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Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of ∼24 h in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here, we used affinity purification coupled with mass spectrometry (APMS) to identify time-of-day–specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD-REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day–specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways.

Indoor path loss models characterize the attenuation of signals between a transmitting and receiving antenna for a certain frequency and type of environment. Their use ranges from network coverage planning to joint communication and sensing applications such as localization and crowd counting. The need for this proposed geodesic path model comes forth from attempts at path loss-based localization on ships, for which the traditional models do not yield satisfactory path loss predictions. In this work, we present a novel pathfinding-based path loss model, requiring only a simple binary floor map and transmitter locations as input. The approximated propagation path is determined using geodesics, which are constrained shortest distances within path-connected spaces. However, finding geodesic paths from one distinct path-connected space to another is done through a systematic process of choosing space connector points and concatenating parts of the geodesic path. We developed an accompanying tool and present its algorithm which automatically extracts model parameters such as the number of wall crossings on the direct path as well as on the geodesic path, path distance, and direction changes on the corners along the propagation path. Moreover, we validate our model against path loss measurements conducted in two distinct indoor environments using DASH-7 sensor networks operating at 868 MHz. The results are then compared to traditional floor-map-based models. Mean absolute errors as low as 4.79 dB and a standard deviation of the model error of 3.63 dB is achieved in a ship environment, almost half the values of the next best traditional model. Improvements in an office environment are more modest with a mean absolute error of 6.16 dB and a standard deviation of 4.55 dB.

Yersinia enterocolitica is a food-borne Gram-negative pathogen responsible for several gastrointestinal disorders. Host-specific lytic bacteriophages have been increasingly used recently as an alternative or complementary treatment to combat bacterial infections, especially when antibiotics fail. Here, we describe the proteogenomic characterization and host receptor identification of the siphovirus vB_YenS_phi R2-01 (in short, phi R2-01) that infects strains of several Yersinia enterocolitica serotypes. The phi R2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The phi R2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical phi R2-01 proteins. Label-free mass-spectrometry-based proteomics confirmed the expression of 90 of the phi R2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and phi R2-01 adsorption experiments identified the outer membrane vitamin B12 receptor BtuB as the host receptor. This study provides a proteogenomic characterization of a T5-type bacteriophage and identifies specific Y. enterocolitica strains sensitive to infection with possible future applications of phi R2-01 as a food biocontrol or phage therapy agent.

One of the best-established area within multi-omics is proteogenomics, whereby the underpinning technologies are next-generation sequencing (NGS) and mass spectrometry (MS). Proteogenomics has contributed significantly to genome (re)-annotation, whereby novel coding sequences (CDS) are identified and confirmed. By incorporating in-silico translated genome variants in protein database, single amino acid variants (SAAV) and splice proteoforms can be identified and quantified at peptide level. The application of proteogenomics in cancer research potentially enables the identification of patient-specific proteoforms, as well as the association of the efficacy or resistance of cancer therapy to different mutations. Here, we discuss how NGS/TGS data are analyzed and incorporated into the proteogenomic framework. These sequence data mainly originate from whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. We explain two major strategies for sequence analysis i.e., de novo assembly and reads mapping, followed by construction of customized protein databases using such data. Besides, we also elaborate on the procedures of spectrum to peptide sequence matching in proteogenomics, and the relationship between database size on the false discovery rate (FDR). Finally, we discuss the latest development in proteogenomics-assisted precision oncology and also challenges and opportunities in proteogenomics research.

One of the best-established area within multi-omics is proteogenomics, whereby the underpinning technologies are next-generation sequencing (NGS) and mass spectrometry (MS). Proteogenomics has contributed significantly to genome (re)-annotation, whereby novel coding sequences (CDS) are identified and confirmed. By incorporating in-silico translated genome variants in protein database, single amino acid variants (SAAV) and splice proteoforms can be identified and quantified at peptide level. The application of proteogenomics in cancer research potentially enables the identification of patient-specific proteoforms, as well as the association of the efficacy or resistance of cancer therapy to different mutations. Here, we discuss how NGS/TGS data are analyzed and incorporated into the proteogenomic framework. These sequence data mainly originate from whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. We explain two major strategies for sequence analysis i.e., de novo assembly and reads mapping, followed by construction of customized protein databases using such data. Besides, we also elaborate on the procedures of spectrum to peptide sequence matching in proteogenomics, and the relationship between database size on the false discovery rate (FDR). Finally, we discuss the latest development in proteogenomics-assisted precision oncology and also challenges and opportunities in proteogenomics research.

