Characterization of CD34+ hematopoietic cells in systemic mastocytosis: Potential role in disease dissemination

[Background]: Recent studies show that most systemic mastocytosis (SM) patients, including indolent SM (ISM) with (ISMs+) and without skin lesions (ISMs−), carry the KIT D816V mutation in PB leukocytes. We investigated the potential association between the degree of involvement of BM hematopoiesis by the KIT D816V mutation and the distribution of different maturation-associated compartments of bone marrow (BM) and peripheral blood (PB) CD34+ hematopoietic precursors (HPC) in ISM and identified the specific PB cell compartments that carry this mutation. [Methods]: The distribution of different maturation-associated subsets of BM and PB CD34+ HPC from 64 newly diagnosed (KIT-mutated) ISM patients and 14 healthy controls was analyzed by flow cytometry. In 18 patients, distinct FACS-purified PB cell compartments were also investigated for the KIT mutation. [Results]: ISM patients showed higher percentages of both BM and PB MC-committed CD34+ HPC vs controls, particularly among ISM cases with MC-restricted KIT mutation (ISMMC); this was associated with progressive blockade of maturation of CD34+ HPC to the neutrophil lineage from ISMMC to multilineage KIT-mutated cases (ISMML). Regarding the frequency of KIT-mutated cases and cell populations in PB, variable patterns were observed, the percentage of KIT-mutated PB CD34+ HPC, eosinophils, neutrophils, monocytes and T cells increasing from ISMs−MC and ISMs+MC to ISMML patients. [Conclusion]: The presence of the KIT D816V mutation in PB of ISM patients is associated with (early) involvement of circulating CD34+ HPC and multiple myeloid cell subpopulations, KIT-mutated PB CD34+ HPC potentially contributing to early dissemination of the disease.This work was supported by Fondo de Investigaciones Sanitarias—FIS—of the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain (grant numbers PI11/02399 and PI16/00642, FEDER); Consejería de Educación (Regional Government of Castilla y León, Spain; grant number SA013U16); Biomedical Research Networking Centre Consortium–CIBER-CIBERONC (CB16/12/00400) of the Instituto de Salud Carlos III, Madrid, Spain; and Fundacion Ramon Areces, Madrid, Spain (grant CIVP16A1806). AM was supported by a RTICC (Red Tematica de Investigacion Cooperativa en Cancer) grant (RD12/0036/0048, FIS, FEDER)

Impact of somatic and germline mutations on the outcome of systemic mastocytosis

[EN]Systemic mastocytosis (SM) is a highly heterogeneous disease with indolent and aggressive forms, with the mechanisms leading to malignant transformation still remaining to be elucidated. Here, we investigated the presence and frequency of genetic variants in 34 SM patients with multilineal KIT D816V mutations. Initial screening was performed by targeted sequencing of 410 genes in DNA extracted from purified bone marrow cells and hair from 12 patients with nonadvanced SM and 8 patients with advanced SM, followed by whole-genome sequencing (WGS) in 4 cases. Somatic mutations were further investigated in another 14 patients with advanced SM. Despite the fact that no common mutation other than KIT D816V was found in WGS analyses, targeted next-generation sequencing identified 67 nonsynonymous genetic variants involving 39 genes. Half of the mutations were somatic (mostly multilineal), whereas the other half were germline variants. The presence of ≥1 multilineal somatic mutation involving genes other than KIT D816V, ≥3 germline variants, and ≥1 multilineal mutation in the SRSF2, ASXL1, RUNX1, and/or EZH2 genes (S/A/R/E genes), in addition to skin lesions, splenomegaly, thrombocytopenia, low hemoglobin levels, and increased alkaline phosphatase and β2-microglobulin serum levels, were associated with a poorer patient outcome. However, the presence of ≥1 multilineal mutation, particularly involving S/A/R/E genes, was the only independent predictor for progression-free survival and overall survival in our cohort

Biomarker Discovery by Novel Sensors Based on Nanoproteomics Approaches

During the last years, proteomics has facilitated biomarker discovery by coupling high-throughput techniques with novel nanosensors. In the present review, we focus on the study of label-based and label-free detection systems, as well as nanotechnology approaches, indicating their advantages and applications in biomarker discovery. In addition, several disease biomarkers are shown in order to display the clinical importance of the improvement of sensitivity and selectivity by using nanoproteomics approaches as novel sensors

Chromosome 19 annotations with disease speciation: A first report from the global research consortium

PMCID: PMC3539432; NIHMSID: NIHMS430346.-- et al.A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented (http://www.c-hpp.org). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC-MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks. © 2012 American Chemical Society.This work was supported by grants from the Swedish Academy of Pharmaceutical Sciences who is the core founder our consortium, Swedish Research Council, the Swedish Foundation for Strategic Research, Vinnova, Ingabritt & Arne Lundbergs forskningsstiftelse, the Crafoord Foundation and by Thermo Fisher Scientific for mass spectrometry instrument support. T.E.F. is supported by the Mobilitas Program sponsored by the European Union Social Fund and administered by the Estonian Science Foundation. We gratefully acknowledge financial support to M.F. from Health Institute Carlos III of Spain (ISCIII, FIS PI02114) and M.G.-G. is supported by a PhD scholarship of ISCIII FI08/00721. C.L.N. is supported by the Cancer Prevention and Research Institute of Texas and the University of Texas Medical Branch. [11-0624]; National Cancer Institute [R33 CA 127768-03, R01CA160816, and P50-CA92629]; Sidney Kimmel Center for Prostate and Urologic Cancers; and David H. Koch through the Prostate Cancer Foundation.Peer Reviewe

Extrusive luxation. Therapeutic procedure

Key Clinical Message Repositioning a traumatized tooth involves replacing and stabilizing it. When it is not possible, a method has been developed by an acetate splint. After few weeks, the tooth was aligned and correctly positioned. Abstract Repositioning a traumatized tooth involves, first, replacing and second stabilizing it. Stabilization, on the other hand, usually requires flexible splints. Occasionally the immediate replacement may be impossible being necessary to use other procedure. When complete replacement is not possible

Is small really beautiful? Nanosensors and low abundance biomarkers for personalized medicine

Biomarkers are defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological or pathogenic processes or pharmacological responses to a therapeutic intervention. Over the past few decades, a growing interest in the identification of biomarkers has emerged in order to detect different pathologies in the early stages. Thus, it would be possible to provide a customized treatment to the patient, improving their outcome. Owing this purpose, proteomics has allowed the development of new methodologies and technologies which are able of detecting low-abundance proteins. This is due, in part, to the development of novel promising materials, such as quantum dots or silicon nanowires. Such emerging approaches come with the advantage of their high sensitivity, low-volume requirements or the potential high-throughput applications, among others. However, since huge information has been generated through nanoproteomic techniques, only few applications have arrived to the clinics. Referring to personalized medicine and targeted therapies, it has to be mentioned that the development of more specific drugs can be improved by the use of biomarkers, helping with decisions about dose, schedule and patient population. In this review, we summarize and highlight the utility of biomarkers identified through nanoproteomics for personalized medicine as well as the applications in the clinic and the future perspectives.Peer Reviewe

Protein microarrays: Technological aspects, applications and intellectual property

Over the last decade, proteomics has undergone remarkable progress thanks to the technical advances made in the field. Improvements in the design of the protein microarrays, including more types of chemical groups for surface functionalization, new capture agents and novel detection strategies, among others, have allowed the detection of proteins in a robust, specific, sensitive, real time and high throughput manner. However, there are still problems that hinder the analysis of low abundance proteins or those present in complex samples. For this reason, the development of patents related to the features mentioned above has an important relevance. In this review, we focus on the study of recently approved patents that try to solve the existing problems. Thanks to them, it is expected that the identification of disease biomarkers can be made in a suitable and reliable way, and above all, biocompatible and environmentally friendly. © 2013 Bentham Science Publishers.We gratefully acknowledge financial support from the Carlos III Health Institute of Spain (ISCIII, FIS PI1102114) and JCYL-SAN10. María González-González is supported by a ISCIII FIS08/00721 Ph.D. scholarship.Peer Reviewe

