Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study.

BACKGROUND: We evaluated a new chemoimmunotherapy combination based on the anti-PD1 monoclonal antibody pembrolizumab and the pyrimidine antimetabolite gemcitabine in HER2- advanced breast cancer (ABC) patients previously treated in the advanced setting, in order to explore a potential synergism that could eventually obtain long term benefit in these patients. METHODS: HER2-negative ABC patients received 21-day cycles of pembrolizumab 200 mg (day 1) and gemcitabine (days 1 and 8). A run-in-phase (6 + 6 design) was planned with two dose levels (DL) of gemcitabine (1,250 mg/m2 [DL0]; 1,000 mg/m2 [DL1]) to determine the recommended phase II dose (RP2D). The primary objective was objective response rate (ORR). Tumor infiltrating lymphocytes (TILs) density and PD-L1 expression in tumors and myeloid-derived suppressor cells (MDSCs) levels in peripheral blood were analyzed. RESULTS: Fourteen patients were treated with DL0, resulting in RP2D. Thirty-six patients were evaluated during the first stage of Simon's design. Recruitment was stopped as statistical assumptions were not met. The median age was 52; 21 (58%) patients had triple-negative disease, 28 (78%) visceral involvement, and 27 (75%) ≥ 2 metastatic locations. Progression disease was observed in 29 patients. ORR was 15% (95% CI, 5-32). Eight patients were treated ≥ 6 months before progression. Fourteen patients reported grade ≥ 3 treatment-related adverse events. Due to the small sample size, we did not find any clear association between immune tumor biomarkers and treatment efficacy that could identify a subgroup with higher probability of response or better survival. However, patients that experienced a clinical benefit showed decreased MDSCs levels in peripheral blood along the treatment. CONCLUSION: Pembrolizumab 200 mg and gemcitabine 1,250 mg/m2 were considered as RP2D. The objective of ORR was not met; however, 22% patients were on treatment for ≥ 6 months. ABC patients that could benefit of chemoimmunotherapy strategies must be carefully selected by robust and validated biomarkers. In our heavily pretreated population, TILs, PD-L1 expression and MDSCs levels could not identify a subgroup of patients for whom the combination of gemcitabine and pembrolizumab would induce long term benefit. TRIAL REGISTRATION: ClinicalTrials.gov and EudraCT (NCT03025880 and 2016-001,779-54, respectively). Registration dates: 20/01/2017 and 18/11/2016, respectively.

An optimized MNK1b aptamer, apMNKQ2, and its potential use as a therapeutic agent in breast cancer.

Breast cancer is the most commonly diagnosed and leading cause of cancer death among women worldwide. Mitogen-activated protein kinase-interacting kinases (MNKs) promote the expression of several oncogenic proteins and are overexpressed in several types of cancer. In human cells, there are four isoforms of MNKs. The truncated isoform MNK1b, first described in our laboratory, has a higher basal activity and is constitutively active. Aptamers are emerging in recent years as potential therapeutic agents that show significant advantages over drugs of other nature. We have previously obtained and characterized a highly specific aptamer against MNK1b, named apMNK2F, with a dissociation constant in the nanomolar range, which produces significant inhibition of proliferation, migration, and colony formation in breast cancer cells. Furthermore, its sequence analysis predicted two G-quadruplex structures. In this work, we show the optimization process of the aptamer to reduce its size, improving its stability. The obtained aptamer, named apMNKQ2, is able to inhibit proliferation, colony formation, migration, and invasion in breast cancer cells. In murine models of breast cancer, apMNKQ2 has demonstrated its efficacy in reducing tumor volume and the number of metastases. In conclusion, apMNKQ2 could be used as an anti-tumor drug in the future.

LC-QQQ-MS routine analysis method for new biomarker quantification in plasma aimed at early chronic kidney disease diagnosis.

Pediatric chronic kidney disease (CKD) is currently assessed using glomerular filtration rate (GFR), which is calculated from different equations based on serum creatinine concentration. However, serum creatinine is affected by several factors and is not reliable enough for the diagnosis of CKD, especially at early stages. Recent targeted and untargeted metabolomics studies found 7 new potential biomarkers that could be useful for early pediatric chronic kidney disease diagnosis. Thus, a new LC-QQQ-MS analytical method has been developed and validated aimed at routine analysis of these 7 potential biomarkers: NG,NG'-dimethyl-l-arginine di(p-hydroxyazobenzene-p'-sulfonate) (SDMA), S-adenosyl-l-methionine (SAM), n-butyryl-l-carnitine (nC4), iso-butyryl-l-carnitine (iC4), citrulline (CIT), creatinine (CNN) and d-erytro-sphingosine-1-phosphate (S1P), in addition to creatinine, the classical biomarker for CKD diagnosis. Then, this analytical method has been used for the quantification of plasma samples from a heterogeneous group of CKD pediatric patients and a control pediatric population. Data analysis of these results showed that it is possible to differentiate between CKD and control populations based on the metabolite concentration in plasma.

Untargeted metabolomics for plasma biomarker discovery for early chronic kidney disease diagnosis in pediatric patients using LC-QTOF-MS.

Pediatric chronic kidney disease (CKD) is a clinical syndrome characterized by renal hypofunction occurring due to gradual and irreversible kidney damage that can further progress over time. New biomarkers may help early diagnosis of pediatric patients suffering from CKD and improve the outcome. Untargeted metabolomics based on LC-QTOF-MS has been used to find new biomarkers for the early diagnosis of CKD in plasma from pediatric patients. In order to avoid any bias in the determination of statistically significant entities as a consequence of the data analysis method followed, two different chemometric approaches have been used, Mass Profiler Professional (MPP) software and Matlab R2015a software. Metabolic fingerprints of control and CKD pediatric patients were compared and five metabolites which showed a significant change common to both data analysis procedures were identified. Sphingosine-1-phosphate, n-butyrylcarnitine, cis-4-decenoylcarnitine and an unidentified feature with 126.0930 m/z were found to be increased in plasma from pediatric patients with CKD, whereas bilirubin was significantly decreased. A partial least squares discriminant analysis model built with these 5 entities classified correctly 96% of the samples. In addition, when considering only early CKD patients against controls, a performance of 97% was obtained. Thus, these promising metabolites could be suitable biomarkers for the early diagnosis of pediatric CKD in a clinical setting.

Evaluation of the effect of polymorphism on G-quadruplex-ligand interaction by means of spectroscopic and chromatographic techniques.

Guanine-rich sequences may fold into highly ordered structures known as G-quadruplexes. Apart from the monomeric G-quadruplex, these sequences may form multimeric structures that are not usually considered when studying interaction with ligands. This work studies the interaction of a ligand, crystal violet, with three guanine-rich DNA sequences with the capacity to form multimeric structures. These sequences correspond to short stretches found near the promoter regions of c-kit and SMARCA4 genes. Instrumental techniques (circular dichroism, molecular fluorescence, size-exclusion chromatography and electrospray ionization mass spectrometry) and multivariate data analysis were used for this purpose. The polymorphism of G-quadruplexes was characterized prior to the interaction studies. The ligand was shown to interact preferentially with the monomeric G-quadruplex; the binding stoichiometry was 1:1 and the binding constant was in the order of 105M-1 for all three sequences. The results highlight the importance of DNA treatment prior to interaction studies.

Plasma biomarker discovery for early chronic kidney disease diagnosis based on chemometric approaches using LC-QTOF targeted metabolomics data.

Chronic kidney disease (CKD) is a progressive pathological condition in which renal function deteriorates in time. The first diagnosis of CKD is often carried out in general care attention by general practitioners by means of serum creatinine (CNN) levels. However, it lacks sensitivity and thus, there is a need for new robust biomarkers to allow the detection of kidney damage particularly in early stages. Multivariate data analysis of plasma concentrations obtained from LC-QTOF targeted metabolomics method may reveal metabolites suspicious of being either up-regulated or down-regulated from urea cycle, arginine methylation and arginine-creatine metabolic pathways in CKD pediatrics and controls. The results show that citrulline (CIT), symmetric dimethylarginine (SDMA) and S-adenosylmethionine (SAM) are interesting biomarkers to support diagnosis by CNN: early CKD samples and controls were classified with an increase in classification accuracy of 18% when using these 4 metabolites compared to CNN alone. These metabolites together allow classification of the samples into a definite stage of the disease with an accuracy of 74%, being the 90% of the misclassifications one level above or below the CKD stage set by the nephrologists. Finally, sex-related, age-related and treatment-related effects were studied, to evaluate whether changes in metabolite concentration could be attributable to these factors, and to correct them in case a new equation is developed with these potential biomarkers for the diagnosis and monitoring of pediatric CKD.

Viability qPCR, a new tool for Legionella risk management.

BACKGROUND: Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. METHODS: The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. RESULTS: In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. CONCLUSIONS: The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy.

Use of Schizosaccharomyces strains for wine fermentation-Effect on the wine composition and food safety.

Schizosaccharomyces was initially considered as a spoilage yeast because of the production of undesirable metabolites such as acetic acid, hydrogen sulfide, or acetaldehyde, but it currently seems to be of great value in enology.o ced Nevertheless, Schizosaccharomyces can reduce all of the malic acid in must, leading to malolactic fermentation. Malolactic fermentation is a highly complicated process in enology and leads to a higher concentration of biogenic amines, so the use of Schizosaccharomyces pombe can be an excellent tool for assuring wine safety. Schizosaccharomyces also has much more potential than only reducing the malic acid content, such as increasing the level of pyruvic acid and thus the vinylphenolic pyranoanthocyanin content. Until now, few commercial strains have been available and little research on the selection of appropriate yeast strains with such potential has been conducted. In this study, selected and wild Sc. pombe strains were used along with a Saccharomyces cerevisiae strain to ferment red grape must. The results showed significant differences in several parameters including non-volatile and volatile compounds, anthocyanins, biogenic amines and sensory parameters.

Classification of patients undergoing weaning from mechanical ventilation using the coherence between heart rate variability and respiratory flow signal.

Weaning from mechanical ventilation is still one of the most challenging problems in intensive care. Unnecessary delays in discontinuation and weaning trials that are undertaken too early are both undesirable. This study investigated the contribution of spectral signals of heart rate variability (HRV) and respiratory flow, and their coherence to classifying patients on weaning process from mechanical ventilation. A total of 121 candidates for weaning, undergoing spontaneous breathing tests, were analyzed: 73 were successfully weaned (GSucc), 33 failed to maintain spontaneous breathing so were reconnected (GFail), and 15 were extubated after the test but reintubated within 48 h (GRein). The power spectral density and magnitude squared coherence (MSC) of HRV and respiratory flow signals were estimated. Dimensionality reduction was performed using principal component analysis (PCA) and sequential floating feature selection. The patients were classified using a fuzzy K-nearest neighbour method. PCA of the MSC gave the best classification with the highest accuracy of 92% classifying GSucc versus GFail patients, and 86% classifying GSucc versus GRein patients. PCA of the respiratory flow signal gave the best classification between GFail and GRein patients (79% accuracy). These classifiers showed a good balance between sensitivity and specificity. Besides, the spectral coherence between HRV and the respiratory flow signal, in patients on weaning trial process, can contribute to the extubation decision.

Carbohydrate quality, weight change and incident obesity in a Mediterranean cohort: the SUN Project.

