Carlos Álvarez

BRAF mutation analysis in circulating free tumor DNA of melanoma patients treated with BRAF inhibitors

BRAFV600E is a unique molecular marker for metastatic melanoma, being the most frequent somatic point mutation in this malignancy. Detection of BRAFV600E in blood could have prognostic and predictive value and could be useful for monitoring response to BRAF-targeted therapy. We developed a rapid, sensitive method for the detection and quantification of BRAFV600E in circulating free DNA (cfDNA) isolated from plasma and serum on the basis of a quantitative 5′-nuclease PCR (Taqman) in the presence of a peptide−nucleic acid. We validated the assay in 92 lung, colon, and melanoma archival serum and plasma samples with paired tumor tissue (40 wild-type and 52 BRAFV600E). The correlation of cfDNA BRAFV600E with clinical parameters was further explored in 22 metastatic melanoma patients treated with BRAF inhibitors. Our assay could detect and quantify BRAFV600E in mixed samples with as little as 0.005% mutant DNA (copy number ratio 1u2009:u200920u2009000), with a specificity of 100% and a sensitivity of 57.7% in archival serum and plasma samples. In 22 melanoma patients treated with BRAF inhibitors, the median progression-free survival was 3.6 months for those showing BRAFV600E in pretreatment cfDNA compared with 13.4 months for those in whom the mutation was not detected (P=0.021). Moreover, the median overall survival for positive versus negative BRAFV600E tests in pretreatment cfDNA differed significantly (7 vs. 21.8 months, P=0.017). This finding indicates that the sensitive detection and accurate quantification of low-abundance BRAFV600E alleles in cfDNA using our assay can be useful for predicting treatment outcome.

The use of sub-critical water hydrolysis for the recovery of peptides and free amino acids from food processing wastes. Review of sources and main parameters.

Food industry processing wastes are produced in enormous amounts every year, such wastes are usually disposed with the corresponding economical cost it implies, in the best scenario they can be used for pet food or composting. However new promising technologies and tools have been developed in the last years aimed at recovering valuable compounds from this type of materials. In particular, sub-critical water hydrolysis (SWH) has been revealed as an interesting way for recovering high added-value molecules, and its applications have been broadly referred in the bibliography. Special interest has been focused on recovering protein hydrolysates in form of peptides or amino acids, from both animal and vegetable wastes, by means of SWH. These recovered biomolecules have a capital importance in fields such as biotechnology research, nutraceuticals, and above all in food industry, where such products can be applied with very different objectives. Present work reviews the current state of art of using sub-critical water hydrolysis for protein recovering from food industry wastes. Key parameters as reaction time, temperature, amino acid degradation and kinetic constants have been discussed. Besides, the characteristics of the raw material and the type of products that can be obtained depending on the substrate have been reviewed. Finally, the application of these hydrolysates based on their functional properties and antioxidant activity is described.

Production of porcine hemoglobin peptides at moderate temperature and medium pressure under a nitrogen stream. Functional and antioxidant properties.

A new hydrolysis method for producing peptides from porcine hemoglobin has been developed. Current processes are based on the use of expensive enzymes or high hydrostatic pressures. In the present study, a cheap and effective process has been assayed to produce peptides from purified porcine hemoglobin. A solution of purified hemoglobin is heated at different temperatures and pressurized at 4 MPa while a stream of nitrogen is injected into the reactor. A total of 82% of initial hemoglobin was transformed into peptides presenting an average size of 3.2 kDa. Some preferential hydrolyzed bonds have been detected. The peptide size distribution was evaluated at different times and temperatures. It has been demonstrated that this technique produces large amounts of peptides possessing good antioxidant properties. Furthermore, functional properties are conserved, and a desirable decrease in color (80%) is achieved.

Extraction and characterization of protein from Irish brown seaweed Ascophyllum nodosum.

This study investigates traditional and non-conventional methods of extraction of protein from Irish brown seaweed A. nodosum. Acid, alkali, combined acid-alkali with and without ultrasound pretreatment were investigated for extraction of protein from A. nodosum. Molecular weight of protein was determined using high performance size exclusion chromatography and amino acid profiling was carried out using an amino acid analyzer. Combination of first acid and then alkali extraction was found to be the most efficient method of extraction among all methods investigated (59% of recovery); followed by single step of alkali extraction assisted with ultrasound (68.4μm) which was able to extract 57% of total protein. Alkaline extraction was shown to yield the best protein/algae liquefaction ratio (1.28). This can be attributed to the release of polysaccharide complexes first by acid and then solubilization of proteins by alkali solvent. The molecular weight of extracted protein was found to be relatively low, in the range of 2-4kDa average MW. The alkali method of extraction was found to be optimum for extraction of amino acids from A. nodosum.

