Search

Background A biconcave polyethylene (PE) bearing was developed for mobile-bearing unicompartmental knee arthroplasties (UKA) to reduce PE dislocation. Methods A modification of the BalanSys Unicompartmental knee system with a biconcave PE and convex tibial component was used in 32 prostheses in 28 patients. Clinical outcomes and five cases of PE fracture are reported and extensively analyzed ex vivo. Results Visual Analogue Score of pain and satisfaction, and Knee Society Scores improved for all patients. The passive range of motion was 130°. No PE bearings dislocated. Five bearings fractured with oxidation, cracking and delamination at the thinnest central region of the PE. The combination of increased stress and decreased poly thickness was associated with increased creep. PE oxidation caused embrittlement and contributed to fractures in the thin waist of the implant. Conclusions The unforeseen consequence of a novel design of a UKA that resulted in a specific mechanical and tribological mode of failure is reported. We conclude the PE failed due to a biconcave design that increased stress on the implant at its region of risk combined with a decrease of the polyethylene thickness by 1 mm in the central area.

Diabetic cardiomyopathy is a progressive disease in diabetic patients, and myocardial insulin resistance contributes to its pathogenesis through incompletely-defined mechanisms. Striated muscle preferentially expressed protein kinase (SPEG) has two kinase-domains and is a critical cardiac regulator. Here we show that SPEG is phosphorylated on Ser2461/Ser2462/Thr2463 by protein kinase B (PKB) in response to insulin. PKB-mediated phosphorylation of SPEG activates its second kinase-domain, which in turn phosphorylates sarcoplasmic/endoplasmic reticulum calcium-ATPase 2a (SERCA2a) and accelerates calcium re-uptake into the SR. Cardiac-specific deletion of PKBα/β or a high fat diet inhibits insulin-induced phosphorylation of SPEG and SERCA2a, prolongs SR re-uptake of calcium, and impairs cardiac function. Mice bearing a Speg3A mutation to prevent its phosphorylation by PKB display cardiac dysfunction. Importantly, the Speg3A mutation impairs SERCA2a phosphorylation and calcium re-uptake into the SR. Collectively, these data demonstrate that insulin resistance impairs this PKB-SPEG-SERCA2a signal axis, which contributes to the development of diabetic cardiomyopathy., Molecular mechanisms linking myocardial insulin resistance to diabetic cardiomyopathy are incompletely understood. Here the authors show that myocardial insulin resistance impairs a PKB-SPEG-SERCA2a signaling axis, which contributes to the development of diabetic cardiomyopathy.

Objective Adrenal vein sampling (AVS) and computed tomography (CT) often show confusingly discordant lateralisation results in primary aldosteronism (PA). We tested a biochemical algorithm using AVS data to detect cortisol cosecretion as a potential explanation for discordant cases. Design Retrospective analysis from a large PA + AVS database. Patients All patients with PA and AVS, 2005-2020. Measurements An algorithm using biochemical data from paired AVS + CT images was devised from physiological first principles and informed by data from unilateral, AVS-CT concordant patients. The algorithm involved calculations based upon the expectation that low cortisol levels exist in adrenal vein effluent opposite an aldosterone-and-cortisol-producing adrenal mass and may reverse lateralisation due to inflated aldosterone/cortisol ratios. Main outcomes The algorithm was applied to cases with discordant CT-AVS lateralisation to determine whether this might be a common or explanatory finding. Clinical and biochemical characteristics of identified cases were collected via chart review and compared to CT-AVS concordant cases to detect evidence of biological plausibility for cortisol cosecretion. Results From a total of 588 AVS cases, 141 AVS + CT pairs were clear unilateral PA cases, used to develop the three-step algorithm for AVS interpretation. Applied to 88 AVS + CT discordant pairs, the algorithm suggested possible cortisol cosecretion in 40%. Case review showed that the proposed cortisol cosecretors, as identified by the algorithm, had low/suppressed adrenocorticotropic hormone levels, larger average nodule size and lower plasma aldosterone. Conclusions Pending external validation and outcome verification by surgery and tissue immunohistochemistry, cortisol cosecretion from aldosteronomas may be a common explanation for discordant CT-AVS results in PA.

High-throughput metabolomics can be used to optimize cell growth for enhanced production or for monitoring cell health in bioreactors. It has applications in cell and gene therapies, vaccines, biologics, and bioprocessing. NMR metabolomics is a method that allows for fast and reliable experimentation, requires only minimal sample preparation, and can be set up to take online measurements of cell media for bioreactor monitoring. This type of application requires a fully automated metabolite quantification method that can be linked with high-throughput measurements. In this review, we discuss the quantifier requirements in this type of application, the existing methods for NMR metabolomics quantification, and the performance of three existing quantifiers in the context of NMR metabolomics for bioreactor monitoring.

This paper describes an operational evaluation of the US Environmental Protection Agency’s (EPA) Air Pollution Exposure Model (APEX). APEX simulations for a multipollutant ambient air mixture, i.e. ozone (O3), carbon monoxide (CO), and particulate matter 2.5 microns in diameter or less (PM2.5), were performed for two seasons in three study areas in central Los Angeles. APEX predicted microenvironmental concentrations were compared with concentrations of these three pollutants monitored in the Exposure Classification Project (ECP) study during the same periods. The ECP was designed expressly for evaluating exposure models and measured concentrations inside and outside 40 microenvironments. This evaluation study identifies important uncertainties in APEX inputs and model predictions useful for guiding further exposure model input data and algorithm development efforts. This paper also presents summaries of the concentrations in the different microenvironments.

The nonlinear dimer obtained through the nonlinear Schrödinger equation has been a workhorse for the discovery the role nonlinearity plays in strongly interacting systems. While the analysis of the stationary states demonstrates the onset of a symmetry broken state for some degree of nonlinearity, the full dynamics maps the system into an effective [Formula: see text] model. In this later context, the self-trapping transition is an initial condition-dependent transfer of a classical particle over a barrier set by the nonlinear term. This transition that has been investigated analytically and mathematically is expressed through the hyperbolic limit of Jacobian elliptic functions. The aim of this work is to recapture this transition through the use of methods of Artificial Intelligence (AI). Specifically, we used a physics motivated machine learning model that is shown to be able to capture the original dynamic self-trapping transition and its dependence on initial conditions. Exploitation of this result in the case of the nondegenerate nonlinear dimer gives additional information on the more general dynamics and helps delineate linear from nonlinear localization. This work shows how AI methods may be embedded in physics and provide useful tools for discovery.