BACKGROUND/OBJECTIVES: To evaluate the association between the carbohydrate quality index (CQI) and weight change or incident overweight/obesity (body mass index ⩾25 kg/m(2)) in the 'Seguimiento Universidad de Navarra (SUN)' cohort. SUBJECTS/METHODS: A total of 8741 participants who were initially free of overweight/obesity were followed up for a median of 7.9 years. We evaluated at baseline the CQI according to four criteria: dietary fiber intake, glycemic index, whole grains/total grains ratio and solid carbohydrates/total carbohydrates ratio. Subjects were classified into quintiles according to CQI. Weight was recorded at baseline and updated every 2 years during follow-up. RESULTS: Increasing CQI of diet was not significantly associated with lower weight gain, although participants in the highest quintile had the lowest average crude weight gain (+211 g/year). We observed 1862 incident cases of overweight/obesity during follow-up. CQI was significantly associated (P for trend 0.006) with a lower risk of overweight/obesity; adjusted odds ratio for the fourth and fifth quintiles were 0.81 (95% confidence interval (CI): 0.66-0.99) and 0.74 (95% CI: 0.60-0.92), respectively. CONCLUSIONS: In this Mediterranean cohort, CQI showed a significant inverse association with the incidence of overweight/obesity, which highlights that carbohydrate intake guidelines related to obesity prevention should be focused on improving the CQI of the diet.

Selection of appropriate Schizosaccharomyces strains for winemaking.

This paper describes the selection of Schizosaccharomyces yeasts with adequate oenological suitability and high capacity for the degradation of malic acid. Despite the almost non-existent number of commercial strains, the use of this yeast genus has recently been recommended by the International Organisation of Vine and Wine (OIV, in French). Thus, in the present study, a large number of Schizosaccharomyces strains were isolated using a selective differential medium. Initially, classic parameters of oenological interest for the use of fermentative strains of Saccharomyces cerevisiae (the most frequently used type of yeast) were assessed. Only five strains of moderate acetic acid production lower than 0.4 g/L were obtained at the end of fermentation. Other, more specific features of this yeast genus' physiology were also studied, including urease activity and the production of pyruvic acid and glycerol. Finally, oenological suitability was determined by comparing selected strains with other Schizosaccharomyces reference and S cerevisiae control strains. Schizosaccharomyces strains produced 80% less urea content, four times higher pyruvic acid levels and 1 g higher glycerol contents than the Saccharomyces reference strains. The results confirmed that it is possible to perform selective processes on microorganisms from the genus Schizosaccharomyces using methodology developed in this work to obtain strains of industrial interest.

Reduction of 4-ethylphenol production in red wines using HCDC+ yeasts and cinnamyl esterases.

Hydroxycinnamate decarboxylase (HCDC) activity has been evaluated in several commercial yeast strains. The combined effect of using cinnamyl esterases (CE) and HCDC+ Saccharomyces cerevisiae strains has been studied in the formation of vinylphenolic pyranoanthocyanins (VPAs) during fermentation, analysing the kind and concentration of pigments formed according to the yeast strain used. Wines fermented with yeasts HCDC+ were contaminated with Dekkera bruxellensis and afterwards analysed to evaluate the formation of ethylphenols (EPs). The musts treated with CE and later fermented with HCDC+ yeast strains showed lower contents of 4-ethylphenol than those fermented with HCDC- strains. This reduction in the EP content is due to the transformation of hydroxycinnamic acids in stable VPAs pigments. The associated use of CEs and HCDC+ Saccharomyces strains is a natural strategy to reduce the formation of EPs in wines contaminated by Dekkera/Brettanomyces.

Formation of pyranoanthocyanins by Schizosaccharomyces pombe during the fermentation of red must.

Schizosaccharomyces pombe is a non-Saccharomyces yeast strain that can ferment grape musts with high sugar contents - but it also has other metabolic and physiological properties that render it of great interest to wine biotechnologists. This work compares the production of pyranoanthocyanins by S. pombe, Saccharomyces cerevisiae and Saccharomyces uvarum during fermentation. Total pyranoanthocyanins ranged from 11.9 to 19.4 mg/l depending on the strain of S. pombe used. On average, S. pombe produced more pyruvic acid than did either Saccharomyces species; as a consequence it also formed more vitisin A-type pigments. S. pombe 938 produced the largest quantity of vitisin A (11.03±0.82 mg/l). The formation of large amounts of pyranoanthocyanins intensifies the post-fermentation colour of wines somewhat, a phenomenon that helps them maintain their colour over ageing as the natural grape anthocyanins become degraded. Some of the S. pombe strains showed hydroxycinnamate decarboxylase activity, which favours the formation of vinylphenolic pyranoanthocyanins. Fermentation with S. pombe therefore provides an interesting way of increasing the overall pyranoanthocyanin content of red wines, and of stabilising their colour during ageing.

