Purification of Parvalbumin from Carp: A Protocol That Avoids Heat Treatment

Parvalbumin from carp, a major allergen,was purified to homogeneity using ion exchange chromatography and size exclusion chromatography (estimated purity \u3e 95% to 98% based on SDS-PAGE and native PAGE) with a yield of 318 mg, and a number of basic biochemical characteristics were determined. The identity was confirmed by peptide-mass fingerprinting, and IgE-binding was demonstrated. The UV/Vis absorbance spectra were explained using the previously published amino acid sequences. Far UV-CD spectroscopy was used to confirm the folding character of parvalbumin. We conclude that parvalbumin from carp can be purified on a comparatively large (hundreds of milligrams) scale using a purification protocol that does not include denaturing steps. The purified protein resembles biochemical characteristics as were earlier published for carp parvalbumin, that is, a molecular weight of approximately 12 kDa, amino acid sequence identity and a secondary structure containing α-helices and β-structures. The described method provides a yield sufficient to produce and characterize antibodies to construct immunochemical methods to detect parvalbumin in food as well as for use as a standard calibrator for such assays. Practical Application: Parvalbumin is a major allergen from fish. Here,we have purified a comparatively large quantity from carp that can be used to develop antisera for use in an assay method to detect fish allergens

Publisher correction:Resource landscapes explain contrasting patterns of aggregation and site fidelity by red knots at two wintering sites

[This corrects the article DOI: 10.1186/s40462-018-0142-4.].</p

Candidate biomarkers for treatment benefit from sunitinib in patients with advanced renal cell carcinoma using mass spectrometry-based (phospho)proteomics

The tyrosine kinase inhibitor sunitinib is an effective first-line treatment for patients with advanced renal cell carcinoma (RCC). Hypothesizing that a functional read-out by mass spectrometry-based (phospho, p-)proteomics will identify predictive biomarkers for treatment outcome of sunitinib, tumor tissues of 26 RCC patients were analyzed. Eight patients had primary resistant (RES) and 18 sensitive (SENS) RCC. A 78 phosphosite signature (p &lt; 0.05, fold-change &gt; 2) was identified; 22 p-sites were upregulated in RES (unique in RES: BCAR3, NOP58, EIF4A2, GDI1) and 56 in SENS (35 unique). EIF4A1/EIF4A2 were differentially expressed in RES at the (p-)proteome and, in an independent cohort, transcriptome level. Inferred kinase activity of MAPK3 (p = 0.026) and EGFR (p = 0.045) as determined by INKA was higher in SENS. Posttranslational modifications signature enrichment analysis showed that different p-site-centric signatures were enriched (p &lt; 0.05), of which FGF1 and prolactin pathways in RES and, in SENS, vanadate and thrombin treatment pathways, were most significant. In conclusion, the RCC (phospho)proteome revealed differential p-sites and kinase activities associated with sunitinib resistance and sensitivity. Independent validation is warranted to develop an assay for upfront identification of patients who are intrinsically resistant to sunitinib.</p

Identification of protein biomarkers for prediction of response to platinum-based treatment regimens in patients with non-small cell lung cancer

The majority of patients with resected stage II-IIIA non-small cell lung cancer (NSCLC) are treated with platinum-based adjuvant chemotherapy (ACT) in a one-size-fits-all approach. However, a significant number of patients do not derive clinical benefit, and no predictive patient selection biomarker is currently available. Using mass spectrometry-based proteomics, we have profiled tumour resection material of 2 independent, multi-centre cohorts of in total 67 patients with NSCLC who underwent ACT. Unsupervised cluster analysis of both cohorts revealed a poor response/survival sub-cluster composed of ~ 25% of the patients, that displayed a strong epithelial-mesenchymal transition signature and stromal phenotype. Beyond this stromal sub-population, we identified and validated platinum response prediction biomarker candidates involved in pathways relevant to the mechanism of action of platinum drugs, such as DNA damage repair, as well as less anticipated processes such as those related to the regulation of actin cytoskeleton. Integration with pre-clinical proteomics data supported a role for several of these candidate proteins in platinum response prediction. Validation of one of the candidates (HMGB1) in a third independent patient cohort using immunohistochemistry highlights the potential of translating these proteomics results to clinical practice.</p

IGF1R signaling induces epithelial-mesenchymal plasticity via ITGAV in cutaneous carcinoma

BackgroundEarly cutaneous squamous cell carcinomas (cSCCs) generally show epithelial differentiation features and good prognosis, whereas advanced cSCCs present mesenchymal traits associated with tumor relapse, metastasis, and poor survival. Currently, the mechanisms involved in cSCC progression are unclear, and the established markers are suboptimal for accurately predicting the clinical course of the disease.MethodsUsing a mouse model of cSCC progression, expression microarray analysis, immunofluorescence and flow cytometry assays, we have identified a prognostic biomarker of tumor relapse, which has been evaluated in a cohort of cSCC patient samples. Phosphoproteomic analysis have revealed signaling pathways induced in epithelial plastic cancer cells that promote epithelial-mesenchymal plasticity (EMP) and tumor progression. These pathways have been validated by genetic and pharmacological inhibition assays.ResultsWe show that the emergence of epithelial cancer cells expressing integrin alpha V (ITGAV) promotes cSCC progression to a mesenchymal state. Consistently, ITGAV expression allows the identification of patients at risk of cSCC relapse above the currently employed clinical histopathological parameters. We also demonstrate that activation of insulin-like growth factor-1 receptor (IGF1R) pathway in epithelial cancer cells is necessary to induce EMP and mesenchymal state acquisition in response to tumor microenvironment-derived factors, while promoting ITGAV expression. Likewise, ITGAV knockdown in epithelial plastic cancer cells also blocks EMP acquisition, generating epithelial tumors.ConclusionsOur results demonstrate that ITGAV is a prognostic biomarker of relapse in cSCCs that would allow improved patient stratification. ITGAV also collaborates with IGF1R to induce EMP in epithelial cancer cells and promotes cSCC progression, revealing a potential therapeutic strategy to block the generation of advanced mesenchymal cSCCs.Graphical AbstractDuring cSCC progression, cancer cells evolve from the epithelial to the mesenchymal state, which is associated with poor prognosis. The current investigation reveals that, at intermediate cSCC stages (MD/PD-SCC), epithelial cancer cells activate IGF1R and ITGAV signaling to acquire EMP and progress to the aggressive mesenchymal state in response to TME-derived factors. In addition, ITGAV allows the identification of these epithelial plastic cancer cells and functions as a prognostic biomarker of tumor relapse

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy

BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. RESULTS: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.</p

Invertebrate abundance increases with vegetation productivity across natural and agricultural wader breeding habitats in Europe

Grassland breeding waders have been steadily declining across Europe. Recent studies indicating a dramatic decline in grassland invertebrates' abundance and biomass, the key food of most grassland wader chicks, suggest a likely driver of the demise of waders. While agricultural intensification is generally inferred as the main cause for arthropod decline there is surprisingly little information on the relationship between land use intensity and total arthropod abundance in grasslands. Here, we explored those relationships across several key wader breeding habitats by surveying ground-active, aerial and soil-dwelling invertebrate communities in five European countries that range from natural undisturbed bogs to intensively managed grasslands. Using maximum vegetation growth and soil moisture content we investigated how they shape the size of the invertebrate community within and across different countries. We found predominantly positive relationships between grassland invertebrate abundance, biomass and body weight with increasing vegetation growth and soil moisture. Maximum vegetation growth was strongly positively related to ground-active invertebrate abundance and biomass and abundance of soil dwelling invertebrates (mainly earthworms). Body weight of aerial invertebrates furthermore increased with increasing maximum vegetation growth. Our results provide little support for the hypothesis that agricultural practices associated with intensification of grassland management result in an abundance decline of invertebrate prey for wader chicks. Conservation practices aiming to enhance wader chick survival require a careful balancing act between maintaining habitat productivity to secure high prey abundance, and keeping productivity low enough to maintain open swards that do not need to be cut before chicks have fledged

Novel diagnostic cerebrospinal fluid biomarkers for pathologic subtypes of frontotemporal dementia identified by proteomics

Introduction: Reliable cerebrospinal fluid (CSF) biomarkers enabling identification of frontotemporal dementia (FTD) and its pathologic subtypes are lacking. Methods: Unbiased high-resolution mass spectrometry-based proteomics was applied on CSF of FTD patients with TAR DNA-binding protein 43 (TDP-43, FTD-TDP, n = 12) or tau pathology (FTD-tau, n = 8), and individuals with subjective memory complaints (SMC, n = 10). Validation was performed by applying enzyme-linked immunosorbent assay (ELISA) or enzymatic assays, when available, in a larger cohort (FTLD-TDP, n = 21, FTLD-tau, n = 10, SMC, n = 23) and in Alzheimer's disease (n = 20), dementia with Lewy bodies (DLB, n = 20), and vascular dementia (VaD, n = 18). Results: Of 1914 identified CSF proteins, 56 proteins were differentially regulated (fold change >1.2, P <.05) between the different patient groups: either between the two pathologic subtypes (10 proteins), or between at least one of these FTD subtypes and SMC (47 proteins). We confirmed the differential expression of YKL-40 by ELISA in a partly independent cohort. Furthermore, enzyme activity of catalase was decreased in FTD subtypes compared with SMC. Further validation in a larger cohort showed that the level of YKL-40 was twofold increased in both FTD pathologic subtypes compared with SMC and that the levels in FTLD-tau were higher compared to Alzheimer's dementia (AD), DLB, and VaD patients. Clinical validation furthermore showed that the catalase enzyme activity was decreased in the FTD subtypes compared to SMC, AD and DLB. Discussion: We identified promising CSF biomarkers for both FTD differential diagnosis and pathologic subtyping. YKL-40 and catalase enzyme activity should be validated further in similar pathology defined patient cohorts for their use for FTD diagnosis or treatment development