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Bastian Welz,* Rolf Bikker,* Leonie Hoffmeister, Mareike Diekmann, Martin Christmann, Korbinian Brand,* René Huber* Institute of Clinical Chemistry, Hannover Medical School, Hannover, 30625, Germany*These authors contributed equally to this workCorrespondence: Korbinian BrandInstitute of Clinical Chemistry, Hannover Medical School, Carl-Neuberg Str. 1, Hannover, 30625, GermanyTel +49 511 532 6614Fax +49 511 532 8614Email brand.korbinian@mh-hannover.deBackground: Termination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context.Materials and Methods: For the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/β-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays.Results: Constitutive GSK3β and PKCβ expression and GSK3α/β-Ser21/9 and PKCα/βII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/β-knockdown modestly increased, whereas GSK3α/β-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/β-Ser176/180 phosphorylation were not affected. Moreover, PKCβ-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects.Conclusion: Our results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 – either pathophysiologically or pharmacologically induced – may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.Keywords: TNF, GSK3, PKC, staurosporine, IL-8, NF-κB, termination of TNF-induced signaling, termination of inflammation

High resolution imaging of model organisms allows the quantification of important physiological measurements. In the case of fish with transparent embryos, these videos can visualise key physiological processes, such as heartbeat. High throughput systems can provide enough measurements for the robust investigation of developmental processes as well as the impact of system perturbations on physiological state. However, few analytical schemes have been designed to handle thousands of high-resolution videos without the need for some level of human intervention. We developed a software package, named FEHAT, to provide a fully automated solution for the analytics of large numbers of heart rate imaging datasets obtained from developing Medaka fish embryos in 96 well plate format imaged on an Acquifer machine. FEHAT uses image segmentation to define regions of the embryo showing changes in pixel intensity over time, followed by the classification of the most likely position of the heart and Fourier Transformations to estimate the heart rate. Here we describe some important features of the FEHAT software, showcasing its performance across a large set of medaka fish embryos and compare its performance to established, less automated solutions. FEHAT provides reliable heart rate estimates across a range of temperature-based perturbations and can be applied to tens of thousands of embryos without the need for any human intervention., Availability: Data used in this manuscript will be made available on request., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2024. Published by Oxford University Press.)

Motivation: Crosses among inbred lines are a fundamental tool for the discovery of genetic loci associated with phenotypes of interest. In organisms for which large reference panels or SNP chips are not available, imputation from low-pass whole-genome sequencing is an effective method for obtaining genotype data from a large number of individuals. To date, a structured analysis of the conditions required for optimal genotype imputation has not been performed., Results: We report a systematic exploration of the effect of several design variables on imputation performance in F2 crosses of inbred medaka lines using the imputation software STITCH. We determined that, depending on the number of samples, imputation performance reaches a plateau when increasing the per-sample sequencing coverage. We also systematically explored the trade-offs between cost, imputation accuracy, and sample numbers. We developed a computational pipeline to streamline the process, enabling other researchers to perform a similar cost-benefit analysis on their population of interest., Availability and Implementation: The source code for the pipeline is available at https://github.com/birneylab/stitchimpute. While our pipeline has been developed and tested for an F2 population, the software can also be used to analyse populations with a different structure., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitous protein mediating versatile effects in a variety of cell types, including actin crosslinking, signal transduction, and intracellular transport processes. MARCKS's functional role in monocyte/macrophages, however, has not yet been adequately addressed. Thus, the aim of this study was to further elucidate the impact of MARCKS on central cellular functions of monocytic cells. To address this topic, we generated monocytic THP-1 (Tohoku Hospital Pediatrics-1)-derived MARCKS wildtype and knockout (KO) cells using the CRISPR/Cas9 technique. Remarkably, in the absence of MARCKS, both total and intracellular reactive oxygen species (ROS) production were strongly suppressed but restored following transient MARCKS re-transfection. In contrast, proliferation, differentiation, cytokine expression, and phagocytosis remained unaltered. A complete inhibition of ROS production could also be achieved in THP-1-derived PKCβ KO cells or in PKC inhibitor Staurosporine-treated primary human monocytes. MARCKS deficiency also involved reduced basal Akt phosphorylation and delayed re-phosphorylation. Further analyses indicated that long-term TNF pre-incubation strongly enhances monocytic ROS production, which was completely blocked in MARCKS and PKCβ KO cells. Collectively, our study demonstrates that MARCKS is an essential molecule enabling ROS production by monocytic cells and suggests that MARCKS is part of a signal cascade involved in ROS formation.

Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka ( Oryzias latipes ) and zebrafish ( Danio rerio ), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3 , ube2b , usp44 , and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies., Competing Interests: AC, JG, BW, JM, TT, JW No competing interests declared, (© 2022, Cornean et al.)

Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straightforward purification. Immediate activity is usually granted by administration of preassembled protein/RNA complexes. We present the 'hei-tag ( h igh e ff i ciency-tag)' which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc-tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T (cytosine-to-thymine) base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level., Competing Interests: TT, TT, JG, JW patent application pending (EP21166099.8) related to the findings described, AC, RM, BW, MF No competing interests declared, (© 2022, Thumberger et al.)

Background: The teleost medaka (Oryzias latipes) is a well-established vertebrate model system, with a long history of genetic research, and multiple high-quality reference genomes available for several inbred strains. Medaka has a high tolerance to inbreeding from the wild, thus allowing one to establish inbred lines from wild founder individuals., Results: We exploit this feature to create an inbred panel resource: the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel. This panel of 80 near-isogenic inbred lines contains a large amount of genetic variation inherited from the original wild population. We use Oxford Nanopore Technologies (ONT) long read data to further investigate the genomic and epigenomic landscapes of a subset of the MIKK panel. Nanopore sequencing allows us to identify a large variety of high-quality structural variants, and we present results and methods using a pan-genome graph representation of 12 individual medaka lines. This graph-based reference MIKK panel genome reveals novel differences between the MIKK panel lines and standard linear reference genomes. We find additional MIKK panel-specific genomic content that would be missing from linear reference alignment approaches. We are also able to identify and quantify the presence of repeat elements in each of the lines. Finally, we investigate line-specific CpG methylation and performed differential DNA methylation analysis across these 12 lines., Conclusions: We present a detailed analysis of the MIKK panel genomes using long and short read sequence technologies, creating a MIKK panel-specific pan genome reference dataset allowing for investigation of novel variation types that would be elusive using standard approaches., (© 2022. The Author(s).)

Background: Unraveling the relationship between genetic variation and phenotypic traits remains a fundamental challenge in biology. Mapping variants underlying complex traits while controlling for confounding environmental factors is often problematic. To address this, we establish a vertebrate genetic resource specifically to allow for robust genotype-to-phenotype investigations. The teleost medaka (Oryzias latipes) is an established genetic model system with a long history of genetic research and a high tolerance to inbreeding from the wild., Results: Here we present the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel: the first near-isogenic panel of 80 inbred lines in a vertebrate model derived from a wild founder population. Inbred lines provide fixed genomes that are a prerequisite for the replication of studies, studies which vary both the genetics and environment in a controlled manner, and functional testing. The MIKK panel will therefore enable phenotype-to-genotype association studies of complex genetic traits while allowing for careful control of interacting factors, with numerous applications in genetic research, human health, drug development, and fundamental biology., Conclusions: Here we present a detailed characterization of the genetic variation across the MIKK panel, which provides a rich and unique genetic resource to the community by enabling large-scale experiments for mapping complex traits., (© 2022. The Author(s).)

The determination of total fluorine content using high-resolution graphite furnace continuum source molecular absorption spectrometry (HR- MAS) has been employed in a variety of samples for over 10 years. However, most of the samples analysed by HR- MAS are rich in fluoride, with negligible levels of organic fluorinated species. With an increase in concern surrounding per- and polyfluoroalkyl substances (PFASs), new methods to measure total fluorine of organofluorine using different techniques have been developed. However, no studies focused on PFASs behaviour in HR-MAS have been performed. As these compounds encompass a wide range of different structures, boiling points, decomposition temperatures and matrix interactions, a loss of accuracy can occur when an aqueous external calibration is performed using only one compound. To overcome this issue, an investigation into permanent modifiers for the graphite furnace was performed. After optimisation similar sensitivity for different PFCA was achieved when 400 μg of W was used as a permanent modifier together with an optimised temperature program. The relative deviation between the different PFCA standard slopes relative to the PFOA slope was lower than 15%. The instrumental limit of detection and quantification (LOD and LOQ, respectively) of total fluorine as total PFCA was 0.1 mg L -1 and 0.3 mg L -1 , respectively, while the method LOD and LOQ (using solid phase extraction) was 0.3 μg L -1 and 1.0 μg L -1 , respectively. The developed method gave satisfactory recoveries for the spiked PFCA into seawater, river water and effluent using PFOA calibration standards. The optimised method is useful for measuring extractable organofluorines (EOF) when only ionic PFASs such as PFCA are expected. When other organofluorines are expected, the results using HR GF-MAS should be taken with caution., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ulva ohnoi is a green macroalga with fast growth and high rates of nitrogen and phosphorus absorption. Recently, this species has been recorded in several places with record green tide formation in some of them. Using molecular tools, we herein report the first occurrence of this species in Brazil and demonstrate its potential for phytoremediation in typical environmental concentrations of Cd (0.625-15 µg L -1 ). Similarly, the effects of physicochemical parameters (salinity and temperature) on the toxicity and uptake efficiency of this species were evaluated. Molecular analysis of two sequences (1141 bp) obtained corroborates another 34 sequences for U. ohnoi obtained from GenBank. The addition of Cd in the medium affected photosynthetic parameters and reduced growth rate. U. ohnoi showed resistance to Cd when cultivated at 18 °C, S15 and 18-25 °C, S35, at concentrations between 0.625 and 2.5 μg. L -1 of Cd; yet, positive growth rate was maintained. Dose-dependent accumulation was observed in all combinations of factors used with a maximum value of 4.20 μg Cd per gram of dry seaweed at 15 μg. L -1 of Cd at 18 °C and S35. Maximum value of the concentration factor was 81.3 ± 1.1% of Cd added at the concentration of 0.625 μg. L -1 to S15 and 18 °C. Our results demonstrate the potential of using U. ohnoi in the phytoremediation of Cd in saltwater or brackish water., (Copyright © 2019 Elsevier Inc. All rights reserved.)

To better understand the inflammation-associated mechanisms modulating and terminating tumor necrosis factor (TNF-)induced signal transduction and the development of TNF tolerance, we analyzed both the proteome and the phosphoproteome in TNF long term-incubated (i.e., 48 h) primary human monocytes using liquid chromatography-mass spectrometry. Our analyses revealed the presence of a defined set of proteins characterized by reproducible changes in expression and phosphorylation patterns in long term TNF-treated samples. In total, 148 proteins and 569 phosphopeptides were significantly regulated (103 proteins increased, 45 proteins decreased; 377 peptides with increased and 192 peptides with decreased phosphorylation). A variety of these proteins are associated with the non-canonical nuclear factor κB (NF-κB) pathway (nuclear factor κB (NFKB) 2, v-rel reticuloendotheliosis viral oncogene homolog (REL) B, indolamin-2,3-dioxygenase (IDO), kynureninase (KYNU)) or involved in the negative regulation of the canonical NF-κB system. Within the phosphopeptides, binding motifs for specific kinases were identified. Glycogen synthase kinase (GSK) 3 proved to be a promising candidate, since it targets NF-κB inhibiting factors, such as CCAAT/enhancer binding protein (C/EBP) β. Our experiments demonstrate that both proteome and phosphoproteome analysis can be effectively applied to study protein/phosphorylation patterns of primary monocytes. These results provide new regulatory candidates and evidence for a complex network of specific but synergistically acting/cooperating mechanisms enabling the affected cells to resist sustained TNF exposure and resulting in the resolution of inflammation.

Soils around coal-fired thermal power plants based on coal combustion can present high concentrations of arsenic. This fact has a direct effect on the food chain. Arsenic can be absorbed by plants and vegetables through the soil, which will then serve as food for different animals, spreading the contamination. A method has been developed using high-resolution continuum source graphite furnace atomic absorption spectrometry (HR-CS GFAAS) for direct determination of arsenic in solid soil samples. Different chemical modifiers were tested to suppress the matrix effects observed. Among them, the modifier that showed the best results was the Zr, used as a permanent modifier. The optimized pyrolysis and atomization temperatures were 1000 °C and 2200 °C, respectively. A calibration curve was established using aqueous standard solutions which was linear up to 16 ng of arsenic. The characteristic mass and limit of detection were 22 pg and 73 pg As, respectively. The accuracy of the method was verified using two certified reference materials and comparison with results obtained for samples after microwave-assisted digestion. Eleven soil samples were collected around the power plant Complex Jorge Lacerda-Tractebel Suezin, in the south of Santa Catarina, Brazil. The concentration of As ranged from 3.4 mg kg -1 to 9.7 mg kg -1 , which is within the limits allowed by Brazilian legislation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Thermochemical processes can convert the biomass into fuels, such as bio-oil. The biomass submitted to pyrolysis process, such as fibers, are generally rich in silicon, an element that can lead to damages in an engine when there is high concentration in a fuel. High-resolution continuum source atomic absorption spectrometry (HR-CS AAS) is an interesting alternative for Si determination in the products and byproducts of the pyrolysis process because, besides the flame (F) and graphite furnace (GF) atomizers, it has enhanced the application of direct analysis of solid samples (SS) within GF. This study aimed the development of methods to determine Si in biomass samples, their products and byproducts using HR-CS AAS. A high-resolution continuum source atomic absorption spectrometer contrAA 700 equipped with F and GF atomizers was used throughout the study. HR-CS F AAS (λ = 251.611nm, 1 detection pixel, N 2 O/C 2 H 2 flame) was used to evaluate Si content in biomass and ash, after a microwave-assisted acid digestion with HNO 3 and HF. HR-CS GF AAS (T pyr = 1400°C, T atom = 2650°C) has evaluated Si in pyrolysis water and bio-oil at 251.611nm, and in peach pit biomass and ash at 221.174nm using SS, both wavelengths with 1 detection pixel. Rhodium (300μg) was applied as permanent modifier and 10μgPd + 6μg Mg were pipetted onto the standards/samples at each analysis. Three different biomass samples were studied: palm tree fiber, coconut fiber and peach pit, and three certified reference materials (CRM) were used to verify the accuracy of the methods. The figures of merit were LOD 0.09-20mgkg -1 , and LOQ 0.3-20mgkg -1 , considering all the methods. There were no significant differences between the CRM certified values and the determined ones, using a Student t-test with a confidence interval of 95% (n = 5). Si concentration ranged from 0.11-0.92% mm -1 , 1.1-1.7mgkg -1 , 3.3-13mgkg -1 , and 0.41-1.4%mm -1 , in biomass, bio-oil, pyrolysis water and ash, respectively. Si remained mostly in the ash, leading to a mass fraction of up to 103%, even when the Si loss is not considered. Silicon concentration in bio-oil was below 1.7mgkg -1 , which is suitable for its application as a fuel. The developed methods using HR-CS AAS are suitable for Si determination in biomass, bio-oil, pyrolysis water, and ash. The application of bio-oil as an alternative fuel would be possible evaluating its Si content due to its low levels. The mass balance for Si has proved to be an important tool in order to evaluate the correct disposal of pyrolysis process byproducts., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The chemical composition of complex inorganic materials, such as copper concentrate, may influence the economics of their further processing because most smelters, and particularly the producers of high-purity electrolyte copper, have strict limitations for the permissible concentration of impurities. These components might be harmful to the quality of the products, impair the production process and be hazardous to the environment. The goal of the present work is the development of a method for the determination of fluorine in copper concentrate using high-resolution graphite furnace molecular absorption spectrometry and direct solid sample analysis. The molecular absorption of the diatomic molecule CaF was measured at 606.440nm. The molecule CaF was generated by the addition of 200µg Ca as the molecule-forming reagent; the optimized pyrolysis and vaporization temperatures were 900°C and 2400°C, respectively. The characteristic mass and limit of detection were 0.5ng and 3ng, respectively. Calibration curves were established using aqueous standard solutions containing the major components Cu, Fe, S and the minor component Ag in optimized concentrations. The accuracy of the method was verified using certified reference materials. Fourteen copper concentrate samples from Chile and Australia were analyzed to confirm the applicability of the method to real samples; the concentration of fluorine ranged from 34 to 5676mgkg -1 . The samples were also analyzed independently at Analytik Jena by different operators, using the same equipment, but different target molecules, InF and GaF, and different operating conditions; but with a few exceptions, the results agreed quite well. The results obtained at Analytik Jena using the GaF molecule and our results obtained with CaF, with one exception, were also in agreement with the values informed by the supplier of the samples, which were obtained using ion selective electrode potentiometry after alkaline fusion. A comparison will also be made for the three target molecules and the three independently developed methods for the determination of fluorine, although all three methods used direct solid sample analysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Following the acute phase of an inflammatory reaction, a strictly controlled resolution of inflammation is necessary. A dysregulation of this process leads to hyperinflammation, chronic inflammatory disease, or immune paralysis. Different mechanisms participate in the coordinated termination of the inflammatory process, e.g. the expression of antiinflammatory molecules and different forms of tolerance. To better understand the processes which mediate resolution of TNF-dependent inflammation and induce tolerance, it is necessary to characterize the signal transduction quality during TNF long-term (pre)incubation. Within a time frame from 12 to 48h, designated as phase III of the TNF response, we measured an ongoing, constitutive activation of TNFR1/NF-κB-dependent pathways in monocytic cells. Phase III signalling which was also named "constitutive signaling in TNF tolerant cells" induces the expression of low- and high-sensitive target genes including A20 which is differentially regulated by transcriptional and proteolytic events. A20 strictly controls TNF long-term constitutive signalling in an IκB kinase complex- and partially RIP-dependent manner supported by adjuvant ABIN1. In addition, CYLD proteins participate in the regulation of this late-phase signal transduction, whereas downstream molecules such as Bcl3 and p50 are not involved. A20 and CYLD are expressed with different mRNA kinetics resulting in a strong or only a modest increase in protein levels, respectively. The identification of mechanisms which contribute to the termination of inflammation will provide additional diagnostic and therapeutic aspects to specifically diagnose certain aspects of inflammation and specifically modulate them., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tumor necrosis factor (TNF) tolerance in monocytes and macrophages means that preexposure to TNF reduces the sensitivity in these cells to a subsequent restimulation with this cytokine. Differential effects arise following preincubation with both low and high doses of TNF resulting in absolute as well as induction tolerance affecting specific immunologically relevant gene sets. In this review article, we summarize the relevance of TNF tolerance in vivo and the molecular mechanisms underlying these forms of tolerance including the role of transcription factors and signaling systems. In addition, the characteristics of cross-tolerance between TNF and lipopolysaccharide (LPS) as well as pathophysiological aspects of TNF tolerance are discussed. We conclude that TNF tolerance may represent a protective mechanism involved in the termination of inflammation and preventing excessive or prolonged inflammation. Otherwise, tolerance may also be a trigger of immune paralysis thus contributing to severe inflammatory diseases such as sepsis. An improved understanding of TNF tolerance will presumably facilitate the implementation of diagnostic or therapeutic approaches to more precisely assess and treat inflammation-related diseases.

