacked graphene sheets offer improved heat conductance contrast between "ON" and "OFF" switching states and that a >10× higher modulation is achieved between isolated graphene resonators than for parallel graphene sheets. In all cases, we find that carrier mobility is a crucial parameter for the performance of a radiative thermal switch. Furthermore, we derive shape-agnostic analytical approximations for the resonant heat transfer that provide general scaling laws and allow for direct comparison between different resonator geometries dominated by a single mode. The presented scheme is relevant for active thermal management and energy harvesting as well as probing excited-state dynamics at the nanoscale.

Synovial inflammation plays a greater role in post-traumatic osteoarthritis compared to idiopathic osteoarthritis in the Hartley guinea pig knee.

BACKGROUND: The objective of this study was to evaluate the extent of stromal cell-derived factor-1's (SDF-1) involvement in the pathogenesis of idiopathic versus post-traumatic OA by comparing differences in synovial membrane morphology, SDF-1 synovial fluid (SF) concentrations, and matrix metalloproteinase-13 (MMP-13) SF concentrations. METHODS: Thirty-six 3-month-old Hartley guinea pigs were obtained and divided into 6 groups. Upon sacrifice, India Ink staining was used to evaluate gross morphology, Safranin O/Fast green staining was used to assess cartilage damage, H/E staining was employed to visualize the synovium, and SF samples were obtained for biochemical analyses. Sandwich ELISA was used to quantify the SF concentrations of SDF-1 and MMP-13. RESULTS: 12 month-old, idiopathic OA guinea pigs and 5.5 month-old ACLT animals had comparable cartilage damage when evaluated by the Modified Mankin Score. SDF-1 and MMP-13 concentrations were not statistically different between the two groups. The synovial membrane of the 5.5 month ACLT group had severe synovitis compared to the idiopathic OA group. CONCLUSION: In this study, it was found that synovial inflammation, independent of cartilage morphology, SDF-1 concentration, and MMP-13 concentration, was markedly different between idiopathic and post-traumatic OA. These results highlight the differing morphological and biochemical profiles of post-traumatic versus idiopathic osteoarthritis and calls for a more thorough examination of the sole of the synovial membrane in the pathogenesis of post-traumatic osteoarthritis.

New free-exchange model of EmrE transport.

EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ∆pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux.