Assessment of Different Expression Strategies for the Production of a Recombinant Lipoprotein Vaccine in Plants

The ability of plants to serve as a production system for bacterial lipoprotein vaccines has been investigated. First, the effect of high-level expression of the Borrelia burgdorferi outer membrane protein A (OspA), a prototype vaccine against Lyme disease, has been examined by a proteomics approach. Analysis by 2D-PAGE of wild type tobacco plants and transplastomic plants accumulating recombinant OspA showed no apparent differences in protein pattern except for OspA. However, presence of the bacterial signal sequence limits transgene accumulation. As an alternative approach OspA was produced in Nicotiana benthamiana plants by transient expression via a deconstructed tobacco mosaic virus-based system. While rapid expression of OspA could be achieved, no palmitoylation occurred with the genuine bacterial sequence. In contrast, modification of the N-terminus with an eukaryotic sequence motif resulted in palmitoylation of OspA. This study shows that plants provide multiple expression strategies and could serve as a versatile production platform for recombinant lipidated subunit vaccines

Platform independent protein-based cell-of-origin subtyping of diffuse large B-cell lymphoma in formalin-fixed paraffin-embedded tissue

Diffuse large B-cell lymphoma (DLBCL) is commonly classified by gene expression profiling according to its cell of origin (COO) into activated B-cell (ABC)-like and germinal center B-cell (GCB)-like subgroups. Here we report the application of label-free nano-liquid chromatography - Sequential Window Acquisition of all THeoretical fragment-ion spectra - mass spectrometry (nanoLC-SWATH-MS) to the COO classification of DLBCL in formalin-fixed paraffin-embedded (FFPE) tissue. To generate a protein signature capable of predicting Affymetrix-based GCB scores, the summed log(2)-transformed fragment ion intensities of 780 proteins quantified in a training set of 42 DLBCL cases were used as independent variables in a penalized zero-sum elastic net regression model with variable selection. The eight-protein signature obtained showed an excellent correlation (r=0.873) between predicted and true GCB scores and yielded only 9 (21.4%) minor discrepancies between the three classifications: ABC, GCB, and unclassified. The robustness of the model was validated successfully in two independent cohorts of 42 and 31 DLBCL cases, the latter cohort comprising only patients aged >75 years, with Pearson correlation coefficients of 0.846 and 0.815, respectively, between predicted and NanoString nCounter based GCB scores. We further show that the 8-protein signature is directly transferable to both a triple quadrupole and a Q Exactive quadrupole-Orbitrap mass spectrometer, thus obviating the need for proprietary instrumentation and reagents. This method may therefore be used for robust and competitive classification of DLBCLs on the protein level

N-cadherin promoter polymorphisms and risk of osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis. It is characterized by cartilage destruction and bone remodeling, mediated in part by synovial fibroblasts (SFs). Given the functional significance of cadherins in these cells, we aimed at determining the role of genetic variants of N-cadherin (CDH2) in OA of the knee and hip. Six single-nucleotide polymorphisms in the genomic region of the CDH2 gene were genotyped in 312 patients with OA and 259 healthy control subjects. Gene expression of CDH2 was analyzed by qRT-PCR. Liquid chromatography-mass spectrometry was used to identify a transcription factor isolated by DNA pulldown. Its potential for binding to gene variants was examined by electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, and chromatin immunoprecipitation. Genetic analysis identified a polymorphism located in the CDH2 promoter region to be associated with risk of OA. The minor allele of rs11564299 had a protective effect against OA. Compared to carriers of the major allele, carriers of the minor allele of rs11564299 displayed increased N-cadherin levels in SFs. Based on in silico analysis, the minor allele was predicted to generate a novel transcription factor binding site, Direct-binding assays and mass spectrometric analysis identified hnRNP K as binding selectively to the minor allele. In summary, a CDH2 promoter polymorphism influences the risk of OA, and hnRNP K was found to be involved in the regulation of elevated N-cadherin expression in patients with OA carrying the minor allele of rs11564299

SU(3) Predictions for Weak Decays of Doubly Heavy Baryons -- including SU(3) breaking terms

