Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers.

The utility of cancer whole genome and transcriptome sequencing (cWGTS) in oncology is increasingly recognized. However, implementation of cWGTS is challenged by the need to deliver results within clinically relevant timeframes, concerns about assay sensitivity, reporting and prioritization of findings. In a prospective research study we develop a workflow that reports comprehensive cWGTS results in 9 days. Comparison of cWGTS to diagnostic panel assays demonstrates the potential of cWGTS to capture all clinically reported mutations with comparable sensitivity in a single workflow. Benchmarking identifies a minimum of 80× as optimal depth for clinical WGS sequencing. Integration of germline, somatic DNA and RNA-seq data enable data-driven variant prioritization and reporting, with oncogenic findings reported in 54% more patients than standard of care. These results establish key technical considerations for the implementation of cWGTS as an integrated test in clinical oncology.

Concurrent loss of the PTEN and RB1 tumor suppressors attenuates RAF dependence in melanomas harboring (V600E)BRAF.

Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring (V600E)BRAF mutations. RB1 alterations were mutually exclusive with loss of p16(INK4A), suggesting that whereas p16(INK4A) and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.

Gene Expression Differences between Enriched Normal and Chronic Myelogenous Leukemia Quiescent Stem/Progenitor Cells and Correlations with Biological Abnormalities.

In comparing gene expression of normal and CML CD34+ quiescent (G0) cell, 292 genes were downregulated and 192 genes upregulated in the CML/G0 Cells. The differentially expressed genes were grouped according to their reported functions, and correlations were sought with biological differences previously observed between the same groups. The most relevant findings include the following. (i) CML G0 cells are in a more advanced stage of development and more poised to proliferate than normal G0 cells. (ii) When CML G0 cells are stimulated to proliferate, they differentiate and mature more rapidly than normal counterpart. (iii) Whereas normal G0 cells form only granulocyte/monocyte colonies when stimulated by cytokines, CML G0 cells form a combination of the above and erythroid clusters and colonies. (iv) Prominin-1 is the gene most downregulated in CML G0 cells, and this appears to be associated with the spontaneous formation of erythroid colonies by CML progenitors without EPO.

The self-association coiled-coil domain of PML is sufficient for the oncogenic conversion of the retinoic acid receptor (RAR) alpha.

In acute promyelocytic leukemia (APL) the retinoic acid receptor alpha (RARα) becomes an oncogene through the fusion with several partners, mostly with promyelocytic leukemia protein (PML), all of which have in common the presence of a self-association domain. The new fusion proteins, therefore, differently from the wild-type RARα, which forms only heterodimers with retinoic X receptor alpha, are also able to homo-oligomerize. The presence of such a domain has been suggested to be crucial for the leukemogenic potential of the chimeric proteins found in APL blasts. Whether or not any self-association domain is sufficient to bestow a leukemogenic activity on RARα is still under investigation. In this work, we address this question using two different X-RARα chimeras, where X represents the coiled-coil domain of PML (CC-RARα) or the oligomerization portion of the yeast transcription factor GCN4 (GCN4-RARα). We demonstrate that in vitro both proteins have transforming potential, and recapitulate the main PML-RARα biological properties, but CC-RARα is uniquely able to disrupt PML nuclear bodies. Indeed, in vivo only the CC-RARα chimera induces efficiently APL in a murine transplantation model. Thus, the PML CC domain represents the minimal structural determinant indispensable to transform RARα into an oncogenic protein.

Phase II trial of neoadjuvant temozolomide in resectable melanoma patients.

BACKGROUND: We treated melanoma patients with temozolomide (TMZ) in the neoadjuvant setting and collected cryopreserved tumor samples before and after treatment. The primary objective was to determine whether the response proportion was higher than previously reported in widely metastatic patients. A secondary objective was to test the feasibility of obtaining adequate tissue before and after treatment for genetic testing. MATERIALS AND METHODS: Chemotherapy-naive melanoma patients who were candidates for surgical resection were eligible. TMZ was administered orally at 75 mg/m(2)/day for 6 weeks of every 8-week cycle. Cycles were repeated until complete response (CR), progression, or stable disease (SD) for two cycles. RESULTS: Of 19 assessable patients, 2 had CRs and 1 had partial response. Four patients had SD; 12 progressed. Tumor O-6-methylguanine-DNA methyltransferase (MGMT) promoter was unmethylated in all nine patients analyzed including from the two CR patients. Pretreatment tumor microarray results were obtained in 16 of 19 patients. CONCLUSIONS: The response proportion to TMZ in the neoadjuvant setting was 16%, not different than in the metastatic setting. Responses were seen even in tumors with a methylated MGMT promoter. Pretreatment cryopreserved tumor adequate for microarray analysis could be obtained in most, but not all, patients. Post-treatment tumor was unavailable in complete responders.