In the past decade, calcium phosphate (CaP) ceramics have emerged as alternatives to autologous bone grafts for the treatment of large, critical-sized bone defects. In order to be effective in the regeneration of such defects, ceramics must show osteoinductive behaviour, defined as the ability to induce de novo heterotopic bone formation. While a set of osteoinductive CaP ceramics has been developed, the exact processes underlying osteoinduction, and the role of the physical and chemical properties of the ceramics, remain largely unknown. Previous studies have focused on the role of the transcriptome to shed light on the mechanism of osteoinduction at the mRNA level. To complement these studies, a proteomic analysis was performed to study the behaviour of hMSCs on osteoinductive and non-osteoinductive CaPs. The results of this analysis suggest that plasma cell glycoprotein 1 (PC-1), encoded by the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, plays a key role in the process of osteoinduction by CaP ceramics. Validation experiments have confirmed that indeed, the mRNA expression of ENPP1 and the production of PC-1 are higher on osteoinductive than on non-osteoinductive CaP ceramics, a trend that was also observed for other osteogenic markers such as bone morphogenetic protein 2 (BMP2) and osteopontin (OPN), but not for alkaline phosphatase (ALP). Our results also showed that the expression of PC-1 is restricted to those cells which are in direct contact with the CaP ceramic surface, plausibly due to the localised depletion of calcium and inorganic phosphate ions from the supersaturated cell culture medium as CaP crystallises on the ceramic surface. Replicating the surface of the osteoinductive ceramic in polystyrene resulted in a significant decrease in ENPP1 expression, suggesting that surface structural properties alone are not sufficient to induce ENPP1 expression. Finally, knocking down ENPP1 expression in hMSCs resulted in increased BMP2 expression, both at the mRNA and protein level, suggesting that ENPP1 is a negative regulator of BMP-2 signalling. Taken together, this study shows, for the first time, that ENPP1/PC-1 plays an important role in CaP-induced osteogenic differentiation of hMSCs and thus possibly osteoinduction by CaP ceramics. Furthermore, we have identified a crucial role for the interfacial (chemical) events occurring on the CaP ceramic surface in the process of osteoinduction. This knowledge can contribute to the development of new bone graft substitutes, with improved osteoinductive potential.

Understanding the pathogenic mechanisms of disease mutations is critical to advancing treatments. ALS-associated mutations in the gene encoding the microtubule motor KIF5A result in skipping of exon 27 (KIF5AΔExon27) and the encoding of a protein with a novel 39 amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C terminus of mutant KIF5A results in a constitutively active state. Furthermore, mutant KIF5A possesses altered protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis. ispartof: CELL REPORTS vol:39 issue:1 ispartof: location:United States status: published

The control of ovulation helps guarantee the success of reproduction and as such, contributes to the fitness of a species. In mammals, two types of ovulation are observed: induced and spontaneous ovulation. Recent work on camelids, that are induced ovulators, highlighted the role of a factor present in seminal plasma, beta Nerve Growth Factor (β-NGF), as the factor that triggers ovulation in a GnRH dependent manner. In the present work, we characterized alpaca β-NGF (aβ-NGF) and its 3D structure and compared it with human recombinant β-NGF (hβ-NGF). We showed that the β-NGF enriched fraction of alpaca semen and the human recombinant protein, both stimulated spontaneous electrical activity of primary GnRH neurons derived from mouse embryonic olfactory placodes. This effect was dose-dependent and mediated by p75 receptor signaling. P75 receptors were found expressed in vitro by olfactory ensheathing cells (OEC) in close association with GnRH neurons and in vivo by tanycytes in close vicinity to GnRH fibers in adult mouse. Altogether, these results suggested that β-NGF induced ovulation through an increase in GnRH secretion provoked by a glial dependent P75 mediated mechanism.

We developed a simple algorithm to classify clouds based on global radiation and cloud base height measured by pyranometer and ceilometer, respectively. We separated clouds into seven different classes (stratus, stratocumulus, cumulus, nimbostratus, altocumulus + altostratus, cirrus + cirrocumulus + cirrostratus and clear sky + cirrus). We also included classes for cumulus and cirrus clouds causing global radiation enhancement, and we classified multilayered clouds, when captured by the ceilometer, based on their height and characteristics (transmittance, patchiness and uniformity). The overall performance of the algorithm was nearly 70 % when compared with classification by an observer using total-sky images. The performance was best for clouds having well-distinguishable effects on solar radiation: nimbostratus clouds were classified correctly in 100 % of the cases. The worst performance corresponds to cirriform clouds (50 %). Although the overall performance of the algorithm was good, it is likely to miss the occurrences of high and multilayered clouds. This is due to the technical limits of the instrumentation: the vertical detection range of the ceilometer and occultation of the laser pulse by the lowest cloud layer. We examined the use of clearness index, which is defined as a ratio between measured global radiation and modeled radiation at the top of the atmosphere, as an indicator of clear-sky conditions. Our results show that cumulus, altocumulus, altostratus and cirriform clouds can be present when the index indicates clear-sky conditions. Those conditions have previously been associated with enhanced aerosol formation under clear skies. This is an important finding especially in the case of low clouds coupled to the surface, which can influence aerosol population via aerosol–cloud interactions. Overall, caution is required when the clearness index is used in the analysis of processes affected by partitioning of radiation by clouds.

In recent years, synthetic calcium phosphate (CaP) ceramics have emerged as an alternative to bone grafts in the treatment of large critical-sized bone defects. To successfully substitute for bone grafts, materials must be osteoinductive, that is, they must induce osteogenic differentiation and subsequent bone formation in vivo. Although a set of osteoinductive CaP ceramics has been developed, the precise biological mechanism by which a material directs cells toward osteogenesis and the role of individual chemical and physical properties in this mechanism remain incompletely understood. Here, we used proteomics to compare serum protein adsorption to two CaP ceramics with different osteoinductive potential, namely an osteoinductive β-tricalcium phosphate (TCP) and a non-osteoinductive hydroxyapatite (HA). Moreover, we analyzed the protein profiles of human mesenchymal stromal cells (hMSCs) cultured on these two ceramics. The serum protein adsorption experiments in the absence of cells highlighted the proteins that are highly abundant in the serum and/or have a high affinity to CaP. The extent of adsorption was suggested to be affected by the available surface area for binding and by the ion exchange dynamics on the surface. Several proteins were uniquely expressed by hMSCs on TCP and HA surfaces. Proteins identified as enriched on TCP were involved in processes related to wound healing, cell proliferation, and the production of extracellular matrix. On the other hand, proteins that were enriched on HA were involved in processes related to protein production, translation, localization, and secretion. In addition, we performed a separate proteomics analysis on TCP, HA, and two biphasic calcium phosphates with known osteoinductive potential and performed a clustering analysis on a combination of a set of proteins found to be enriched on osteoinductive materials with a set of proteins already known to be involved in osteogenesis. This yielded two protein networks potentially involved in the process of osteoinduction – one consisting of collagen fragments and collagen-related enzymes and a second consisting of endopeptidase inhibitors and regulatory proteins. The results of this study show that protein profiling can be a useful tool to help understand the effect of biomaterial properties on the interactions between a biomaterial and a biological system. Such understanding will contribute to the design and development of improved biomaterials for (bone) regenerative therapies., Graphical abstract Image 1

Differential analysis of MS-data to identify biomarkers or to understand biology is a cornerstone in proteomics. DEqMS is a robust method for analysis of both labelled and label-free MS-data. The method takes into account the inherent dependence of protein variance on the number of PSMs or peptides used for quantification, thereby providing a more accurate variance estimation. Compared to current methods, DEqMS achieves better accuracy and statistical power in quantitative proteomics. The tool is available as user-friendly R package., Graphical Abstract Highlights DEqMS is a method for statistical analysis of quantitative MS-data. Variance estimates based on the actual MS-data structure. Improved statistical power and accuracy in protein differential analysis. DEqMS is available as a user-friendly R package in Bioconductor., Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all data sets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several data sets including E. coli proteome spike-in data, using both label-free and TMT-labeled quantification. Compared with previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared with other statistical methods in both label-free and labeled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.

Hunter’s syndrome (mucopolysaccharidosis type II) is a rare X-linked lysosomal storage disorder caused by mutations in the iduronate 2-sulfatase (IDS) gene. Motivated by the case of a child affected by this syndrome, we compared the intracellular fate of wild type IDS (IDSWT) and of four nonsense mutations of IDS (IDSL482X, IDSY452X, IDSR443X and IDSW337X) generating progressively shorter forms of IDS associated with mild to severe forms of the disease. Our analyses revealed formylation of all forms of IDS at cysteine 84, which is a pre-requisite for enzymatic activity. After formylation, IDSWT was transported within lysosomes, where it was processed in the mature form of the enzyme. The length of disease-causing deletions correlated with gravity of the folding and transport phenotype, which was anticipated by molecular dynamics analyses. The shortest form of IDS, IDSW337X, was retained in the endoplasmic reticulum (ER) and degraded by the ubiquitin-proteasome system. IDSR443X, IDSY452X, IDSL482X passed ER quality control, were transported to the lysosomes, but failed lysosomal quality control resulting in their rapid clearance and in loss-of-function phenotype. Failure of ER quality control inspection is an established cause of loss-of-function observed in protein misfolding diseases. Our data reveal that fulfillment of ER requirements might not be sufficient, highlight lysosomal quality control as the distal station to control lysosomal enzymes fitness and pave the way for alternative therapeutic interventions.

Particle precipitation is a central aspect of space weather, as it strongly couples the magnetosphere and the ionosphere and can be responsible for radio signal disruption at high latitudes. We present the first hybrid-Vlasov simulations of proton precipitation in the polar cusps. We use two runs from the Vlasiator model to compare cusp proton precipitation fluxes during southward and northward interplanetary magnetic field (IMF) driving. The simulations reproduce well-known features of cusp precipitation, such as a reverse dispersion of precipitating proton energies, with proton energies increasing with increasing geomagnetic latitude under northward IMF driving, and a nonreversed dispersion under southward IMF driving. The cusp is also found more polewards in the northward IMF simulation than in the southward IMF simulation. In addition, we find that the bursty precipitation during southward IMF driving is associated with the transit of flux transfer events in the vicinity of the cusp. In the northward IMF simulation, dual lobe reconnection takes place. As a consequence, in addition to the high-latitude precipitation spot associated with the lobe reconnection from the same hemisphere, we observe lower-latitude precipitating protons which originate from the opposite hemisphere’s lobe reconnection site. The proton velocity distribution functions along the newly closed dayside magnetic field lines exhibit multiple proton beams travelling parallel and antiparallel to the magnetic field direction, which is consistent with previously reported observations with the Cluster spacecraft. In both runs, clear electromagnetic ion cyclotron waves are generated in the cusps and might further increase the calculated precipitating fluxes by scattering protons to the loss cone in the low-altitude cusp. Global kinetic simulations can improve the understanding of space weather by providing a detailed physical description of the entire near-Earth space and its internal couplings.

Background The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the "vulnerable plaque," necessitating an improved approach for predicting onset of symptoms. Methods We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. Results Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. Conclusion The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. Funding UK: British Heart Foundation (BHF); King's BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy's and St Thomas' NHS Foundation Trust and King's College London in partnership with King's College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.