We find expressions for the weak decay amplitudes of baryons containing two b quarks (or one b and one c quark -- many relationship are the same) in terms of unknown reduced matrix elements. This project was originally motivated by the request of the FNAL Run II b Physics Workshop organizers for a guide to experimentalists in their search for as yet unobserved hadrons. We include an analysis of linear SU(3) breaking terms in addition to relationships generated by unbroken SU(3) symmetry, and relate these to expressions in terms of the complete set of possible reduced matrix elements.Comment: 49 page

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity

A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis

A novel, high-affinity sucrose transporter identified in the plasma membrane of the plant pathogen Ustilago maydis is essential for fungal virulence and successful infection of maize

Interruption of bile acid uptake by hepatocytes after acetaminophen overdose ameliorates hepatotoxicity.

Background & aimsAcetaminophen (APAP) overdose remains a frequent cause of acute liver failure, which is generally accompanied by increased levels of serum bile acids (BAs). However, the pathophysiological role of BAs remains elusive. Herein, we investigated the role of BAs in APAP-induced hepatotoxicity.MethodsWe performed intravital imaging to investigate BA transport in mice, quantified endogenous BA concentrations in the serum of mice and patients with APAP overdose, analyzed liver tissue and bile by mass spectrometry and MALDI-mass spectrometry imaging, assessed the integrity of the blood-bile barrier and the role of oxidative stress by immunostaining of tight junction proteins and intravital imaging of fluorescent markers, identified the intracellular cytotoxic concentrations of BAs, and performed interventions to block BA uptake from blood into hepatocytes.ResultsPrior to the onset of cell death, APAP overdose causes massive oxidative stress in the pericentral lobular zone, which coincided with a breach of the blood-bile barrier. Consequently, BAs leak from the bile canaliculi into the sinusoidal blood, which is then followed by their uptake into hepatocytes via the basolateral membrane, their secretion into canaliculi and repeated cycling. This, what we termed 'futile cycling' of BAs, led to increased intracellular BA concentrations that were high enough to cause hepatocyte death. Importantly, however, the interruption of BA re-uptake by pharmacological NTCP blockage using Myrcludex B and Oatp knockout strongly reduced APAP-induced hepatotoxicity.ConclusionsAPAP overdose induces a breach of the blood-bile barrier which leads to futile BA cycling that causes hepatocyte death. Prevention of BA cycling may represent a therapeutic option after APAP intoxication.Lay summaryOnly one drug, N-acetylcysteine, is approved for the treatment of acetaminophen overdose and it is only effective when given within ∼8 hours after ingestion. We identified a mechanism by which acetaminophen overdose causes an increase in bile acid concentrations (to above toxic thresholds) in hepatocytes. Blocking this mechanism prevented acetaminophen-induced hepatotoxicity in mice and evidence from patients suggests that this therapy may be effective for longer periods after ingestion compared to N-acetylcysteine

Proteomics of yeast mitochondria

Because virtually all cellular processes are based on proteins, detailed knowledge of the mitochondrial proteome represents an integral part of understanding mitochondrial function. The analysis of very complex protein mixtures such as entire cell organelles makes high demands on analysis techniques in order to ensure integrity of the obtained data set. The use of Saccharomyces cerevisiae as a model system allows the isolation of mitochondria of utmost purity in large amounts. Various approaches can be accomplished in the subsequent analysis to achieve the most complete overview possible. Combinations of orthogonal proteomics techniques include two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS-PAGE), and nano-LC-MS/MS (nano-liquid chromatography with tandem mass spectrometry) as well as multidimensional high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The inherent limitations of the individual methods can be countervailed by parallel application of these approaches

Modificomics: posttranslational modifications beyond protein phosphorylation and glycosylation

Posttranslational modifications of proteins possess key functions in the regulation of various cellular processes. While they facilitate fast, location-specific and transient reactions to changing conditions in the first place they enhance the already high complexity of a cellular proteome by orders of magnitude. Furthermore, they can utterly alter the properties of the modified protein, thus making a timely analysis even more difficult. While several standardized methods for the analysis of protein phosphorylation and glycosylation have been established most other modifications require tailor-made solutions for a comprehensive analysis. Therefore, we will provide guidelines for the analysis of some important posttranslational modifications that are underrepresented in contemporary literature