High-resolution continuum source graphite furnace atomic absorption spectrometry (HR-CS GF AAS) has been applied for the development of a method for the determination of total As in fish oil samples using direct analysis. The method does not use any sample pretreatment, besides dilution with 1-propanole, in order to decrease the oil viscosity. The stability and sensitivity of As were evaluated using ruthenium and iridium as permanent chemical modifiers and palladium added in solution over the sample. The best results were obtained with ruthenium as the permanent modifier and palladium in solution added to samples and standard solutions. Under these conditions, aqueous standard solutions could be used for calibration for the fish oil samples diluted with 1-propanole. The pyrolysis and atomization temperatures were 1400 °C and 2300 °C, respectively, and the limit of detection and characteristic mass were 30 pg and 43 pg, respectively. Accuracy and precision of the method have been evaluated using microwave-assisted acid digestion of the samples with subsequent determination by HR-CS GF AAS and ICP-MS; the results were in agreement (95% confidence level) with those of the proposed method., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An analytical method for the determination of sulfur, as the tin mono-sulfide (SnS) molecule, in crude oil using high-resolution continuum source graphite furnace molecular absorption spectrometry (HR-CS GF MAS) has been developed. The molecular absorbance of the SnS has been measured using the wavelength at 271.624 nm and the crude oil samples were prepared as micro-emulsions due to their high viscosity. Several chemical modifiers (Ir, Pd, Ru, Zr) were tested and palladium was chosen, because it exhibited the best performance. The heating program was optimized by comparing the pyrolysis and vaporization curves obtained for an aqueous sulfur standard and a micro-emulsion of a crude oil certified reference material (CRM). The optimum pyrolysis and vaporization temperatures were found to be 600 and 2000°C, respectively. The limit of detection and the characteristic mass using micro-emulsion analysis of crude oil samples were 5.8 and 13.3 ng S. Accuracy and precision of the method has been evaluated using two crude oil CRM (NIST 2721 and NIST 2722), showing good agreement with the informed or certified values., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A method has been developed for the determination of lead in biomass, bio-oil, pyrolysis aqueous phase, and biomass ashes by high-resolution continuum source graphite furnace atomic absorption spectrometry (HR-CS GF AAS) and direct solid or liquid sample analysis. All measurements were performed without chemical modifier and calibration could be carried out using aqueous standard solutions. A pyrolysis temperature of 800°C and an atomization temperature of 2200°C were applied. The limits of detection and quantification were, respectively, 0.5 µg kg(-1) and 2 µg kg(-1) using the analytical line at 217.001 nm and 6 µg kg(-1) and 19 µg kg(-1) at 283.306 nm. The precision, expressed as relative standard deviation, was between 3% and 10%, which is suitable for direct analysis. The lead concentrations found for the solid samples varied between 0.28 and 1.4 mg kg(-1) for biomass and between 0.25 and 2.3 mg kg(-1) for ashes, these values were much higher than those found for bio-oil (2.2-16.8 µg kg(-1)) and pyrolysis aqueous phase (3.2-18.5 µg kg(-1)). After the determination of lead in the samples, it was possible to estimate the relative distribution of this element in the fractions of the pyrolysis products, and it was observed that most of the lead present in the biomass was eliminated to the environment during the pyrolysis process, with a significant portion retained in the ashes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation.

