Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells ...the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na⁺/H⁺ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We integrate existing and new geologic data REtreating TRench, Extension, and Accretion Tectonics (RETREAT project), particularly on the origin, growth, and activity of the mountain front at Bologna, ...Italy, into a new model that explains Apennine orogenesis in the context of a slab rollback ‐ upper plate retreat process. The Bologna mountain front is an actively growing structure driving rock uplift ∼1 mm/year, cored by a midcrustal flat‐ramp structure that accommodates ongoing shortening driven by Adria subduction at a rate of ∼2.5 mm/year. The data we use are assembled from river terraces and associated Pleistocene growth strata, geodesy including releveling surveys, reinterpretation of published reflection lines, and a new high‐resolution reflection line. These data constrain a simple trishear model that inverts for blind thrust ramp depth, dip, and slip. Apennine extension is recognized both in the foreland, as high‐angle normal faults and modest stretching in the carapace of the growing mountain front, and in the hinterland, with larger normal faults that accomplish some crustal thinning as the upper plate retreats. This coevolution of extension and shortening shares some notable characteristics with other basement‐involved collisional orogens including the early Tertiary Laramide orogeny in the American West and the Oligocene to Miocene evolution of the Alps. We propose a possible relationship between underplating and the development of the Po as a sag basin as a Quaternary phenomenon that may also apply to past periods of Apennine deformation (Tortonian). Continued shortening on the structure beneath the Bologna mountain front represents by far the most important and underappreciated seismogenic source in the front of the northern Apennines.
Protein aggregation is mostly viewed as deleterious and irreversible causing several pathologies. However, reversible protein aggregation has recently emerged as a novel concept for cellular ...regulation. Here, we characterize stress-induced, reversible aggregation of yeast pyruvate kinase, Cdc19. Aggregation of Cdc19 is regulated by oligomerization and binding to allosteric regulators. We identify a region of low compositional complexity (LCR) within Cdc19 as necessary and sufficient for reversible aggregation. During exponential growth, shielding the LCR within tetrameric Cdc19 or phosphorylation of the LCR prevents unscheduled aggregation, while its dephosphorylation is necessary for reversible aggregation during stress. Cdc19 aggregation triggers its localization to stress granules and modulates their formation and dissolution. Reversible aggregation protects Cdc19 from stress-induced degradation, thereby allowing cell cycle restart after stress. Several other enzymes necessary for G1 progression also contain LCRs and aggregate reversibly during stress, implying that reversible aggregation represents a conserved mechanism regulating cell growth and survival.
Anomalous patterns of the sedimentary architecture have been recognized in passive margins, and only recently they have been associated with plate reorganization or compressional deformations ...propagating from distant margins. With the aim of discussing the sedimentary architecture and the potential tectonic perturbations to the passive margin pattern, we present the revision of the stratigraphy of a fossil passive margin, involved in the retrobelt of the Alpine orogeny. The main events at the transition from rifted to passive margin have been controlled by palaeoceanography, i.e. the trophic state of surface waters that hampered the carbonate photozoan productivity for a long period between Toarcian and Callovian. Toward the latest Bajocian–earliest Bathonian, the platform productivity increased, dominated by ooids. A regressional trend up to the Middle Bathonian allowed the rapid infilling of the previous rift basin. The successive aggradation in the platform was still dominated by non-skeletal grains until the Early Oxfordian. The Middle Oxfordian to Early Kimmeridgian was a time of recovery of the palaeoceanographic conditions allowing the establishment of a hydrozoan/coral rich platform. The sedimentation rates in the platform increased at the margin of the productive Friuli–Adriatic Platform. From Late Kimmeridgian on, the sedimentation rates at the platform margin returned to the pre-Oxfordian values. At the scale of the whole Adriatic Platform, the Middle Oxfordian to Early Kimmeridgian interval is variable in thickness from 0 to 800 m, and it depicts a couple of folds of around 80–100 km of wavelength. The subsidence analysis of wells and composite logs from literature suggests this interval as a perturbation to the passive margin trend of around 3 Myr of duration. We interpret this folding event, superimposed to the passive margin subsidence, as the far field expression of the transition from intraoceanic to continental obduction, occurred at the eastern Adria active margin.
The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific ...pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.
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•Human SRMAtlas: 166,174 proteotypic peptides representing the human proteome•Resource of verified high-resolution spectra and multiplexed SRM assays•Supports proteome-scale quantification as well as hypothesis-driven research•Web database with free unlimited access
This resource enables the accurate detection and quantification of any known or predicted human protein from complex biological samples.
