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In the skin-healing field, porcine models are regarded as a useful analogue for human skin due to their numerous anatomical and physiological similarities. Despite the widespread use of porcine models in skin healing studies, the initial origin, recruitment and transition of fibroblasts to matrix-secreting contractile myofibroblasts are not well defined for this model. In this review, we discuss the merit of the pig as an animal for studying myofibroblast origin, as well as the challenges associated with assessing their contributions to skin healing. Although a variety of wound types (incisional, partial thickness, full thickness, burns) have been investigated in pigs in attempts to mimic diverse injuries in humans, direct comparison of human healing profiles with regards to myofibroblasts shows evident differences. Following injury in porcine models, which often employ juvenile animals, myofibroblasts are described in the developing granulation tissue at 4 days, peaking at Days 7-14, and persisting at 60 days post-wounding, although variations are evident depending on the specific pig breed. In human wounds, the presence of myofibroblasts is variable and does not correlate with the age of the wound or clinical contraction. Our comparison of porcine myofibroblast-mediated healing processes with those in humans suggests that further validation of the pig model is essential. Moreover, we identify several limitations evident in experimental design that need to be better controlled, and standardisation of methodologies would be beneficial for the comparison and interpretation of results. In particular, we discuss anatomical location of the wounds, their size and depth, as well as the healing microenvironment (wet vs. moist vs. dry) in pigs and how this could influence myofibroblast recruitment. In summary, although a widespread model used in the skin healing field, further research is required to validate pigs as a useful analogue for human healing with regards to myofibroblasts.

This review is a summary of the most up-to-date knowledge regarding assessment of atmospheric deposition of reactive nitrogen (Nr) pollutants across complex terrain in the UK. Progress in the understanding of the mechanisms and quantification of Nr deposition in areas of complex topography is slow, as no concerted attempts to measure the components of Nr in complex terrain have been made in the last decade. This is likely due to the inherent complexity of the atmospheric processes and chemical interactions which contribute to deposition in these areas. More than 300 studies have been reviewed, and we have consulted with a panel of international experts which we assembled for that purpose. We report here on key findings and knowledge gaps identified regarding measurement and modelling techniques used to quantify deposition of Nr across complex terrain in the UK, which depending on definition, may represent up to 60% of land coverage across Great Britain. The large body of peer reviewed papers, reports and other items reviewed in this study has highlighted both the strengths and weaknesses in the tools available to scientists, regulators and policy makers. This review highlights that there is no coherent global research effort to constrain the uncertainties in Nr deposition over complex terrain, despite the clearly identified risk of N deposition to ecosystems and water quality. All evidence identified that enhanced Nr deposition across complex terrain occurs, and magnitude of the enhancement is not known; however, there are major uncertainties particularly in the differences between modelled and measured wet deposition in complex terrain and representing accurate surface interactions in models. Using simplified estimates for Nr deposition, based on current understanding of current measurement and model approaches, an enhancement across UK complex terrain in the range of a factor of 1.4–2.5 (i.e. 40–150% larger than current estimates) is likely over complex upland terrain. If at the upper limits of this, then significantly more ecosystems in the UK would be at a direct risk of degradation, and the potential for long-term non-remediable water quality issues increased.

Assessing nutrient critical load exceedances requires complete and accurate atmospheric deposition budgets for reactive nitrogen (Nr). The exceedance is the total amount of Nr deposited to the ecosystem in excess of the critical load, which is the amount of Nr input below which harmful effects do not occur. Total deposition includes all forms of Nr (i.e., organic and inorganic) deposited to the ecosystem by wet and dry pathways. Here we present results from the Southern Appalachian Nitrogen Deposition Study (SANDS), in which a combination of measurements and field-scale modeling was used to develop a complete annual Nr deposition budget for a deciduous forest at the Coweeta Hydrologic Laboratory. Wet deposition of ammonium, nitrate, nitrite, and bulk organic N were measured directly. The dry deposited Nr fraction was estimated using a bidirectional resistance-based model driven with speciated measurements of Nr air concentrations (e.g., ammonia, ammonium aerosol, nitric acid, nitrate aerosol, bulk organic N in aerosol, total alkyl nitrates, and total peroxy nitrates), micrometeorology, canopy structure, and biogeochemistry. Total annual deposition was ∼ 6.7 kg N ha−1 yr−1, which is on the upper end of Nr critical load estimates recently developed for similar ecosystems in the nearby Great Smoky Mountains National Park. Of the total (wet + dry) budget, 51.1 % was contributed by reduced forms of Nr (NHx = ammonia + ammonium), with oxidized and organic forms contributing ∼ 41.3 % and 7.6 %, respectively. Our results indicate that reductions in NHx deposition would be needed to achieve the lowest estimates (∼ 3.0 kg N ha−1 yr−1) of Nr critical loads in southern Appalachian forests.

We previously showed that α cell function and gene expression in islets from donors with type 1 diabetes (T1D) was significantly compromised. The reason (s) for these intrinsic α cell changes are unknown but may include loss of α-to-β cell contact, chronic hyperglycemia, and/or repeated hypoglycemic events. To test these concurrent hypotheses, we modeled human T1D islets using α cell-enriched (“T1D-like") human pseudoislets. The synchronous acquisition of intracellular Ca2+ and hormone response showed impaired intracellular Ca2+ signaling and glucagon secretion in T1D-like pseudoislets compared to controls containing both α and β cells. For modeling of chronic hyperglycemia, we transplanted pseudoislets into Nod-SCID-IL2Rγnull; RIP-Diphtheria Toxin Receptor mice made diabetic by diphtheria toxin (DT) -induced β cell depletion. In DT-treated mice, α cells in T1D-like pseudoislets maintained expression of MAFB and ARX but began to express the β cell-enriched transcription factor NKX6.1 (control vs. T1D-like, 3.43±2.49 vs. 54.8±20.8% NKX6.1+ α cells; P=0.046, N=3-5 mice, 2 donors/group) . To mimic repeated hypoglycemic events, we intermittently exposed T1D-like pseudoislets to low glucose (1.7 mM) (3 events x 3hr each) in vitro, using those remaining in basal glucose (5 mM) as control. Following multiple low glucose (MLG) exposures, T1D-like pseudoislets had reduced glucagon secretion in the presence of both 1.7 mM (control vs. MLG, 1.51±0.18 vs. 0.79±0.13 % glucagon content; P=0.011, N=4 donors) and 16.7 mM glucose (control vs. MLG, 0.79±0.13 vs. 0.39±0.% glucagon content; P=0.023, N=4) . Native human islets exposed to MLG had similar changes in glucagon response (control vs. MLG, 1.7mM: 0.63±0.vs. 0.22±0.% glucagon content, P=0.007; 16.7mM: 0.18±0.vs. 0.05±0.% glucagon content, P=0.029; N=4 donors) . These data suggest that loss of α-to-β cell contact and repeated exposure to low glucose impair α cell function. Further, exposure to chronic hyperglycemia may lead to changes in α cell identity state after β cell loss. Disclosure Y.D.Pettway: None. C.Dai: None. T.M.Richardson: None. J.T.Walker: None. R.Aramandla: None. G.Poffenberger: None. A.Bradley: None. A.C.Powers: None. M.Brissova: None.

