Proteome Profiling of Wheat Shoots from Different Cultivars

Wheat is a cereal grain and one of the world's major food crops. Recent advances in wheat genome sequencing are by now facilitating its genomic and proteomic analyses. However, little is known about possible differences in total protein levels of hexaploid versus tetraploid wheat cultivars, and also knowledge of phosphorylated wheat proteins is still limited. Here, we performed a detailed analysis of the proteome of seedling leaves from two hexaploid wheat cultivars (Triticum aestivum L. Pavon 76 and USU-Apogee) and one tetraploid wheat (T. turgidum ssp. durum cv. Senatore Cappelli). Our shotgun proteomics data revealed that, whereas we observed some significant differences, overall a high similarity between hexaploid and tetraploid varieties with respect to protein abundance was observed. In addition, already at the seedling stage, a small set of proteins was differential between the small (USU-Apogee) and larger hexaploid wheat cultivars (Pavon 76), which could potentially act as growth predictors. Finally, the phosphosites identified in this study can be retrieved from the in-house developed plant PTM-Viewer (bioinformatics.psb.ugent.be/webtools/ptm_viewer/), making this the first searchable repository for phosphorylated wheat proteins. This paves the way for further in depth, quantitative (phospho) proteome-wide differential analyses upon a specific trigger or environmental change

Sequence-specific protein aggregation generates defined protein knockdowns in plants

Protein aggregation is determined by short (5-15 amino acids) aggregation-prone regions (APRs) of the polypeptide sequence that self-associate in a specific manner to form beta-structured inclusions. Here, we demonstrate that the sequence specificity of APRs can be exploited to selectively knock down proteins with different localization and function in plants. Synthetic aggregation-prone peptides derived from the APRs of either the negative regulators of the brassinosteroid (BR) signaling, the glycogen synthase kinase 3/Arabidopsis SHAGGY-like kinases (GSK3/ASKs), or the starch-degrading enzyme alpha-glucan water dikinase were designed. Stable expression of the APRs in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) induced aggregation of the target proteins, giving rise to plants displaying constitutive BR responses and increased starch content, respectively. Overall, we show that the sequence specificity of APRs can be harnessed to generate aggregation-associated phenotypes in a targeted manner in different subcellular compartments. This study points toward the potential application of induced targeted aggregation as a useful tool to knock down protein functions in plants and, especially, to generate beneficial traits in crops

Up-to-date workflow for plant (phospho)proteomics identifies differential drought-responsive phosphorylation events in maize leaves

Protein phosphorylation is one of the most common post-translational modifications (PTMs), which can regulate protein activity and localization as well as proteinprotein interactions in numerous cellular processes. Phosphopeptide enrichment techniques enable plant researchers to acquire insight into phosphorylation-controlled signaling networks in various plant species. Most phosphoproteome analyses of plant samples still involve stable isotope labeling, peptide fractionation, and demand a lot of mass spectrometry (MS) time. Here, we present a simple workflow to probe, map, and catalogue plant phosphoproteomes, requiring relatively low amounts of starting material, no labeling, no fractionation, and no excessive analysis time. Following optimization of the different experimental steps on Arabidopsis thaliana samples, we transferred our workflow to maize, a major monocot crop, to study signaling upon drought stress. In addition, we included normalization to protein abundance to identify true phosphorylation changes. Overall, we identified a set of new phosphosites in both Arabidopsis thaliana and maize, some of which are differentially phosphorylated upon drought. All data are available via ProteomeXchange with identifier PXD003634, but to provide easy access to our model plant and crop data sets, we created an online database, Plant PTM Viewer (bioinformatics.psb.ugent.be/webtools/ptm_viewer/), where all phosphosites identified in our study can be consulted

Clinical examination, critical care ultrasonography and outcomes in the critically ill : cohort profile of the Simple Intensive Care Studies-I

Purpose In the Simple Intensive Care Studies-I (SICS-I), we aim to unravel the value of clinical and haemodynamic variables obtained by physical examination and critical care ultrasound (CCUS) that currently guide daily practice in critically ill patients. We intend to (1) measure all available clinical and haemodynamic variables, (2) train novices in obtaining values for advanced variables based on CCUS in the intensive care unit (ICU) and (3) create an infrastructure for a registry with the flexibility of temporarily incorporating specific (haemodynamic) research questions and variables. The overall purpose is to investigate the diagnostic and prognostic value of clinical and haemodynamic variables. Participants The SICS-I includes all patients acutely admitted to the ICU of a tertiary teaching hospital in the Netherlands with an ICU stay expected to last beyond 24 hours. Inclusion started on 27 March 2015. Findings to date On 31 December 2016, 791 eligible patients fulfilled our inclusion criteria of whom 704 were included. So far 11 substudies with additional variables have been designed, of which six were feasible to implement in the basic study, and two are planned and awaiting initiation. All researchers received focused training for obtaining specific CCUS images. An independent Core laboratory judged that 632 patients had CCUS images of sufficient quality. Future plans We intend to optimise the set of variables for assessment of the haemodynamic status of the critically ill patient used for guiding diagnostics, prognosis and interventions. Repeated evaluations of these sets of variables are needed for continuous improvement of the diagnostic and prognostic models. Future plans include: (1) more advanced imaging; (2) repeated clinical and haemodynamic measurements; (3) expansion of the registry to other departments or centres; and (4) exploring possibilities of integration of a randomised clinical trial superimposed on the registry. Study registration number NCT02912624; Pre-results.Peer reviewe

The diagnostic accuracy of clinical examination for estimating cardiac index in critically ill patients:the Simple Intensive Care Studies-I

PurposeClinical examination is often the first step to diagnose shock and estimate cardiac index. In the Simple Intensive Care Studies-I, we assessed the association and diagnostic performance of clinical signs for estimation of cardiac index in critically ill patients.MethodsIn this prospective, single-centre cohort study, we included all acutely ill patients admitted to the ICU and expected to stay>24h. We conducted a protocolised clinical examination of 19 clinical signs followed by critical care ultrasonography for cardiac index measurement. Clinical signs were associated with cardiac index and a low cardiac index (4.5s, or skin mottling over the knee.ConclusionsSeven out of 19 clinical examination findings were independently associated with cardiac index. For estimation of cardiac index, clinical examination was found to be insufficient in multivariable analyses and in diagnostic accuracy tests. Additional measurements such as critical care ultrasonography remain necessary

Allosteric modulation of a tyrosine kinase receptor by an extracellularly acting small-molecule chemical compound: inhibition of (lymph)angiogenesis in fish,frogs and mice by a multi-FGFR inhibitor

De Receptor Tyrosine Kinase (RTK) superfamilie vormt een uiterst belangrijk doelwit in de huidige ontwikkeling van anti-angiogene/kanker geneesmiddelen. De huidige RTK inhibitoren binden aan en blokkeren de orthostere bindingsplaatsen van de endogene liganden of substraten van hun respectieve receptoren of enzymes. Allostere geneesmiddelen (of middelen die binden aan topografisch verschillende locaties) vormen een groep van veelbelovende therapeutica hoewel er tot dusver nog geen enkel klein-molecule, allostere modulator werd ontwikkeld die werkzaam is op het extracellulaire deel van RTKs. In deze studie werd SSR128129E (SSR) naar voor geschoven als de, naar alle waarschijnlijkheid, eerste klein-chemische allostere inhibitor die actief is op het extracellulaire deel van de FGFR familie. Aan de hand van een groot aantal verschillende biofysische testen, heeft deze studie aangetoond dat SSR bindt aan het extracellulaire deel van verscheidene FGFRs, echter, zonder in competitie te treden met de binding van de FGF-ligand aan zijn receptor. Bovendien induceert de binding van SSR een conformationele verandering in de receptorstructuur waardoor de FGFR downstream signaling wordt gemoduleerd. Opmerkelijk was dat niet alle downstream signaling even fel werd geïnhibeerd door SSR: terwijl signaling via de MAPK reactieweg compleet werd geblokkeerd, bleef de PLCg reactieweg onaangetast. Dit fenomeen, beter gekend als signal pathway-bias, was reeds gekend voor allostere compounds die inwerken op de G-proteïne gekoppelde receptoren (GPCRs). De multi-FGFR inhibitor interfereert ook met de vasculaire ontwikkeling en regeneratie in zebravis en kikkervisembryo s, en inhibeert bloedvatgroei en de infiltratie van inflammatoire cellen in muismodellen voor arthritis en kanker. Daarenboven inhibeert SSR ook FGF-gestuurde lymphangiogenese in zebravis en kikkervisembryo s en in arthritis en kanker bij muizen. Orale toediening van SSR reduceerde tumorgroei, zelfs bij hardnekkige tumoren die resistent zijn tegen de standaard anti-VEGFR2 behandeling. Dit toont aan dat beide therapiëen complementair werken. Oraal toedienbare, klein-molecule allostere modulators van RTKs, die een extracellulaire conformationele verandering induceren, met daarenboven een modulerend effect op de signaaltransductie pathways, zijn niet alleen bruikbaar om nieuwe mechanismen te ontdekken in RTK biologie. Ze zorgen eveneens voor ongeziene mogelijkheiden om angiogene aandoeningen te behandelen via nieuwe strategieën.nrpages: 167status: publishe

Functional Precision Oncology: The Next Frontier to Improve Glioblastoma Outcome?

Glioblastoma remains the most malignant and intrinsically resistant brain tumour in adults. Despite intensive research over the past few decades, through which numerous potentially druggable targets have been identified, virtually all clinical trials of the past 20 years have failed to improve the outcome for the vast majority of GBM patients. The observation that small subgroups of patients displayed a therapeutic response across several unsuccessful clinical trials suggests that the GBM patient population probably consists of multiple subgroups that probably all require a distinct therapeutic approach. Due to extensive inter- and intratumoral heterogeneity, assigning the right therapy to each patient remains a major challenge. Classically, bulk genetic profiling would be used to identify suitable therapies, although the success of this approach remains limited due to tumor heterogeneity and the absence of direct relationships between mutations and therapy responses in GBM. An attractive novel strategy aims at implementing methods for functional precision oncology, which refers to the evaluation of treatment efficacies and vulnerabilities of (ex vivo) living tumor cells in a highly personalized way. Such approaches are currently being implemented for other cancer types by providing rapid, translatable information to guide patient-tailored therapeutic selections. In this review, we discuss the current state of the art of transforming technologies, tools and challenges for functional precision oncology and how these could improve therapy selection for GBM patients