Data-Independent Acquisition (DIA) is a method to improve consistent identification and precise quantitation of peptides and proteins by mass spectrometry (MS). The targeted data analysis strategy in DIA relies on spectral assay libraries that are generally derived from a priori measurements of peptides for each species. Although Escherichia coli (E. coli) is among the best studied model organisms, so far there is no spectral assay library for the bacterium publicly available. Here, we generated a spectral assay library for 4,014 of the 4,389 annotated E. coli proteins using one- and two-dimensional fractionated samples, and ion mobility separation enabling deep proteome coverage. We demonstrate the utility of this high-quality library with robustness in quantitation of the E. coli proteome and with rapid-chromatography to enhance throughput by targeted DIA-MS. The spectral assay library supports the detection and quantification of 91.5% of all E. coli proteins at high-confidence with 56,182 proteotypic peptides, making it a valuable resource for the scientific community. Data and spectral libraries are available via ProteomeXchange (PXD020761, PXD020785) and SWATHAtlas (SAL00222-28)., Measurement(s) Proteome • database type spectral library Technology Type(s) SWATH MS protein profiling assay • mass spectrometry • Data-Independent Acquisition Sample Characteristic - Organism Escherichia coli Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.13078733

An ozone abatement strategy for the South Coast Air Basin (SoCAB) has been proposed by the South Coast Air Quality Management District (SCAQMD) and the California Air Resources Board (ARB). The proposed emissions reduction strategy is focused on the reduction of nitrogen oxide (NOx) emissions by the year 2030. Two high PM2.5 concentration episodes with high ammonium nitrate compositions occurring during September and November 2008 were simulated with the Community Multi-scale Air Quality model (CMAQ). All simulations were made with same meteorological files provided by the SCAQMD to allow them to be more directly compared with their previous modeling studies. Although there was an overall under-prediction bias, the CMAQ simulations were within an overall normalized mean error of 50%; a range that is considered acceptable performance for PM modeling. A range of simulations of these episodes were made to evaluate sensitivity to NOx and ammonia emissions inputs for the future year 2030. It was found that the current ozone control strategy will reduce daily average PM2.5 concentrations. However, the targeted NOx reductions for ozone were not found to be optimal for reducing PM2.5 concentrations. Ammonia emission reductions reduced PM2.5 and this might be considered as part of a PM2.5 control strategy. Implications: The SCAQMD and the ARB have proposed an ozone abatement strategy for the SoCAB that focuses on NOx emission reductions. Their strategy will affect both ozone and PM2.5. Two episodes that occurred during September and November 2008 with high PM2.5 concentrations and high ammonium nitrate composition were selected for simulation with different levels of nitrogen oxide and ammonia emissions for the future year 2030. It was found that the ozone control strategy will reduce maximum daily average PM2.5 concentrations but its effect on PM2.5 concentrations is not optimal.

Nitrous oxide (N2O) is a prominent greenhouse gas. Our understanding of environmental controls on N2O fluxes has mainly come from small-scale experiments, for example, static chamber measurements on plots or lab incubations. However, studies of the environmental controls for N2O fluxes at ecosystem scales have been limited. Using eddy covariance (EC) measurements, this study evaluated the environmental drivers of N2O fluxes for a one-year period at a farm grazed year-round by dairy cows in the Waikato region, New Zealand. We identified an optimum soil moisture/temperature zone that favours maximal N2O emissions, demonstrating maximum N2O fluxes at ∼70% water-filled pore space (WFPS) and moderate soil temperatures. Our measurements consistently identified significant N2O flux pulses associated with rainfall following grazing events in warm-dry months. In contrast, during cold-wet months when WFPS was consistently high, pulses after rainfall did not occur. A clear positive temperature response for N2O fluxes was observed above 70% WFPS while a negative relationship was detected when WFPS was less than 70%. Distinctive diurnal flux patterns emerged in both pulses and background fluxes, implying that soil temperature regulates N2O fluxes at sub-daily timescales. Over the annual period, N2O emissions were 6.5 kg N2O-N ha−1. We found the highest cumulative rates (maximum 35.7 g N2O-N ha−1 day−1) in autumn but the rates were low during both summer and winter. Our results highlighted the combined effects of environmental factors on N2O fluxes, and quantified N2O flux variations at seasonal and daily scales, suggesting that continuous measurement techniques, such as EC, could serve as an alternative in national N2O inventories.

The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.

Contemporary measurements of carbon (C) accumulation rates in peatlands around the world often show the C sink to be stronger on average than at times in the past. Alteration of global nutrient cycles could be contributing to elevated carbon accumulation in the present day. Here we examine the effect of volcanic inputs of nutrients on peatland C accumulation in Moanatuatua Bog, New Zealand, by examining a high-resolution Late Holocene C accumulation record during which powerful volcanic eruptions occurred, depositing two visible rhyolitic tephra layers (Taupo, 232 ± 10 CE; Kaharoa, 1314 ± 12 CE). Carbon accumulation rates since c. 50 CE, well before any human presence, increased from a background rate of 23 g C m−2 yr−1 up to 110 g C m−2 yr−1 following the deposition of the Taupo Tephra, and 84 g C m−2 yr−1 following the deposition of the Kaharoa Tephra. Smaller but nevertheless marked increases in C accumulation additionally occurred in association with the deposition of three andesitic-dacitic cryptotephras (each ≤ ∼1 mm thick) of the Tufa Trig Formation between the Taupo and Kaharoa events. These five periods of elevated C uptake, especially those associated with the relatively thick Taupo and Kaharoa tephras, were accompanied by shifts in nutrient stoichiometry, indicating that there was greater availability of phosphorus (P) relative to nitrogen (N) and C during the period of high C uptake. Such P was almost certainly derived from volcanic sources, with P being present in the volcanic glass at Moanatuatua, and many of the eruptions described being associated with the local deposition of the P rich mineral apatite. We found peatland C accumulation to be tightly coupled to N and P accumulation, suggesting nutrient inputs exert a strong control on rates of peat accumulation. Nutrient stoichiometry indicated a strong ability to recover P within the ecosystem, with C:P ratios being higher than most other peatlands in the literature. We conclude that nutrient inputs, deriving from volcanic eruptions, have been very important for C accumulation rates in the past. Therefore, the elevated nutrient inputs occurring in the present day could offer a more plausible explanation, as opposed to a climatic component, for observed high contemporary C accumulation in New Zealand peatlands.

