The inclusive electron neutrino charged-current cross section is measured in the NOvA near detector using 8.02×10^{20} protons-on-target in the NuMI beam. The sample of GeV electron neutrino interactions is the largest analyzed to date and is limited by ≃17% systematic rather than the ≃7.4% statistical uncertainties. The double-differential cross section in final-state electron energy and angle is presented for the first time, together with the single-differential dependence on Q^{2} (squared four-momentum transfer) and energy, in the range 1  GeV≤E_{ν}<6  GeV. Detailed comparisons are made to the predictions of the GENIE, GiBUU, NEUT, and NuWro neutrino event generators. The data do not strongly favor a model over the others consistently across all three cross sections measured, though some models have especially good or poor agreement in the single differential cross section vs Q^{2}.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH-gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures such as Coomassie, silver staining, or fluorescence labeling. This article gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.

Classical 2D-PAGE allows comparison and quantitation of proteomes by visualization of protein patterns using gel stains and comparative image analysis. The introduction of fluorescent reagents for protein labeling (difference in-gel electrophoresis or DIGE) has brought substantial improvement in this field. It provides multiplexing of up to three samples in one gel, higher sensitivity compared to normal protein staining methods, and a higher linear range for quantitation. This article gives detailed protocols for 2D-DIGE, including both minimal and saturation labeling.

Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer's disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations (e.g. in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction Pm showed overall occupancies of ∼8 Pi (± 5) with a bell-shaped distribution; the highly phosphorylated fraction Ph had 14 Pi (± 6). The distribution of sites was highly asymmetric (with 71% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit (e.g. acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.

Lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria provide the first line of defense against antibiotics and other harmful compounds. LPS biosynthesis critically depends on LpxC catalyzing the first committed enzyme in this process. In Escherichia coli, the cellular concentration of LpxC is adjusted in a growth rate-dependent manner by the FtsH protease making sure that LPS biosynthesis is coordinated with the cellular demand. As a result, LpxC is stable in fast-growing cells and prone to degradation in slow-growing cells. One of the factors involved in this process is the alarmone guanosine tetraphosphate (ppGpp) but previous studies suggested the involvement of yet unknown factors in LpxC degradation. We established a quantitative proteomics approach aiming at the identification of proteins that are associated with LpxC and/or FtsH at high or low growth rates. The identification of known LpxC and FtsH interactors validated our approach. A number of proteins involved in fatty acid biosynthesis and degradation, including the central regulator FadR, were found in the LpxC and/or FtsH interactomes. Another protein associated with LpxC and FtsH was WaaH, a LPS-modifying enzyme. When overproduced, several members of the LpxC/FtsH interactomes were able to modulate LpxC proteolysis. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS production by involving multiple enzymes in fatty acid metabolism, phospholipid biosynthesis and LPS modification.

Background/Aims: An estimated 80 million people worldwide are infected with viremic hepatitis C virus (HCV). Even after eradication of HCV with direct acting antivirals (DAAs), hepatic fibrosis remains a risk factor for hepatocarcinogenesis. Recently, we confirmed the applicability of microfibrillar-associated protein 4 (MFAP4) as a serum biomarker for the assessment of hepatic fibrosis. The aim of the present study was to assess the usefulness of MFAP4 as a biomarker of liver fibrosis after HCV eliminating therapy with DAAs. Methods: MFAP4 was measured using an immunoassay in 50 hepatitis C patients at baseline (BL), the end-of-therapy (EoT), and the 12-week follow-up (FU) visit. Changes in MFAP4 from BL to FU and their association with laboratory parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), platelets, the AST to platelet ratio index (APRI), fibrosis-4 score (FIB-4), and albumin were analyzed. Results: MFAP4 serum levels were representative of the severity of hepatic fibrosis at BL and correlated well with laboratory parameters, especially APRI (Spearman correlation, R-2 = 0.80). Laboratory parameters decreased significantly from BL to EoT. MFAP4 serum levels were found to decrease from BL and EoT to FU with high statistical significance (Wilcoxon P<0.001 for both). Conclusions: Our findings indicate that viral eradication resulted in reduced MFAP4 serum levels, presumably representing a decrease in hepatic fibrogenesis or fibrosis. Hence, MFAP4 may be a useful tool for risk assessment in hepatitis C patients with advanced fibrosis after eradication of the virus.

Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen. Finally, the PTS1-receptor is monoubiquitinated on the conserved cysteine 6 in Saccharomyces cerevisiae. The monoubiquitinated Pex5p is recognized by the peroxisomal export machinery and is retrotranslocated into the cytosol for further rounds of protein import. However, the functional relevance of deubiquitination has not yet been addressed. In this study, we have analyzed a Pex5p-truncation lacking Cys6 [(Δ6)Pex5p], a construct with a ubiquitin-moiety genetically fused to the truncation [Ub-(Δ6)Pex5p], as well as a construct with a reduced susceptibility to deubiquitination [Ub(G75/76A)-(Δ6)Pex5p]. While the (Δ6)Pex5p-truncation is not functional, the Ub-(Δ6)Pex5p chimeric protein can facilitate matrix protein import. In contrast, the Ub(G75/76A)-(Δ6)Pex5p chimera exhibits a complete PTS1-import defect. The data show for the first time that not only ubiquitination but also deubiquitination rates are tightly regulated and that efficient deubiquitination of Pex5p is essential for peroxisomal biogenesis.

