Single-cell DNA-seq depicts clonal evolution of multiple driver alterations in osimertinib-resistant patients.

BACKGROUND: The development of targeted agents, such as osimertinib for EGFR-mutated non-small-cell lung cancer (NSCLC), has drastically improved patient outcome, but tumor resistance eventually always occurs. In osimertinib-resistant NSCLC, the emergence of a second molecular driver alteration (such as ALK, RET, FGFR3 fusions or BRAF, KRAS mutations) has been described. Whether those alterations and the activating EGFR mutations occur within a single cancer cell or in distinct cell populations is largely debated. PATIENTS AND METHODS: Tumor sequencing was used to identify the acquired resistance mechanisms to osimertinib in the MATCH-R trial (NCT0251782). We implemented single-cell next-generation sequencing to investigate tumor heterogeneity on patient's frozen tissues in which multiple alterations have been identified. Patient-derived models, cell lines, and patient-derived xenografts were exposed to specific inhibitors to investigate combination treatment strategies. RESULTS: Among the 45 patients included in MATCH-R who progressed on osimertinib, 9 developed a second targetable alteration (n = 2 FGFR3-TACC3, n = 1 KIF5B-RET, n = 1 STRN-ALK fusions; n = 2 BRAFV600E, n = 1 KRASG12V, n = 1 KRASG12R, n = 1 KRASG12D mutations). Single-cell analysis revealed that the two driver alterations coexist within one single cancer cell in the four patients whose frozen samples were fully contributive. A high degree of heterogeneity within samples and sequential acquisitions of molecular events were highlighted. A combination treatment concomitantly targeting the two driver alterations was required on the corresponding patient-derived models to restore cell sensitivity, which was consistent with clinical data showing efficacy of brigatinib in the patient with ALK fusion after progression to osimertinib and crizotinib administered sequentially. CONCLUSIONS: Distinct molecular driver alterations at osimertinib resistance coexist with initial EGFR mutations in single cancer cells. The clonal evolution of cancer cell populations emphasized their heterogeneity leading to osimertinib relapse. Combining two targeted treatments is effective to achieve clinical benefit.

Sensitivity analysis and reduction of a dynamic model of a bioproduction of fructo-oligosaccharides.

Starting from a relatively detailed model of a bioprocess producing fructo-oligosaccharides, a set of experimental data collected in batch and fed-batch experiments is exploited to estimate the unknown model parameters. The original model includes the growth of the fungus Aureobasidium pullulans which produces the enzymes responsible for the hydrolysis and transfructosylation reactions, and as such contains 25 kinetic parameters and 16 pseudo-stoichiometric coefficients, which are not uniquely identifiable with the data at hand. The aim of this study is, therefore, to show how sensitivity analysis and quantitative indicators based on the Fisher information matrix can be used to reduce the detailed model to a practically identifiable model. Parametric sensitivity analysis can indeed be used to progressively simplify the model to a representation involving 15 kinetic parameters and 8 pseudo-stoichiometric coefficients. The reduced model provides satisfactory prediction and can be convincingly cross validated.

Multiresidue analysis and evaluation of the matrix effect on 20 pesticides in Brazilian maize (Zea mays L.) flour.

Maize consists of a cereal widely used in the preparation of different food products. Brazil is one of the world's largest maize producers. Several types of pesticides have been applied in maize crop, which can lead to the contamination of the derived products. The present work aims at the validation of multiresidue method to analyze the matrix effect and level of pesticides in maize flour. Twenty residues were investigated in samples commercialized in the state of Ceará, Brazil. The method was satisfactorily validated, according to parameters recommended by European Union. About 55% of the pesticides had an intense negative matrix effect. Multiresidue analyzes showed the presence of traces of fenitrotion in 20% of maize flour samples. Detected levels were below maximum residue limits recommended for maize. The results indicate that maize products need continuous monitoring to ensure food security.

Polarization-dependent loss characterization method based on optical frequency beat.

Characterization of the polarization-dependent loss (PDL) of optical components is fundamental for the reliable operation of fiber-optic communication systems. Here we present a method for determining the PDL of optical devices based on optical frequency beating and spectral analysis. Depending on the beat note between components of two orthogonally polarized probe signals modulated at different frequencies, the PDL value and its axis can be determined from a single sweep of an optical spectrum analyzer. Our proposal represents an alternative high-speed option for PDL characterization.

Immature Stages, Natural History, Systematics and Conservation of an Endangered Neotropical Butterfly: the Case of Scada karschina delicata (Nymphalidae: Ithomiini).

The endangered butterfly Scada karschina delicata Talbot, 1932 (Nymphalidae: Danainae: Ithomiini) is endemic to northeastern Brazil, occurring in very few forest remnants of the 'Pernambuco Center of Endemism'. Larvae feed on Solanaceae and are very similar to those of other species in the subtribe Mechanitina, with lateral projections on body, one of the main synapomorphies of this subtribe. Based on molecular data, S. k. delicata clustered together with S. karschina karschina, as a monophyletic group sister to the amazon clade of S. reckia. Based on all available data, S. k. delicata is known from only five localities of mid- to high-altitude forests (from 500 to 1000 m of altitude) in northeastern Brazil. Grounded on available data, a new assessment of extinction risk is proposed, and S. k. delicata is now considered Vulnerable (VU) taxon.

Amazon deforestation and climate change.

A coupled numerical model of the global atmosphere and biosphere has been used to assess the effects of Amazon deforestation on the regional and global climate. When the tropical forests in the model were replaced by degraded grass (pasture), there was a significant increase in surface temperature and a decrease in evapotranspiration and precipitation over Amazonia. In the simulation, the length of the dry season also increased; such an increase could make reestablishment of the tropical forests after massive deforestation particularly difficult.

Amazon river discharge and climate variability: 1903 to 1985.

Reconstruction of an 83-year record (1903 to 1985) of the discharge of the Amazon River shows that there has been no statistically significant change in discharge over the period of record and that the predominant interannual variability occurs on the 2- to 3-year time scale. Oscillations of river discharge predate significant human influences in the Amazon basin and reflect both extrabasinal and local factors. Cross-spectrum analyses of Amazon flow anomalies with indicators of the El Niño-Southern Oscillation phenomenon suggest that the oscillations in the hydrograph are coupled to the tropical Pacific climate cycle.

One-step co-culture fermentation strategy to produce high-content fructo-oligosaccharides.

An integrated process enabling the simultaneous production and purification of fructo-oligosaccharides (FOS) was explored. A co-culture fermentation with Aspergillus ibericus (used as FOS producer strain) and Saccharomyces cerevisiae YIL162 W (for small saccharides removal) was optimized. Inoculation conditions of S. cerevisiae, fermentative broth composition, temperature and pH were optimized by experimental design. Yeast extract concentration and temperature were the most significant variables affecting FOS purity. Co-culture fermentations with simultaneously inoculation of the strains, run under 30 °C, initial pH 6.0 and 17 g L-1 yeast extract led to FOS mixtures with 97.4 ± 0.2% (w/w) purity. The fermentations conducted in bioreactor, at a 0.8 vvm aeration rate, yielded 0.70 ± 0.00 gFOS.ginitial GF-1 at 45 h fermentation, with a FOS content of 133.7 ± 0.1 g L-1. A purity of FOS up to 93.8 ± 0.7% (w/w) was achieved. The one-step fermentation proved to be efficient, economical and fast.

Glycerol-dimethacrylate as alternative hydrophilic monomer for HEMA replacement in simplified adhesives.

OBJECTIVES: Hydroxyethyl-methacrylate (HEMA) is still widely used in simplified adhesives. Indeed, several shortcomings occur with this monomer, such as water uptake and formation of linear polymers. This study aimed to compare the effects of HEMA replacement by glycerol-dimethacrylate (GDMA) on selected physicochemical properties and bonding performance of simplified model adhesives. MATERIALS AND METHODS: Experimental simplified etch-and-rinse and self-etch adhesives were formulated containing 20 wt% HEMA or GDMA. Three-point bending test was used to obtain the elastic modulus of bar-shaped specimens, and water sorption and solubility were attained by ISO-4049 (ISO, 2009) method. Degree of conversion was surveyed by Micro-Raman spectroscopy, and microtensile bond strength was tested after 24 h or 6 months simulated pulpal pressure aging. Statistical analysis was realized with two-way ANOVA and Tukey's test (p < 0.05). RESULTS: GDMA promoted higher elastic modulus to the self-etch adhesive, and GDMA-containing etch-and-rinse adhesive achieved overall lower water sorption and solubility. The degree of conversion was statistically higher for GDMA adhesives than for HEMA etch-and-rinse one. All bond strengths dropped significantly after aging, except that of GDMA self-etch adhesive. The nanoleakage was higher and gaps were found in the interface of HEMA-containing adhesives, which were less present in GDMA equivalents. CONCLUSIONS: GDMA is a feasible hydrophilic dimethacrylate monomer to replace HEMA in simplified adhesives, thereby providing better polymerization, mechanical properties and dentin adhesion as well as lower water uptake and solubility.

