Post-processed data and graphical tools for a CONUS-wide eddy flux evapotranspiration dataset.

Large sample datasets of in situ evapotranspiration (ET) measurements with well documented data provenance and quality assurance are critical for water management and many fields of earth science research. We present a post-processed ET oriented dataset at daily and monthly timesteps, from 161 stations, including 148 eddy covariance flux towers, that were chosen based on their data quality from nearly 350 stations across the contiguous United States. In addition to ET, the data includes energy and heat fluxes, meteorological measurements, and reference ET downloaded from gridMET for each flux station. Data processing techniques were conducted in a reproducible manner using open-source software. Most data initially came from the public AmeriFlux network, however, several different networks (e.g., the USDA-Agricultural Research Service) and university partners provided data that was not yet public. Initial half-hourly energy balance data were gap-filled and aggregated to daily frequency, and turbulent fluxes were corrected for energy balance closure error using the FLUXNET2015/ONEFlux energy balance ratio approach. Metadata, diagnostics of energy balance, and interactive graphs of time series data are included for each station. Although the dataset was developed primarily to benchmark satellite-based remote sensing ET models of the OpenET initiative, there are many other potential uses, such as validation for a range of regional hydrologic and atmospheric models.

Liquid biopsy for cancer diagnosis using vibrational spectroscopy: systematic review.

BACKGROUND: Vibrational spectroscopy (VS) is a minimally invasive tool for analysing biological material to detect disease. This study aimed to review its application to human blood for cancer diagnosis. METHODS: A systematic review was undertaken using a keyword electronic database search (MEDLINE, Embase, PubMed, TRIP and Cochrane Library), with all original English-language manuscripts examining the use of vibrational spectral analysis of human blood for cancer detection. Studies involving fewer than 75 patients in the cancer or control group, animal studies, or where the primary analyte was not blood were excluded. RESULTS: From 1446 results, six studies (published in 2010-2018) examining brain, bladder, oral, breast, oesophageal and hepatic cancer met the criteria for inclusion, with a total population of 2392 (1316 cancer, 1076 control; 1476 men, 916 women). For cancer detection, reported mean sensitivities in each included study ranged from 79·3 to 98 per cent, with specificities of 82·8-95 per cent and accuracies between 81·1 and 97·1 per cent. Heterogeneity in reporting strategies, methods and outcome measures made meta-analysis inappropriate. CONCLUSION: VS shows high potential for cancer diagnosis, but until there is agreement on uniform standard reporting methods and studies with adequate sample size for valid classification models have been performed, its value in clinical practice will remain uncertain.

la espectroscopia vibracional (vibrational spectroscopy, VS) es un dispositivo mínimamente invasivo para analizar material biológico y detectar enfermedad. Este estudio se propuso revisar su aplicación en la sangre humana para el diagnóstico de cáncer. MÉTODOS: Se llevó a cabo una revisión sistemática utilizando una búsqueda con palabras claves en bases de datos electrónicas (MEDLINE, EMBASE, PubMed, TRIP, Cochrane Library), de todos los manuscritos originales publicados en inglés que examinaban la utilización del análisis espectral vibracional de la sangre humana para la detección del cáncer. Se excluyeron estudios que incluían menos de 75 pacientes en los grupos cáncer/control, estudios en animales o cuando la muestra principal no fuera la sangre. RESULTADOS: De los 1.446 resultados, 6 estudios publicados en 2010-2018, y que examinaban cánceres del cerebro, vejiga, oral, mama, esófago e hígado cumplieron los criterios de inclusión, con una población total de 2.506 casos (1.316 cánceres, 1.076 controles; 1.476 varones, 915 mujeres). Para la detección del cáncer, las sensibilidades medias descritas en cada uno de los estudios incluidos variaron entre 79,3-98,0%, con especificidades entre 82,8-95,0%, y exactitudes diagnósticas entre 81,1% y 97,1%. La heterogeneidad en las estrategias descritas, métodos y medidas de resultados hicieron que el metaanálisis fuera inapropiado. CONCLUSIÓN: La VS muestra un alto potencial para el diagnóstico del cáncer, pero hasta que no se llegue a un acuerdo en los métodos para informar de los resultados y se hayan efectuado estudios con un tamaño muestral adecuado para una clasificación válida de los modelos, su valor en la práctica clínica sigue siendo incierto.

Potocytosis. Robert Feulgen Lecture.

Potocytosis represents a mechanism by which small and large molecules as well as macromolecular complexes are sequestered and transported by caveolae. Caveolae are flask-shaped plasma membrane specializations characterized by a filamentous coat consisting of caveolins that decorates the inside surface of each caveola membrane. They have endocytotic functions that differ from the clathrin-coated pit pathway. Ligands bound to receptors that are internalized by caveolae can be delivered to four different locations in the cell bypassing the lysosome and at least four different caveolae membrane traffic patterns during potocytosis can be distinguished. Hence, cells have two endocytic machines and each is designed to accomplish different tasks. This review provides a brief summary of the discovery of caveolae and of potocytosis, and focuses on recent discoveries of the unique endocytic capabilities of caveolae in a variety of different cells.

High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase.

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

Modified atmosphere treatments as a potential disinfestation technique for arthropod pests in greenhouses.

Incidental transport of arthropods on plant material can be a significant mode of pest entry into greenhouses. We evaluated the use of controlled atmosphere treatments as a potential way to eliminate arthropod pests on plant propagules (i.e., cuttings or small rooted plants). Lethal exposures to CO2 or N2 were determined for common greenhouse pests including fungus gnat larvae, Bradysia sp.; green peach aphid, Myzus persicae (Sulzer); sweetpotato whitefly, Bemisia sp.; twospotted spider mite, Tetranychus urticae Koch; and western flower thrips, Frankliniella occidentalis (Pergande). We also studied the effect of pest species, life stage, and presence or absence of plants on efficacy of modified atmosphere treatments. Finally, effects of modified atmospheres on plant quality were evaluated for several bedding plant species including begonia, Begonia semperflorens-cultorum Hort. 'Cocktail Series', chrysanthemum, Dendranthema grandiflora Tzvelev., geranium, Pelargonium X hortorum L.H. Bailey, and impatiens, Impatiens wallerana Hook f., and among cultivars of geranium and chrysanthemum. Exposure for 12-18 h to >99% N2 or CO2 caused complete mortality of aphids, mites, thrips, and whiteflies. Fungus gnat larvae were more tolerant of hypoxic conditions. Adult mites and eggs were equally susceptible. For most pests, there was no difference in response to atmospheres modified by CO2 or N2. However, there was variation in response among plant species and cultivars, with effects ranging from delayed flowering to mortality. Despite the possibility of adverse effects on some plants, this work indicates that use of modified atmospheres has potential to eliminate arthropod pests on plant propagules before they are introduced into greenhouses.

Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria.

In commonly used tissue culture cells, caveolin-1 is embedded in caveolae membranes. It appears to reach this location after being cotranslationally inserted into ER membranes, processed in the Golgi and shipped to the cell surface. We now report that caveolae are not the preferred location for caveolin-1 in all cell types. Skeletal muscle cells and keratinocytes target caveolin-1 to the cytosol while in exocrine and endocrine cells it accumulates in the secretory pathway. We also found that airway epithelial cells accumulate caveolin-1 in modified mitochondria. The cytosolic and the secreted forms appear to be incorporated into a soluble, lipid complex. We conclude that caveolin-1 can be targeted to a variety of intracellular destinations, which suggests a novel mechanism for the intracellular traffic of this protein.

Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine.

Synaptotagmin 1 probably functions as a Ca2+ sensor in neurotransmitter release via its two C2-domains, but no common Ca2+-dependent activity that could underlie a cooperative action between them has been described. The NMR structure of the C2B-domain now reveals a beta sandwich that exhibits striking similarities and differences with the C2A-domain. Whereas the bottom face of the C2B-domain has two additional alpha helices that may be involved in specialized Ca2+-independent functions, the top face binds two Ca2+ ions and is remarkably similar to the C2A-domain. Consistent with these results, but in contrast to previous studies, we find that the C2B-domain binds phospholipids in a Ca2+-dependent manner similarly to the C2A-domain. These results suggest a novel view of synaptotagmin function whereby the two C2-domains cooperate in a common activity, Ca2+-dependent phospholipid binding, to trigger neurotransmitter release.