The Atlantic mackerel, Scomber scombrus, is one of the most fished species in the world, but it is still largely used for low-value products, such as bait, mainly for crustacean fishery. This resource could be transformed into products of high value and may offer new opportunities for the discovery of bioactive molecules. Mackerel hydrolysate was investigated to discover antibacterial peptides with biotechnological potential. The proteolytic process generated a hydrolysate composed of 96% proteinaceous compounds with molecular weight lower than 7 kDa. From the whole hydrolysate, antibacterial activity was detected against both Gram-negative and Gram-positive bacteria. After solid phase extraction, purification of the active fraction led to the identification of 4 peptide sequences by mass spectrometry. The peptide sequence N-KVEIVAINDPFIDL-C, called Atlantic Mackerel GAPDH-related peptide (AMGAP), was selected for chemical synthesis to confirm the antibacterial activity and to evaluate its stability through in vitro digestibility. Minimal inhibitory concentrations of AMGAP revealed that Listeria strains were the most sensitive, suggesting potential as food-preservative to prevent bacterial growth. In addition, in vitro digestibility experiments found rapid (after 20 min) and early digestibility (stomach). This study highlights the biotechnological potential of mackerel hydrolysate due to the presence of the antibacterial AMGAP peptide.

The interactions between oviductal fluid (OF) proteins and spermatozoa play major roles in sperm selection, storage and capacitation before fertilization. However, only a few sperm-interacting proteins in the OF has been identified and very little is known about the regulation of sperm-oviduct interactions across the estrous cycle. Samples of bovine frozen-thawed sperm from three bulls were incubated with OF at pre-, post-ovulatory stages (Pre-/Post-ov) or luteal phase (LP) of the estrous cycle (7 mg/mL proteins, treated groups) or with a protein-free media (control). The proteomes of sperm cells were assessed by nanoLC–MS/MS and quantified by label-free methods. A total of 27 sperm-interacting proteins originating in the OF were identified. Among those, 14 were detected at all stages, eight at Post-ov and LP and five only at LP. The sperm-interacting proteins detected at all stages or at LP and Post-ov were on average more abundant at LP than at other stages (P

With the advent of mass spectrometry based proteomics, the identification of thousands of proteins has become commonplace in biology nowadays. Increasingly, efforts have also been invested toward the detection and localization of posttranslational modifications. It is furthermore common practice to quantify the identified entities, a task supported by a panel of different methods. Finally, the results can also be enriched with functional knowledge gained on the proteins, detecting for instance differentially expressed gene ontology terms or biological pathways. In this study, we review the resources, methods and tools available for the researcher to achieve such a quantitative functional analysis. These include statistics for the post-processing of identification and quantification results, online resources and public repositories. With a focus on free but user-friendly software, preferably also open-source, we provide a list of tools designed to help the researcher manage the vast amount of data generated. We also indicate where such applications currently remain lacking. Moreover, we stress the eventual pitfalls of every step of such studies. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan. acceptedVersion

Brassinosteroid (BR) hormones are primarily perceived at the cell surface by the leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1 (BRI1). In Arabidopsis thaliana, BRI1 has two close homologs, BRI1-LIKE1 (BRL1) and BRL3, respectively, which are expressed in the vascular tissues and regulate shoot vascular development. Here, we identify novel components of the BRL3 receptor complex in planta by immunoprecipitation and mass spectrometry analysis. Whereas BRI1 ASSOCIATED KINASE1 (BAK1) and several other known BRI1 interactors coimmunoprecipitated with BRL3, no evidence was found of a direct interaction between BRI1 and BRL3. In addition, we confirmed that BAK1 interacts with the BRL1 receptor by coimmunoprecipitation and fluorescence microscopy analysis. Importantly, genetic analysis of brl1 brl3 bak1-3 triple mutants revealed that BAK1, BRL1, and BRL3 signaling modulate root growth and development by contributing to the cellular activities of provascular and quiescent center cells. This provides functional relevance to the observed protein–protein interactions of the BRL3 signalosome. Overall, our study demonstrates that cell-specific BR receptor complexes can be assembled to perform different cellular activities during plant root growth, while highlighting that immunoprecipitation of leucine-rich repeat receptor kinases in plants is a powerful approach for unveiling signaling mechanisms with cellular resolution in plant development., S.D.C., T.E.N., and M.B.G. are funded by grants from the U.S. National Science Foundation (MCB-1021363 and DBI-1126244). N.F. is funded by an Formació de personal Investigador PhD fellowship from the Generalitat de Catalunya. A.I.C.-D. and S.d.V. are recipients of a Marie-Curie Initial Training Network “BRAVISSIMO” (Grant PITN-GA-2008-215118). S.d.V. was a sabbatical professor funded by AGAUR (Generalitat de Catalunya) in A.I.C.-D.'s lab. A.I.C.-D. is funded by a grant from the Spanish Ministry of Education and Science (BIO2010/00505).

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of Nglycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

Proteogenomics is a multi-omics research field that has the aim to efficiently integrate genomics, transcriptomics and proteomics. With this approach it is possible to identify new patient-specific proteoforms that may have implications in disease development, specifically in cancer. Understanding the impact of a large number of mutations detected at the genomics level is needed to assess the effects at the proteome level. Proteogenomics data integration would help in identifying molecular changes that are persistent across multiple molecular layers and enable better interpretation of molecular mechanisms of disease, such as the causal relationship between single nucleotide polymorphisms (SNPs) and the expression of transcripts and translation of proteins compared to mainstream proteomics approaches. Identifying patient-specific protein forms and getting a better picture of molecular mechanisms of disease opens the avenue for precision and personalized medicine. Proteogenomics is, however, a challenging interdisciplinary science that requires the understanding of sample preparation, data acquisition and processing for genomics, transcriptomics and proteomics. This chapter aims to guide the reader through the technology and bioinformatics aspects of these multi-omics approaches, illustrated with proteogenomics applications having clinical or biological relevance.