Rationale: SPEG (Striated muscle preferentially expressed protein kinase) has 2 kinase-domains and is critical for cardiac development and function. However, it is not clear how these 2 kinase-domains function to maintain cardiac performance. Objective: To determine the molecular functions of the 2 kinase-domains of SPEG. Methods and Results: A proteomics approach identified SERCA2a (sarcoplasmic/endoplasmic reticulum calcium ATPase 2a) as a protein interacting with the second kinase-domain but not the first kinase-domain of SPEG. Furthermore, the second kinase-domain of SPEG could phosphorylate Thr 484 on SERCA2a, promote its oligomerization and increase calcium reuptake into the sarcoplasmic/endoplasmic reticulum in culture cells and primary neonatal rat cardiomyocytes. Phosphorylation of SERCA2a by SPEG enhanced its calcium-transporting activity without affecting its ATPase activity. Depletion of Speg in neonatal rat cardiomyocytes inhibited SERCA2a-Thr 484 phosphorylation and sarcoplasmic reticulum calcium reuptake. Moreover, overexpression of SERCA2a Thr484Ala mutant protein also slowed sarcoplasmic reticulum calcium reuptake in neonatal rat cardiomyocytes. In contrast, domain mapping and phosphorylation analysis revealed that the first kinase-domain of SPEG interacted and phosphorylated its recently identified substrate JPH2 (junctophilin-2). An inducible heart-specific Speg knockout mouse model was generated to further study this SPEG-SERCA2a signal nexus in vivo. Inducible deletion of Speg decreased SERCA2a-Thr 484 phosphorylation and its oligomerization in the heart. Importantly, inducible deletion of Speg inhibited SERCA2a calcium-transporting activity and impaired calcium reuptake into the sarcoplasmic reticulum in cardiomyocytes, which preceded morphological and functional alterations of the heart and eventually led to heart failure in adult mice. Conclusions: Our data demonstrate that the 2 kinase-domains of SPEG may play distinct roles to regulate cardiac function. The second kinase-domain of SPEG is a critical regulator for SERCA2a. Our findings suggest that SPEG may serve as a new target to modulate SERCA2a activation for treatment of heart diseases with impaired calcium homeostasis.

P.Z.H. developed the theory, wrote the paper, and performed the DFT calculations. A.S.R. contributed to the analytical work. H.S.P., D.K.C., and A.C.H.N. supervised the research. We thank Vitor M. Pereira for useful comments and discussions. P.Z.H., H.S.P., and D.K.C. acknowledge support by the Boston University Materials Science and Engineering Innovation Grants. P.Z.H, H.S.P., and D.K.C are grateful for computing resources provided by the Boston University Shared Computing Cluster. (Boston University Materials Science and Engineering Innovation Grants)

The casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signalling. It is therefore critically important to understand how their activity is controlled in cells. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A–H) interacted with the α and α-like isoforms of CK1; FAM83A, -B, -E, and -H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member with the related CK1γ1, -2 and -3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterises the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization of both the FAM83 proteins and their CK1 binding partners and interfered with the cellular functions of both families of proteins. Based on its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 (PACK1) domain.

In vitro bioassays to estimate biotransformation rate constants of contaminants in fish are currently being investigated to improve bioaccumulation assessments of hydrophobic contaminants. The present study investigates the relationship between chemical substrate concentration and in vitro biotransformation rate of 4 environmental contaminants (9-methylanthracene, pyrene, chrysene, and benzo[a]pyrene) in rainbow trout (Oncorhynchus mykiss) liver S9 fractions and methods to determine maximum first-order biotransformation rate constants. Substrate depletion experiments using a series of initial substrate concentrations showed that in vitro biotransformation rates exhibit strong concentration dependence, consistent with a Michaelis-Menten kinetic model. The results indicate that depletion rate constants measured at initial substrate concentrations of 1 μM (a current convention) could underestimate the in vitro biotransformation potential and may cause bioconcentration factors to be overestimated if in vitro biotransformation rates are used to assess bioconcentration factors in fish. Depletion rate constants measured using thin-film sorbent dosing experiments were not statistically different from the maximum depletion rate constants derived using a series of solvent delivery-based depletion experiments for 3 of the 4 test chemicals. Multiple solvent delivery-based depletion experiments at a range of initial concentrations are recommended for determining the concentration dependence of in vitro biotransformation rates in fish liver fractions, whereas a single sorbent phase dosing experiment may be able to provide reasonable approximations of maximum depletion rates of very hydrophobic substances.

There are still large uncertainties in peatland methane flux dynamics and insufficient understanding of how biogeochemical processes scale to ecosystems. New Zealand bogs differ from Northern Hemisphere ombrotrophic systems in climatic setting, hydrology, and dominant vegetation, offering an opportunity to evaluate our knowledge of peatland methane biogeochemistry gained primarily from northern bogs and fens. We report eddy covariance methane fluxes from a raised bog in New Zealand over 2.5 years. Annual total methane flux in 2012 was 29.1 g CH4 m−2 yr−1, whereas during a year with a severe drought (2013) it was 20.6 g CH4 m−2 yr−1, both high compared to Northern Hemisphere bogs and fens. Drier conditions led to a decrease in fluxes from ~100 mg CH4 m−2 d−1 to ~20 mg CH4 m−2 d−1, and subsequent slow recovery of flux after postdrought water table rise. Water table depth regulated the temperature sensitivity of methane fluxes, and this sensitivity was greatest when the water table was within 100 mm of the surface, corresponding to the shallow rooting zone of the dominant vegetation. A correlation between daytime CO2 uptake and methane fluxes emerged during times with shallow water tables, suggesting that controls on methane production were critical in determining fluxes, more so than oxidation. Water table recession through this shallow zone led to increasing methane fluxes, whereas changes in temperature during these periods were not correlated. Models of methane fluxes should consider drought-induced lags in seasonal flux recovery that depend on drought characteristics and location of the critical zone for methane production.

Trypanosomatid protozoa are unusual eukaryotes that are well known for having unusual ways of controlling their gene expression. The lack of a refined mode of transcriptional control in these organisms is compensated by several post-transcriptional control mechanisms, such as control of mRNA turnover and selection of mRNA for translation, that may modulate protein synthesis in response to several environmental conditions found in different hosts. In other eukaryotes, selection of mRNA for translation is mediated by the complex eIF4F, a heterotrimeric protein complex composed by the subunits eIF4E, eIF4G, and eIF4A, where the eIF4E binds to the 5′-cap structure of mature mRNAs. In this review, we present and discuss the characteristics of six trypanosomatid eIF4E homologs and their associated proteins that form multiple eIF4F complexes. The existence of multiple eIF4F complexes in trypanosomatids evokes exquisite mechanisms for differential mRNA recognition for translation.