Different functions are used to account for turbulence strength in the atmospheric boundary layer for different stability regimes. These functions are one of the sources for differences among different atmospheric models' predictions and associated biases. Also, turbulence strength is underrepresented in some of the resistance formulations. To address these issues with dry deposition, firstly we take advantage of three-dimensional (3-D) turbulence information in estimating resistances by proposing and validating a 3-D turbulence velocity scale that is relevant for different stability regimes of boundary layer. Secondly, we hypothesize and validate that friction velocity measured by 3-D sonic anemometer can be effectively replaced by the new turbulence velocity scale multiplied by the von Karman constant. Finally, we (1) present a set of resistance formulations for ozone (O

Clinical and pathologic heterogeneity in type 1 diabetes is increasingly being recognized. Findings in the islets and pancreas of a 22-year-old male with 8 years of type 1 diabetes were discordant with expected results and clinical history (islet autoantibodies negative, hemoglobin A1c 11.9%) and led to comprehensive investigation to define the functional, molecular, genetic, and architectural features of the islets and pancreas to understand the cause of the donor’s diabetes. Examination of the donor’s pancreatic tissue found substantial but reduced β-cell mass with some islets devoid of β cells (29.3% of 311 islets) while other islets had many β cells. Surprisingly, isolated islets from the donor pancreas had substantial insulin secretion, which is uncommon for type 1 diabetes of this duration. Targeted and whole-genome sequencing and analysis did not uncover monogenic causes of diabetes but did identify high-risk human leukocyte antigen haplotypes and a genetic risk score suggestive of type 1 diabetes. Further review of pancreatic tissue found islet inflammation and some previously described α-cell molecular features seen in type 1 diabetes. By integrating analysis of isolated islets, histological evaluation of the pancreas, and genetic information, we concluded that the donor’s clinical insulin deficiency was most likely the result autoimmune-mediated β-cell loss but that the constellation of findings was not typical for type 1 diabetes. This report highlights the pathologic and functional heterogeneity that can be present in type 1 diabetes.

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled >40,000 cells from normal human islets by scRNA-seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells co-expressingARX/MAFB(α cells) andMAFA/MAFB(β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-seq,MAFA/MAFBco-expressing β cells showed enhanced electrophysiological activity. Thus, these results indicate combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.

Waters impounded behind dams (i.e. reservoirs) are important sources of greenhouses gases, especially methane (CH4), but their contribution is not well constrained due to high spatial and temporal variability, limitations in monitoring methods to characterize hot spot and hot moment emissions, and the limited number of studies that investigate diurnal, seasonal, and interannual patterns in emissions. In this study, we investigate the temporal patterns and biophysical drivers of CH4 emissions from Acton Lake, a small eutrophic reservoir, using a combination of methods: eddy covariance monitoring, continuous warm-season ebullition measurements, spatial emission surveys, and measurements of key drivers of CH4 production and emission. We used an artificial neural network to gap-fill the eddy covariance time series and to explore the relative importance of biophysical drivers on the inter-annual timescale. Acton Lake had cumulative areal emission rates of 40.6 ± 5.9 and 71.4 ±  4.2 g CH4 m−2 in 2017 and 2018, respectively, or 97.4 ± 14 and 171 ± 10 Mg CH4 in 2017 and 2018 across the whole 2.4 km2 area of the lake. The main difference between years was a period of elevated emissions lasting less than two weeks in the spring of 2018, which contributed 17 % of the total annual emissions, and was likely due to favourable sediment temperature and algal carbon substrate availability in 2018 compared to 2017. CH4 emissions only displayed diurnal patterns 18.5 % of the monitoring period, suggesting that factors that do not follow a diurnal pattern (e.g. substrate availability) may be driving emissions. Combining spatially extensive measurements with temporally continuous monitoring enabled us to quantify aspects of the spatial and temporal variability in CH4 emission. We found that the relationships between CH4 emissions and sediment T depended on location within the reservoir and observed a clear spatio-temporal offset in maximum CH4 emissions as a function of reservoir depth. These findings suggest a strong spatial pattern in CH4 biogeochemistry within this relatively small (2.4 km2) reservoir. In addressing the need for a better understanding of GHG emissions from reservoirs, there is a trade-off in intensive measurement of one water body versus short-term and/or spatially limited measurements in many water bodies. The insights from multi-year, continuous, spatially extensive studies like this one can be used to inform both the study design and emission upscaling from spatially or temporally limited results, specifically the importance of trophic status and intra-lake variability in assumptions about upscaling CH4 emissions.

This review focuses on the human pancreatic islet—including its structure, cell composition, development, function, and dysfunction. After providing a historical timeline of key discoveries about human islets over the past century, we describe new research approaches and technologies that are being used to study human islets and how these are providing insight into human islet physiology and pathophysiology. We also describe changes or adaptations in human islets in response to physiologic challenges such as pregnancy, aging, and insulin resistance and discuss islet changes in human diabetes of many forms. We outline current and future interventions being developed to protect, restore, or replace human islets. The review also highlights unresolved questions about human islets and proposes areas where additional research on human islets is needed.