Genome-wide association studies (GWASs) have identified many SNPs underlying variations in plasma-lipid levels. We explore whether additional loci associated with plasma-lipid phenotypes, such as high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TGs), can be identified by a dense gene-centric approach. Our meta-analysis of 32 studies in 66,240 individuals of European ancestry was based on the custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) covering ∼2,000 candidate genes. SNP-lipid associations were replicated either in a cohort comprising an additional 24,736 samples or within the Global Lipid Genetic Consortium. We identified four, six, ten, and four unreported SNPs in established lipid genes for HDL-C, LDL-C, TC, and TGs, respectively. We also identified several lipid-related SNPs in previously unreported genes: DGAT2, HCAR2, GPIHBP1, PPARG, and FTO for HDL-C; SOCS3, APOH, SPTY2D1, BRCA2, and VLDLR for LDL-C; SOCS3, UGT1A1, BRCA2, UBE3B, FCGR2A, CHUK, and INSIG2 for TC; and SERPINF2, C4B, GCK, GATA4, INSR, and LPAL2 for TGs. The proportion of explained phenotypic variance in the subset of studies providing individual-level data was 9.9% for HDL-C, 9.5% for LDL-C, 10.3% for TC, and 8.0% for TGs. This large meta-analysis of lipid phenotypes with the use of a dense gene-centric approach identified multiple SNPs not previously described in established lipid genes and several previously unknown loci. The explained phenotypic variance from this approach was comparable to that from a meta-analysis of GWAS data, suggesting that a focused genotyping approach can further increase the understanding of heritability of plasma lipids. © 2012 The American Society of Human Genetics.

Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (

Wave-front analysis data from the human eye are commonly presented using the aberration coefficient c(4)(0) (primary spherical aberration) together with an overall measure of all higher-order aberrations. If groups of subjects are compared, however, the relevance of an observed difference cannot easily be assessed from these summary aberration measures. A method was developed for estimating various optical variables relevant to visual perception from summary wave-front analysis data. These variables were the myopic shift (the difference in the optimal focus between high and low spatial frequencies, a threat to the simultaneous in-focus viewing of fine and coarse patterns), the depth-of-focus (at 8 cpd), and the modulation transfer at high (16 cpd; reading small print) and low (4 cpd; edge detection) spatial frequencies. The depth-of-focus was defined in two ways: using a relative measure (the full width at half-height of the through-focus curve) and an absolute measure (the range where the through-focus curve exceeds a predefined modulation transfer value). The method was shown to be accurate by using previously published contrast sensitivity data and wave-front analysis data. The applicability of the method was illustrated by applying the method to wave-front analysis measurements performed in pseudophakic patients with aspheric and spherical intraocular lenses. (C) 2010 Optical Society of America

Supernatants from cell cultures (also called conditioned media, CMs) are commonly analyzed to study the pool of secreted proteins (secretome). To reduce the exogenous protein background, serum-free media are often used to obtain CMs. Serum deprivation, however, can severely affect cell viability and phenotype, including protein secretion. We present a strategy to analyze the proteins secreted by cells in fetal bovine serum-containing CMs, which combines the advantage of metabolic labeling and protein concentration linearization techniques. Incubation of CMs with a hexapeptide ligand library was used to reduce the dynamic range of the samples and led to the identification of 3 times more proteins than in untreated CM samples. Labeling with a deuterated amino acid was used to distinguish between cellular proteins and homologous bovine proteins contained in the medium. Application of the strategy to two breast cancer cell lines led to the identification of proteins secreted in different amounts and which could correlate with their varying degree of aggressiveness. Selected reaction monitoring (SRM)-based quantitation of three proteins of interest in the crude samples yielded data in good agreement with the results from concentration-equalized samples.

Transport of solutes between the cytosol and the vacuolar lumen is of crucial importance for various functions of vacuoles, including ion homeostasis; detoxification; storage of different molecules such as amino acids, phosphate, and calcium ions; and proteolysis. To identify proteins that catalyze solute transport across the vacuolar membrane, the membrane proteome of purified Saccharomyces cerevisiae vacuoles was analyzed. Subtractive proteomics was used to distinguish contaminants from true vacuolar proteins by comparing the relative abundances of proteins in pure and crude preparations. A robust statistical analysis combining enrichment ranking with the double boundary iterative group analysis revealed that 148 proteins were significantly enriched in the pure vacuolar preparations. Among these proteins were well characterized vacuolar proteins, such as the subunits of the vacuolar H(+)-ATPase, but also proteins that had not previously been assigned to a cellular location, many of which are likely novel vacuolar membrane transporters, e. g. for nucleosides and oligopeptides. Although the majority of contaminating proteins from other organelles were depleted from the pure vacuolar membranes, some proteins annotated to reside in other cellular locations were enriched along with the vacuolar proteins. In many cases the enrichment of these proteins is biologically relevant, and we discuss that a large group is involved in membrane fusion and protein trafficking to vacuoles and may have multiple localizations. Other proteins are degraded in vacuoles, and in some cases database annotations are likely to be incomplete or incorrect. Our work provides a wealth of information on vacuolar biology and a solid basis for further characterization of vacuolar functions. Molecular & Cellular Proteomics 8:380-392, 2009.