Biology and especially systems biology projects increasingly require the capability to detect and quantify specific sets of proteins across multiple samples, for example the components of a ...biological pathway through a set of perturbation-response experiments. Targeted proteomics based on selected reaction monitoring (SRM) has emerged as an ideal tool to this purpose, and complements the discovery capabilities of shotgun proteomics methods. SRM experiments rely on the development of specific, quantitative mass spectrometric assays for each protein of interest and their application to the quantification of the protein set in various biological samples. SRM measurements are multiplexed, namely, multiple proteins can be quantified simultaneously, and are characterized by a high reproducibility and a broad dynamic range. We provide here a practical guide to the development of SRM assays targeting a set of proteins of interest and to their application to complex biological samples.
Selected reaction monitoring (SRM) is a targeted mass spectrometric method that is increasingly used in proteomics for the detection and quantification of sets of preselected proteins at high ...sensitivity, reproducibility and accuracy. Currently, data from SRM measurements are mostly evaluated subjectively by manual inspection on the basis of ad hoc criteria, precluding the consistent analysis of different data sets and an objective assessment of their error rates. Here we present mProphet, a fully automated system that computes accurate error rates for the identification of targeted peptides in SRM data sets and maximizes specificity and sensitivity by combining relevant features in the data into a statistical model.
During interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear ...envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle. Here, we show that both CDK1 and polo-like kinase 1 (PLK1) support mitotic NPC disintegration by hyperphosphorylation of Nup98, the gatekeeper nucleoporin, and Nup53, a central nucleoporin linking the inner NPC scaffold to the pore membrane. Multisite phosphorylation of Nup53 critically contributes to its liberation from its partner nucleoporins, including the pore membrane protein NDC1. Initial steps of NPC disassembly in semi-permeabilized cells can be reconstituted by a cocktail of mitotic kinases including cyclinB-CDK1, NIMA, and PLK1, suggesting that the unzipping of nucleoporin interactions by protein phosphorylation is an important principle underlying mitotic NE permeabilization.
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•PLK1 localizes to NPCs during prophase and promotes mitotic NPC disassembly•CDK1 and PLK1 mediate hyperphosphorylation of the central linker nucleoporin Nup53•Multisite phosphorylation of Nup53 promotes the disintegration of the central NPC•Mitotic kinases induce NPC disassembly in a reconstituted system
The disassembly of nuclear pore complexes (NPCs) is a key process for nuclear envelope breakdown at the onset of open mitosis in metazoan cells. Linder et al. show that multisite phosphorylation of the key interconnecting nucleoporin Nup53 by CDK1 and PLK1 is required for the timely disassembly of the central NPC framework.
•Development of a physical model that allows to predict soiling (reflectance) losses.•The size distribution of airborne dust influences significantly its deposition.•The concentration of airborne ...dust influences significantly the soiling losses.•Model prediction of the reflectance trend validated with experimental data.
Addressing soiling-related losses of glazed surfaces in solar power technologies is one of the most critical issues to improve the competitiveness of solar power plants in the current energy market. Reliable predictions of soiling rates and performance losses would enable optimisation of cleaning strategies and add a valuable assessment criterion for the evaluation of potential future power plant sites. However, currently available soiling models are data-driven and thus specific to the site and do not allow for reliable extrapolation to different sites and environmental conditions. To address this gap, this paper details the development of a physical soiling model for the prediction of the deposition of airborne dust onto the surface of solar collectors and the subsequent loss of performance (i.e. reflectance in the specific case of heliostats). The model inputs are the measured airborne dust concentration and estimated size distribution, the position of the mirrors, and the recorded wind speed and air temperature. The outputs of the model have been validated using experiments performed at the Queensland University of Technology. A reflectometer was used to measure the reflectance of the mirrors on an almost-daily basis while meteorological and dust data were obtained through a dust sensor and a weather station. The agreement between the experimental data and the simulated results demonstrates the effectiveness of the model in dry conditions. For example, for a mirror tilted at 45° with a daily loss of reflectance between 0.5%/day and 2.1%/day, the model reflectance loss predictions have an average relative error of 14%. Furthermore, the visual trends of the model predictions agree well with those observed in the experimental data. Finally, the model is used to investigate scenarios at three candidate sites to assess reflectance losses in different conditions.
The identification of specific biomarkers will improve the early diagnosis of disease, facilitate the development of targeted therapies, and provide an accurate method to monitor treatment response. ...A major challenge in the process of verifying biomarker candidates in blood plasma is the complexity and high dynamic range of proteins. This article reviews the current, targeted proteomic strategies that are capable of quantifying biomarker candidates at concentration ranges where biomarkers are expected in plasma (i.e. at the ng/ml level). In addition, a workflow is presented that allows the fast and definitive generation of targeted mass spectrometry-based assays for most biomarker candidate proteins. These assays are stored in publicly accessible databases and have the potential to greatly impact the throughput of biomarker verification studies.
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