Decellularized adipose tissue (DAT) scaffolds represent a promising cell-instructive platform for soft tissue engineering. While recent work has highlighted that mesenchymal stromal cells, including adipose-derived stromal cells (ASCs), can be combined with decellularized scaffolds to augment tissue regeneration, the mechanisms involved require further study. The objective of this work was to probe the roles of syngeneic donor ASCs and host-derived macrophages in tissue remodeling of DAT scaffolds within an immunocompetent mouse model. Dual transgenic reporter mouse strains were employed to track and characterize the donor ASCs and host macrophages within the DAT implants. More specifically, ASCs isolated from dsRed mice were seeded on DAT scaffolds, and the seeded and unseeded control scaffolds were implanted subcutaneously into MacGreen transgenic mice for up to 8 weeks. ASC seeding was shown to augment cell infiltration into the DAT implants at 8 weeks, and this was linked to significantly enhanced angiogenesis relative to the unseeded controls. Immunohistochemical staining demonstrated long-term retention of the syngeneic donor ASCs over the duration of the 8-week study, providing evidence that the DAT scaffolds are a cell-supportive delivery platform. Notably, newly formed adipocytes within the DAT implants were not dsRed

Nitrogen loss from fertilized turf has been a concern for decades, with most research focused on inorganic (NO 3 − ) leaching. The present work examined both inorganic and organic N species in leachate and soil N 2 O emissions from intact soil cores of a bermudagrass chronosequence (1, 15, 20, and 109 years old) collected in both winter and summer. Measurements of soil N 2 O emissions were made daily for 3 weeks, while leachate was sampled once a week. Four treatments were established to examine the impacts of fertilization and temperature: no N, low N at 30 kg N ha −1 , and high N at 60 kg N ha −1 , plus a combination of high N and temperature (13 °C in winter or 33 °C in summer compared to the standard 23 °C). Total reactive N loss generally showed a “cup” pattern of turf age, being lowest for the 20 years old. Averaged across all intact soil cores sampled in winter and summer, organic N leaching accounted for 51% of total reactive N loss, followed by inorganic N leaching at 41% and N 2 O-N efflux at 8%. Proportional loss among the fractions varied with grass age, season, and temperature and fertilization treatments. While high temperature enhanced total reactive N loss, it had little influence on the partitioning of loss among dissolved organic N, inorganic N and N 2 O-N when C availability was expected to be high in summer due to rhizodeposition and root turnover. This effect of temperature was perhaps due to higher microbial turnover in response to increased C availability in summer. However when C availability was low in winter, warming might mainly affect microbial growth efficiency and therefore partitioning of N. This work provides a new insight into the interactive controls of warming and substrate availability on dissolved organic N loss from turfgrass systems.

This study investigates the composition of organic particulate matter in PM2.5 in a remote montane forest in the southeastern US, focusing on the role of organic nitrogen (N) in sulfur-containing secondary organic aerosol (nitrooxy-organosulfates) and aerosols associated with biomass burning (nitro-aromatics). Bulk water-soluble organic N (WSON) represented ∼ 14 % w∕w of water-soluble total N (WSTN) in PM2.5 on average across seasonal measurement campaigns conducted in the spring, summer, and fall of 2015. The largest contributions of WSON to WSTN were observed in spring ( ∼ 18 % w∕w) and the lowest in the fall ( ∼ 10 % w∕w). On average, identified nitro-aromatic and nitrooxy-organosulfate compounds accounted for a small fraction of WSON, ranging from ∼ 1 % in spring to ∼ 4 % in fall, though were observed to contribute as much as 28 % w∕w of WSON in individual samples that were impacted by local biomass burning. The highest concentrations of oxidized organic N species occurred during summer (average of 0.65 ng N m−3) along with a greater relative abundance of higher-generation oxygenated terpenoic acids, indicating an association with more aged aerosol. The highest concentrations of nitro-aromatics (e.g., nitrocatechol and methyl-nitrocatechol), levoglucosan, and aged SOA tracers were observed during fall, associated with aged biomass burning plumes. Nighttime nitrate radical chemistry is the most likely formation pathway for nitrooxy-organosulfates observed at this low NOx site (generally x conditions, was the most abundant individual organosulfate. Concentration-weighted average WSON ∕ WSOC ratios for nitro-aromatics + organosulfates + terpenoic acids were 1 order of magnitude lower than the overall aerosol WSON ∕ WSOC ratio, indicating the presence of other uncharacterized higher-N-content species. Although nitrooxy-organosulfates and nitro-aromatics contributed a small fraction of WSON, our results provide new insight into the atmospheric formation processes and sources of these largely uncharacterized components of atmospheric organic N, which also helps to advance the atmospheric models to better understand the chemistry and deposition of reactive N.

Increasing air temperature is expected to extend growing season length in temperate, broadleaf forests, leading to potential increases in evapotranspiration and net carbon uptake. However, other key processes affecting water and carbon cycles are also highly temperature-dependent. Warmer temperatures may result in higher ecosystem carbon loss through respiration and higher potential evapotranspiration through increased atmospheric demand for water. Thus, the net effects of a warming planet are uncertain and highly dependent on local climate and vegetation. We analyzed five years of data from the Coweeta eddy covariance tower in the southern Appalachian Mountains of western North Carolina, USA, a highly productive region that has historically been underrepresented in flux observation networks. We examined how leaf phenology and climate affect water and carbon cycling in a mature forest in one of the wettest biomes in North America. Warm temperatures in early 2012 caused leaf-out to occur two weeks earlier than in cooler years and led to higher seasonal carbon uptake. However, these warmer temperatures also drove higher winter ecosystem respiration, offsetting much of the springtime carbon gain. Interannual variability in net carbon uptake was high (147 to 364 g C m(−2) y(−1)), but unrelated to growing season length. Instead, years with warmer growing seasons had 10% higher respiration and sequestered ~40% less carbon than cooler years. In contrast, annual evapotranspiration was relatively consistent among years (coefficient of variation = 4%) despite large differences in precipitation (17%, range = 800 mm). Transpiration by the evergreen understory likely helped to compensate for phenologically-driven differences in canopy transpiration. The increasing frequency of high summer temperatures is expected to have a greater effect on respiration than growing season length, reducing forest carbon storage.

A member of the lectin family, galectin-3 is a 250 amino-acid protein that contains a C-terminus carbohydrate recognition domain (CRD) that recognizes β-galactosides. Considered to have certain common properties associated with matricellular proteins, galectin-3 is expressed in the dermis and epidermis in healthy skin and is upregulated in skin healing, peaking at day 1 post wounding in mice. Galectin-3 has been implicated in several processes central to the wound healing response, specifically in the regulation of inflammation, macrophage polarization, angiogenesis, fibroblast to myofibroblast transition and re-epithelialization. However, it appears that many of the effects of Galectin-3 are highly tissue specific and context dependent. Genetic deletion of galectin-3 shows different effects in skin compared to lung, heart, and kidney remodeling. In this review, we will compare galectin-3 functions in these tissues. Furthermore, we will discuss, based on its identified regulation of cell processes, whether in an exogenous form, galectin-3 could represent a novel therapeutic for impaired skin healing.

Land application of compost has been a promising remediation strategy for soil health and environmental quality, but substantial emissions of greenhouse gases, especially N2O, need to be controlled during making and using compost of high N-load wastes, such as chicken manure. Biochar as a bulking agent for composting has been proposed as a novel approach to solve this issue, due to large surface area and porosity, and thus high ion exchange and adsorption capacity. Here, we compared the impacts of biochar-chicken manure co-compost (BM) and chicken manure compost (M) on soil biological properties and processes in a 120-d microcosm experiment at the soil moisture of 60% water-filled pore space. Our results showed that BM and M addition significantly enhanced soil total C and N, inorganic and KCl-extractable organic N, microbial biomass C and N, cellulase enzyme activity, abundance of N2O-producing bacteria and fungi, and gas emissions of N2O and CO2. However, compared to the M treatment, BM significantly reduced soil CO2 and N2O emissions by 35% and 27%, respectively, over the experimental period. The 15N-N2O site preference, i.e., difference between 15N-N2O in the center position (δ15Nα) and the end position (δ15Nβ), was ~17‰ for M and ~26‰ for BM during the first week of incubation, suggesting that BM suppressed N2O from bacterial denitrification and/or nitrifier denitrification. This inference was well aligned with the observation that soil glucosaminidase activity and nirK gene abundance were lower in BM than M treatment. Further, soil peroxidase activity was greater in BM than M treatment, implying soil organic C was more stable in BM treatment. Our data demonstrated that the biochar-chicken manure co-compost could substantially reduce soil N2O emissions compared to chicken manure compost, via controls on soil organic C stabilization and the activities of microbial functional groups, especially bacterial denitrifiers.

Selective inhibitors of sodium glucose cotransporter-2 (SGLT2) are widely used for the treatment of type 2 diabetes and act primarily to lower blood glucose by preventing glucose reabsorption in the kidney. However, it is controversial whether these agents also act on the pancreatic islet, specifically the α cell, to increase glucagon secretion. To determine the effects of SGLT2 on human islets, we analyzed SGLT2 expression and hormone secretion by human islets treated with the SGLT2 inhibitor dapagliflozin (DAPA) in vitro and in vivo. Compared to the human kidney, SLC5A2 transcript expression was 1600-fold lower in human islets and SGLT2 protein was not detected. In vitro, DAPA treatment had no effect on glucagon or insulin secretion by human islets at either high or low glucose concentrations. In mice bearing transplanted human islets, 1 and 4 weeks of DAPA treatment did not alter fasting blood glucose, human insulin, and total glucagon levels. Upon glucose stimulation, DAPA treatment led to lower blood glucose levels and proportionally lower human insulin levels, irrespective of treatment duration. In contrast, after glucose stimulation, total glucagon was increased after 1 week of DAPA treatment but normalized after 4 weeks of treatment. Furthermore, the human islet grafts showed no effects of DAPA treatment on hormone content, endocrine cell proliferation or apoptosis, or amyloid deposition. These data indicate that DAPA does not directly affect the human pancreatic islet, but rather suggest an indirect effect where lower blood glucose leads to reduced insulin secretion and a transient increase in glucagon secretion.

Posttransplantation diabetes mellitus (PTDM) is a common and significant complication related to immunosuppressive agents required to prevent organ or cell transplant rejection. To elucidate the effects of 2 commonly used agents, the calcineurin inhibitor tacrolimus (TAC) and the mTOR inhibitor sirolimus (SIR), on islet function and test whether these effects could be reversed or prevented, we investigated human islets transplanted into immunodeficient mice treated with TAC or SIR at clinically relevant levels. Both TAC and SIR impaired insulin secretion in fasted and/or stimulated conditions. Treatment with TAC or SIR increased amyloid deposition and islet macrophages, disrupted insulin granule formation, and induced broad transcriptional dysregulation related to peptide processing, ion/calcium flux, and the extracellular matrix; however, it did not affect regulation of β cell mass. Interestingly, these β cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced β cell dysfunction and partially prevented SIR-induced β cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human β cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.

SUMMARYG-protein-coupled-receptors (GPCRs) modulate insulin secretion from β cells and glucagon secretion from α cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. We studied the Gi and Gq GPCR pathways by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles and showed that the dual effects for Gq signaling occur through changes in intracellular Ca2+. By combining pseudoislets with a microfluidic system, we co-registered intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells.

© 2019, Mary Ann Liebert, Inc., publishers 2019. Impaired skin wounds represent a significant and growing complication associated with type II diabetes and effective clinical strategies to reproducibly induce wound resolution do not exist. As a class of secreted nonstructural matrix proteins that modulate many cellular aspects of skin healing, matricellular proteins could represent a novel mechanism to promote skin healing. Based on their role in the regulation of the proliferative phase of healing, we hypothesized that local delivery of periostin and CCN2 could suppress inflammation and progress wounds into the proliferative and remodeling phases of repair. Using electrospinning, scaffolds composed of collagen type I and periostin or CCN2 were fabricated. Delivery of periostin/collagen and CCN2/collagen electrospun scaffolds increased excisional wound closure rates in a murine db/db diabetic model compared with collagen alone or untreated wounds. Reduced neutrophil infiltration was evident in the presence of periostin/collagen and CCN2/collagen scaffolds, concomitant with an increase in mesenchymal cell infiltration versus empty and collagen scaffolds alone at day 7. Microarray analysis demonstrated scaffold-dependent upregulation of gene clusters associated with wound contraction, cell differentiation, and suppression of PPARγsignaling at day 7; however, no changes in mRNA levels of extracellular matrix genes were observed compared with controls. At day 11, significantly increased vascularization of the wound bed was evident. Local delivery of periostin- and CCN2-based biomaterials may represent a novel strategy to induce the proliferative phase of repair and correction of the healing process in impaired skin wounds. Nonhealing skin wounds remain a significant burden on health care systems, with diabetic patients 20 times as likely to undergo a lower extremity amputation due to impaired healing. Novel treatments that suppress the proinflammatory signature and induce the proliferative and remodeling phases are needed clinically. We demonstrate that the addition of periostin and CCN2 in a scaffold form increases closure rates of full-thickness skin wounds in diabetic mice, concomitant with enhanced angiogenesis. Our results demonstrate the efficacy of periostin- and CCN2-containing biomaterials to stimulate wound closure, which could represent a novel method for the treatment of diabetic skin wounds.

Frameworks for limiting ecosystem exposure to excess nutrients and acidity require accurate and complete deposition budgets of reactive nitrogen (Nr). While much progress has been made in developing total Nr deposition budgets for the U.S., current budgets remain limited by key data and knowledge gaps. Analysis of National Atmospheric Deposition Program Total Deposition (NADP/TDep) data illustrates several aspects of current Nr deposition that motivate additional research. Averaged across the continental U.S., dry deposition contributes slightly more (55%) to total deposition than wet deposition and is the dominant process (> 90%) over broad areas of the Southwest and other arid regions of the West. Lack of dry deposition measurements imposes a reliance on models, resulting in a much higher degree of uncertainty relative to wet deposition which is routinely measured. As nitrogen oxide (NO(x)) emissions continue to decline, reduced forms of inorganic nitrogen (NH(x) = NH(3) + NH(4)(+)) now contribute > 50% of total Nr deposition over large areas of the U.S. Expanded monitoring and additional process-level research are needed to better understand NH(x) deposition, its contribution to total Nr deposition budgets, and the processes by which reduced N deposits to ecosystems. Urban and suburban areas are hotspots where routine monitoring of oxidized and reduced Nr deposition is needed. Finally, deposition budgets have incomplete information about the speciation of atmospheric nitrogen; monitoring networks do not capture important forms of Nr such as organic nitrogen. Building on these themes, we detail the state of the science of Nr deposition budgets in the U.S. and highlight research priorities to improve deposition budgets in terms of monitoring and flux measurements, leaf- to regional-scale modeling, source apportionment, and characterization of deposition trends and patterns.

Determination of the amount of reactive nitrogen (Nr) deposition in excess of the ecosystem critical load (CL) requires an estimate of total deposition. Because the CL exceedance is used to inform policy decisions, uncertainty in both the CL and the exceedance itself must be understood. In this paper we review the state of the science with respect to the sources of uncertainty in total Nr deposition budgets used for CLs assessments in North America and put forth recommendations for research and monitoring to improve deposition measurements and models. In the absence of methods to rigorously quantify uncertainty in total Nr deposition, a simple weighted deposition uncertainty metric (WDUM) is introduced as a tool for scientists and decision makers to use in assessing CL exceedances. Maps of the WDUM applied to National Atmospheric Deposition Program (NADP) Total Deposition (TDep) estimates show greater uncertainty in areas of the U.S. where dry deposition makes a larger contribution to the deposition budget, particularly ammonia (NH3) in agricultural areas and oxidized nitrogen (NOx) in urban areas. Organic N deposition is an important source of uncertainty over much of the U.S. Our analysis illustrates how the WDUM can be used to assess spatial patterns of deposition uncertainty and inform actions to improve deposition budgets for CL assessments at the local scale.

Efforts to advance our understanding of human islet biology are hindered by a paucity of approaches to effectively manipulate gene expression in human islets. To address this, we developed a pseudoislet system of human islet dispersion and reaggregation via modified hanging drop with 2500 cells/drop to generate 150-200 μm diameter pseudoislets (n=9 normal donors; 26-73 years). Pseudoislet morphology, dithizone uptake, and expression of markers of α and/or β cell identity (PDX1, NKX6.1, ARX, MAFB, NKX2.2, and PAX6) were similar to native islets; in paired comparisons from the same donor, pseudoislets had slightly fewer β cells and more α cells (53% β, 44% α, 3% δ cells) than native islets (62% β, 35% α, 3% δ cells; p90% β cell transduction) formed functioning pseudoislets similar to untransduced controls. Using this approach, we virally expressed hM3Dq, a Gq-coupled designer receptor exclusively activated by a designer drug (DREADD), which selectively responds to the otherwise inert ligand clozapine-n-oxide (CNO) and signals through PLC/IP3. In low glucose (2mM G + 10μM CNO), hM3Dq pseudoislets secreted more insulin than control mCherry pseudoislets (19.2 vs. 1.0 ng/100IEQ/hr; p0.05), indicating that activation of Gq signaling does not potentiate insulin secretion in high glucose. These data demonstrate how manipulation of gene expression in pseudoislets can provide insight into human islet biology and suggest that activation of Gq signaling in β cells elicits robust insulin secretion that appears to be uncoupled from glucose metabolism. Disclosure J.T. Walker: None. R. Haliyur: None. R. Aramandla: None. G. Poffenberger: None. H.A. Nelson: None. J. Luchsinger: None. P. Wang: None. A. Garcia-Ocaña: None. M. Brissova: None. A.C. Powers: None.