In this paper, two research approaches to specify the relation between land use types and their explanatory factors are applied to the same modelling framework. The two approaches are used to construct land suitability maps, which are used as inputs in two model applications. The first is an inductive approach that uses regression analysis. The second applies a theoretical, actor decision framework to derive relations deductively using detailed field data. Broadly speaking, this classification coincides with the distinction between empirical and theoretical models and the distinction between deriving process from pattern and pattern from process. The two modelling approaches are illustrated by a scenario analysis for a case study in a municipality in the Philippines. Goodness-of-fit of the deductive approach in predicting current land use is slightly lower compared to the inductive approach. Resulting land use projections from the modelling exercise for the two approaches differ in 15 percent of the cells, which is caused by differences in the specification of the suitability maps. The paper discusses the assumptions underlying the two approaches as well as the implications for the applicability of the models in policy-oriented research. The deductive approach describes processes explicitly and can therefore better handle discontinuities in land use processes. This approach allows the user to evaluate a wide range of scenarios, which can also include new land use types. The inductive approach is easily reproducible by others but cannot guarantee causality. Therefore, the inductive approach is less suitable to handle discontinuities or additional land use types, but is well able to rapidly identify hotspots of land use change. It is concluded that both approaches have their advantages and drawbacks for different purposes. Generally speaking, the inductive approach is applicable in situations with relatively small land use changes, without introduction of new land use types, whereas the deductive approach is more flexible. The choice of modelling approach should therefore be based on the research and policy questions for which it is used.

Neutron-induced cross sections of short-lived nuclei are important in various fields such as fundamental physics, astrophysics or nuclear energy. However, these cross sections are often extremely difficult to measure due to high radioactivity of the targets involved. The surrogate-reaction method is an indirect way to determine neutron-induced cross sections of short-lived nuclei. In order to study the validity of the method, we have measured for the very first time in a surrogate-reaction experiment simultaneously fission and gamma-decay probabilities for the actinides 236U, 238U, 237Np and 238Np. This is challenging because one has to remove the gamma rays emitted by the fission fragments. The measurement was performed at the Oslo cyclotron.Our results show that for a given excitation energy, our gamma-decay probabilities are several times higher than neutron-induced probabilities, which can be attributed to differences in spin distribution between the two types of reactions. On the other hand, our fission probabilities are in good agreement with neutron-induced data. Statistical-model calculations applied with standard parameters cannot repro duce the weak spin sensibility to variations of the angular momentum observed for the fission probabilities. However, it is possible to repro duce the experimental observations by considering a stronger increase of the moment of inertia of the fissionning nucleus with deformation. Further theoretical efforts are needed to improve the understanding of our results., Les sections efficaces induites par neutrons des noyaux de courte durée de vie jouent un rôle important dans des domaines variés parmi la physique fondamentale, l'astrophysique ou l'énergie nucléaire. Malheureusement de nombreuses contraintes liées à la radiotoxicité des cibles rendent la mesure de ces sections efficaces souvent très difficiles. La méthode de substitution est une technique de mesure indirecte de sections efficaces neutroniques de noyaux radioactifs qui à l'avantage de s'affranchir de ces contraintes. Pour la première fois dans ce type d'expérience, les probabilités de fission et d'émission gamma sont mesurées simultanément, pour les actinides 236U, 238U, 237Np et 238Np dans le but d'étudier la validité de la méthode. Une des difficultés provient de la soustraction des gammas des fragments de fission et cette mesure constitue en cela un véritable dé . Cette expérience de mesure simultanée a été effectuée au cyclotron d'Oslo.A une énergie d'excitation fixée du noyau formé, les résultats montrent que les probabilités de fission de substitution sont en bon accord avec celles induites par neutron alors que les probabilités d'émission gamma mesurées sont plusieurs fois plus élevées. Ces écarts sont liés à la différence distribution spin peuplée par le noyau entre les deux méthodes. Des calculs de modèles statistiques avec des paramètres standards n'ont pas permis de reproduire cette insensibilité de la probabilité de fission vis à vis du spin du noyau. La reproduction des observations expérimentales devient possible en considérant un moment d'inertie du noyau fissionnant qui augmente plus rapidement avec la déformation du noyau que ne le préconisent les paramètres standards. De nouveaux efforts théoriques sont à fournir pour améliorer la compréhension de nos résultats.

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterisation of the total secretome of this zoonotic parasite. Fasciola secretes at least two sub-populations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialised cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic datasets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular machinery required for EV biogenesis is constitutively expressed across the intra-mammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

Ensconsin cooperates with its binding partner, Kinesin-1, during interphase to trigger centrosome separation, but it promotes microtubule polymerization independently of Kinesin-1 to control spindle length during mitosis., The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications., The (seco)iridoids and their monoterpenoid indole alkaloid (MIA) derivatives are plant-derived compounds with pharmaceutical applications. Here, the authors identify the last four missing steps of the (seco)iridoid pathway, which they reconstitute in an alternative plant host to produce the complex MIA, strictosidine.

WOS: 000344636500065 PubMed ID: 25210790 Metal based chemotherapeutic drugs are widely used as an effective method to defeat various cancers. In this study, the mechanism of action of a novel therapeutic agent, [Pd(sac)(terpy)](sac)center dot 4H(2)O (sac = saccharinate, and terpy = 2,2':6',2 ''-terpyridine) was studied. To better understand the proteomic changes in response to this agent, we performed nano LC-MS/MS analyses in human breast cancer cells (MDA-MB-231). Thirty proteins were identified to be differentially expressed more than 40% after drug treatment. Many cellular pathways were affected, including proteins involved in DNA repair, apoptosis, energy metabolism, protein folding, cytoskeleton, pre-mRNA maturation, or protein translation. The changes in protein expression were further verified for XRCC5, which plays a role in double strand break (DSB) repair, and ubiquitin, which is involved in protein degradation and apoptosis. The elevated XRCC5 levels were suggestive of increased DSBs. The presence of DSBs was confirmed by smearing of plasmid DNA in vitro and induction of gamma H2AX foci in vivo. There was also increased intracellular reactive oxygen species (ROS) formation, as detected by 2',7'-dichlorofluorescein diacetate (DCFDA) staining. Scavenging ROS by N-acetylcysteine rescued cell death in response to Pd(II) treatment, potentially explaining how the Pd(II) complex damaged the DNA. The details of this analysis and the significance will be discussed during the scope of this work.

Our study deals with the ultramicroscopic structure of the yellow pigmented cuticular stripes on the abdomen of the oriental hornet Vespa orientalis (Hymenoptera, Vespinae). The abdominal cuticle is composed of numerous (more than 25) lamellae which progressively attenuate as one proceeds from the outside interiorly. On a logarithmic scale the pattern of lamellar attenuation is a linear drop to almost asymptotic thickness with the innermost lamellae at least one order of magnitude less than that of the outermost one and describable by a mathematic equation. Each lamella is a composite structure consisting of two primary elements, namely, a skeleton and filling material. The skeleton is composed of chitin and comprised of parallel 'plates' which visibly separate between the lamellae. These 'plates' are interconnected by: (1) perpendicular 'pillars' which are distributed intermittently at more or less uniform intervals; and (2) by a layer of chitin shaped like a horizontal helix or at times even a double helix whose diameter equals the distance between two successive lamellae. In the space within the skeletal element there is a filling material of variable nature, mostly proteinaceous, which dissolves out and vacates the cuticle during its processing for microscopic observation, leaving behind the skeletal element. Such structure of the cuticle, wherein two different materials combine serially into one duplex structure is typical of composite materials. This duplex structuring of the lamellae, combined with their gradual attenuation and compacting towards the interior, lends the cuticle in the abdominal region the shape of a thin-walled laminated beam endowed with a wide array of mechanical, thermal, electric, optic, acoustic and probably numerous other properties which render the cuticle efficient for multiple purposes. (C) 1998 Elsevier Science Inc. All rights reserved.

This work presents an analysis of Dense Multipath Components (DMC) in an industrial workshop for shipping container restoration. Radio channel sounding experiments with a vector network analyzer and virtual antenna arrays were carried out. Specular and dense multipath components were estimated from channel sounding data by means of an iterative maximum-likelihood algorithm (RiMAX). Two main parameters of DMC are studied: the distribution of radio channel power between specular and dense multipath, and the DMC reverberation time in the time-delay domain. The DMC power is found to account for 23 to 70% of the total channel pow er. Statistically significant difference between DMC powers in line-of-sight and non-line-of-sight situations is discovered, and this difference can be largely attributed to the power of the line-of-sight multipath component. In agreement with the theory of room electromagnetics, the DMC reverberation time is found to be nearly constant and thus independent of transmitter/receiver location or link shadowing conditions. Overall, DMC in the industrial workshop appears to be more important, i.e., larger fraction of channel power and longer reverberation time, than in office environments: this is explained by the highly cluttered and metallic nature of the industrial environment.

Purpose Studies in the field of cataract and refractive surgery often report only summary wave-front analysis data data that are too condensed to allow for a retrospective calculation of metrics relevant to visual perception. The aim of this study was to develop a tool that can be used to estimate these metrics from summary wave-front analysis data. Methods The core of the tool is a two-parameter (primary spherical aberration and an overall measure of all other higher-order aberrations) model for the statistical description of the optical aberrations of the human eye. Modulation transfer through-focus curves were calculated for realistic ranges of spherical aberration, other higher-order aberrations and pupil diameters, for both white and monochromatic light. A series of commonly used metrics (modulation transfer, Strehl ratio, visual Strehl ratio and width of line spread function) was derived from these through-focus curves, with corresponding location of optimal focus and depth-of-focus. Results Published wave-front analysis data from healthy subjects served as input for the tool. The output was compared to published measurements of the included metrics, performed in the same or in comparable samples. Agreement between output of the tool and measurements was within the accuracy of the data. Conclusions The tool is able to relate accurately summary wave-front analysis data from groups of patients to metrics relevant to visual perception and can thus be used to estimate these metrics in situations where their calculation from raw wave-front analysis data is not possible.

Background Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates, and plays an important role in a number of cellular processes. The cellular functions of Plasmodium falciparum CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2α, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK2β1 and PfCK2β2. Results We now show that the genes encoding both regulatory PfCK2 subunits (PfCK2β1 and PfCK2β2) cannot be disrupted. Using immunofluorescence and electron microscopy, we examined the intra-erythrocytic stages of transgenic parasite lines expressing hemagglutinin (HA)-tagged catalytic and regulatory subunits (HA-CK2α, HA-PfCK2β1 or HA-PfCK2β2), and localized all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2β1- and PfCK2β2-containing complexes, which were analyzed by mass spectrometry. The recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2β1 and PfCK2β2 precipitates, implicating PfCK2 in chromatin dynamics. We also found that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are phosphorylated by PfCK2α in vitro. Conclusions Our reverse-genetics data show that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.

The (n,gamma) cross section of (96)Zr has been investigated at the CERN n_TOF spallation neutron source. High-resolution time-of-flight measurements using an enriched ZrO(2) sample allowed us to analyze 15 resonances below 40 keV with improved accuracy. On average, the capture widths were found to be 25% smaller than reported in earlier experiments. If complemented with the contribution by direct radiative capture, the derived Maxwellian averaged cross sections are consistent with activation data at kT = 25 keV. The present results confirm the astrophysical implications for the s-process branching at (95)Zr. Physical Review C

Contains fulltext : 97420.pdf (Publisher’s version ) (Closed access) Recent studies have established ciliary dysfunction as the underlying cause of a broad range of multi-organ phenotypes, known as 'ciliopathies'. Ciliopathy-associated proteins have a common site of action in the cilium, however, their overall importance for ciliary function differs, as implied by the extreme variability in ciliopathy phenotypes. The aim of this study was to gain more insight in the function of two ciliopathy-associated protein homologs, RPGR interacting protein 1 (RPGRIP1) and RPGRIP1-like protein (RPGRIP1L). Mutations in RPGRIP1 lead to the eye-restricted disease Leber congenital amaurosis, while mutations in RPGRIP1L are causative for Joubert and Meckel syndrome, which affect multiple organs and are at the severe end of the ciliopathy spectrum. Using tandem affinity purification in combination with mass spectrometry, we identified Nek4 serine/threonine kinase as a prominent component of both the RPGRIP1- as well as the RPGRIP1L-associated protein complex. In ciliated cells, this kinase localized to basal bodies, while in ciliated organs, the kinase was predominantly detected at the ciliary rootlet. Down-regulation of NEK4 in ciliated cells led to a significant decrease in cilium assembly, pointing to a role for Nek4 in cilium dynamics. We now hypothesize that RPGRIP1 and RPGRIP1L function as cilium-specific scaffolds that recruit a Nek4 signaling network which regulates cilium stability. Our data are in line with previously established roles in the cilium of other members of the Nek protein family and define NEK4 as a ciliopathy candidate gene.

Neutron resonance analyses have been performed for the capture cross sections of (186)Os, (187)Os, and (188)Os measured at the n_TOF facility at CERN. Resonance parameters have been extracted up to 5, 3, and 8 keV, respectively, using the SAMMY code for a full R-matrix fit of the capture yields. From these results average resonance parameters were derived by a statistical analysis to provide a comprehensive experimental basis for modeling of the stellar neutron capture rates of these isotopes in terms of the Hauser-Feshbach statistical model. Consistent calculations for the capture and inelastic reaction channels are crucial for the evaluation of stellar enhancement factors to correct the Maxwellian averaged cross sections obtained from experimental data for the effect of thermally populated excited states. These factors have been calculated for the full temperature range of current scenarios of s-process nucleosynthesis using the combined information of the experimental data in the region of resolved resonances and in the continuum. The consequences of this analysis for the s-process component of the (187)Os abundance and the related impact on the evaluation of the time duration of galactic nucleosynthesis via the Re/Os cosmochronometer are discussed. Physical Review C

The neutron capture cross sections of the Zr isotopes are relevant to studies in nuclear structure, nuclear astrophysics, and nuclear technology. The valence neutron of (91)Zr with respect to the neutron magic nucleus (90)Zr has interesting implications for the statistical analysis in the proximity of shell closures. In stellar nucleosynthesis, the Zr isotopes are important for the s-process reaction flow between the Fe seeds and the heavier isotopes. Because of its relatively small (n, gamma) cross sections, Zr represents also an interesting structural material for nuclear reactors. For the same reason, these cross sections are difficult to measure and reliable data are sparse. Therefore, the (n, gamma) cross sections of the Zr isotopes have been remeasured at the CERN n_TOF facility. Thanks to its high instantaneous flux, good energy resolution, and low background, this facility is particularly suited for the determination of small, resonance-dominated cross sections. In this work, results for the (91)Zr(n, gamma) (92)Zr reaction are reported in the neutron energy range from thermal to 26 keV. In this region, accurate data of 157 resonances could be obtained, 33 of these resonances are not present in the main databases and/or were observed for the first time. Physical Review C

Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M. S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M. S. and non-M. S. patients using ELISA. ispartof: Journal of proteome research vol:5 issue:7 pages:1647-1657 ispartof: location:United States status: published

Excitation functions for Fr208-211 isotopes produced in the O-18+Au-197 fusion-evaporation reaction have been measured at E-lab=75-130 MeV via characteristic alpha decays by means of an electrostatic deflector and a semiconductor detector. Data have been compared with calculations giving barrier-passing (capture) cross sections and probabilities of the compound nucleus decay into different channels according to the standard statistical model.