Exposure to crack cocaine causes adverse effects on marine mussels Perna perna.

Our study aimed to evaluate crack cocaine effects in different life stages of the marine mussel Perna perna. For this purpose, fertilization rate, embryo-larval development, lysosomal membrane stability and DNA strand breaks were assessed. Effect concentrations in gametes and in larval development were found after 1h (IC50=23.53mg·L-1) and 48h (IC50=16.31mg·L-1), respectively. The highest tested concentration showing no acute toxicity (NOEC) was 10mg·L-1, while the lowest observed effect concentration (LOEC) was 20mg·L-1. NOEC concerning embryo-larval development was 0.625mg·L-1, while the LOEC was 1.25mg·L-1. Cyto-genotoxic effects were evidenced in mussels exposed to crack cocaine concentrations ranging from 5 to 500µg·L-1. Our results report the first data on effects of an illicit drug to marine organisms and should encourage further ecotoxicological studies of these contaminants of emerging concern in coastal ecosystems.

Strategies for the production of high-content fructo-oligosaccharides through the removal of small saccharides by co-culture or successive fermentation with yeast.

Fructo-oligosaccharides (FOS) obtained by fermentation of sucrose may be purified at large-scale by continuous chromatography (Simulated Moving Bed: SMB). In order to improve the efficiency of the subsequent SMB purification, the optimization of the fermentative broth composition in salts and sugars was investigated. Fermentations conducted at reduced amount of salts, using Aureobasidium pullulans whole cells, yielded 0.63 ± 0.03 g of FOS per gram of initial sucrose. Additionally, a microbial treatment was proposed to reduce the amount of small saccharides in the mixture. Two approaches were evaluated, namely a co-culture of A. pullulans with Saccharomyces cerevisiae; and a two-step fermentation in which FOS were first synthesized by A. pullulans and then the small saccharides were metabolized by S. cerevisiae. Assays were performed in 100mL shaken flasks and further scaled-up to a 3 L working volume bioreactor. Fermentations in two-step were found to be more efficient than the co-culture ones. FOS were obtained with a purity of 81.6 ± 0.8% (w/w), on a dry weight basis, after the second-step fermentation with S. cerevisiae. The sucrose amount was reduced from 13.5 to 5.4% in total sugars, which suggests that FOS from this culture broth will be more efficiently separated by SMB.

Periodontal disease detection in primary and mixed dentitions.

AIM: This was to compare the periodontal status of children with primary and mixed dentition at the time of their first consultation. METHODS: Children (200), aged 0-12 years (156 with mixed and 44 with primary dentition), were examined by assessing their simplified plaque index (PI) and simplified periodontal record (PSR). Statistical analysis (Chi-square test) was performed with appropriate software to find any significant associations between sex, type of dentition and PI with the PSR codes (0, 1, 2, 3 and 4). RESULTS: There was no statistically significant difference with regard to gender (p = 0.82). Generalised PI was associated more significantly with mixed dentition (p = 0.025 and higher PSR scores (p < 0.001). There was no significant relationship between sex and PSR codes (p = 0.82). CONCLUSION: Children presenting with a mixed dentition had worse PI values and PSR scores. It is important to perform periodontal examination in children to diagnose and prevent future periodontal disease and maintain their dentition as well as to identify any associated systemic conditions.

Assessment of microplastic toxicity to embryonic development of the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea).

Apart from the physiological impacts on marine organisms caused by ingesting microplastics, the toxicity caused by substances leaching from these particles into the environment requires investigation. To understand this potential risk, we evaluated the toxicity of virgin (raw) and beach-stranded plastic pellets to the development of embryos of Lytechinus variegatus, simulating transfers of chemical compounds to interstitial water and water column by assays of pellet-water interface and elutriate, respectively. Both assays showed that virgin pellets had toxic effects, increasing anomalous embryonic development by 58.1% and 66.5%, respectively. The toxicity of stranded pellets was lower than virgin pellets, and was observed only for pellet-water interface assay. These results show that (i) plastic pellets act as a vector of pollutants, especially for plastic additives found on virgin particles; and that (ii) the toxicity of leached chemicals from pellets depends on the exposure pathway and on the environmental compartment in which pellets accumulate.

Knowledge extraction: a comparison between symbolic and connectionist methods.

The use of a linguistic representation for expressing knowledge acquired by learning systems is an important issue as regards to user understanding. Under this assumption, and to make sure that these systems will be welcome and used, several techniques have been developed by the artificial intelligence community, under both the symbolic and the connectionist approaches. This work discusses and investigates three knowledge extraction techniques based on these approaches. The first two techniques, the C4.5 and CN2 symbolic learning algorithms, extract knowledge directly from the data set. The last technique, the TREPAN algorithm extracts knowledge from a previously trained neural network. The CN2 algorithm induces if...then rules from a given data set. The C4.5 algorithm extracts decision trees, although it can also extract ordered rules, from the data set. Decision trees are also the knowledge representation used by the TREPAN algorithm.

Evaluation of commercial resins for fructo-oligosaccharide separation.

Fructo-oligosaccharides (FOS) produced by fermentative processes are obtained in mixtures containing significant amounts of salts and other non-prebiotic sugars. A demineralisation process using a mixture of a cationic and an anionic resin was proposed. The separation of FOS from a mixture of fructose, glucose and sucrose was evaluated. Experiments were conducted with several commercial cationic exchange resins in calcium, sodium and potassium forms packed in preparative columns (7cm×2.2cm length×diameter). Resins in potassium form obtained the higher retention factor values for sugars when compared to the other ionic forms. However, when compared to calcium and sodium ones, resins in potassium cationic forms were shown to be the less efficient separating sugar mixtures. The resin with best separation performance was the Diaion UBK535Ca. A recovery yield of 92% (w/w) of FOS with 90% (w/w) of purity was obtained from batch experiments conducted in a single column loaded with the Diaion UBK535Ca resin at 25°C. The temperature shown did not influence the separation performance significantly. By increasing the column length, the purity of FOS increased to 92% (w/w), however the recovery yield decreased to 88% (w/w).

Fructo-oligosaccharides purification from a fermentative broth using an activated charcoal column.

In this study, a simple and efficient process to purify fructo-oligosaccharides (FOS) from a fermentative broth was proposed using a single activated charcoal column. The FOS adsorption onto the activated charcoal was modeled by a pseudo-second order model. Several volumes and concentrations of water/ethanol were studied to optimize the selective desorption of sugars from the broth mixture at 25°C. Mixtures containing 50.6% (w/w) of FOS (FOS content in the fermentative broth) were purified to 92.9% (w/w) with a FOS recovery of 74.5% (w/w). Moreover, with the proposed process, fractions with purity up to 97% (w/w) of FOS were obtained. This purification process was also found to be efficient in the desalting of the fermentative broth.

Comparison of adsorption equilibrium of fructose, glucose and sucrose on potassium gel-type and macroporous sodium ion-exchange resins.

Adsorption equilibrium of fructose, glucose and sucrose was evaluated on sulfonated poly(styrene-co-divinylbenzene) cation-exchange resins. Two types of resins were used: potassium (K+) gel-type and sodium (Na+) macroporous resins. Influence of the cation and effect of the resin structure on adsorption were studied. The adsorption isotherms were determined by the static method in batch mode for mono-component and multi-component sugar mixtures, at 25 and 40 degrees C, in a range of concentrations between 5 and 250 g L(-1). All adsorption isotherms were fitted by a linear model in this range of concentrations. Sugars were adsorbed in both resins by the following order: fructose > glucose > sucrose. Sucrose was more adsorbed in the Na+ macroporous resin, glucose was identically adsorbed, and fructose was more adsorbed in the K+ gel-type resin. Data obtained from the adsorption of multi-component mixtures as compared to the mono-component ones showed a competitive effect on the adsorption at 25 degrees C, and a synergetic effect at 40 degrees C. The temperature increase conducted to a decrease on the adsorption capacity for mono-component sugar mixtures, and to an increase for the multi-component mixtures. Based on the selectivity results, K+ gel-type resin seems to be the best choice for the separation of fructose, glucose and sucrose, at 25 degrees C.

Hydro-climate and ecological behaviour of the drought of Amazonia in 2005.

In 2005, southwestern Amazonia experienced the effects of an intense drought that affected life and biodiversity. Several major tributaries as well as parts of the main river itself contained only a fraction of their normal volumes of water, and lakes were drying up. The consequences for local people, animals and the forest itself are impossible to estimate now, but they are likely to be serious. The analyses indicate that the drought was manifested as weak peak river season during autumn to winter as a consequence of a weak summertime season in southwestern Amazonia; the winter season was also accompanied by rainfall that sometimes reached 25% of the climatic value, being anomalously warm and dry and helping in the propagation of fires. Analyses of climatic and hydrological records in Amazonia suggest a broad consensus that the 2005 drought was linked not to El Niño as with most previous droughts in the Amazon, but to warming sea surface temperatures in the tropical North Atlantic Ocean.