Proteomic analysis of exosomes derived from human mature milk and colostrum of mothers with term, late preterm, or very preterm delivery.

The growth and development of the human brain is a long and complex process that requires a precise sequence of genetic and molecular events. This begins in the third week of gestation with the differentiation of neural progenitor cells and extends at least until late adolescence, possibly for life. One of the defects of this development is that we know very little about the signals that modulate this sequence of events. The first 3 years of life, during breastfeeding, is one of the critical periods in brain development. In these first years of life, it is believed that neurodevelopmental problems may be the molecular causes of mental disorders. Therefore, we herein propose a new hypothesis, according to which the chemical signals that could modulate this entire complex sequence of events appear in this early period, and the molecular level study of human breast milk and colostrum of mothers who give birth to children in different gestation periods could give us information on proteins influencing this process. In this work, we collected milk and colostrum samples (term, late preterm and moderate/very preterm) and exosomes were isolated. The samples of exosomes and complete milk from each fraction were analyzed by LC-ESI-MS/MS. In this work, we describe proteins in the different fractions of mature milk and colostrum of mothers with term, late preterm, or very preterm delivery, which could be involved in the regulation of the nervous system by their functions. We describe how they differ in different types of milk, paving the way for the investigation of possible new neuroregulatory pathways as possible candidates to modulate the nervous system.

Colorectal cancer screening and diagnosis: omics-based technologies for development of a non-invasive blood-based method.

Introduction: Colorectal cancer (CRC) is one of the most important health problems in the Western world. In order to reduce the burden of the disease, two strategies are proposed: screening and prompt detection in symptomatic patients. Although diagnosis and prevention are mainly based on colonoscopy, fecal hemoglobin detection has been widely implemented as a noninvasive strategy. Various studies aiming to discover blood-based biomarkers have recently emerged.Areas covered: The burgeoning omics field provides diverse high-throughput approaches for CRC blood-based biomarker discovery. In this review, we appraise the most robust and commonly used technologies within the fields of genomics, transcriptomics, epigenomics, proteomics, and metabolomics, together with their targeted validation approaches. We summarize the transference process from the discovery phase until clinical translation. Finally, we review the best candidate biomarkers and their potential clinical applicability.Expert opinion: Some available biomarkers are promising, especially in the field of epigenomics: DNA methylation and microRNA. Transference requires the joint collaboration of basic researchers, intellectual property experts, technology transfer officers and clinicians. Blood-based biomarkers will be selected not only based on their diagnostic accuracy and cost but also on their reliability, applicability to clinical analysis laboratories and their acceptance by the population.

Proteomic Profiling for Colorectal Cancer Biomarker Discovery.

Nowadays, the ideal biomarker for colorectal cancer (CRC) has not been found. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) are suitable techniques for searching new biomarkers. In this chapter, we describe methodology for biomarker discovery based on a proteomic approach. In addition, special attention is given to the sample preparation, including protein extraction, fractionation, and cleanup, as we consider this a critical step. Comparing the proteomic profile of tumor and mucosa, we identified the nucleoside diphosphate kinase A (NDKA) protein as a candidate biomarker for CRC. Finally, we validated NDKA with an ELISA kit using serum samples from individuals of a screening cohort. Our results suggest that serum NDKA is a potential biomarker for screening of CRC and premalignant advanced adenomas (AA).

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum.

Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

Proteomics for discovery of candidate colorectal cancer biomarkers.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in Europe and other Western countries, mainly due to the lack of well-validated clinically useful biomarkers with enough sensitivity and specificity to detect this disease at early stages. Although it is well known that the pathogenesis of CRC is a progressive accumulation of mutations in multiple genes, much less is known at the proteome level. Therefore, in the last years many proteomic studies have been conducted to find new candidate protein biomarkers for diagnosis, prognosis and as therapeutic targets for this malignancy, as well as to elucidate the molecular mechanisms of colorectal carcinogenesis. An important advantage of the proteomic approaches is the capacity to look for multiple differentially expressed proteins in a single study. This review provides an overview of the recent reports describing the different proteomic tools used for the discovery of new protein markers for CRC such as two-dimensional electrophoresis methods, quantitative mass spectrometry-based techniques or protein microarrays. Additionally, we will also focus on the diverse biological samples used for CRC biomarker discovery such as tissue, serum and faeces, besides cell lines and murine models, discussing their advantages and disadvantages, and summarize the most frequently identified candidate CRC markers.

Proteomic characterization of the hemolymph of Octopus vulgaris infected by the protozoan parasite Aggregata octopiana.