Trypanosomatids are parasitic protozoans characterized by several unique structural and metabolic processes that include exquisite mechanisms associated with gene expression and regulation. During the initiation of protein synthesis, for instance, mRNA selection for translation seems to be mediated by different eIF4F-like complexes, which may play a significant role in parasite adaptation to different hosts. In eukaryotes, the heterotrimeric eIF4F complex (formed by eIF4E, eIF4G, and eIF4A) mediates mRNA recognition and ribosome binding and participates in various translation regulatory events. Six eIF4Es and five eIF4Gs have been described in trypanosomatids with several of these forming different eIF4F-like complexes. This has raised questions about their role in differential mRNA translation. Here we have studied further TbEIF4E2, the least known eIF4E homologue from Trypanosoma brucei, and found that it is not associated with an eIF4G homolog. It is, however, associated with mature mRNAs and binds to a histone mRNA stem-loop-binding protein (SLBP), one of two Trypanosoma SLBP homologs (TbSLBP1 and TbSLBP2). TbSLBP1 is more similar to the mammalian counterpart while TbSLBP2 is exclusive to trypanosomatids and related organisms. TbSLBP2 binds to TbEIF4E2 through a conserved central region missing in other SLBP homologs. Both SLBPs, as well as TbEIF4E2, were found to localize to the cytoplasm. TbEIF4E2 and TbSLBP2 are differentially expressed during cell culture, being more abundant in early-log phase, with TbSLBP2 also showing cell-cycle dependent expression. The new data reinforce unique aspects of the trypanosomatid eIF4Es, with the TbEIF4E2–TbSLBP complex possibly having a role in differential selection of mRNAs containing stem-loop structures.

Peatlands are one of the largest terrestrial stores of carbon. Carbon exchange in peatlands is often assessed solely by measurement of contemporary fluxes; however, these fluxes frequently indicate a much stronger sink strength than that measured by the rate of C accumulation in the peat profile over longer timescales. Here we compare profile-based measurements of C accumulation with the published net ecosystem C balance for the largest peatland area in Britain, the Flow Country of northern Scotland. We estimate the long-term rate of C accumulation to be 15.4 g C m−2 yr−1 for a site where a recent eddy covariance study has suggested contemporary C uptake more than six times greater (99.37 g C m−2 yr−1). Our estimate is supported by two further long-term C accumulation records from nearby sites which give comparable results. We demonstrate that a strong contemporary C sink strength may not equate to a strong long-term sink and explore reasons for this disparity. We recommend that contemporary C sequestration should be viewed in the context of the long-term ecological drivers, such as fires, ecohydrological feedbacks and the changing quality of litter inputs.

Estimates of regional and national nitrous oxide (N2O) emissions rely on emission factors (EFs) commonly derived from measurements using static chambers. These measurements can include high uncertainties and might obscure the quantification of N2O fluxes. Advances in micrometeorological eddy covariance technique (EC) now allow direct measurements of N2O fluxes at the field scale. Here, we compared N2O emissions calculated from site-specific EFs with N2O flux data derived from year-round EC measurements on an intensively grazed dairy pasture in the Waikato region, NZ. Annual N2O emissions of 7.30 kg N2O-N ha−1 yr−1 determined using gap-filled EC flux data were greater than N2O estimates of 3.82 kg N2O-N ha−1 yr−1 based on site-specific EFs for cattle urine (1.53%), cattle dung (0.24%) and urea fertiliser (0.16%). Likely reasons for this difference were that the EF approach did not take into account the seasonal variability of EFs, the effect of supplementary feed on cattle nitrogen (N) excretion and background N2O emissions (BNE). Including calculated emissions from supplementary feed N (0.92 kg N2O-N ha−1 yr−1) and BNE (1.09 kg N2O-N ha−1 yr−1) increased annual EF-based emissions to 5.83 kg N2O-N ha−1 yr−1. The site-specific EFs were established in spring 2017 and may not have adequately represented summer, winter and particularly autumn N2O emissions. The EF approach, therefore, did not fully account for the seasonal variability of N2O fluxes as measured by EC but, if quantified, could have led to further agreement between measurements. Using EC measurements to complement static chambers and EF approaches altered annual N2O emissions estimates from intensively grazed pastoral land. Hence, we conclude that N2O budgets derived from EFs need to better capture the effect of seasonal variability, supplementary feed and BNE.

Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.

Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

Precise homoeostasis of the intracellular concentration of Cl− is achieved via the co-ordinated activities of the Cl− influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+–K+ ion co-transporters), also promote inhibition of the KCCs (K+–Cl− co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl− influx, we propose that the targeting of WNK–SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl− extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states., WNK-regulated SPAK/OSR1 act as direct phosphorylators and major regulators of the KCC isoforms, which explains how activation of the WNK signalling pathway can co-ordinately regulate Cl− influx and efflux by reciprocally controlling the SLC12A family N[K]CC and KCC isoforms.

Many pharmaceuticals used to treat cancer target the cell cycle or mitotic spindle dynamics, such as the anti-tumor drug, paclitaxel, which stabilizes microtubules. Here we show that, in cells arrested in mitosis with the spindle toxins, nocodazole, or paclitaxel, the endogenous protein phosphatase 4 (Ppp4) complex Ppp4c-R2-R3A is phosphorylated on its regulatory (R) subunits, and its activity is inhibited. The phosphorylations are blocked by roscovitine, indicating that they may be mediated by Cdk1-cyclin B. Endogenous Ppp4c is enriched at the centrosomes in the absence and presence of paclitaxel, nocodazole, or roscovitine, and the activity of endogenous Ppp4c-R2-R3A is inhibited from G 1/S to the G 2/M phase of the cell cycle. Endogenous γ-tubulin and its associated protein, γ-tubulin complex protein 2, both of which are essential for nucleation of microtubules at centrosomes, interact with the Ppp4 complex. Recombinant γ-tubulin can be phosphorylated by Cdk1-cyclin B or Brsk1 and dephosphorylated by Ppp4c-R2-R3A in vitro. The data indicate that Ppp4c-R2-R3A regulates microtubule organization at centrosomes during cell division in response to stress signals such as spindle toxins, paclitaxel, and nocodazole, and that inhibition of the Ppp4 complex may be advantageous for treatment of some cancers.

