Improvement of Robusta coffee aroma by modulating flavor precursors in the green coffee bean with enzymatically treated spent coffee grounds: A circular approach.

Spent coffee grounds (SCG) are by-products obtained from the industrial process of instant coffee production or alternatively after brewing of coffee at the point of consumption. This solid residue represents one of the largest waste materials worldwide, making this fraction a rational target for valorization. The composition of SCG varies significantly depending on the brewing and extraction methods. However, this by-product is mainly composed of cellulose, hemicellulose polysaccharides and lipids. Here, we report on the enzymatic hydrolysis of industrial SCG by the use of a combination of specific carbohydrate active enzymes, enabling sugar extraction yield of 74.3 %. The generated sugar-rich extract, primarily composed of glucose (8.41 ± 1.00 % of total SCG mass) and mannose (2.88 ± 0.25 % of total SCG mass), is separated from hydrolyzed grounds and soaked with green coffee. After drying and roasting, the coffee soaked with SCG enzymatic extract displayed lower earthy, burnt and rubbery notes as well as smoother and more acidic notes in the flavor profile as compared to untreated reference. Aroma profiling performed by SPME-GC-MS corroborated the sensorial effect, with a 2-fold increase in the generation of sugar-derived molecules such as Strecker aldehydes and diketones after soaking and roasting and a 45 % and respectively 37 % reduction in phenolic compounds and pyrazines. This novel technology could represent an innovative in situ valorization stream for the coffee industry, coupled with sensory improvement of the final cup.

Automated segmentation of lungs and lung tumors in mouse micro-CT scans.

Here, we have developed an automated image processing algorithm for segmenting lungs and individual lung tumors in in vivo micro-computed tomography (micro-CT) scans of mouse models of non-small cell lung cancer and lung fibrosis. Over 3000 scans acquired across multiple studies were used to train/validate a 3D U-net lung segmentation model and a Support Vector Machine (SVM) classifier to segment individual lung tumors. The U-net lung segmentation algorithm can be used to estimate changes in soft tissue volume within lungs (primarily tumors and blood vessels), whereas the trained SVM is able to discriminate between tumors and blood vessels and identify individual tumors. The trained segmentation algorithms (1) significantly reduce time required for lung and tumor segmentation, (2) reduce bias and error associated with manual image segmentation, and (3) facilitate identification of individual lung tumors and objective assessment of changes in lung and individual tumor volumes under different experimental conditions.

EHMT2 methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation.

Lung development, integrity and repair rely on precise Wnt signaling, which is corrupted in diverse diseases, including cancer. Here, we discover that EHMT2 methyltransferase regulates Wnt signaling in the lung by controlling the transcriptional activity of chromatin-bound ß-catenin, through a non-histone substrate in mouse lung. Inhibition of EHMT2 induces transcriptional, morphologic, and molecular changes consistent with alveolar type 2 (AT2) lineage commitment. Mechanistically, EHMT2 activity functions to support regenerative properties of KrasG12D tumors and normal AT2 cells-the predominant cell of origin of this cancer. Consequently, EHMT2 inhibition prevents KrasG12D lung adenocarcinoma (LUAD) tumor formation and propagation and disrupts normal AT2 cell differentiation. Consistent with these findings, low gene EHMT2 expression in human LUAD correlates with enhanced AT2 gene expression and improved prognosis. These data reveal EHMT2 as a critical regulator of Wnt signaling, implicating Ehmt2 as a potential target in lung cancer and other AT2-mediated lung pathologies.

SEMMs: Somatically Engineered Mouse Models. A New Tool for In Vivo Disease Modeling for Basic and Translational Research.