Direct solid sample analysis with graphite furnace atomic absorption spectrometry (SS-GF AAS) was investigated initially with the intention of developing a method for the determination of total As in fish and other seafood. A mixture of 0.1% Pd+0.06% Mg+0.06% Triton X-100 was used as the chemical modifier, added in solution over the solid samples, making possible the use of pyrolysis and atomization temperatures of 1200 °C and 2400 °C, respectively. The sample mass had to be limited to 0.25 mg, as the integrated absorbance did not increase further with increasing sample mass. Nevertheless, the recovery of As from several certified reference materials was of the order of 50% lower than the certified value. Strong molecular absorption due to the phosphorus monoxide molecule (PO) was observed with high-resolution continuum source AAS (HR CS AAS), which, however, did not cause any spectral interference. A microwave-assisted digestion with HNO3/H2O2 was also investigated to solve the problem; however, the results obtained for several certified reference materials were statistically not different from those found with direct SS-GF AAS. Accurate values were obtained using inductively coupled plasma mass spectrometry (ICP-MS) to analyze the digested samples, which suggested that organic As compounds are responsible for the low recoveries. HPLC-ICP-MS was used to determine the arsenobetaine (AB) concentration. Accurate results that were not different from the certified values were obtained when the AB concentration was added to the As concentration found by SS-GF AAS for most certified reference materials (CRM) and samples, suggesting that SS-GF AAS could be used as a fast screening procedure for inorganic As determination in fish and seafood., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A new method for the direct analysis of coal using electrothermal vaporization inductively coupled plasma mass spectrometry and direct solid sample analysis was developed, aiming at the determination of Br and Cl. The procedure does not require any significant sample pretreatment and allows simultaneous determination of both elements to be carried out, requiring small mass aliquots of sample (about 0.5 mg). All operating parameters, including carrier gas flow-rate and RF power, were optimized for maximum sensitivity. The use of modifiers/aerosol carriers (Pd, Pd+Al and Pd+Ca) was evaluated, and the mixture of Pd and Ca was chosen, allowing pyrolysis and vaporization temperatures of 700°C and 1900°C, respectively. Chlorine was accurately determined using calibration against solid standards, whereas Br could also be determined using calibration against aqueous standard solutions. The limits of quantification were 0.03 μg g(-1) for Br and 7 μg g(-1) for Cl, and no spectral interferences were observed., (Copyright © 2014 Elsevier B.V. All rights reserved.)