Emergence of Pseudomonas aeruginosa strains producing metallo-beta-lactamases of the IMP-15 and VIM-2 types in Mexico.

Eighty-six carbapenem non-susceptible Pseudomonas aeruginosa isolates collected in the National Institute of Respiratory Diseases of Mexico City were screened for the presence of metallo-beta-lactamase (MBL) activity using both E-test strips and a microbiological assay with EDTA-imipenem. Genomic comparisons and sequence analyses conducted with these isolates revealed the presence of bla(VIM-2) in two clonally related isolates, and bla(IMP-15) in a clonally unrelated isolate. Both genes were found to be carried by class 1 integrons, and bla(IMP-15) was additionally present on a broad host-range plasmid. This is the first report of co-existing P. aeruginosa strains producing different MBLs in a Mexican hospital, highlighting the necessity of appropriate surveillance to prevent dissemination of carbapenem resistance.

Hyperpolarized agents for advanced MRI investigations.

The strong signal enhancement attainable by hyperpolarization methods has allowed the detection of heteronuclei in magnetic resonance imaging (MRI), allowing to obtain high quality images with very high signal to noise ratios in few seconds. The four methods to produce hyperpolarized molecules, i.e. the "brute force" approach, optical pumping of noble gases, parahydrogen induced polarization (PHIP) and dynamic nuclear polarization (DNP), are reported. The applications of hyperpolarized probes to MRI range from vascular imaging to interventional imaging and perfusion studies, up to the emerging and challenging field of molecular/metabolic imaging. In fact, the high signal intensities achievable by using hyperpolarized molecules make it possible to detect and image the metabolic products formed upon the administration of the hyperpolarized agent. The most striking examples are surveyed, including the use of hyperpolarized 13C-pyruvate in tumor diagnosis and stadiation, and in myocardium perfusion and activity studies, as well as the recently reported proposal of using 13C-bicarbonate as agent for pH-mapping in vivo.

Microbial dehalogenation of polychlorinated biphenyls in aerobic conditions.

From soils contaminated with polychlorinated biphenyls (PCBs) a strain of Alcaligenes sp. able to grow in a mineral medium with a commercial mixture of PCBs as carbon source was isolated. This strain consumed up to 200 ppm in seven days in laboratory conditions. After 24 h of incubation, some new congeners of PCBs could be recognized by mass spectrometry. Through the identification of these compounds it was possible to postulate examples of possible transformations by dechlorinations of penta- and tetra-chlorinated congeners into tri-chlorinated ones. The properties of the isolated strain are appropriate for bioremediation of contaminated areas and also for using in bioreactors in order to remove the xenobiotic chemical.

Assessment of molecularly imprinted sol-gel materials for selective room temperature phosphorescence recognition of nafcillin.

Sol-gel imprinted materials were prepared against nafcillin, a semisynthetic beta-lactamic antibiotic employed in the treatment of serious infections caused by penicillinase-producing staphylococci. Two approaches were addressed for preparation of the imprinted materials and the controls: as conventional monoliths, which were ground and sieved to a desired particle size for rebinding analysis, and as films on supporting glass slides. The specific binding sites that are created during the imprinting process are analyzed via selective room temperature phosphorescence (RTP) (sol-gel films) measurements as well as via competitive room temperature phosphorescence ligand assay. Results demonstrated the importance of the physical configuration of the imprinted material for minimizing non-specific binding. The close similarities between the structures of different beta-lactamic antibiotics made it possible to interpret the roles of the template structure on specific molecular recognition. In this article, we introduce the use of room temperature phosphorescence as selective transduction method for the template. The imprinted sol-gel films displayed enhanced specific binding characteristics respect to the monolithic sol-gel and can be envisaged for the use as recognition matrices for the screening and rapid selection of antibiotics from a combinatorial library or for the rapid control of nafcillin in biological samples (e.g. milk, serum, urine).

Microbial dehalogenation of polychlorinated biphenyls in aerobic conditions / Dehalogenación microbiana de bifenilos policlorados en condiciones aeróbicas

From soils contaminated with polychlorinated biphenils (PCBs) a strain of Alcaligenes sp. able to grow in a mineral medium with a commercial mixture of PCBs as carbon source was isolated. This strain consumed up to 200 ppm in seven days in laboratory conditions. After 24 h of incubation, some new congeners of PCBs could be recognized by mass spectrometry. Through the identification of these compounds it was possible to postulate examples of possible transformations by dechlorinations of penta- and tetra-chlorinated congeners into tri-chlorinated ones. The properties of the isolated strain are appropriate for bioremediation of contaminated areas and also for using in bioreactors in order to remove the xenobiotic chemical.

A partir de suelos contaminados con bifenilos policlorados (PCBs) se aisló una cepa de Alcaligenes sp. capaz de crecer en medio mineral con una mezcla comercial de PCBs como fuente de carbono. Esta cepa consumió hasta 200 ppm de PCBs en siete días de incubación en condiciones de laboratorio. En 24 horas de incubación se han podido detectar nuevos congéneres de PCBs mediante espectrometria de masa. La identificación de estos compuestos ha permitido postular transformaciones de congéneres penta- y tetra-clorados que originarían derivados triclorados. Las propiedades de la cepa aislada son apropiadas para biorremediación y para su uso en biorreactores para eliminar estos compuestos xenobióticos.

Microbial dehalogenation of polychlorinated biphenyls in aerobic conditions.

From soils contaminated with polychlorinated biphenyls (PCBs) a strain of Alcaligenes sp. able to grow in a mineral medium with a commercial mixture of PCBs as carbon source was isolated. This strain consumed up to 200 ppm in seven days in laboratory conditions. After 24 h of incubation, some new congeners of PCBs could be recognized by mass spectrometry. Through the identification of these compounds it was possible to postulate examples of possible transformations by dechlorinations of penta- and tetra-chlorinated congeners into tri-chlorinated ones. The properties of the isolated strain are appropriate for bioremediation of contaminated areas and also for using in bioreactors in order to remove the xenobiotic chemical.

Optimization of a culture medium for ligninolytic enzyme production and synthetic dye decolorization using response surface methodology.

A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation. Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared. The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels. The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes. Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process. The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization. The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R. This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes. It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.

How respiratory system mechanics may help in minimising ventilator-induced lung injury in ARDS patients.

The main supportive therapy in acute respiratory distress syndrome patients is mechanical ventilation. As with any therapy, mechanical ventilation has side-effects, and may induce lung injury (ventilator-induced lung injury (VILI)/ventilator-associated lung injury). The mechanical factors responsible for VILI are thought to be related to tidal recruitment/derecruitment of previously collapsed alveoli and/or pulmonary overdistension. The volume/pressure (V/P) curve of the respiratory system in patients as well as in animal models of acute lung injury (ALI) has a characteristic sigmoid shape, with a lower inflection point (LIP) corresponding to the pressure/end-expiratory volume required to initiate recruitment of collapsed alveoli, and an upper inflection point (UIP) corresponding to the pressure/end inspiratory volume at which alveolar overdistension occurs. "Protective" ventilatory approaches have therefore set out to minimise mechanical injury by using the V/P curve to individualise positive end-expiratory pressure (PEEP) (PEEP above the LIP) and tidal volume (by setting end-inspiratory V/P below the UIP) since a large number of experimental studies correlate P/V curves to histological and biological manifestations of VILI and two randomised trials showed that protective ventilatory strategy individually tailored to the P/V curve minimised pulmonary and systemic inflammation and decreased mortality in patients with ALI. However, despite the fact that several studies have: 1) proposed new techniques to perform pressure/volume curves at the bedside, 2) confirmed that the lower inflection point and upper inflection point correspond to computed tomography scan evidence of atelectasis and overdistension, and 3) demonstrated the ability of the pressure/volume curve to estimate alveolar recruitment with positive end-expiratory pressure, no large studies have assessed whether such measurement can be performed in all intensive care units as a monitoring tool to orient ventilator therapy. Preliminary experimental and clinical studies show that the shape of the dynamic inspiratory pressure/time profile during constant flow inflation (stress index), allows prediction of a ventilatory strategy that minimises the occurrence of ventilator-induced lung injury.

Degradation of environmental pollutants by Trametes trogii.