Protein arrays as tool for studies at the host-pathogen interface

Pathogens and parasites encode a wide spectrum of multifunctional proteins interacting to and modifying proteins in host cells. However, the current lack of a reliable method to unveil the protein-protein interactions (PPI) at the host-pathogen interface is retarding our understanding of many important pathogenic processes. Thus, the identification of proteins involved in host-pathogen interactions is important for the elucidation of virulence determinants, mechanisms of infection, host susceptibility and/or disease resistance. In this sense, proteomic technologies have experienced major improvements in recent years and protein arrays are a powerful and modern method for studying PPI in a high-throughput format. This review focuses on these techniques analyzing the state-of-the-art of proteomic technologies and their possibilities to diagnose and explore host-pathogen interactions. Major technical advancements, applications and protocol concerns are presented, so readers can appreciate the immense progress achieved and the current technical options available for studying the host-pathogen interface. Finally, future uses of this kind of array-based proteomic tools in the fight against infectious and parasitic diseases are discussed. © 2013 Elsevier B.V.RM-R is funded by the JAEDoc program (CSIC-FSE). VD-M is funded by the JAEPreDoc program (CSIC-FSE). This work has been supported by grants from the Fondo de Investigación Sanitaria (FIS) of the Instituto de Salud Carlos III (ISCIII), Madrid, Spain (ISCIII, FIS PI11 02114) and the Junta Castilla y Leon SA198A12-2.Peer Reviewe

High-throughgput phage-display screening in array format

et al.Emerging technologies for the design and generation of human antibodies require improved approaches enabling their screening, characterization and validation. Currently, strategies based on ELISA or western blot are used to that aim. However, the ever increasing number of novel antibodies generated would benefit from the development of new high-throughput (HT) platforms facilitating rapid antibody identification and characterization. Herein, we describe a protein chip bearing recombinant phage particles and based on a large phage antibody library. In this paper we have set forth a novel implementation which provides a powerful and simple methodology enabling the identification of single-chain variable fragments (scFv). As a proof-of-principle of this method, we tested it with recombinant antigen (human recombinant interleukin 8). Additionally, we developed a novel bioinformatics tool that serves to compare this novel strategy with traditional methods. The method described here, together with associated informatics tools, is robust, relatively fast and represents a step-forward in protocols including phage library screenings.We gratefully acknowledge financial support from the Carlos III Health Institute of Spain (ISCIII, FIS PI11/02114)-Fondos FEDER and Junta Castilla-León SA198A12-2. The Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. Paula Díez and Noelia Dasilva are supported by a JCYLEDU/346/2013 Ph.D., scholarship. María González-González and Raquel Bartolomé are supported by ISCIII FIS08/00721 Ph.D., scholarship and MEC 2009 scholarship, respectively. J. Casado-Vela is a JAE-DOC (CSIC) holder supported by Ministerio de Economía y Competitividad, Spain, co-funded by the European Social Fund (FEDER).Peer Reviewe

Evaluation of homo- and hetero-functionally activated glass surfaces for optimized antibody arrays

et al.Antibody arrays hold great promise for biomedical applications, but they are typically manufactured using chemically functionalized surfaces that still require optimization. Here, we describe novel hetero-functionally activated glass surfaces favoring oriented antibody binding for improved performance in protein microarray applications. Antibody arrays manufactured in our facility using the functionalization chemistries described here proved to be reproducible and stable and also showed good signal intensities. As a proof-of-principle of the glass surface functionalization protocols described in this article, we built antibody-based arrays functionalized with different chemistries that enabled the simultaneous detection of 71 human leukocyte membrane differentiation antigens commonly found in peripheral blood mononuclear cells. Such detection is specific and semi-quantitative and can be performed in a single assay under native conditions. In summary, the protocol described here, based on the use of antibody array technology, enabled the concurrent detection of a set of membrane proteins under native conditions in a specific, selective, and semi-quantitative manner and in a single assay.This work was supported by grants FIS PI081884 and PI1102114 from the Instituto de Salud Carlos III (ISCIII; Ministerio de Ciencia e Innovación, Madrid, Spain) and grant JCYL-SA198A12-2 from the Consejería de Educación de la Junta de Castilla y León (Spain). María González-González is supported by a PhD scholarship from the ISCIII (FI08/00721), Raquel Bartolome is supported by a PhD scholarship from the Ministerio de Educación (FPU, AP2009-2633), and Noelia Dasilva is supported by a PhD scholarship from JCyL (Orden EDU 346/2013). Juan Casado-Vela is a JAE–DOC (CSIC) holder supported by the Ministerio de Economía y Competitividad (Spain), cofunded by the European Social Fund.Peer Reviewe