Single cell RNA-sequencing (scRNA-seq) approaches are continuously improving at the point of data collection and analysis to simultaneously profile all cell types within islet microorgans to better understand heterogeneity and phenotype-function relationships and how these change with aging and disease. To better understand the advantages and limitations of scRNA-seq, we used the ChromiumTMplatform to profile human islets with a robust insulin and glucagon secretion by perifusion (5 nondiabetic donors, ages 14-66 years) and compared these results to scRNA-seq analysis of purified α and β cells. In context of the same donor (n=2), purified α and β cells (using NTPDase3 marker) showed high correlation (r=0.99) with the Seurat-identified α and β subsets originating from dispersed whole islets. Based on the analysis of a very large pool of single cells (79,881 cells; 2316 genes/cell), the Seurat-guided analysis identified cell types that segregated into eight different clusters (17% β, 66% α, 4% δ, 4% ductal, 4% mesenchymal, 2% acinar, 2% endothelial, and 1% immune). Interstingly, in this large single cell context, we did not identify the four β cell subsets previously reported on the basis of alternative cell surface markers, but found a β cell cluster with highly expressed ER stress-associated genes and an α cell cluster with higher cell cycle regulation marker expression. We concurrently investigated expression of functionally important MAFA and MAFB genes, which are expressed only in a fraction of β cells by histological analysis, and found that PERK-mediated UPR transcripts were enriched in the MAFB-expressing β cells, while the MAFA-expressing β cells differentially expressed genes associated with β cell function. Overall, our study indicates high fidelity of α and β cell clustering in dispersed and sorted islet cells and points to a different pattern of β cell heterogeneity when β cells are purified using NTPDase3 expression compared to other cell surface markers. Disclosure S. Shrestha: None. D.C. Saunders: None. J.T. Walker: None. R. Haliyur: None. G. Poffenberger: None. R. Aramandla: None. A. Jones: None. N. Prasad: None. S.E. Levy: None. A.C. Powers: None. M. Brissova: None.

With the goal of establishing a new clinically-relevant bioscaffold format to enable the delivery of high densities of human adipose-derived stromal cells (ASCs) for applications in soft tissue regeneration, a novel "cell-assembly" method was developed to generate robust 3-D scaffolds comprised of fused networks of decellularized adipose tissue (DAT)-derived beads. In vitro studies confirmed that the assembly process was mediated by remodelling of the extracellular matrix by the seeded ASCs, which were well distributed throughout the scaffolds and remained highly viable after 8 days in culture. The ASC density, sulphated glycosaminoglycan content and scaffold stability were enhanced under culture conditions that included growth factor preconditioning. In vivo testing was performed to compare ASCs delivered within the cell-assembled DAT bead foams to an equivalent number of ASCs delivered on a previously-established pre-assembled DAT bead foam platform in a subcutaneous implant model in athymic nude mice. Scaffolds were fabricated with human ASCs engineered to stably co-express firefly luciferase and tdTomato to enable long-term cell tracking. Longitudinal bioluminescence imaging showed a significantly stronger signal associated with viable human ASCs at timepoints up to 7 days in the cell-assembled scaffolds, although both implant groups were found to retain similar densities of human ASCs at 28 days. Notably, the infiltration of CD31+ murine endothelial cells was enhanced in the cell-assembled implants at 28 days. Moreover, microcomputed tomography angiography revealed that there was a marked reduction in vascular permeability in the cell-assembled group, indicating that the developing vascular network was more stable in the new scaffold format. Overall, the novel cell-assembled DAT bead foams represent a promising platform to harness the pro-regenerative paracrine functionality of human ASCs and warrant further investigation as a clinically-translational approach for volume augmentation.

A coupled photosynthesis-stomatal conductance model with single layer sunlit and shaded leaf canopy scaling is developed for the Pleim-Xiu land surface model (LSM) option in the meteorology and air quality modeling system - WRF/CMAQ (Weather Research and Forecast model and Community Multiscale Air Quality model). The photosynthesis-based model for the PX LSM (PX PSN) is implemented and evaluated in a diagnostic box model that has evapotranspiration and ozone deposition components taken directly from WRF/CMAQ. We evaluate PX PSN for latent heat (LH) estimation at four FLUXNET sites with different vegetation types and landscape characteristics and at one FLUXNET site with ozone flux measurements against the simple Jarvis approach used in the current PX LSM. Overall, the PX PSN simulates LH as well as the PX Jarvis approach. The PX PSN, however, shows distinct advantages over the PX Jarvis approach on grassland that likely results from its treatment of C3 and C4 plants for CO2 assimilation estimation. Simulations using Moderate Resolution Imaging Spectroradiometer (MODIS) LAI rather than LAI observations assess how the model would perform with the grid averaged data available in the Eulerian grid model (WRF/CMAQ). While MODIS LAI generally follows the seasonality of the observed LAI, it cannot capture the extreme highs and lows of the site measurements. MODIS LAI estimates degrade model performance at all sites but one site having old and tall trees. Ozone deposition velocity and ozone flux along with LH are simulated especially well by PX PSN as compared to significant PX Jarvis overestimation.

Isotopocule analysis by quantum cascade laser spectrometry (QCL) is a promising approach for in situ , real-time tracking of the biological sources of soil N 2 O emissions. However, background atmospheric N 2 O is an important source of variability in the measurement of isotopocule ratios (i.e., 14 N 15 N 16 O/ 14 N 14 N 16 O and 15 N 14 N 16 O/ 14 N 14 N 16 O) of gas samples. Here, a method based on Keeling plot for determining the intramolecular 15 N distribution in N 2 O is introduced. The sensitivity and reliability of this method are examined against N 2 O of known 15 N site preference (SP), and N 2 O produced from fungal and bacterial isolates, soils with different moisture contents and organic amendments, and a soil chamber under field conditions. The isotopocules of N 2 O determined by standard gases supported that the Keeling plot method was more reliable than the averaging method. Using this method, SP of N 2 O was greater in fungal than bacterial denitrifiers, as well as in soil at 60% water filled pore space (WFPS) than 80–100% WFPS. This method also determined that the SP of N 2 O was distinct between soils of different substrate complexity. Further, we observed a N 2 O SP between −18.9‰ and 2.2% in a soil flux chamber deployed in a corn field after 2 d of rainfalls that is consistent with the SP of N 2 O produced from bacterial denitrification and nitrifier denitrification. Our data demonstrate that this Keeling plot method provides accurate discrimination of biological sources when N 2 O is analyzed by the QCL system.