Prolonged Clinical Benefit of Everolimus Therapy in the Management of High-Grade Pancreatic Neuroendocrine Carcinoma

Treatment options for patients with high-grade pancreatic neuroendocrine tumors (pNET) are limited, especially for those with progressive disease and for those who experience treatment failure. Everolimus, an oral inhibitor of mammalian target of rapamycin (mTOR), has been approved for the treatment of patients with low- or intermediate-grade advanced pNET. In the randomized phase III RADIANT-3 study in patients with low- or intermediate-grade advanced pNET, everolimus significantly increased progression-free survival (PFS) and decreased the relative risk for disease progression by 65% over placebo. This case report describes a heavily pretreated patient with high-grade pNET and liver and peritoneal metastases who achieved prolonged PFS, clinically relevant partial radiologic tumor response, and resolution of constitutional symptoms with improvement in Karnofsky performance status while receiving a combination of everolimus and octreotide long-acting repeatable (LAR). Radiologic and clinical responses were maintained for 19 months, with minimal toxicity over the course of treatment. This case supports the findings that the combination of everolimus plus octreotide LAR may be considered for use in patients with high-grade pNET and progressive disease. Although behavior and aggressiveness are different between low- or intermediate-grade and high-grade pNET, some high-grade pNET may express mTOR; hence, everolimus should be considered in a clinical trial.

Classification and target compounds

Abstract Unsurprisingly, food production leads to the generation of many co-/by-products and waste material along the chain from collection through to various stages of processing and final manufacture. Such cogenerated products can pose environmental and economic problems thus reducing the profitability of the food industry. However, within the problem lies the solution, as these coproducts are rich in a variety of biomolecules of high relevance to the food and nonfood (e.g. pharmaceutical, cosmetic) industries and can command high value for the agro-food industry. In this chapter an overview is presented of some of the high added-value biomolecules identified in the different by-products generated by the most important industries (cereals, roots, tubers, pulses and oil crops, fruits, vegetables, meat, fish, and milk); the corresponding target compounds in each case as well as their potential applications in key sectors such as food, pharmaceutical, or biomedical are also outlined. The reader is guided to a number of relevant published articles for more in-depth descriptions, which are beyond the scope of this chapter.

The yield of peptides and amino acids following acid hydrolysis of haemoglobin from porcine blood

Animal blood is the most important waste product from the meat industry due to the huge volumes produced and its pollutant power. Different methods are currently employed to process this by-product, such as drying, incineration or enzymatic hydrolysis. All these techniques are expensive, do not result in revalorisation or are not applicable at an industrial scale. In this paper, chemical hydrolysis is presented as an alternative to recover and increase the value of purified haemoglobin, the most abundant protein in blood. Non-enzymatic hydrolysis of haemoglobin is a good method for obtaining peptides due to its low cost, ease of control and the large amount of peptides produced, as well as being suitable for industrial applications. This paper presents a study of the use of two acids (sulfuric and hydrochloric) for this purpose under different experimental conditions. From the analysis of the kinetics of the hydrolysis process, four fractions can be defined: unbroken haemoglobin, soluble peptides, non-soluble peptides and free amino acids. A kinetic model was developed to simulate the hydrolysis mechanisms, providing a good fit to the experimental results. Both sulfuric and hydrochloric acid at concentrations of 6 M can hydrolyse the haemoglobin completely, but the average peptide size is lower for sulfuric than for hydrochloric acid.

Alternative uses for co-products: Harnessing the potential of valuable compounds from meat processing chains

Opportunities for exploiting the inherent value of protein-rich meat processing co-products, in the context of increased global demand for protein and for sustainable processing systems, are discussed. While direct consumption maybe the most profitable route for some, this approach is influenced greatly by local and cultural traditions. A more profitable and sustainable approach may be found in recognizing this readily available and under-utilised resource can provide high value components, such as proteins, with targeted high value functionality of relevance to a variety of sectors. Applications in food & beverages, petfood biomedical and nutrition arenas are discussed. Utilization of the raw material in its entirety is a necessary underlying principle in this approach to help maintain minimum waste generation. Understanding consumer attitudes to these products, in particular when used in food or beverage systems, is critical in optimizing commercialization strategies.

Extraction of biomolecules from seaweeds

Abstract Seaweeds are an excellent source of phenolics, polysaccharides, proteins, peptides, and pigments. The seaweed extraction technologies currently employed are energy intensive and require extended extraction times. Thus, there is a need to develop efficient and environmentally friendly technologies for biomolecule extraction from seaweeds. Recently, novel extraction technologies such as enzyme-assisted extraction, microwave-assisted extraction, ultrasound-assisted extraction, supercritical fluid extraction, and pressurized liquid extraction have been demonstrated to have potential application for extraction of biomolecules from seaweeds. Advantages of these novel technologies include increased yield and reduced extraction time as compared to traditional solvent extraction techniques. This chapter outlines recent advances in novel technologies for seaweed extraction applications.

Inert and Oxidative Subcritical Water Hydrolysis of Insoluble Egg Yolk Granular Protein, Functional Properties, and Comparison to Enzymatic Hydrolysis

The use of enzymes to recover soluble peptides with functional properties from insoluble proteins could prove to be very expensive, implying high reaction times and low yields. In this study, the insoluble granular protein, previously delipidated, was hydrolyzed using enzymes (trypsin) as a comparison to the proposed alternative method: subcritical water hydrolysis (SWH) using both nitrogen and oxygen streams. The result of the hydrolysis was characterized in terms of the yield and peptide size distribution as well as different functional properties. The SWH of the delipidated granules resulted in a higher recovery yield than that obtained by enzymatic hydrolysis in half of the time. The foaming capacity of the peptides obtained by SWH was higher than that obtained by trypsin hydrolysis, although the foam stability was lower. Slight differences were detected between these peptides in terms of their emulsifying properties.