One cross-linked polyethylene (XLPE) liner is manufactured using a lower dose of radiation, 5 Mrad, which may result in less cross-linking. The reported in vivo wear rate of this XLPE liner in patients undergoing THA has varied, and has included some patients in each reported cohort who had greater than 0.1 mm/year of wear, which is an historical threshold for osteolysis. Previous studies have measured wear on plain radiographs, an approach that has limited sensitivity.We therefore measured the amount and direction of wear at 6 years using Radiostereometric analysis (RSA) in patients who had THAs that included a cross-linked polyethylene liner manufactured using 5 Mrad radiation.We prospectively reviewed wear in 30 patients who underwent primary THAs with the same design of cross-linked acetabular liner and a 28-mm articulation. Tantalum markers were inserted during surgery and all patients had RSA radiographic examinations at 1 week, 6 months, 1, 2, and 6 years postoperatively.The mean proximal, two-dimensional (2-D) and three-dimensional (3-D) wear rates calculated between 1 year and 6 years were 0.014, 0.014, and 0.018 mm/per year, respectively. The direction of the head penetration recorded between 1 week and 6 years was in a proximal direction for all patients, proximolateral for 16 of 24 patients, and proximomedial for eight of 24 patients.The proximal, 2-D and 3-D wear of a XLPE liner produced using 5 Mrad of radiation was low but measurable by RSA after 6 years. No patients had proximal 2-D or 3-D wear rates exceeding 0.1 mm/year. Further followup is needed to evaluate the effect of XLPE wear particles on the development of long-term osteolysis.

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (Ctx (R) ) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in Ctx (R) clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all Ctx (R) clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of Ctx (R) cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting.

Skeletal muscle development is influenced by myogenic regulatory factors, including the expression of MyoD and myogenin. Our objective was to use the C2C12 cell culture model to test the hypothesis that both MyoD and myogenin were required for agrin-induced acetylcholine receptor (AChR) clustering and the fusion of myoblasts into myotubes. We induced fusion of myoblasts into myotubes by switching from growth medium (GM) to differentiation medium (DM). During myotube formation AChRs cluster spontaneously, but treatment with motor neuron derived agrin increases clustering of AChRs and other postsynaptic components of the neuromuscular synapse. We examined the normal expression pattern of MyoD and myogenin in C2C12 cell culture using immunofluorescence. MyoD was highly expressed while myoblasts were in GM, but expression declined within 72 hours after cell cultures were switched to DM. Myogenin expression was low in GM, but increased when cell cultures were switched to DM. Next we used antibodies to decrease MyoD and/or myogenin function. Fluorescence microscopy images were captured and then analyzed to assess agrin-induced AChR clustering with or without antibody treatment. Finally we calculated the proportion of nuclei in myotubes and myoblasts by creating digital overlays of phase contrast and DAPI stained microscopy images. This allowed the comparison of myotube formation with or without antibody treatment. We report that antibody to either MyoD or myogenin decreases the frequency of agrin-induced AChR clustering without affecting myotube formation. We conclude that agrin-induced AChR clustering requires both MyoD and myogenin.

Organ-enriched blood proteins, those produced primarily in one organ and secreted or exported to the blood, potentially afford a powerful and specific approach to assessing diseases in their cognate organs. In this paper, we demonstrate that quantification of organ-enriched proteins in the blood offers a new strategy to find biomarkers for diagnosis and assessment of drug-induced liver injury (and presumably the assessment of other liver diseases). We used selected reaction monitoring (SRM) mass spectrometry to quantify 81 liver-enriched proteins plus three aminotransferases (ALT1, AST1, and AST2) in plasma of C57BL/6J and NOD/ShiLtJ mice exposed to acetaminophen or carbon tetrachloride. Plasma concentrations of 49 liver-enriched proteins were perturbed significantly in response to liver injury induced by one or both toxins. We validated four of these toxin-responsive proteins (ALDOB, ASS1, BHMT and GLUD1) by Western blotting. By both assays, these four proteins constitute liver injury markers superior to currently employed markers such as ALT and AST. A similar approach was also successful in human serum where we had analyzed 66 liver-enriched proteins in acetaminophen overdose patients. Of these, 23 proteins were elevated in patients; 15 of 23 overlapped with the concentration-increased proteins in the mouse study. A combination of 5 human proteins, AGXT, ALDOB, CRP, FBP1, and MMP9, provides the best diagnostic performance to distinguish acetaminophen overdose patients from controls (sensitivity: 0.85, specificity: 0.84, accuracy: 85%). These five blood proteins are candidates for detecting acetaminophen-induced liver injury using next-generation diagnostic devices (e.g, microfluidic ELIZA assays).

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001, eLife digest During digestion, proteins are broken down into their constituent parts called amino acids. Amino acids are transported in the bloodstream and are used to build up new cells and repair old ones. Optimal regulation of the cellular rates of amino acid uptake and protein synthesis is critical to the overall health of our bodies. Inside each of our cells is a molecule called mammalian target of rapamycin (mTOR for short), which acts as a controller that receives information about amino acid availability. mTOR also senses how much of each amino acid the cell needs and calibrates the cell’s amino acid uptake and protein synthesis machineries accordingly. When investigating an enzyme named ZNRF2, Hoxhaj et al. discovered that it interacts with mTOR on membranes inside cells. This raised questions about how ZNRF2 might work with mTOR to sense amino acid supplies and regulate cell growth. Hoxhaj et al. found that when cells are provided with amino acids and growth-stimulating hormones, mTOR is activated and attaches a phosphate group to ZNRF2. This chemical modification promotes the release of ZNRF2 from membranes so that ZNRF2 separates from mTOR. In contrast, when cells are starved of amino acids, this phosphate group is removed from ZNRF2, which then returns to the membranes. On membranes, ZNRF2 also influences the activity of a pump called V-ATPase, which controls the internal acidity of the membrane-enclosed vesicles named lysosomes that help to recycle amino acids inside cells. The action of ZNRF2 on the pump may help to prime mTOR so that it is ready to sense amino acids. These findings by Hoxhaj et al. suggest that ZNRF2 and mTOR may ‘tune’ each other, making constant to-and-fro adjustments to help ensure that levels of amino acid uptake and cell growth are set just right. However, many questions about ZNRF2 still remain to be addressed. For example, are genetic mutations in ZNRF2 involved in cancers, developmental disorders or growth syndromes? Is ZNRF2 most important in the brain, where it is particularly abundant? And how does ZNRF2 affect acidity within the lysosomes? DOI: http://dx.doi.org/10.7554/eLife.12278.002