AAA+ proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsHtrap) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsHtrap or FtsHwt (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA+ proteases.

Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP-dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super-SILAC (stable isotope labeling with amino acids in cell culture) mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon-dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon-associated proteins were identified by label-free LC-MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example, the superoxide stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism.

Targeted genome editing in blood and immune cells enable new therapeutic applications, especially for infectious diseases. We present a GMP-compliant protocol to manufacture CCR5-edited CD34+ hematopoietic stem and precursor cells (HSPCs) with the goal to cure patients suffering from chronic infection with human immunodeficiency virus type 1 (HIV1). We hypothesize that genetic disruption of the CCR5 gene, which encodes the major HIV1 co-receptor, in HSPCs will give rise to an HIV-resistant immune system after transplantation. We have developed engineered nucleases based on transcription activator-like effector nucleases (TALENs) targeting CCR5. Electroporation of CD4+ T-cells and CD34+ HSPCs with mRNAs encoding TALENs revealed disruption of up to 80% of CCR5 alleles in CD4+ T-cells and over 90% of alleles in HSPCs. The high gene editing frequencies in T-cells and HSPCs were confirmed by deep sequencing, and no cleavage activity above background levels were detected at the top 20 predicted off-target sites. CCR5-edited CD4+ cells preserved their proliferation capacity and their biological function. Importantly, these cells showed significantly reduced CCR5 expression and became resistant to infection with the R5-tropic HIV-1JR-FL virus. The CCR5-edited HSPCs maintained their proliferation potential and their capacity to differentiate into the various blood lineages in vitro and in vivo, and clonal analysis revealed bi-allelic CCR5 disruption in more than 75% of cells. In summary, our developed protocol enables highly efficient and GMP-compliant knockout of the CCR5 locus in clinically relevant cells, so forming the foundation for a planned phase I/II clinical study. Gautron: Cellectis SA: Employment. Busser:Cellectis: Employment, Patents & Royalties: Cellectis. Smith:Cellectis. Inc: Employment, Patents & Royalties. Duchateau:Cellectis: Employment, Patents & Royalties: Cellectis. Cathomen:TRACR Hematology: Consultancy; Cellectis: Research Funding; Miltenyi Biotec: Research Funding. Cornu:Cellectis: Research Funding; Miltenyi Biotec: Research Funding.

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

Background: Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-g2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFa on tumor cells. Results: Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFa with an increased expression of all three chains encoding laminin-332; coincubation with TGFb and TNFa leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFa can induce expression of the g2 and b3 but not the a3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of g2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble g2 is associated with increased cell migration. Conclusions: Loss of Smad4 may lead to uncoupled induction of laminin-g2 in response to TNFa and may therefore represent one of the mechanisms which underlie accumulation of laminin-g2 at the invasive margin of a tumor. The finding, that g2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor

Lipocalin-2 (LCN2) is a secreted adipokine that transports small hydrophobic molecules such as fatty acids and steroids. LCN2 limits bacterial growth by sequestering iron-containing siderophores and in mammalian liver protects against inflammation, infection, injury and other stressors. Because LCN2 modulates hepatic fat metabolism and homeostasis, we performed a comparative profiling of proteins and lipids of wild type (WT) and Lcn2-deficient mice fed either standard chow or a methionine- and choline-deficient (MCD) diet. Label-free proteomics and 2D-DIGE protein expression profiling revealed differential expression of BRIT1/MCPH1, FABP5, HMGB1, HBB2, and L-FABP, results confirmed by Western blotting. Gene ontology enrichment analysis identified enrichment for genes associated with mitochondrial membrane permeabilization and metabolic processes involving carboxylic acid. Measurements of mitochondrial membrane potential, mitochondrial chelatable iron pool, intracellular lipid peroxidation, and peroxisome numbers in primary hepatocytes confirmed that LCN2 regulates mitochondrial and peroxisomal integrity. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry imaging identified significant changes to sphingomyelins, triglycerides, and glycerophospholipids in livers of mice fed an MCD diet regardless of LCN2 status. However, two arachidonic acid-containing glycerophospholipids were increased in Lcn2-deficient livers. Thus, LCN2 influences peroxisomal and mitochondrial biology in the liver to maintain triglyceride balance, handle oxidative stress, and control apoptosis.

Quantitative mass spectrometry approaches are used for absolute and relative quantification in global proteome studies. To date, relative and absolute quantification techniques are available that differ in quantification accuracy, proteome coverage, complexity and robustness. This review focuses on most common relative or absolute quantification strategies exemplified by three experimental studies. A label-free relative quantification approach was performed for the investigation of the membrane proteome of sensory cilia to the depth of olfactory receptors in Mus musculus . A SILAC-based relative quantification approach was successfully applied for the identification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae . Furthermore, AQUA using stable isotopes was exemplified to unraveling the prenylome influenced by novel prenyltransferase inhibitors. Characteristic enrichment and fragmentation strategies for a robust quantification of the prenylome are also summarized.

Extracellular vesicles (EV) are secreted by most cells and are present in every body fluid. They can be seen as major players in the regulation and communication of cells. They are adressed as therapeutic and diagnostic tool for many disorders including neurodegenerative diseases. Distinct subpopulations of platelet derived EVs have been shown to contain high concentrations of Alzheimer’s disease(AD)-associated proteins e. g. APP, APOE, lipids and miRNAs and thus might contribute actively to the progression of the disease.

The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are β-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression.