Detection of rare and novel EGFR mutations in NSCLC patients: Implications for treatment-decision.

OBJECTIVES: Mutations in the gene that encodes epidermal growth factor receptor (EGFR) are biomarkers that predict how non-small cell lung cancer (NSCLC) patients respond to EGFR-targeted therapies collectively known as tyrosine kinase inhibitors (TKIs). Thus, EGFR genotyping provides crucial information for treatment decision. Both Sanger sequencing and real-time PCR methodologies are used for EGFR genotyping. However, methods based on real-time PCR have limitations, as they may not detect rare or novel mutations. The aim of this study was to determine the prevalence of rare mutations in the tyrosine kinase domain (exons 18-21) of the EGFR gene not targeted by the most frequently used real-time PCR approaches, i.e., the cobas® EGFR Mutation Test, and the Idylla™ EGFR Mutation Assay. METHODS: A total of 1228 NSCLC patients were screened for mutations in exons 18-21 of the EGFR gene using Sanger sequencing. RESULTS: We observed that 252 patients (∼20%) had at least one mutation in the EGFR gene, and 38 (∼3%) carried uncommon genetic alterations that would not be identified by the cobas® or the Idylla™ tests. We further found six new single mutations and seven previously unreported compound mutations. Clinical information and patient outcome are presented for these cases. CONCLUSIONS: This study highlights the value of sequencing-based approaches to identify rare mutations. Our results add to the inventory of known EGFR mutations, thus contributing to improved lung cancer precision treatment.

Real-world data from the Portuguese Nivolumab Expanded Access Program (EAP) in previously treated Non Small Cell Lung Cancer (NSCLC).

OBJECTIVE: The main aim of the study was to evaluate the efficacy and safety profile of Nivolumab, an immune-checkpoint-inhibitor antibody, in advanced, previously treated, Non-Small Cell Lung Cancer (NSCLC) patients, in a real world setting. METHODS: We performed a retrospective, multicentre data analysis of patients who were included in the Portuguese Nivolumab Expanded Access Program (EAP). Eligibility criteria included histologically or citologically confirmed NSCLC, stage IIIB and IV, evaluable disease, sufficient organ function and at least one prior line of chemotherapy. The endpoints included Overall Response Rate (ORR), Disease Control Rate (DCR), Progression Free Survival (PFS) and Overall Survival (OS). Safety analysis was performed with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 4.0, and immune-related Adverse Events (irAEs) were treated according to protocol treatment guidelines. Tumour response was assessed using the Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1. Data was analysed using SPSS, version 21.0 (IBM Statistics). RESULTS: From June 2015 to December 2016, a total of 229 patients with advanced NSCLC were enrolled at 30 Portuguese centres. Clinical data were collected up to the end of July 2018. The baseline median age was 64 years (range 37-83) and the majority of patients were males (70.3%) and former/current smokers (69.4%). Patients with non-squamous histology predominated (88.1%), and 67.6% of the patients had received 2 or more prior lines of chemotherapy. Out of 229 patients, data was available for 219 patients (3 patients did not start treatment, while data was unavailable in 7 patients); of the 219 patients, 15.5% were not evaluated for radiological tumour assessment, 1.4% had complete response (CR), 21% partial response (PR), 31% stable disease (SD) and 31.1% progressive disease (PD). Thus, the ORR was 22.4% and DCR was 53.4% in this population. At the time of survival analysis the median PFS was 4.91 months (95% CI, 3.89-6.11) and median OS was 13.21 months (95% CI, 9.89-16.53). The safety profile was in line with clinical trial data. CONCLUSIONS: Efficacy and safety results observed in this retrospective analysis were consistent with observations reported in clinical trials and from other centres.

Salivary gland metabolism in an animal model of chronic kidney disease.

OBJECTIVE: The aim of this study was to determine the effects of experimental CKD into the metabolism of parotid and submandibular glands of rats. CKD was induced by 5/6 nephrectomy. DESIGN: Serum analyses of BUN (Blood Urea Nitrogen) and creatinine concentrations were performed. Major salivary glands metabolism was investigated in vivo, both at rest and during salivary stimulation conditions by NMR isotopomer analysis, using [U-13C]glucose as metabolic tracer. RESULTS: CKD increases BUN and serum creatinine concentrations (p < 0.001). Multiple metabolic alterations were detected in the parotid glands of this animal model, including decreased concentrations of alanine (p < 0.05) and creatine (p < 0.05) and increased lactate/alanine ratios (p < 0.05). The salivary stimulus fostered accumulations of acetate at both analyzed glands of the CKD model (p < 0.05), indicative of disruption of the oxidative metabolic process. CONCLUSIONS: Experimental CKD induced by 5/6 nephrectomy altered the parotid salivary gland function, since glucose metabolism is clearly affected after stimulation for salivation in this gland.

Digestible methionine+cysteine in the diet of commercial layers and its influence on the performance, quality, and amino acid profile of eggs and economic evaluation.

This work aimed at evaluating the effects of 4 digestible Met+Cys levels on the diet of commercial layers and their influence on the productive performance, quality, and amino acid profile of eggs and economic viability of the activity. A total of 576 white Lohmann LSL-Lite layers was distributed into 6 replicates of 24 birds for each diet. The experimental design was completely randomized, with 4 treatments defined by levels evaluated in the feed (0.465, 0.540, 0.581, and 0.647%). The productive performance was measured for 30 weeks. The quality (34 and 50 wk old) and the amino acid profile of eggs (43 wk old) also were evaluated. A linear positive response was observed at higher Met+Cys levels for feed intake, number of eggs per housed bird, and digestible Met+Cys intake. Egg production, egg weight, egg mass, feed efficiency, and weight gain had their optimal values determined by the quadratic regression model at 0.638, 0.654, 0.647, 0.644, and 0.613% digestible Met+Cys, respectively. In the 34th wk, eggshell thickness decreased linearly at higher Met+Cys levels. In the 50th week, the optimal levels detected for eggshell thickness and percentage were 0.571 and 0.570% digestible Met+Cys, respectively. The percentages of proteins, branched-chain amino acids (leucine, isoleucine, and valine), histidine, and proline in eggs (albumen+yolk) showed a linear negative response in function of higher Met+Cys levels. Higher digestible Met+Cys levels (>0.630%) led to a good performance of layers, while lower Met+Cys levels improved the eggshell quality of layers in peak production. Optimal Met+Cys levels may change according to the price of the synthetic amino acid.

Development of a reusable and sustainable biocatalyst by immobilization of soybean peroxidase onto magnetic adsorbent.

In this work we synthesized an activated carbon/magnetite composite by a simple co-precipitation method. The activated carbon (AC) was synthesized from the solid waste obtained in the extraction process of the peroxidase enzyme and the magnetic composite was used as support for the immobilization of soybean peroxidase (SP). After the determination of the optimal immobilization parameters, a 100% yield was achieved under the following conditions: support:enzyme proportion of 1.0:0.05 g, equilibration time of 7 h, pH 3.0 (citrate buffer phosphate 0.1 mol L-1) and temperature of 50 °C. The determination of pH to the point of zero charge was also done to assist in the understanding of the immobilization process at different pH values. Several characterization techniques were used, such as thermogravimetric analysis, elemental analysis composition, X-ray powder diffraction, Fourier transform infrared spectroscopy and Scanning electron microscopy. The biocatalyst presented excellent operational stability and was reused for 11 consecutive cycles. The magnetic properties inserted in the AC contributed to the removal of the biocatalyst from the reaction medium without interfering in the adsorptive characteristics of the AC. Thus, the activated carbon/magnetite composite can be applied to different research fields with high performance.

Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures.

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L-1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.

Antiphospholipid Syndrome On Cloud (APSOnCloud): web-based monitoring of oral anticoagulation.

Antiphospholipid Syndrome (APS) is an autoimmune disease characterized by recurrent thromboses and fetal losses with the presence of antiphospholipid antibodies. The main treatment to prevent recurrent thrombotic events is oral anticoagulation with vitamin K antagonist (VKA), which requires frequent monitoring and dosage adjustments. Outpatient anticoagulation monitoring has its limitations, such as patients spending long hours between the testing procedure and waiting for the results to be adjusted. To optimize this adjustment and to improve APS patients-doctors relationship, we developed a website to help monitor APS patients, called Antiphospholipid Syndrome On Cloud or APSOnCloud. To test it, since March 2014 to March 2016, we registered 20 patients with APS that have inserted 132 international normalized ratio (INR) values. Sixty two percent were out of range and it took on average 7 hours for the doctor in charge to adjust these values. The mean time in therapeutic range was 58.1%. Our preliminary experience in monitoring VKA oral anticoagulation on APSOnCloud suggests that patients with APS might benefit from this web-based monitoring.

GeLC-MS-based proteomics of Chromobacterium violaceum: comparison of proteome changes elicited by hydrogen peroxide.

Chromobacterium violaceum is a free-living bacillus with several genes that enables it survival under different harsh environments such as oxidative and temperature stresses. Here we performed a label-free quantitative proteomic study to unravel the molecular mechanisms that enable C. violaceum to survive oxidative stress. To achieve this, total proteins extracted from control and C. violaceum cultures exposed during two hours with 8 mM hydrogen peroxide were analyzed using GeLC-MS proteomics. Analysis revealed that under the stress condition, the bacterium expressed proteins that protected it from the damage caused by reactive oxygen condition and decreasing the abundance of proteins responsible for bacterial growth and catabolism. GeLC-MS proteomics analysis provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress ultimately aggregating knowledge of the response of this organism to environmental stress. This study identified approximately 1500 proteins, generating the largest proteomic coverage of C. violaceum so far. We also detected proteins with unknown function that we hypothesize to be part of new mechanisms related to oxidative stress defense. Finally, we identified the mechanism of clustered regularly interspaced short palindromic repeats (CRISPR), which has not yet been reported for this organism.

Lithium Induces Glycogen Accumulation in Salivary Glands of the Rat.

Lithium is administered for the treatment of mood and bipolar disorder. The aim of this study was to verify whether treatment with different concentrations of lithium may affect the glycogen metabolism in the salivary glands of the rats when compared with the liver. Mobilization of glycogen in salivary glands is important for the process of secretion. Two sets of experiments were carried out, that is, in the first, the rats received drinking water supplemented with LiCl (38,25 and 12 mM of LiCl for 15 days) and the second experiment was carried out by intraperitoneal injection of LiCl solution (12 mg/kg and 45 mg LiCl/kg body weight) for 3 days. The active form of glycogen phosphorylase was not affected by treatment with LiCl considering the two experiments. The active form of glycogen synthase presented higher activity in the submandibular glands of rats treated with 25 and 38 mM LiCl and in the liver, with 25 mM LiCl. Glycogen level was higher than that of control in the submandibular glands of rats receiving 38 and 12 mM LiCl, in the parotid of rats receiving 25 and 38 mM, and in the liver of rats receiving 12 mM LiCl. The absolute value of glycogen for the submandibular treated with 25 mM LiCl, and the liver treated with 38 mM LiCl, was higher than the control value, although not statistically significant for these tissues. No statistically significant difference was found in the submandibular and parotid salivary glands for protein concentration when comparing experimental and control groups. We concluded that LiCl administered to rats influences the metabolism of glycogen in salivary glands.

Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination.

Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods. However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays. Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process. Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions. These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase. Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents. On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants. As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of false-positive results in highly sensitive molecular fungal detection assays.

Materials Design On-the-Fly.

The dream of any solid-state theorist is to be able to predict new materials with tailored properties from scratch, i.e., without any input from experiment. Over the past decades, we have steadily approached this goal. Recent developments in the field of high-throughput calculations focused on finding the best material for specific applications. However, a key input for these techniques still had to be obtained experimentally, namely, the crystal structure of the materials. Here, we give a step further and show that one can indeed optimize material properties using as a single starting point the knowledge of the periodic table and the fundamental laws of quantum mechanics. This is done by combining state-of-the-art methods of global structure prediction that allow us to obtain the ground-state crystal structure of arbitrary materials, with an evolutionary algorithm that optimizes the chemical composition for the desired property. As a first showcase demonstration of our method, we perform an unbiased search for superhard materials and for transparent conductors. We stress that our method is completely general and can be used to optimize any property (or combination of properties) that can be calculated in a computer.

New Insights on Coffea miRNAs: Features and Evolutionary Conservation.

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5' RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.

Epidemiological surveillance of colonising group B Streptococcus epidemiology in the Lisbon and Tagus Valley regions, Portugal (2005 to 2012): emergence of a new epidemic type IV/clonal complex 17 clone.

This study presents the serotype distribution and the antibiotic resistance profile of 953 colonising group B Streptococcus (GBS) recovered from women of child bearing age (15 to 49 years) between 2005 and 2012 in the Lisbon and Tagus Valley region, Portugal. Overall, serotypes Ia, II, III, and V were the most common, accounting 752 of the 953 isolates (about 80%). However, there were changes in GBS distribution, in particular in the two last years of the study. Of note, the proportion of serotype IV isolates increased from 1% (2/148) in 2006 to 20% (19/97) in 2012. Also, considerable proportions of serotype IV isolates from 2010 to 2012 were respectively resistant to erythromycin (9/43; 21%) or clindamycin (6/43; 14%). The identification of nine serotype IV isolates presenting a novel association with the clonal complex (CC) 17 lineage, involving a putative capsular switch, may accentuate their virulence potential and ecological success. Molecular analysis of this subgroup of isolates revealed the presence of rib, IS (insertion sequence) 861 and GBSi1 group II intron within the C5a peptidase gene (scpB) ­ laminin-binding protein gene (lmb) region, reflecting high clonality and a putative common origin. A close surveillance of the emergent type IV/CC17 isolates is crucial considering the potential impact over GBS treatment guidelines and capsular vaccine development.

Altered acetylcholine release in the hippocampus of dystrophin-deficient mice.

Mild cognitive impairments have been described in one-third of patients with Duchenne muscle dystrophy (DMD). DMD is characterized by progressive and irreversible muscle degeneration caused by mutations in the dystrophin gene and lack of the protein expression. Previously, we have reported altered concentrations of α7- and ß2-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal membranes of dystrophic (mdx) mice. This suggests that alterations in the central cholinergic synapses are associated with dystrophin deficiency. In this study, we examined the release of acetylcholine (ACh) and the level of the vesicular ACh transporter (VAChT) using synaptosomes isolated from brain regions that normally have a high density of dystrophin (cortex, hippocampus and cerebellum), in control and mdx mice at 4 and 12months of age. ACh release evoked by nicotinic stimulation or K(+) depolarization was measured as the tritium outflow from superfused synaptosomes preloaded with [(3)H]-choline. The results showed that the evoked tritium release was Ca(2+)-dependent and mostly formed by [(3)H]-ACh. ß2-containing nAChRs were involved in agonist-evoked [(3)H]-ACh release in control and mdx preparations. In hippocampal synaptosomes from 12-month-old mdx mice, nAChR-evoked [(3)H]-ACh release increased by 57% compared to age-matched controls. Moreover, there was a 98% increase in [(3)H]-ACh release compared to 4-month-old mdx mice. [(3)H]-ACh release evoked by K(+) depolarization was not altered, while the VAChT protein level was decreased (19%) compared to that of age-matched controls. In cortical and cerebellar preparations, there was no difference in nAChR-evoked [(3)H]-ACh release and VAChT levels between mdx and age-matched control groups. Our previous findings and the presynaptic alterations observed in the hippocampi of 12-month-old mdx mice indicate possible dysfunction of nicotinic cholinergic synapses associated with dystrophin deficiency. These changes may contribute to the cognitive and behavioral abnormalities described in dystrophic mice and patients with DMD.

Controlling the depth of anesthesia by a novel positive control strategy.

In this paper a positive control law is designed for multi-input positive systems that ensures asymptotic tracking of a desired output reference value. This control law can be viewed as a generalization of another one proposed in the literature for the control of the total mass in SISO compartmental systems, but is suitable for a wider class of positive systems. The controller proposed here is applied to the control of the depth of anesthesia (DoA), by means of the administration of propofol and remifentanil, when using a parameter parsimonious Wiener model recently introduced in the literature. Its performance is illustrated by realistic simulations.