Non-linear oscillatory rheological properties of a generic continuum foam model: comparison with experiments and shear-banding predictions.

The occurrence of shear bands in a complex fluid is generally understood as resulting from a structural evolution of the material under shear, which leads (from a theoretical perspective) to a non-monotonic stationary flow curve related to the coexistence of different states of the material under shear. In this paper we present a scenario for shear-banding in a particular class of complex fluids, namely foams and concentrated emulsions, which differs from other scenarios in two important ways. First, the appearance of shear bands is shown to be possible both without any intrinsic physical evolution of the material (e.g. via a parameter coupled to the flow such as concentration or entanglements) and without any finite critical shear rate below which the flow does not remain stationary and homogeneous. Secondly, the appearance of shear bands depends on the initial conditions, i.e. the preparation of the material. In other words, it is history dependent. This behaviour relies on the tensorial character of the underlying model (2D or 3D) and is triggered by an initially inhomogeneous strain distribution in the material. The shear rate displays a discontinuity at the band boundary whose amplitude is history dependent and thus depends on the sample preparation.

Wettability of carbon nanofiber layers on nickel foils.

Carbon nanofiber (CNF) layers have been directly synthesized on nickel foils by chemical vapor deposition at 450°C using different H(2) concentrations and reaction times. The addition of 5% H(2) produces thicker, rougher and more porous CNF layers than when 1% H(2) is used. The roughness and porosity increases with reaction time when 5%, 10% or 20% H(2) are used; however, this effect is less pronounced when 1% H(2) is used. CNFs are 50-55 nm in diameter and have a fishbone type structure. We have studied the influence of CNF layer thickness, porosity and surface roughness on the interaction with water by measuring the contact angle. The water wetting properties of the samples are more significantly influenced by the CNF layer thickness than both surface roughness and porosity. When the CNF layer is thicker than ca. 20 µm, the surface is hydrophobic and the contact angle increases with surface roughness and porosity. When the CNF layer is thinner than ca. 20 µm, the surface is hydrophilic and the contact angle decreases with increasing surface roughness and porosity. This behavior is attributed to penetration of water, making contact with the hydrophilic C layer between the CNF layer and the foil.

Egg consumption and risk of cardiovascular disease in the SUN Project.

BACKGROUND/OBJECTIVES: Egg consumption has been associated with the risk of cardiovascular diseases (CVDs), but evidence is scarce and inconsistent. Our aim was to examine the association between egg consumption and incidence of CVD in a prospective dynamic Mediterranean cohort of 14,185 university graduates. SUBJECTS/METHODS: Egg intake was assessed using a 136-item-validated food-frequency questionnaire. Baseline consumption was categorized into no consumption or <1 egg/week, 1 egg/week, 2-4 eggs/week and >4 eggs/week. The presence of cardiovascular risk factors was assessed by questionnaire at baseline, and the incidence of CVD was assessed using biennial assessments. The median follow-up was 6.1 years. Cox regression models were fitted to estimate multivariable-adjusted hazard ratios (HRs) for CVD (myocardial infarction, revascularization procedures or stroke). Outcomes were confirmed by review of medical records. RESULTS: During a median follow-up of 6.1 years, 91 new confirmed cases of CVD were observed. No association was found between egg consumption and the incidence of CVD (HR: 1.10, 95% confidence interval: 0.46-2.63) for the highest versus the lowest category of egg consumption after adjusting for age, sex, total energy intake, adherence to the Mediterranean food pattern and other cardiovascular risk factors. Results were robust to different analytical scenarios. CONCLUSIONS: No association between egg consumption and the incidence of CVD was found in this Mediterranean cohort.