Most experimental oncology therapies fail during clinical development despite years of preclinical testing rationalizing their use. This begs the question of whether the current preclinical models used for evaluating oncology therapies adequately capture patient heterogeneity and response to therapy. Most of the preclinical work is based on xenograft models where tumor mis-location and the lack of the immune system represent a major limitation for the translatability of many observations from preclinical models to patients. Genetically engineered mouse models (GEMMs) hold great potential to recapitulate more accurately disease models but their cost and complexity have stymied their widespread adoption in discovery, early or late drug screening programs. Recent advancements in genome editing technology made possible by the discovery and development of the CRISPR/Cas9 system has opened the opportunity of generating disease-relevant animal models by direct mutation of somatic cell genomes in an organ or tissue compartment of interest. The advent of CRISPR/Cas9 has not only aided in the production of conventional GEMMs but has also enabled the bypassing of the construction of these costly strains. In this review, we describe the Somatically Engineered Mouse Models (SEMMs) as a new category of models where a specific oncogenic signature is introduced in somatic cells of an intended organ in a post-natal animal. In addition, SEMMs represent a novel platform to perform in vivo functional genomics studies, here defined as DIVoS (Direct In Vivo Screening).

Força muscular, distância percorrida e capacidade pulmonar na Doença Pulmonar Obstrutiva Crônica / Muscle strength, distance traveled and lung capacity in Chronic Obstructive Pulmonary Disease

Objetivo: Avaliar a força do músculo quadríceps e sua relação com a capacidade pulmonar em pacientes com Doença Pulmonar Obstrutiva Crônica (DPOC). Assim como a capacidade funcional destes pacientes e sua repercussão sobre a funcionalidade desses indivíduos. Métodos: Quarenta voluntários sendo vinte com diagnóstico de DPOC (grupo 1) e vinte saudáveis (grupo 2). Foi mensurado o pico de força (PF) do músculo quadríceps através do dinamômetro portátil MicroFETâ 2 (Hoggan Health Industries, West Jordan, UT, EUA), modo High Threshold. A capacidade pulmonar foi avaliada através do Teste de Caminhada de 6 minutos (TC6m). Resultados: Os pacientes com DPOC classificaram-se em média (61%) como GOLD II de acordo com os valores de VEF1/CVF. A idade média da amostra foi de 61±7 anos e IMC de 25±4 kg.m-2. Apenas 10% do grupo 1 pratica atividade física, e consequentemente obtiveram valores menor de PF (106±33)N comparado com o grupo 2 (163±45)N (p<0,05). O mesmo se repetiu na distância percorrida do TC6m, onde o grupo 1 percorreu uma média de 356±58m e o grupo 2 380±106m (p<0,05). Conclusões: Os pacientes com DPOC apresentam fraqueza muscular do quadríceps desde estágios iniciais da doença (GOLD II). Assim como percorreram uma distância menor do que os participantes saudáveis no TC6m, o que demonstra uma capacidade pulmonar limitada com declínio funcional e baixa tolerância ao exercício.

UV-C inactivation of foodborne bacterial and viral pathogens and surrogates on fresh and frozen berries.

Outbreaks of foodborne illness associated with berries often involve contamination with hepatitis A virus (HAV) and norovirus but also bacteria such as Escherichia coli O157:H7 and parasites such as Cyclospora caytanensis. We evaluated the applicability of UV-C to the inactivation of pathogens on strawberries, raspberries and blueberries. Our three-step approach consisted of assessing the chemical safety of UV-C-irradiated berries, evaluating the sensory quality after UV-C treatment and finally studying the inactivation of the target microorganisms. Treatments lasting up to 9 min (4000 mJ cm-2) did not produce detectable levels of furan (<5 µg/kg), a known photolysis product of fructose with genotoxic activity and thus were assessed to be toxicologically safe. No effect on taste or appearance was observed, unless treatment was excessively long. 20 s of treatment (an average fluence of ~ 212 mJ cm-2) reduced active HAV titer by >1 log10 unit in 95% of cases except on frozen raspberries, while 120 s were required to inactivate murine norovirus to this extent on fresh blueberries. The mean inactivation of HAV and MNV was greater on blueberries (2-3 log10) than on strawberries and raspberries (<2 log10). MNV was more sensitive on fresh than on frozen berries, unlike HAV. Inactivation of Salmonella, E. coli O157:H7 and Listeria monocytogenes was poor on all three berries, no treatment reducing viable counts by >1 log10 unit. In most matrices, prolonging the treatment did not improve the result to any significant degree. The effect was near its plateau after 20 s of treatment. These results provide insight into the effectiveness of UV-C irradiation for inactivating bacterial and viral pathogens and surrogates on fresh and frozen berries having different surface types, under different physical conditions and at different levels of contamination. Overall they show that UV-C as single processing step is unsuitable to inactivate significant numbers of foodborne pathogens on berries.