Estrogen receptor alpha and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae.

Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) alpha-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ERalpha and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E(2), 10(-8) mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ERalpha antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ERalpha displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ERalpha plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ERalpha protein in caveolae, and E(2) caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E(2) on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ERalpha is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org.

Presence of oxidized cholesterol in caveolae uncouples active platelet-derived growth factor receptors from tyrosine kinase substrates.

Platelet-derived growth factor receptor beta (PDGFRbeta) in fibroblasts is concentrated in caveolae where it controls the tyrosine phosphorylation of multiple proteins. Caveolae are enriched in cholesterol and sphingolipids, but the role of these lipids in PDGFR signal transduction is unknown. We report that introduction of cholest-4-en-3-one into caveolae membranes uncouples PDGFR autophosphorylation from tyrosine phosphorylation of neighboring proteins. Cholest-4-en-3-one appears to interfere with the normal interaction between PDGFR and its partners. The results suggest that tightly packed caveolae lipids form a membrane platform that functions as a lipid scaffold for organizing the molecular interactions of multiple signaling pathways.

Synaptotagmin regulation of coated pit assembly.

Synaptotagmins bind clathrin AP-2 with high affinity via their second C(2) domain, which indicates they are involved in coated pit function. We now report that expression of synaptotagmins lacking either the second C(2) domain or the entire cytoplasmic region potently inhibit endocytosis. Inhibition was dependent on two intramembrane cysteine residues that were found to be essential for synaptotagmin oligomerization. Cells expressing the wild-type, but not the mutant, truncated synaptotagmin fragment had a reduced number of clathrin-coated pits. These results suggest that the formation of synaptotagmin multimers is an important step in the regulation of coated pit assembly.

Multiple domains in caveolin-1 control its intracellular traffic.

Caveolin-1 is an integral membrane protein of caveolae that is thought to play an important role in both the traffic of cholesterol to caveolae and modulating the activity of multiple signaling molecules at this site. The molecule is synthesized in the endoplasmic reticulum, transported to the cell surface, and undergoes a poorly understood recycling itinerary. We have used mutagenesis to determine the parts of the molecule that control traffic of caveolin-1 from its site of synthesis to the cell surface. We identified four regions of the molecule that appear to influence caveolin-1 traffic. A region between amino acids 66 and 70, which is in the most conserved region of the molecule, is necessary for exit from the endoplasmic reticulum. The region between amino acids 71 and 80 controls incorporation of caveolin-1 oligomers into detergent-resistant regions of the Golgi apparatus. Amino acids 91-100 and 134-154 both control oligomerization and exit from the Golgi apparatus. Removal of other portions of the molecule has no effect on targeting of newly synthesized caveolin-1 to caveolae. The results suggest that movement of caveolin-1 among various endomembrane compartments is controlled at multiple steps.

A requirement for ankyrin binding to clathrin during coated pit budding.

Recent studies suggest that the mobility of clathrin-coated pits at the cell surface are restricted by an actin cytoskeleton and that there is an obligate reduction in the amount of spectrin on membranes during coated pit budding. The spectrin-actin cytoskeleton associates with membranes primarily through ankyrins, which interact with the cytoplasmic region of numerous integral membrane proteins. We now report that the fourth repeat domain (D4) of ankyrin(R) binds to the N-terminal domain of clathrin heavy chain with high affinity. Addition of peptides containing the D4 region inhibited clathrin-coated pit budding in vitro. In addition, microinjection of D4 containing peptides blocked the endocytosis of fluorescent low density lipoprotein (LDL). Ankyrin(R) peptides that contained repeat domains other than D4 had no effect on either in vitro budding or internalization of LDL. Finally, immunofluorescence shows that ankyrin is uniformly associated with endosomes that contain fluorescent LDL. These results suggest that ankyrin plays a role in the budding of clathrin-coated pits during endocytosis.