Mobile-source air toxic (MSAT) levels increase in confining microenvironments (MEs) with numerous emission sources of vehicle exhaust or evaporative emissions or during high-load and cold-start conditions. Reformulated fuels are expected to reduce MSAT and ozone precursor emissions. This study, required under the Clean Air Act Section 211b, evaluated high-end exposures in cities using reformulated (methyl tertiary-butyl ether [MTBE] or ethanol [EtOH]) fuels and conventional gasoline blends. The study investigates 13 high-end MEs, sampling under enhanced exposure conditions expected to result in maximal fuel and exhaust component exposures to carbon monoxide (CO), carbon dioxide (CO2), BTEX (benzene, toluene, ethylbenzene, xylenes), MTBE, 1,3-butadiene (1,3-BD), EtOH,formaldehyde (HCHO), and acetaldehyde (CH3CHO). The authors found that day-to-day ME variations in high-end benzene, 1,3-BD, HCHO, and CO concentrations are substantial, but independent of gasoline composition and season, and related to the activity and emission rates of ME sources, which differ from day to day.

The precision, accuracy, and sampling rates of Radiello and Ogawa passive samplers were evaluated in the laboratory using a flow-through chamber and under field conditions prior to their use in the 2007 Harbor Community Monitoring Study (HCMS), a saturation monitoring campaign in the communities adjacent to the Ports of Los Angeles and Long Beach. Passive methods included Radiello samplers for volatile organic compounds (benzene, toluene, ethylbenzene, xylenes, 1,3-butadiene), aldehydes (formaldehyde, acetaldehyde, acrolein) and hydrogen sulfide, and Ogawa samplers for nitrogen oxides and sulfur dioxide. Additional experiments were conducted to study the robustness of the passive sampling methods under variable ambient wind speed, sampling duration, and storage time before analysis. Our experimentally determined sampling rates were in agreement with the rates published by Radiello and Ogawa with the following exceptions: we observed a diffusion rate of 22.4 ± 0.1 mL/min for benzene and 37.4 ± 1.5 mL/min for ethylbenzene compared to the Radiello published values of 27.8 and 25.7 mL/min, respectively. With few exceptions, the passive monitoring methods measured one-week average ambient concentrations of selected pollutants with sensitivity and precision comparable to conventional monitoring methods averaged over the same period. Radiello Carbograph 4 VOC sampler is not suitable for the collection of 1,3-butadiene due to backdiffusion. Results for the Radiello aldehyde sampler were inconclusive due to lack of reliable reference methods for all carbonyl compounds of interest.

The 5' cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5'-5' triphosphate bond followed by 2'-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2'-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N(7) methylation of the guanosine cap nor 2'-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2'-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.

A new series of NS3/4A protease boronic acid inhibitors is described. The compounds show good biochemical potency and cellular activity. The peptidomimetic inhibitors were evaluated against proteases from different HCV genotypes and clinically relevant NS3/4A mutants. Compound 28 displayed subnanomolar to single digit nanomolar potencies in the enzymatic assays and an EC50 of 25 nM in the replicon cell-based assay.

The E3 ubiquitin ligase Pellino can be activated by phosphorylation in vitro, catalyzed by IL-1 receptor-associated kinase 1 (IRAK1) or IRAK4. Here, we show that phosphorylation enhances the E3 ligase activity of Pellino 1 similarly with any of several E2-conjugating enzymes (Ubc13-Uev1a, UbcH4, or UbcH5a/5b) and identify 7 amino acid residues in Pellino 1 whose phosphorylation is critical for activation. Five of these sites are clustered between residues 76 and 86 (Ser-76, Ser-78, Thr-80, Ser-82, and Thr-86) and decorate a region of antiparallel β-sheet, termed the “wing,” which is an appendage of the forkhead-associated domain that is thought to interact with IRAK1. The other 2 sites are located at Thr-288 and Ser-293, just N-terminal to the RING-like domain that carries the E3 ligase activity. Unusually, the full activation of Pellino 1 can be achieved by phosphorylating any one of several different sites (Ser-76, Thr-86, Thr-288, or Ser-293) or a combination of other sites (Ser-78, Thr-80, and Ser-82). These observations imply that dephosphorylation of multiple sites is required to inactivate Pellino 1, which could be a device for prolonging Pellino's E3 ubiquitin ligase activity in vivo.

IntroductionA proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented in this paper, using a mouse model for skin cancer as an example.Materials and MethodsBlood plasma was collected from ten control mice and ten mice having a mutation in the p19ARFgene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists.Results and DiscussionsWe assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins are also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localization, transport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application.ConclusionThese results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort.

Background Bone cement impregnated with high doses of aminoglycosides is used to treat infection associated with joint arthroplasty. Aim To examine serum tobramycin and creatinine concentrations in patients who received high-dose tobramycin and vancomycin impregnated bone cement during revision joint arthroplasty. Method This was a prospective cohort case series of 10 patients aged 58 to 82 years undergoing revision joint arthroplasty with high-dose tobramycin and vancomycin impregnated bone cement. Serum tobramycin and creatinine concentrations were monitored post surgery. Results Sterile tobramycin 3.6 g and vancomycin 1.0 g powder was mixed into each 40 g of Palacos bone cement, resulting in total maximum doses of tobramycin 7.2 g and vancomycin 2.0 g. At the time of hospital discharge renal function was unchanged in 9 patients. A 75-year-old male patient with preexisting renal impairment developed significant and persistent worsening of his renal function. Conclusion Bone cement impregnated with high doses of aminoglycosides should be used with caution in patients with a history of renal impairment.

Osteocytes are essential regulators of bone homeostasis. However, they are difficult to study due to their location within the bone mineralised matrix. Although several techniques have been published for the isolation of osteocytes from mouse bone, no such technique has been described for human osteocytes. We have therefore developed a protocol for the isolation of osteocytes from human trabecular bone samples acquired during surgery. The cells were digested from the bone matrix by sequential collagenase and ethylenediaminetetraacetic acid (EDTA) digestions and the cells from later digests displayed characteristic dendritic osteocyte morphology when cultured ex vivo. Furthermore, the cells expressed characteristic osteocyte marker genes, such as E11, dentin matrix protein 1 (DMP1), SOST, matrix extracellular phosphoglycoprotein (MEPE) and phosphate regulating endopeptidase homologue, X-linked (PHEX). In addition, genes associated with osteocyte perilacunar remodelling, including matrix metallopeptidase-13 (MMP13), cathepsin K (CTSK) and carbonic anhydrase 2 (CAR2) were expressed. The cells also responded to parathyroid hormone (PTH) by downregulating SOST mRNA expression and to 1α,25-dihydroxyvitamin D3 (1,25D) by upregulating fibroblast growth factor 23 (FGF23) mRNA expression. Therefore, the cells behave in a similar manner to osteocytes in vivo. These cells represent an important tool in enhancing current knowledge in human osteocyte biology.