Inactivation of viruses and bacteria on strawberries using a levulinic acid plus sodium dodecyl sulfate based sanitizer, taking sensorial and chemical food safety aspects into account.

The efficacy of levulinic acid (LVA) in combination with sodium dodecyl sulfate (SDS) in removal of foodborne viruses, enteric bacterial pathogens and their surrogates on fresh strawberries was investigated. Inoculated strawberries were treated with potable water, sodium hypochlorite solution (50ppm), 0.5% LVA plus 0.5% SDS solution, and 5% LVA plus 2% SDS solution respectively for 2min, followed by spray-rinsing with potable water. Water washing removed at least 1.0-log of the tested viral and bacterial strains from the strawberries' surfaces. The 50ppm chlorine wash induced 3.4, 1.5 and 2.1-log reductions for hepatitis A virus (HAV), murine norovirus-1 (MNV-1) and MS2 bacteriophage, respectively. In comparison, the tested bacterial strains showed uniform reductions around 1.6-log CFU/ml. The 0.5% LVA plus 0.5% SDS wash induced 2.7, 1.4 and 2.4-log reductions for HAV, MNV-1 and MS2, which were comparable with the reductions induced by chlorine (P>0.05). For bacteria, over 2.0-log reductions were obtained for Enterococcus faecium, Listeria monocytogenes and Salmonella, while Escherichia coli O157:H7 and Escherichia coli P1 showed reductions of 1.9 and 1.8-log CFU/ml. Higher concentration of LVA plus SDS showed no significantly higher reductions (P>0.05). Sensory tests of washed strawberries and chemical residue analysis of LVA on strawberries after washing were also performed. In conclusion, this study demonstrates good performance of 0.5% LVA plus 0.5% SDS to reduce the levels of enteric pathogens if present on strawberries without altering taste and introducing chemical safety issues.

Quantification of Tumor Burden in a Genetically Engineered Mouse Model of Lung Cancer by Micro-CT and Automated Analysis.

Genetically engineered mouse models (GEMMs) of lung cancer closely recapitulate the human disease but suffer from the difficulty of evaluating tumor growth by conventional methods. Herein, a novel automated image analysis method for estimating the lung tumor burden from in vivo micro-computed tomography (micro-CT) data is described. The proposed tumor burden metric is the segmented soft tissue volume contained within a chest space region of interest, excluding an estimate of the heart volume. The method was validated by comparison with previously published manual analysis methods and applied in two therapeutic studies in a mutant K-ras GEMM of non-small cell lung carcinoma. Mice were imaged by micro-CT pre-treatment and stratified into four treatment groups: an antibody inhibiting vascular endothelial growth factor (anti-VEGF), chemotherapy, combination of anti-VEGF and chemotherapy, or control antibody. In the first study, post-treatment imaging was performed 4 weeks later. In the second study, mice were scanned serially on a high-throughput scanner every 2 weeks for 8 weeks during treatment. In both studies, the automated tumor burden estimates were well correlated with manual metrics (r value range: 0.83-0.93, P < .0001) and showed a similar, significant reduction in tumor growth in mice treated with anti-VEGF alone or in combination with chemotherapy. Given the fully automated nature of this technique, the proposed analysis method can provide a valuable tool in preclinical drug research for screening and randomizing animals into treatment groups and evaluating treatment efficacy in mouse models of lung cancer in a highly robust and efficient manner.

Anti-EGFL7 antibodies enhance stress-induced endothelial cell death and anti-VEGF efficacy.

Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix-associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF-mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non-small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.

Anti-VEGF antibody therapy does not promote metastasis in genetically engineered mouse tumour models.

Resistance to anti-angiogenic therapy can occur via several potential mechanisms. Unexpectedly, recent studies showed that short-term inhibition of either VEGF or VEGFR enhanced tumour invasiveness and metastatic spread in preclinical models. In an effort to evaluate the translational relevance of these findings, we examined the consequences of long-term anti-VEGF monoclonal antibody therapy in several well-validated genetically engineered mouse tumour models of either neuroendocrine or epithelial origin. Anti-VEGF therapy decreased tumour burden and increased overall survival, either as a single agent or in combination with chemotherapy, in all four models examined. Importantly, neither short- nor long-term exposure to anti-VEGF therapy altered the incidence of metastasis in any of these autochthonous models, consistent with retrospective analyses of clinical trials. In contrast, we observed that sunitinib treatment recapitulated previously reported effects on tumour invasiveness and metastasis in a pancreatic neuroendocrine tumour (PNET) model. Consistent with these results, sunitinib treatment resulted in an up-regulation of the hypoxia marker GLUT1 in PNETs, whereas anti-VEGF did not. These results indicate that anti-VEGF mediates anti-tumour effects and therapeutic benefits without a paradoxical increase in metastasis. Moreover, these data underscore the concept that drugs targeting VEGF ligands and receptors may affect tumour metastasis in a context-dependent manner and are mechanistically distinct from one another.

Assessing therapeutic responses in Kras mutant cancers using genetically engineered mouse models.

The low rate of approval of novel anti-cancer agents underscores the need for better preclinical models of therapeutic response as neither xenografts nor early-generation genetically engineered mouse models (GEMMs) reliably predict human clinical outcomes. Whereas recent, sporadic GEMMs emulate many aspects of their human disease counterpart more closely, their ability to predict clinical therapeutic responses has never been tested systematically. We evaluated the utility of two state-of-the-art, mutant Kras-driven GEMMs--one of non-small-cell lung carcinoma and another of pancreatic adenocarcinoma--by assessing responses to existing standard-of-care chemotherapeutics, and subsequently in combination with EGFR and VEGF inhibitors. Standard clinical endpoints were modeled to evaluate efficacy, including overall survival and progression-free survival using noninvasive imaging modalities. Comparisons with corresponding clinical trials indicate that these GEMMs model human responses well, and lay the foundation for the use of validated GEMMs in predicting outcome and interrogating mechanisms of therapeutic response and resistance.

Improvement in seizure control and quality of life in medically refractory epilepsy patients converted from polypharmacy to monotherapy.

In a retrospective chart review, we identified 35 patients with medically refractory epilepsy (MRE) who had been converted from polypharmacy to monotherapy and maintained on monotherapy for at least 12 months. None of the 35 patients had worsening of their seizure frequency after the conversion to monotherapy. Fourteen of the 35 patients (40%) became seizure-free. Nine of 35 patients (26%) had a 50% reduction in seizure frequency. Five of 35 patients (14%) had a 75% reduction in seizure frequency. Twenty-eight (80%) of 35 patients participated in a quality-of-life questionnaire. Quality of life was rated as better on monotherapy as compared with polypharmacy in a number of domains: memory loss, concern over medication long-term effects, difficulty in taking the medications, trouble with leisure time activities, and overall state of health. This improvement reached statistical significance. Conversion to monotherapy in patients with MRE may be successful in achieving a reduction in seizure frequency and an improvement in quality-of-life parameters. A prospective, randomized trial is necessary to validate these findings.