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.

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.

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.

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.

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.

Adherence to Mediterranean diet and risk of developing diabetes: prospective cohort study.

OBJECTIVE: To assess the relation between adherence to a Mediterranean diet and the incidence of diabetes among initially healthy participants. DESIGN: Prospective cohort study with estimates of relative risk adjusted for sex, age, years of university education, total energy intake, body mass index, physical activity, sedentary habits, smoking, family history of diabetes, and personal history of hypertension. SETTING: Spanish university department. PARTICIPANTS: 13 380 Spanish university graduates without diabetes at baseline followed up for a median of 4.4 years. MAIN OUTCOME MEASURES: Dietary habits assessed at baseline with a validated 136 item food frequency questionnaire and scored on a nine point index. New cases of diabetes confirmed through medical reports and an additional detailed questionnaire posted to those who self reported a new diagnosis of diabetes by a doctor during follow-up. Confirmed cases of type 2 diabetes. RESULTS: Participants who adhered closely to a Mediterranean diet had a lower risk of diabetes. The incidence rate ratios adjusted for sex and age were 0.41 (95% confidence interval 0.19 to 0.87) for those with moderate adherence (score 3-6) and 0.17 (0.04 to 0.75) for those with the highest adherence (score 7-9) compared with those with low adherence (score <3). In the fully adjusted analyses the results were similar. A two point increase in the score was associated with a 35% relative reduction in the risk of diabetes (incidence rate ratio 0.65, 0.44 to 0.95), with a significant inverse linear trend (P=0.04) in the multivariate analysis. CONCLUSION: Adherence to a Mediterranean diet is associated with a reduced risk of diabetes.

A flavonoid-rich diet increases nitric oxide production in rat aorta.

1. Red wine intake is associated with a low risk of cardiovascular disease. This effect has been partly attributed to the action of polyphenolic compounds, which decrease the oxidation of plasma low density lipoproteins. Moreover, nitric oxide ((*)NO) is a vasodilator and polyphenolic compounds induce endothelium-dependent vasorelaxation in vitro. 2. Here we studied whether a diet rich in dealcoholated red wine (DRW) increases acetylcholine-induced vasorelaxation and whether ingestion of DRW-, quercetin- or catechin-rich diets modifies the (*)NO-cyclic guanosine-3',5'-monophosphate (cyclic GMP) pathway and superoxide anion (O2(.-)) release in aorta in a resting state in rats fed semi-purified diets containing either 35% (v w(-1)) DRW, 0.3% (w w(-1)) quercetin or 0.3% (w w(-1)) catechin for 10 days. 3. (*)NO-mediated vasorelaxation induced by acetylcholine was greater in rats fed the DRW-rich diet than in those that received the control diet. 4. Expression of endothelial (*)NO synthase (eNOS) was similar in the four dietary groups. The aortic rings of rats fed either the DRW-, quercetin-, or catechin-rich diets showed higher NOS activity, (*)NO production and cyclic GMP content than those of rats fed the control diet. No changes were observed in O2(.-) production. 5. In summary, diets rich in either DRW, quercetin or catechin induced endothelium-dependent vasorelaxation in rat aorta in a resting state through the enhancement of (*)NO production, without modifying O2(.-) generation, thus the bioavailability of (*)NO was increased. The increase in the (*)NO-cyclic GMP pathway explains the beneficial effect of flavonoids at vascular level.

Dor orofacial - procedimentos clínicos em urgências endodônticas / Orofacial pain - endodontics urgencies

As alterações pulpares são responsáveis por um número significativo de atendimentos odontológicos e um motivo freqüênte de urgência em dor orofacial. Nem sempre seu diagnóstico é fácil pois, dependendo da fase evolutiva da inflamação pulpar, existem diferentes manifestações clíicas, sendo fundamental o diagnóstico diferencial. Durante o período de 12 meses foram atendidos 67 pacientes consecutivos para avaliação endodôntica, os quais resultaram em 46 atendimentos de urgência, sendo 38 casos de urgência endodônticas, 7 casos de urgências periodontais e 1 caso foi de origem sinusal, cuja dor era referida aos dentes. Houve leve predominância dos homens (22 urgências contra 16 nas mulheres); a idade mínima foi de 22 anos e máxima de 81 anos. As urgências endodônticas responderam aos tratamentos convencionais aplicados, exceto em 1 caso que só teve controle da dor após cirurgia periapical e a presença de casos de dor referida, mostra a necessidade de um diagnóstico clínico correto para o sucesso do atendimento. A presença de 1 caso de "flare-up" (urgência após a terapia endodôntica) foi compatível com a literatura

Oral absorption and metabolism of quercetin and sugar-conjugated derivatives in specific transport systems.