The U.S. Department of Energy Gasoline/Diesel PM Split Study examined the sources of uncertainties in using an organic compound-based chemical mass balance receptor model to quantify the contributions of spark-ignition (SI) and compression-ignition (CI) engine exhaust to ambient fine particulate matter (PM2.5). This paper presents the chemical composition profiles of SI and CI engine exhaust from the vehicle-testing portion of the study. Chemical analysis of source samples consisted of gravimetric mass, elements, ions, organic carbon (OC), and elemental carbon (EC) by the Interagency Monitoring of Protected Visual Environments (IMPROVE) and Speciation Trends Network (STN) thermal/optical methods, polycyclic aromatic hydrocarbons (PAHs), hopanes, steranes, alkanes, and polar organic compounds. More than half of the mass of carbonaceous particles emitted by heavy-duty diesel trucks was EC (IMPROVE) and emissions from SI vehicles contained predominantly OC. Although total carbon (TC) by the IMPROVE and STN protocols agreed well for all of the samples, the STN/IMPROVE ratios for EC from SI exhaust decreased with decreasing sample loading. SI vehicles, whether low or high emitters, emitted greater amounts of high-molecular-weight particulate PAHs (benzo[ghi]perylene, indeno[1,2,3-cd]pyrene, and coronene) than did CI vehicles. Diesel emissions contained higher abundances of two- to four-ring semivolatile PAHs. Diacids were emitted by CI vehicles but are also prevalent in secondary organic aerosols, so they cannot be considered unique tracers. Hopanes and steranes were present in lubricating oil with similar composition for both gasoline and diesel vehicles and were negligible in gasoline or diesel fuels. CI vehicles emitted greater total amounts of hopanes and steranes on a mass per mile basis, but abundances were comparable to SI exhaust normalized to TC emissions within measurement uncertainty. The combustion-produced high-molecular-weight PAHs were found in used gasoline motor oil but not in fresh oil and are negligible in used diesel engine oil. The contributions of lubrication oils to abundances of these PAHs in the exhaust were large in some cases and were variable with the age and consumption rate of the oil. These factors contributed to the observed variations in their abundances to total carbon or PM2.5 among the SI composition profiles.

Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.

The soils of the Seabee Hook area of Cape Hallett in northern Victoria Land, Antarctica, were mapped and characterized. Seabee Hook is a low-lying gravel spit of beach deposits built up by coastal currents carrying basalt material from nearby cliffs. Seabee Hook is the location of an Adélie penguin (Pygoscelis adeliae) colony which influences the soils with additions of guano, dead birds, eggshells and feathers. A soil-landscape model was developed and a soil association was identified between the soils formed on mounds (relict beach ridges) favoured by penguins for nests (Typic Haplorthel) and the soils in the areas between the mounds (Typic Haplorthel/Typic Aquorthel). Soils formed on the mounds inhabited by penguins contained guano in the upper 50 cm, overlying sub-rounded beach-deposited gravel and sand. Soils between mounds had a thin veneer (< 5 cm) of guano overlying basaltic gravelly sand similar to that in the lower parts of the mound soils. The soils had high concentrations of nitrogen, organic carbon, phosphorus, cadmium, zinc, copper, and increased electrical conductivity, within horizons influenced by penguin guano. Five buried penguin bones were collected from the base of soil profiles and radiocarbon dated. The dates indicate that Seabee Hook has been colonized by penguins for at least 1000 years.

N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F1 ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.

Investigate whether fecal neopterin concentration (a potential marker of gut inflammation) in Gambian children with enteropathy was associated with growth failure. Secondary outcome measures tested the associations between Giardia lamblia infestation, fecal neopterin and lactulose mannitol absorption ratio(L:M), a measure of intestinal permeability.Seventy-two children had height and weight measured every 6 to 8 weeks until 15 months of age in a rural Gambian village. L:M ratio, a measure of small intestinal permeability and fecal neopterin were measured at these times. Stool was examined by immunofluorescence and light microscope for Giardia cysts.Long-term height and weight gains were negatively associated with mean subject fecal neopterin concentration (r = -0.29 and -0.36, respectively; P0.001). There was no correlation between fecal neopterin and intestinal permeability or history of diarrhea. Of 72 children studied, 19 had Giardia cysts in stool and 38 had negative stool examinations. Infected children had a mean of 0.7 days of diarrhea/week (95% confidence interval [CI], 0.31-1.03) versus 0.8 days/week (95% CI, 0.71-0.85) in uninfected children. No difference in growth was detected between those with positive or negative fecal smears. Mean L:M ratio was the same in both groups (0.31; 95% CI, 0.26-0.34).Consistent with the theory that intestinal inflammation in tropical infants may impair growth, fecal neopterin concentrations were inversely associated with growth. Factors other than Giardia are causing enteropathy and growth failure in Gambian infants.

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.

The Sm-binding site of the kinetoplastid spliced leader RNA has been implicated in accurate spliced leader RNA maturation and trans -splicing competence. In Trypanosoma brucei , RNA interference-mediated knockdown of SmD1 caused defects in spliced leader RNA maturation, displaying aberrant 3′-end formation, partial formation of cap 4, and overaccumulation in the cytoplasm; U 28 pseudouridylation was unaffected.

For at least 30 years, ozone (O3) levels on weekends in parts of California's South Coast (Los Angeles) Air Basin (SoCAB) have been as high as or higher than on weekdays, even though ambient levels of O3 precursors are lower on weekends than on weekdays. A field study was conducted in the Los Angeles area during fall 2000 to test whether proposed relationships between emission sources and ambient nonmethane hydrocarbon (NMHC) and oxides of nitrogen (NOx) levels can account for observed diurnal and day-of-week variations in the concentration and proportions of precursor pollutants that may affect the efficiency and rate of O3 formation. The contributions to ambient NMHC by motor vehicle exhaust and evaporative emissions, estimated using chemical mass balance (CMB) receptor modeling, ranged from 65 to 85% with minimal day-of-week variation. Ratios of ambient NOx associated with black carbon (BC) to NOx associated with carbon monoxide (CO) were approximately 1.25 +/- 0.22 during weekdays and 0.76 +/- 0.07 and 0.52 +/- 0.07 on Saturday and Sunday, respectively. These results demonstrate that lower NOx emissions from diesel exhaust can be a major factor causing lower NOx mixing ratios and higher NMHC/NOx ratios on weekends. Nonmobile sources showed no significant day-of-week variations in their contributions to NMHC. Greater amounts of gasoline emissions are carried over on Friday and Saturday evenings but are, at most, a minor factor contributing to higher NMHC/NOx ratios on weekend mornings.