Identification of caveolin-1 in lipoprotein particles secreted by exocrine cells.

Caveolin-1 is a protein component (of relative molecular mass 22, 000) of the striated coat that decorates the cytoplasmic surface of caveolae membranes. Previous biochemical and molecular tests have indicated that caveolin-1 is an integral membrane protein that is co-translationally inserted into endoplasmic-reticulum membranes of fibroblast and epithelial cells such that its carboxy- and amino-terminal ends are in the cytoplasm. Here we identify caveolin-1 in the secretory pathway of exocrine cells. Secretion of caveolin-1 from pancreatic acinar cells and a transfected exocrine cell line, but not from Chinese hamster ovary cells, is stimulated by the secretagogues secretin, cholecystokinin and dexamethasone. The secreted caveolin-1 co-fractionates with apolipoproteins, indicating that it may be secreted in a complex with lipids.

Regulated migration of epidermal growth factor receptor from caveolae.

In quiescent fibroblasts, epidermal growth factor (EGF) receptors (EGFR) are initially concentrated in caveolae but rapidly move out of this membrane domain in response to EGF. To better understand the dynamic localization of EGFR to caveolae, we have studied the behavior of wild-type and mutant receptors expressed in cells lacking endogenous EGFR. All of the receptors we examined, including those missing the first 274 amino acids or most of the cytoplasmic tail, were constitutively concentrated in caveolae. By contrast, migration from caveolae required EGF binding, an active receptor kinase domain, and at least one of the five tyrosine residues present in the regulatory domain of the receptor. Movement appears to be modulated by Src kinase, is blocked by activators of protein kinase C, and occurs independently of internalization by clathrin-coated pits. Two mutant receptors previously shown to induce an oncogenic phenotype lack the ability to move from caveolae in response to EGF, suggesting that a prolonged residence in this domain may contribute to abnormal cell behavior.

Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.

Spatial organization of EGF receptor transmodulation by PDGF.

Even though the modulation of EGF receptors by PDGF is well documented, it is not known where on the cell surface cross-talk between the two receptor systems takes place. The recent finding that both populations of receptors are concentrated in cell surface caveolae suggestes that the confinement of the two receptors to this space might facilitate their interaction. Here we show that stimulation of PDGF receptors in caveolae with PDGF causes a subpopulation of EGF receptors in the same membrane fraction to become phosphorylated on tyrosine. Coincident with tyrosine phosphorylation, the binding of EGF to its receptor markedly declines. Loss of EGF binding is partially blocked by tyrosine kinase inhibitors. Despite the close proximity of the two receptors in caveolae, we saw no evidence that EGF could stimulated PDGFR tyrosine phosphorylation. These results suggest that these two receptor systems are highly organized in caveolae.

Polarized distribution of endogenous Rac1 and RhoA at the cell surface.

Rac1 and RhoA regulate membrane ruffling and stress fiber formation. Both molecules appear to exert their control from the plasma membrane. In fibroblasts stimulated with platelet-derived growth factor or lysophosphatidic acid, the reorganization of the cytoskeleton begins at specific sites on the cell surface. We now report that endogenous Rac1 and RhoA also have a polarized distribution at the cell surface. Cell fractionation and immunogold labeling show that in quiescent fibroblasts both of these molecules are concentrated in caveolae, which are plasma membrane domains that are associated with actin-rich regions of the cell. Treatment of these cells with platelet-derived growth factor stimulated the recruitment of additional Rac1 and RhoA to caveolae fractions, while lysophosphatidic acid only caused the recruitment of RhoA. We could reconstitute the recruitment of RhoA using either whole cell lysates or purified caveolae. Surprisingly, pretreatment of the lysates with exoenzyme C3 shifted both resident and recruited RhoA from caveolae to noncaveolae membranes. The shift in location was not caused by inactivation of the RhoA effector domain. Moreover, chimeric proteins containing the C-terminal consensus site for Rac1 and RhoA prenylation were constitutively targeted to caveolae fractions. These results suggest that the polarized distribution of Rho family proteins at the cell surface involves an initial targeting of the protein to caveolae and a mechanism for retaining it at this site.