The intestinal transport and metabolism of quercetin and various sugar-conjugates were quantified in in vitro and in vivo model systems. The nature of the sugar moiety at the C3 and C4' position had no significant effect on the rate of transport. At the 10 microM level, quercetin and glycosides with sugars at position 3 were determined to be glucose transport carrier inhibitors.

Phospholipid fatty acid composition affects enzymatic antioxidant defenses in cultured Swiss 3T3 fibroblasts.

The aim of this work was to study the adaptation of enzymatic antioxidant cell defense to the nature of the membrane polyunsaturated fatty acids (PUFA). 3T3 Swiss fibroblasts were grown for 5 days in a medium supplemented with 50 microM linoleic acid (LA) or eicosapentaenoic acid (EPA) and compared to control cells (C). The phospholipid fatty acid content was evaluated: LA were enriched in n-6 PUFA (27.8%) in comparison to C (6.7%) or EPA (5.6%); EPA were enriched in n-3 PUFA (26.2%) in comparison to LA (4.4%) or C (4.6%). The fatty acid double bond index (DBI) increased from C to LA and EPA. The activities of the three key enzymatic antioxidant defenses, SOD, GPx and GST, increased with the degree of unsaturation of the phospholipid fatty acids. In the cells with fatty acids that are very sensitive to oxidative stress, the higher activities of SOD and GPx might act to limit the initiation of lipid peroxidation and the higher activities of GST and GPx to decrease the toxic effects of the various species produced from lipid degradation.

Changes in gentamicin pharmacokinetic profiles induced by mechanical ventilation.

The influence of controlled mechanical ventilation (CMV) on the pharmacokinetic profile of gentamicin has been examined in 23 patients after elective open heart surgery. A parallel design was adopted in two groups of patients; 13 patients requiring CMV for at least 32 h after surgery, all of whom were able to breath spontaneously (SB) after 72 h (study group), and 10 patients who required CMV for only a brief period and who showed SB at 32 h postsurgery. Haemodynamic parameters remained stable throughout the study. Apparent volume of distribution (VZ), half-life (t1/2), total clearance (CL), peak (Cmax") and trough (Cmin") plasma levels at steady-state for target levels (6-8 microgram/ml), were measured. In the study group significant differences between CMV and SB conditions were found in VZ (mean 0.36 and 0.25 l/kg). t1/2 (mean 3.63 and 2.90 h) and Cmax" (mean 4.30 and 5.53 microgram/ml) while Cmin" (mean 1.06 microgram.ml-1 and 0.92 microgram.ml-1) did not change significantly. In contrast, the pharmacokinetics in the control group showed no differences. It appears that CMV leads to an increase in gentamicin Vz which accounts for the fall in Cmax" below the therapeutic dose range (less than 5 microgram/ml) recommended for gentamicin. It seems advisable to use a large dose of gentamicin in patients receiving CMV, even before the level is assessed.

La estenosis subaórtica en la espondilitis anquilosante: una nueva hipótesis de la afectación cardíaca / Subaortic stenosis in ankylosing spondylitis: a new hypothesis of the cardiac involvement

La espondilitis anquilosante es una forma de inflamación crónica reumática, en el curso de cuya evolución se han descrito anomalías cardiovasculares asociadas, fundamentalmente circunscritas al área de la raíz aórtica. De ellas, parece ser patognomónica la proliferación fibrosa del tejido supra y subvalvular. Presentamos dos casos de espondilitis anquilosante estudiados mediante ecocardiografía bidimensional y que muestran una estenosis subaórtica fija en forma de membrana y rodete respectivamente, sin apreciarse alteraciones a nivel de la válvula mitral ni de la válvula aórtica. Ello, a título de hipotesis y en nuestra opinión, podría representar dos estadíos evolutivos distintos de una misma y originaria lesión, la de la membrana subvalvular aórtica, la cual pudiera constituir la alteración inicial de la afectación valvular, misma que suele encontrarse posteriormente en algunos casos de espondilitis anquilosante