Background Although evidence suggests that end-stage renal disease is associated with poor limb salvage and patient survival after arterial revascularization, little is known about the effect of renal transplantation. We analyzed the outcome in patients with renal transplants who underwent infrainguinal bypass procedures. Methods Data prospectively entered into our vascular registry were reviewed for all patients who underwent lower extremity bypass procedures from January 1, 1990, through January 31, 2002. Sixty patients were identified who had a functioning renal allograft at infrainguinal revascularization. Kaplan-Meier survival curves were generated for limb salvage, patency, and patient survival and were compared with the Mantel-Cox log- rank test. Results Sixty patients (40 men, 20 women; mean age, 47.1 years) underwent 76 bypass procedures in 71 limbs. Preoperative demographic data included diabetes (59 of 60 patients, 98.3%), coronary artery disease (26 of 60 patients, 43.3%), and preoperative serum creatinine concentration (SCr) greater than 2.0 mg/dL (9 of 60 patients, 11.7%). Mean follow-up was 25.1 months. Overall major complication rate was 11.8%, and 30-day mortality rate was 1.3%. Survival was 93.3% at 1 year and 66.6% at 5 years. Limb salvage was 87% at 1 year and 78% at 5 years. Primary graft patency was 78% at 1 year and 44% at 5 years. Preoperative SCr less than or equal to 2.0 mg/dL was associated with improved overall patient survival (5-year survival, 73.4% vs 37.5%; P = .01, log-rank test). Limb salvage and patency rates were not significantly affected by preoperative SCr greater than 2.0 mg/dL. Conclusions Lower extremity bypass can be performed safely and effectively in patients who have undergone renal transplantation. However, the importance of a well-functioning renal allograft at surgery is demonstrated by marked improvement in patient survival.

This study examined the effects of varying future reductions in emissions of oxides of nitrogen (NOx) and volatile organic compounds (VOC) on the location and magnitude of peak ozone levels within California's South Coast Air Basin (SoCAB or Basin). As ozone formation is currently VOC-limited in the Basin, model simulations with 2030 baseline emissions (-61% for NOx and -32% for VOC from 2008) predict 10-20% higher peak ozone levels (i.e., NOx disbenefit) in the western and central SoCAB compared with the 2008 base simulation. With additional NOx reductions of 50% beyond the 2030 baseline emissions (-81% from 2008), the predicted ozone levels are reduced by about 15% in the eastern SoCAB but remain comparable to 2008 levels in the western and central Basin. The Basin maximum ozone site shifts westward to more populated areas of the Basin and will result potentially in greater population-weighted exposure to ozone with even a relatively small shortfall in the required NOx reductions unless accompanied by additional VOC reductions beyond 2030 baseline levels. Once committed to a NOx-focused control strategy, NOx reductions exceeding 90% from 2008 levels will be necessary to attain the ozone National Ambient Air Quality Standards (NAAQS). The findings from this study and other recent work that the current VOC emission estimates are underestimated by about 50% suggest that greater future VOC reductions will be necessary to reach the projected 2030 baseline emissions. Increasing the base year VOC emissions by a factor of 1.5 result in higher 2008 baseline ozone predictions, lower relative response factors, and about 20% lower projected design values. If correct, these findings have important implications for the total and optimum mix of VOC and NOx emission reductions that will be required to attain the ozone NAAQS in the SoCAB.Results of this study indicate that ozone levels in the western and central SoCAB would remain the same or increase with even a relatively small shortfall in the projected NOx reductions under planned NOx-focused controls. This possibility, therefore, warrants a rigorous analysis of the costs and effects of varying reductions of VOC and NOx on the formation and combined health impacts of ozone and secondary particles. Given the nonlinearity of ozone formation, such analyses should include the implications of gradually increasing global background ozone concentrations and the Basin's topography and meteorology on the practical limits of alternative emission control strategies.

Lipopolysaccharide (LPS) stimulates production of inflammatory mediators, partly by stabilizing [interleukin-6 (IL-6), cyclooxygenase 2 (COX-2)] and/or stimulating translation [tumour necrosis factor-alpha (TNF-alpha)] of their mRNAs. Such regulation depends on AU-rich elements (AREs) within the 3'-untranslated regions and is partially suppressed by SB 203580 (which inhibits SAPK2a/p38). The LPS-induced production of TNF-alpha and IL-6 is suppressed in MAPKAP-K2-deficient mice (a kinase activated by SAPK2a/p38). Here, we identify 18 macrophage proteins that bind to AREs and show that hnRNP A0 is a major substrate for MAPKAP-K2 in this fraction. MAPKAP-K2 phosphorylated hnRNP A0 at Ser84 in vitro and this residue became phosphorylated in LPS-stimulated cells. Phosphorylation was prevented by SB 203580 and suppressed in macrophages derived from MAPKAP-K2-deficient mice. The mRNAs encoding TNF-alpha, COX-2 and macrophage inflammatory protein-2 (MIP-2) bound to hnRNP A0 in LPS-stimulated macrophages, an interaction prevented by SB 203580. The LPS-induced stabilization of MIP-2 mRNA and production of MIP-2 protein were abolished when macrophages were incubated with SB 203580 plus PD 184352 (which inhibits the classical MAP kinase cascade). Our data suggest that LPS-induced binding of hnRNP A0 to AREs may contribute to the post-transcriptional regulation of specific mRNAs.

We show that oxidation of phosphorene can lead to the formation of a new family of planar (2D) and tubular (1D) oxides and sub-oxides, most of them insulating. This confers to black phosphorus a native oxide that can be used as barrier material and protective layer. Further, the bandgap of phosphorene oxides depends on the oxygen concentration, suggesting that controlled oxidation can be used as a means to engineer the bandgap. For the oxygen saturated composition, P$_2$O$_5$, both the planar and tubular phases have a large bandgap energy of about 8.5eV, and are transparent in the near UV. These two forms of phosphorene oxides are predicted to have the same formation enthalpy as o$^\prime$-P$_2$O$_5$, the most stable of the previously known forms of phosphorus pentoxide., 10 pages, 12 figures