During tissue healing, dynamic and temporal alterations occur in the structure and composition of the extracellular matrix (ECM) that are required for effective repair to occur. Matricellular proteins (MPs) are a group of diverse non-structural ECM components, which bind cell surface receptors mediating interactions between the cell and its microenviroment, effectively regulating adhesion, migration, proliferation, signaling and cell phenotype. Periostin (Postn), a pro-fibrogenic secreted glycoprotein, was defined as a MP based on its expression pattern and regulatory roles during development, healing and in disease processes. Postn consists of a typical signal sequence, an EMI domain responsible for binding to fibronectin, four tandem fasciclin-like domains that are responsible for integrin binding and a C-terminal region where multiple splice variants originate. This review will focus specifically on the role of Postn in wound healing and remodeling, an area of intense research in the last 10 years particularly related to skin healing as well as in myocardium post infarction. Postn interacts with cells through various integrin pairs and is an essential downstream effector of TGF-β superfamily signaling. As will be discussed, across different tissues, Postn is associated with pro-fibrogenic process, specifically, the transition of fibroblasts to myofibroblasts, collagen fibrillogenesis and ECM synthesis. Although the complexity of Postn as a modulator of cell behavior in tissue healing is only beginning to be elucidated, its expression is clearly a defining event in moving wound healing through the proliferative and remodeling phases.

Rapid development of agriculture and fossil fuel combustion greatly increased US reactive nitrogen emissions to the atmosphere in the second half of the 20th century, resulting in excess nitrogen deposition to natural ecosystems. Recent efforts to lower nitrogen oxides emissions have substantially decreased nitrate wet deposition. Levels of wet ammonium deposition, by contrast, have increased in many regions. Together these changes have altered the balance between oxidized and reduced nitrogen deposition. Across most of the United States, wet deposition has transitioned from being nitrate-dominated in the 1980s to ammonium-dominated in recent years. Ammonia has historically not been routinely measured because there are no specific regulatory requirements for its measurement. Recent expansion in ammonia observations, however, along with ongoing measurements of nitric acid and fine particle ammonium and nitrate, permit new insight into the balance of oxidized and reduced nitrogen in the total (wet + dry) US nitrogen deposition budget. Observations from 37 sites reveal that reduced nitrogen contributes, on average, ∼65% of the total inorganic nitrogen deposition budget. Dry deposition of ammonia plays an especially key role in nitrogen deposition, contributing from 19% to 65% in different regions. Future progress toward reducing US nitrogen deposition will be increasingly difficult without a reduction in ammonia emissions.

Evaluation of the semi-continuous Monitor for AeRosols and GAses in ambient air (MARGA, Metrohm Applikon B.V.) was conducted with an emphasis on examination of accuracy and precision associated with processing of chromatograms. Using laboratory standards and atmospheric measurements, analytical accuracy, precision and method detection limits derived using the commercial MARGA software were compared to an alternative chromatography procedure consisting of a custom Java script to reformat raw MARGA conductivity data and Chromeleon (Thermo Scientific Dionex) software for peak integration. Our analysis revealed issues with accuracy and precision resulting from misidentification and misintegration of chromatograph peaks by the MARGA automated software as well as a systematic bias at low concentrations for anions. Reprocessing and calibration of raw MARGA data using the alternative chromatography method lowered method detection limits and reduced variability (precision) between parallel sampler boxes. Instrument performance was further evaluated during a 1-month intensive field campaign in the fall of 2014, including analysis of diurnal patterns of gaseous and particulate water-soluble species (NH3, SO2, HNO3, NH4+, SO42− and NO3−), gas-to-particle partitioning and particle neutralization state. At ambient concentrations below ∼ 1 µg m−3, concentrations determined using the MARGA software are biased +30 and +10 % for NO3− and SO42−, respectively, compared to concentrations determined using the alternative chromatography procedure. Differences between the two methods increase at lower concentrations. We demonstrate that positively biased NO3− and SO42− measurements result in overestimation of aerosol acidity and introduce nontrivial errors to ion balances of inorganic aerosol. Though the source of the bias is uncertain, it is not corrected by the MARGA online single-point internal LiBr standard. Our results show that calibration and verification of instrument accuracy by multilevel external standards is required to adequately control analytical accuracy. During the field intensive, the MARGA was able to capture rapid compositional changes in PM2.5 due to changes in meteorology and air mass history relative to known source regions of PM precursors, including a fine NO3− aerosol event associated with intrusion of Arctic air into the southeastern US.

T2D is characterized by progressive pancreatic islet dysfunction; however, dissecting causes of this dysfunction in humans is challenging due to disease heterogeneity and the difficulty of obtaining relevant pancreatic samples. To address these challenges, we used an integrated approach to functionally and molecularly study the native pancreas and isolated islets from the same human T2D cadaveric donor. Using associated donor clinical information, we focused on donors with short disease duration (n=10, age 47-66 years, 2-7-year duration, oral medications). Compared to age-matched control islets, T2D islets had dysfunctional insulin (decreased) and glucagon (increased) secretion despite similar insulin and glucagon content. Tissue-section analysis of T2D pancreata showed no difference in relative endocrine (α, β and δ) cell composition. To assess for changes in the islet microenvironment, we analyzed macrophages by staining with Iba1 and endothelial cells with Caveolin-1 and found a 2-fold increase in intraislet macrophages (T2D vs. control: 3.01% vs. 1.45% by area) but no change in intraislet endothelial cell area (T2D vs. control: 8.97% vs. 8.21% by area). Interestingly, costaining with Iba1 and thioflavin S demonstrated that T2D islets with amyloid did not have increased macrophage infiltration compared to T2D islets without amyloid although a close spatial association of macrophages and amyloid deposits was noted. RNA-sequencing analysis of FACS-purified α and β cells from T2D islets showed increased inflammatory signaling pathways in both cell types. Specifically, transcripts for cytokine receptors such as IL6R, IL1R1, and IFNGR1 as well as downstream regulators including NFKB1 and NFKB2 were elevated in T2D α and/or β cells. Together, these data suggest that early in the course of T2D, increased macrophages in the islet microenvironment may lead to elevated α and β cell inflammatory signaling and contribute to human α and β cell dysfunction. Disclosure J.T. Walker: None. S. Shrestha: None. N. Prasad: None. T. Richardson: None. R. Aramandla: None. G. Poffenberger: None. R. Bottino: None. M. Brissova: None. A.C. Powers: None.

