The high luminosity upgrade of Large Hadron Collider will increase its luminosity by an order of magnitude increasing with it the density of particles on the detector. For protecting the inner ...detectors of experiments and for monitoring the delivered luminosity, a radiation hard beam monitor is being developed. Text describes the proposed system’s principles of operation and its design. It details on the poly-crystalline chemical vapor deposition diamonds as sensor material, a dedicated radiation hard ASIC, as well as on electronics and data processing. The latter finally offers beam abort and luminosity measurement functionalities of the system.
A crucial stage of the Streptomyces life cycle is the sporulation septation, a process were dozens of cross walls are synchronously formed in the aerial hyphae in a highly coordinated manner. This ...process includes the remodeling of the spore envelopes to make Streptomyces spores resistant to detrimental environmental conditions. Sporulation septation and the synthesis of the thickened spore envelope in S. coelicolor A3(2) involves the Streptomyces spore wall synthesizing complex SSSC. The SSSC is a multi-protein complex including proteins directing peptidoglycan synthesis (MreBCD, PBP2, Sfr, RodZ) and cell wall glycopolymer synthesis (PdtA). It also includes two eukaryotic like serin/threonine protein kinases (eSTPK), PkaI and PkaH, which were shown to phosphorylate MreC. Since unbalancing phosphorylation activity by either deleting eSTPK genes or by expressing a second copy of an eSTPK gene affected proper sporulation, a model was developed, in which the activity of the SSSC is controlled by protein phosphorylation.
C-terminus of HSC70-interacting protein (CHIP) encoded by the gene
is a co-chaperone and E3 ligase that acts as a key regulator of cellular protein homeostasis. Mutations in
cause autosomal recessive ...spinocerebellar ataxia type 16 (SCAR16) with widespread neurodegeneration manifesting as spastic-ataxic gait disorder, dementia and epilepsy. CHIP
mice display severe cerebellar atrophy, show high perinatal lethality and impaired heat stress tolerance. To decipher the pathomechanism underlying SCAR16, we investigated the heat shock response (HSR) in primary fibroblasts of three SCAR16 patients. We found impaired HSR induction and recovery compared to healthy controls.
transcript levels (coding for HSP70) were reduced upon heat shock but HSP70 remained higher upon recovery in patient- compared to control-fibroblasts. As SCAR16 primarily affects the central nervous system we next investigated the HSR in cortical neurons (CNs) derived from induced pluripotent stem cells of SCAR16 patients. We found CNs of patients and controls to be surprisingly resistant to heat stress with high basal levels of HSP70 compared to fibroblasts. Although heat stress resulted in strong transcript level increases of many HSPs, this did not translate into higher HSP70 protein levels upon heat shock, independent of
mutations. Furthermore,
(-/-) neurons generated by CRISPR/Cas9-mediated genome editing from an isogenic healthy control line showed a similar HSR to patients. Proteomic analysis of CNs showed dysfunctional protein (re)folding and higher basal oxidative stress levels in patients. Our results question the role of impaired HSR in SCAR16 neuropathology and highlight the need for careful selection of proper cell types for modeling human diseases.
The chicken egg and foods containing egg components are an important part of human nutrition. Furthermore, eggs are a potential source of bioactive molecules and a potential delivery system for ...therapeutic proteins, explaining the continuing scientific interest in eggs and their components. Using mass spectrometry-based high-throughput proteomic techniques, 119 proteins in egg yolk, 78 proteins in egg white, and 528 proteins in the decalcified eggshell organic matrix have recently been identified. Most of these proteins were identified in their respective egg compartment for the first time. Some of these proteins were already known from chicken tissues or other egg compartments, but many were novel. In the eggshell soluble matrix 39 phosphoproteins containing more than 150 different phosphorylation sites were identified. Twenty-two of the identified phosphoproteins had not been recognized as phosphoproteins previously.
Over the past decade the number and variety of protein post-translational modifications that have been detected and characterized in bacteria have rapidly increased. Most post-translational protein ...modifications occur in a relatively low number of bacterial proteins in comparison with eukaryotic proteins, and most of the modified proteins carry low, substoichiometric levels of modification; therefore, their structural and functional analysis is particularly challenging. The number of modifying enzymes differs greatly among bacterial species, and the extent of the modified proteome strongly depends on environmental conditions. Nevertheless, evidence is rapidly accumulating that protein post-translational modifications have vital roles in various cellular processes such as protein synthesis and turnover, nitrogen metabolism, the cell cycle, dormancy, sporulation, spore germination, persistence and virulence. Further research of protein post-translational modifications will fill current gaps in the understanding of bacterial physiology and open new avenues for treatment of infectious diseases.
Summary
Background
Platelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, ...biogenesis of platelet granules is poorly understood.
Objectives
Serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far.
Methods and Results
Electron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1−/−). In sgk1−/− platelets serotonin content was significantly reduced and activation‐dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3‐induced vascular injury was significantly diminished in sgk1−/− mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1−/− platelets without affecting Rab27b mRNA stability. In MEG‐01 cells transfection with constitutively active S422DSGK1 but not with inactive K127NSGK1 significantly enhanced Rab27b mRNA levels. Sgk1−/− megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1+/+ megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1−/− platelets independent of Rab27b.
Conclusions
The present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes.
Identifying the building blocks of mammalian tissues is a precondition for understanding their function. In particular, global and quantitative analysis of the proteome of mammalian tissues would ...point to tissue-specific mechanisms and place the function of each protein in a whole-organism perspective. We performed proteomic analyses of 28 mouse tissues using high-resolution mass spectrometry and used a mix of mouse tissues labeled via stable isotope labeling with amino acids in cell culture as a “spike-in” internal standard for accurate protein quantification across these tissues. We identified a total of 7,349 proteins and quantified 6,974 of them. Bioinformatic data analysis showed that physiologically related tissues clustered together and that highly expressed proteins represented the characteristic tissue functions. Tissue specialization was reflected prominently in the proteomic profiles and is apparent already in their hundred most abundant proteins. The proportion of strictly tissue-specific proteins appeared to be small. However, even proteins with household functions, such as those in ribosomes and spliceosomes, can have dramatic expression differences among tissues. We describe a computational framework with which to correlate proteome profiles with physiological functions of the tissue. Our data will be useful to the broad scientific community as an initial atlas of protein expression of a mammalian species.
Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR
instruments are capable of even greater accuracy provided ion numbers ...are well controlled. Here we demonstrate sub-ppm mass
accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ
Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap.
Ions forming the MS or MS n spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration
on the âlock massâ by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining
mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over
the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance
and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than
2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High
and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification.
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