Functional analysis of the rodent CK1tau mutation in the circadian clock of a marine unicellular alga

BACKGROUND: Casein Kinase 1 (CK1) is one of few proteins known to affect cellular timekeeping across metazoans, and the naturally occurring CK1(tau) mutation shortens circadian period in mammals. Functional conservation of a timekeeping function for CK1 in the green lineage was recently identified in the green marine unicell Ostreococcus tauri, in spite of the absence of CK1's transcriptional targets known from other species. The short-period phenotype of CK1(tau) mutant in mammals depends specifically on increased CK1 activity against PERIOD proteins. To understand how CK1 acts differently upon the algal clock, we analysed the cellular and proteomic effects of CK1(tau) overexpression in O. tauri. RESULTS: Overexpression of the CK1(tau) in O. tauri induces period lengthening identical to overexpression of wild-type CK1, in addition to resistance to CK1 inhibitor IC261. Label-free quantitative mass spectrometry of CK1(tau) overexpressing algae revealed a total of 58 unique phospho-sites that are differentially responsive to CK1(tau). Combined with CK1 phosphorylation site prediction tools and previously published wild-type CK1-responsive peptides, this study results in a highly stringent list of upregulated phospho-sites, derived from proteins containing ankyrin repeats, kinase proteins, and phosphoinositide-binding proteins. CONCLUSIONS: The identical phenotype for overexpression of wild-type CK1 and CK1(tau) is in line with the absence of critical targets for rodent CK1(tau) in O. tauri. Proteomic analyses reveal that two thirds of previously reported CK1 overexpression-responsive phospho-sites are shared with CK1(tau). These results indicate that the two alleles are functionally indiscriminate in O. tauri, and verify the identified cellular CK1 target proteins in a minimal circadian model organism

Biom Biostat Int J

CC999999/Intramural CDC HHS/United States2016-06-17T00:00:00Z27331197PMC491222

Label-free quantitative analysis of the casein kinase 2-responsive phosphoproteome of the marine minimal model species Ostreococcus tauri

Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild‐type and CK2‐overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2‐responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. All MS data have been deposited in the ProteomeXchange with identifier PXD000975 (http://proteomecentral.proteomexchange.org/dataset/PXD000975)

The reduced kinome of Ostreococcus tauri:core eukaryotic signalling components in a tractable model species

BACKGROUND: The current knowledge of eukaryote signalling originates from phenotypically diverse organisms. There is a pressing need to identify conserved signalling components among eukaryotes, which will lead to the transfer of knowledge across kingdoms. Two useful properties of a eukaryote model for signalling are (1) reduced signalling complexity, and (2) conservation of signalling components. The alga Ostreococcus tauri is described as the smallest free-living eukaryote. With less than 8,000 genes, it represents a highly constrained genomic palette. RESULTS: Our survey revealed 133 protein kinases and 34 protein phosphatases (1.7% and 0.4% of the proteome). We conducted phosphoproteomic experiments and constructed domain structures and phylogenies for the catalytic protein-kinases. For each of the major kinases families we review the completeness and divergence of O. tauri representatives in comparison to the well-studied kinomes of the laboratory models Arabidopsis thaliana and Saccharomyces cerevisiae, and of Homo sapiens. Many kinase clades in O. tauri were reduced to a single member, in preference to the loss of family diversity, whereas TKL and ABC1 clades were expanded. We also identified kinases that have been lost in A. thaliana but retained in O. tauri. For three, contrasting eukaryotic pathways – TOR, MAPK, and the circadian clock – we established the subset of conserved components and demonstrate conserved sites of substrate phosphorylation and kinase motifs. CONCLUSIONS: We conclude that O. tauri satisfies our two central requirements. Several of its kinases are more closely related to H. sapiens orthologs than S. cerevisiae is to H. sapiens. The greatly reduced kinome of O. tauri is therefore a suitable model for signalling in free-living eukaryotes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-640) contains supplementary material, which is available to authorized users

Long-term psychosocial functioning after Ilizarov limb lengthening during childhood: 37 patients followed for 2–14 years

Background and purpose Few studies have been concerned with the patient's perception of the outcome of limb lengthening. We describe the psychological and social functioning after at least 2 years of follow-up in patients who had had a leg length discrepancy and who had undergone an Ilizarov limb lengthening procedure

Diagnosis of childhood febrile illness using a multi-class blood RNA molecular signature

Background: Appropriate treatment and management of children presenting with fever depend on accurate and timely diagnosis, but current diagnostic tests lack sensitivity and specificity and are frequently too slow to inform initial treatment. As an alternative to pathogen detection, host gene expression signatures in blood have shown promise in discriminating several infectious and inflammatory diseases in a dichotomous manner. However, differential diagnosis requires simultaneous consideration of multiple diseases. Here, we show that diverse infectious and inflammatory diseases can be discriminated by the expression levels of a single panel of genes in blood. Methods: A multi-class supervised machine-learning approach, incorporating clinical consequence of misdiagnosis as a ‘‘cost’’ weighting, was applied to a whole-blood transcriptomic microarray dataset, incorporating 12 publicly available datasets, including 1,212 children with 18 infectious or inflammatory diseases. The transcriptional panel identifiedwas further validated in a new RNA sequencing dataset comprising 411 febrile children. Findings: We identified 161 transcripts that classified patients into 18 disease categories, reflecting individual causative pathogen and specific disease, as well as reliable prediction of broad classes comprising bacterial infection, viral infection, malaria, tuberculosis, or inflammatory disease. The transcriptional panel was validated in an independent cohort andbenchmarked against existingdichotomousRNA signatures. Conclusions: Our data suggest that classification of febrile illness can be achieved with a single blood sample and opens the way for a new approach for clinical diagnosis. Funding: European Union’s Seventh Framework no. 279185; Horizon2020 no. 668303 PERFORM; Wellcome Trust (206508/Z/17/Z); Medical Research Foundation (MRF-160-0008-ELP-KAFO-C0801); NIHR Imperial BRC

Transcriptome Kinetics of Circulating Neutrophils during Human Experimental Endotoxemia

Polymorphonuclear cells (neutrophils) play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n = 4) administered a single dose of endotoxin (LPS, 2 ng/kg iv). In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFα, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFα and IL-1α and IL-1β. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. These changes in neutrophil transcriptome suggest a combination of early activation of circulating neutrophils by TNFα and G-CSF and a mobilization of young neutrophils from the bone marrow

Novel VHH-Based Tracers with Variable Plasma Half-Lives for Imaging of CAIX-Expressing Hypoxic Tumor Cells

Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type

Endosonography With or Without Confirmatory Mediastinoscopy for Resectable Lung Cancer:A Randomized Clinical Trial

PURPOSE:Resectable non-small-cell lung cancer (NSCLC) with a high probability of mediastinal nodal involvement requires mediastinal staging by endosonography and, in the absence of nodal metastases, confirmatory mediastinoscopy according to current guidelines. However, randomized data regarding immediate lung tumor resection after systematic endosonography versus additional confirmatory mediastinoscopy before resection are lacking.METHODS:Patients with (suspected) resectable NSCLC and an indication for mediastinal staging after negative systematic endosonography were randomly assigned to immediate lung tumor resection or confirmatory mediastinoscopy followed by tumor resection. The primary outcome in this noninferiority trial (noninferiority margin of 8% that previously showed to not compromise survival, Pnoninferior &lt;.0250) was the presence of unforeseen N2 disease after tumor resection with lymph node dissection. Secondary outcomes were 30-day major morbidity and mortality.RESULTS:Between July 17, 2017, and October 5, 2020, 360 patients were randomly assigned, 178 to immediate lung tumor resection (seven dropouts) and 182 to confirmatory mediastinoscopy first (seven dropouts before and six after mediastinoscopy). Mediastinoscopy detected metastases in 8.0% (14/175; 95% CI, 4.8 to 13.0) of patients. Unforeseen N2 rate after immediate resection (8.8%) was noninferior compared with mediastinoscopy first (7.7%) in both intention-to-treat (Δ, 1.03%; UL 95% CIΔ, 7.2%; Pnoninferior =.0144) and per-protocol analyses (Δ, 0.83%; UL 95% CIΔ, 7.3%; Pnoninferior =.0157). Major morbidity and 30-day mortality was 12.9% after immediate resection versus 15.4% after mediastinoscopy first (P =.4940).CONCLUSION:On the basis of our chosen noninferiority margin in the rate of unforeseen N2, confirmatory mediastinoscopy after negative systematic endosonography can be omitted in patients with resectable NSCLC and an indication for mediastinal staging.</p