The ability of the ligninolytic fungus Trametes trogii to degrade in vitro different xenobiotics (PCBs, PAHs and dyes) was evaluated. Either 200 ppm of a PCB mixture (Aroclor 1150) or 160 ppm of an industrial PAH mixture (10% V/V of PAHs, principal components hexaethylbenzene, naphthalene, 1-methyl naphthalene, acenaphthylene, anthracene, fluorene and phenanthrene), were added to trophophasic and idiophasic cultures growing in a nitrogen limited mineral medium (glucose/asparagine) and in a complex medium (malt extract/glucose). Gas-liquid chromatography proved that within 7 to 12 d more than 90% of the organopollutants added were removed. The decrease in absorbance at 620 nm demonstrated that cultures of this fungus were able to transform 80% of the dye Anthraquinone-blue (added at a concentration of 50 ppm) in 1.5 h. Enzyme estimations indicated high activity of laccase (up to 0.55 U/mL), as well as lower production of manganese-peroxidase. Laccase activity, detected in all the conditions assayed, could be implicated in the degradation of these organopollutants. Considering the results obtained, T. trogii seems promising for detoxification.

Degradation of environmental pollutants by Trametes trogii

The ability of the ligninolytic fungus Trametes trogii to degrade in vitro different xenobiotics (PCBs, PAHs and dyes) was evaluated. Either 200 ppm of a PCB mixture (Aroclor 1150) or 160 ppm of an industrial PAH mixture (10 V/V of PAHs, principal components hexaethylbenzene, naphthalene, 1-methyl naphthalene, acenaphthylene, anthracene, fluorene and phenanthrene), were added to trophophasic and idiophasic cultures growing in a nitrogen limited mineral medium (glucose/asparagine) and in a complex medium (malt extract/glucose). Gas-liquid chromatography proved that within 7 to 12 d more than 90 of the organopollutants added were removed. The decrease in absorbance at 620 nm demonstrated that cultures of this fungus were able to transform 80 of the dye Anthraquinone-blue (added at a concentration of 50 ppm) in 1.5 h. Enzyme estimations indicated high activity of laccase (up to 0.55 U/mL), as well as lower production of manganese-peroxidase. Laccase activity, detected in all the conditions assayed, could be implicated in the degradation of these organopollutants. Considering the results obtained, T. trogii seems promising for detoxification.(AU)

Degradation of environmental pollutants by Trametes trogii

The ability of the ligninolytic fungus Trametes trogii to degrade in vitro different xenobiotics (PCBs, PAHs and dyes) was evaluated. Either 200 ppm of a PCB mixture (Aroclor 1150) or 160 ppm of an industrial PAH mixture (10 V/V of PAHs, principal components hexaethylbenzene, naphthalene, 1-methyl naphthalene, acenaphthylene, anthracene, fluorene and phenanthrene), were added to trophophasic and idiophasic cultures growing in a nitrogen limited mineral medium (glucose/asparagine) and in a complex medium (malt extract/glucose). Gas-liquid chromatography proved that within 7 to 12 d more than 90 of the organopollutants added were removed. The decrease in absorbance at 620 nm demonstrated that cultures of this fungus were able to transform 80 of the dye Anthraquinone-blue (added at a concentration of 50 ppm) in 1.5 h. Enzyme estimations indicated high activity of laccase (up to 0.55 U/mL), as well as lower production of manganese-peroxidase. Laccase activity, detected in all the conditions assayed, could be implicated in the degradation of these organopollutants. Considering the results obtained, T. trogii seems promising for detoxification.

Use of a CO2 electrode to monitor fermentations that use xenobiotic compounds as source of carbon.

Bacterial strains were isolated from contaminated waters, mud or soils. They are capable of growing in mineral medium with different chemicals as carbon source, such as aliphatic or aromatic hydrocarbons and polychlorinated biphenyls (PCBs). Most of these strains tolerate high concentrations (up to 30% v/v) of the xenobiotic substrates. This is particularly important for the development of fermenting processes to treat effluents or residues with a high content of contaminating compounds. An ion-specific potentiometric electrode (CO2) has been developed to measure CO2 production continuously. When the different strains were incubated in a mineral medium and in the presence of the corresponding substrate, a parallel between growth, substrate consumption and CO2 production was found. The developed system is suggested as an efficient and economical alternative to evaluate the potential of biodegradation by different microorganisms.