The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The common octopus Octopus vulgaris has a high economic relevance and potential for aquaculture. However, disease outbreaks provoke serious reductions in production with potentially severe economic losses. In this study, a proteomic approach is used to analyze the immune response of O. vulgaris against the coccidia Aggregata octopiana, a gastrointestinal parasite which impairs the cephalopod nutritional status. The hemocytes and plasma proteomes were compared by 2-DE between sick and healthy octopus. The identities of 12 differentially expressed spots and other 27 spots without significant alteration from hemocytes, and 5 spots from plasma, were determined by mass spectrometry analysis aided by a six reading-frame translation of an octopus hemocyte RNA-seq database and also public databases. Principal component analysis pointed to 7 proteins from hemocytes as the major contributors to the overall difference between levels of infection and so could be considered as potential biomarkers. Particularly, filamin, fascin and peroxiredoxin are highlighted because of their implication in octopus immune defense activity. From the octopus plasma, hemocyanin was identified. This work represents a first step forward in order to characterize the protein profile of O. vulgaris hemolymph, providing important information for subsequent studies of the octopus immune system at molecular level and also to the understanding of the basis of octopus tolerance-resistance to A. octopiana. BIOLOGICAL SIGNIFICANCE: The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The study herein presented is focused to the comprehension of the octopus immune defense against a parasite infection. Particularly, it is centered in the host-parasite relationship developed between the octopus and the protozoan A. octopiana, which induces severe gastrointestinal injuries in octopus that produce a malabsorption syndrome. The common octopus is a commercially important species with a high potential for aquaculture in semi-open systems, and this pathology reduces the condition of the octopus populations on-growing in open-water systems resulting in important economical loses. This is the first proteomic approach developed on this host-parasite relationship, and therefore, the contribution of this work goes from i) ecological, since this particular relationship is tending to be established as a model of host-parasite interaction in natural populations; ii) evolutionary, due to the characterization of immune molecules that could contribute to understand the functioning of the immune defense in these highly evolved mollusks; and iii) to economical view. The results of this study provide an overview of the octopus hemolymph proteome. Furthermore, proteins influenced by the level of infection and implicated in the octopus cellular response are also showed. Consequently, a set of biomarkers for disease resistance is suggested for further research that could be valuable for the improvement of the octopus culture, taken into account their high economical value, the declining of landings and the need for the diversification of reared species in order to ensure the growth of the aquaculture activity. Although cephalopods are model species for biomedical studies and possess potential in aquaculture, their genomes have not been sequenced yet, which limits the application of genomic data to research important biological processes. Similarly, the octopus proteome, like other non-model organisms, is poorly represented in public databases. Most of the proteins were identified from an octopus' hemocyte RNA-seq database that we have performed, which will be the object of another manuscript in preparation. Therefore, the need to increase molecular data from non-model organisms is herein highlighted. Particularly, here is encouraged to expand the knowledge of the genomic of cephalopods in order to increase successful protein identifications. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Selection of putative colorectal cancer markers by applying PCA on the soluble proteome of tumors: NDK A as a promising candidate.

Aiming to find new tumor markers for colorectal cancer (CRC), we applied proteomic methodologies to compare the soluble sub-proteome of healthy and tumoral colorectal mucosa. Out of 91 differentially expressed proteins, 23 were selected by principal component analysis (PCA) as the major contributors to the overall difference detected. After MS/MS analysis, 16 proteins were identified. From those, we chose 14-3-3-zeta/delta, retinoblastoma-binding protein 4 (RBBP-4), DJ-1, and nucleoside diphosphate kinase A (NDK A) for further studies, on the basis of their levels and known implication in cancer. Specific immunodetection demonstrated only the NDK A levels allowed to differentiate healthy mucosa from tumor tissue in all the patients. Hence, we used the colon cancer cell line Caco-2 to study the relationship between NDK A and colon cell tumorigenesis, finding it over-expressed in undifferentiated (tumor-like) cells regarding the differentiated ones. Noticeably, we also found increased levels of the NDK A in the secretome of tumor-like cells and, as expected, indications of higher levels of NDK A in the serum of CRC patients. In conclusion, the four validated proteins could constitute a panel of tissue markers for CRC, being the NDK A the most interesting candidate for further serum biomarker studies.

Identification of hydrophobic proteins as biomarker candidates for colorectal cancer.

Nowadays, colorectal cancer is one of the major causes of cancer death in Western countries. Due to the lack of biomarkers with clinical utility for this pathology, and considering that membrane and hydrophobic proteins have not been studied in depth, we performed a prefractionation of colorectal tissues prior to two-dimensional gel electrophoresis in order to identify hydrophobic proteins differentially expressed in colorectal cancer patients. Fractions enriched in hydrophobic proteins were obtained from healthy mucosa and tumor tissue by a specific extraction method based on temperature-dependent phase partitioning with Triton X-114. Proteins were separated by two-dimensional gel electrophoresis and gels were silver-stained, scanned and compared using the PDQuest software. Those spots presenting significantly different abundance were submitted to mass spectrometry for protein identification. Alterations in the expression of cytoskeletal proteins, including a decrease of vimentin and the absence of desmin, were found. We also detected alterations in antioxidant and transport proteins, chaperones, and in two isoforms of the calcium-binding protein S100A6. On the other hand, vimentin was chosen to corroborate the electrophoretic results by specific immunodetection. Most of the altered proteins have been related to cellular membranes, many of them to lipid rafts microdomains in the plasma membrane, and they have also been implicated in the control of cell proliferation, apoptosis, or metastasis. In conclusion, all the proteins found altered in colorectal tumor samples could be considered as candidates for future studies focused on their utility as markers for colorectal diagnosis and prognosis, or as targets for colorectal cancer therapy.