Article, Recent laboratory chamber studies indicate a significant role for highly oxidized low-volatility organics in new particle formation (NPF), but the actual role of these highly oxidized low-volatility organics in atmospheric NPF remains uncertain. Here, particle size distributions (PSDs) measured in nine forest areas in North America are used to characterize the occurrence and intensity of NPF and to evaluate model simulations using an empirical formulation in which formation rate is a function of the concentrations of sulfuric acid and low-volatility organics from alpha-pinene oxidation (Nucl-Org), and using an ion-mediated nucleation mechanism (excluding organics) (Nucl-IMN). On average, NPF occurred on ~ 70 % of days during March for the four forest sites with springtime PSD measurements, while NPF occurred on only ~ 10 % of days in July for all nine forest sites. Both Nucl-Org and Nucl-IMN schemes capture the observed high frequency of NPF in spring, but the Nucl-Org scheme significantly overpredicts while the Nucl-IMN scheme slightly underpredicts NPF and particle number concentrations in summer. Statistical analyses of observed and simulated ultrafine particle number concentrations and frequency of NPF events indicate that the scheme without organics agrees better overall with observations. The two schemes predict quite different nucleation rates (including their spatial patterns), concentrations of cloud condensation nuclei, and aerosol first indirect radiative forcing in North America, highlighting the need to reduce NPF uncertainties in regional and global earth system models., published version, http://purl.org/eprint/status/PeerReviewed

Lawn management practices that produce aesthetically appealing landscapes may also create environmental conditions that stimulate soil nitrous oxide (N2O) fluxes. The purpose of this exploratory study is to investigate the effects of lawn management practices on N2O fluxes from three non-replicated distinctly different residential landscapes: a high maintenance fescue (Festuca arundinacea) lawn (HMFL), a low maintenance fescue lawn (LMFL), and a mixed hardwood forested residential landscape (FRL) located in Cary, North Carolina. The specific objectives are (1) to measure the N2O fluxes from three residential landscapes within a fixed 24-h period following natural rainfall events; and (2) to determine the effect of lawn maintenance, season, water filled pore space (WFPS%), temperature, and days after fertilization on N2O flux. The mean N2O fluxes for HMFL (14.3 ± 2.28 μg N2O-N m−2 h−1), LMFL (3.14 ± 0.68 μg N2O-N m−2 h−1) and FRL (0.43 ± 0.08 μg N2O-N m−2 h−1) indicate that residential lawns receiving the recommended amount of fertilizer and frequent irrigation exhibit higher N2O fluxes than non-irrigated fertilized lawns or forested landscapes. Patterns of N2O fluxes from the HMFL and LMFL were associated with timing of fertilizer applications, presence or absence of irrigation, and seasonal fescue growth patterns. For the FRL, lower N inputs and the presence of a decomposing litter layer potentially limited N2O production. Our findings suggest that lawn management practices (i.e., fertilizer applications and irrigation) can create differences in the N2O flux following natural rainfall events.

Ammonia (NH3) and carbon monoxide (CO) are primary pollutants emitted to the Earth's atmosphere from common as well as distinct sources associated with anthropogenic and natural activities. The seasonal and global distributions and correlations of NH3 and CO from the Tropospheric Emission Spectrometer (TES) satellite observations and GEOS-Chem model simulations for 2007 are investigated to evaluate how well the global and seasonal pollutant sources are prescribed in the model. Although the GEOS-Chem simulations of NH3 and CO atmospheric mixing ratio values are lower than the TES satellite observations, the global distribution patterns from the model reasonably agree with the observations, indicating that the model represents the general location of the source regions and the seasonal enhancements of NH3 and CO globally over large regional scales. In regions and seasons where biomass burning is the dominant source of both NH3 and CO emissions into the atmosphere, there are strong NH3:CO correlations, which is consistent with the relationship demonstrated by surface measurements over fires. In regions where the enhanced NH3 and CO are known to be produced by different sources, the NH3:CO correlations from TES observations and model simulations are weak or non-existent. For biomass burning regions the NH3:CO ratios are 0.015 (TES) and 0.013 (GEOS-Chem). In regions of high-population density, known heavy traffic, and limited biomass burning sources, such as the rapidly developing areas of South Asia and northern China, which include mixtures of megacities, industrial, and agricultural areas, the two species show weaker but still positive correlations and NH3:CO ratios of 0.051 (TES) and 0.036 (GEOS-Chem). These enhancement ratios of NH3 relative to CO are useful in constraining NH3 emission inventories when CO emission inventories are better known for some events or regions (i.e. biomass burning).

We developed and validated an automated, inexpensive, and continuous multiple-species gas-flux monitoring system that can provide data for a variety of relevant atmospheric pollutants, including O3, CO2, and NOx. Validation consisted of conducting concurrent gas-phase dry-deposition experiments, using both dynamic flux chambers and an eddy-covariance system, in a grassy clearing in the Duke Forest (Chapel Hill, NC). Experiments were carried out in June and September, under a variety of meteorological conditions. Ozone-deposition measurements from the two methods matched very well (4–10% difference in mean flux rate) when the leaf-area index (LAI) inside the chambers was representative of the average LAI in the field. The dynamic flux chambers can be considered an accurate measurement system under these conditions. CO2 measurements were conducted for one 20 h period, and the flux chamber captured the diurnal trend in CO2 flux well, although the quantity of the data was not sufficient to validate chamber performance. Flux-chamber NOx measurements could be calculated when ambient NOx concentrations were above 1 ppb. Unfortunately, the eddy-covariance system for measuring NOx was not available during this field campaign, so comparisons cannot be made. NOx fluxes were in a reasonable range for the field site.