In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47{Delta}B) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts

Neither dust nor black carbon causing apparent albedo decline in Greenland\u27s dry snow zone: Implications for MODIS C5 surface reflectance

Remote sensing observations suggest Greenland ice sheet (GrIS) albedo has declined since 2001, even in the dry snow zone. We seek to explain the apparent dry snow albedo decline. We analyze samples representing 2012–2014 snowfall across NW Greenland for black carbon and dust light-absorbing impurities (LAI) and model their impacts on snow albedo. Albedo reductions due to LAI are small, averaging 0.003, with episodic enhancements resulting in reductions of 0.01–0.02. No significant increase in black carbon or dust concentrations relative to recent decades is found. Enhanced deposition of LAI is not, therefore, causing significant dry snow albedo reduction or driving melt events. Analysis of Collection 5 Moderate Resolution Imaging Spectroradiometer (MODIS) surface reflectance data indicates that the decline and spectral shift in dry snow albedo contains important contributions from uncorrected Terra sensor degradation. Though discrepancies are mostly below the stated accuracy of MODIS products, they will require revisiting some prior conclusions with C6 data

Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450_(BM3) demethylases

Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450_(BM3)) demethylases that provides a highly efficient means to access valuable intermediates, which can be converted to a wide range of substituted monosaccharides and polysaccharides. Demethylases displaying high levels of regioselectivity toward a number of protected monosaccharides were identified using a combination of protein and substrate engineering, suggesting that this approach ultimately could be used in the synthesis of a wide range of substituted mono- and polysaccharides for studies in chemistry, biology, and medicine

Electrochemical reduction of CO2 with an oxide-derived lead nano-coralline electrode in dimcarb

Electroreduction of CO2 in the distillable ionic liquid dimethylammonium dimethylcarbamate (dimcarb) has been investigated with an oxide‐derived lead (od‐Pb) electrode. Compared with unmodified polycrystalline Pb, where H2 is the dominant electrolysis product, od‐Pb possesses impressive catalytic properties for the reduction of CO2 in dimcarb (mixing molar ratio of CO2 and dimethylamine (DMA) >1 : 1.8), with faradaic efficiencies for the generation of H2, CO, and [HCOO]− of approximately 15, 10, and 75 %, respectively. These efficiencies are independent of the applied potential in the range of −1.34 to −3.34 V vs. Cc0/+ (where Cc+=cobaltocenium). Thorough analysis of the properties of od‐Pb, we demonstrate that its intrinsically high catalytic activity towards CO2 reduction compared to bulk Pb is attributable to an increased surface roughness and greater surface area (ca. 10 times higher), rather than the existence of residual metal oxides that are known to suppress the hydrogen evolution reaction, preferred crystal orientation, or the existence of metastable active sites

Differential Requirement for CD70 and CD80/CD86 in Dendritic Cellmediated Activation of Tumor Tolerized CD8 T Cells

A major obstacle to efficacious T cell-based cancer immunotherapy is the tolerizing-tumor microenvironment that rapidly inactivates tumor-infiltrating lymphocytes. In an autochthonous model of prostate cancer, we have previously shown that intratumoral injection of Ag-loaded dendritic cells (DCs) delays T cell tolerance induction as well as refunctionalizes already tolerized T cells in the tumor tissue. In this study, we have defined molecular interactions that mediate the effects of DCs. We show that pretreating Ag-loaded DCs with anti-CD70 Ab abolishes the ability of DCs to delay tumor-mediated T cell tolerance induction, whereas interfering with 4-1BBL, CD80, CD86, or both CD80 and CD86 had no significant effect. In contrast, CD80[superscript −/−] or CD80[superscript −/−]CD86[superscript −/−] DCs failed to reactivate already tolerized T cells in the tumor tissue, whereas interfering with CD70 and 4-1BBL had no effect. Furthermore, despite a high level of programmed death 1 expression by tumor-infiltrating T cells and programmed death ligand 1 expression in the prostate, disrupting programmed death 1/programmed death ligand 1 interaction did not enhance T cell function in this model. These findings reveal dynamic requirements for costimulatory signals to overcome tumor-induced tolerance and have significant implications for developing more effective cancer immunotherapies.American Cancer Society (Postdoctoral Fellowship 12109-PF-11-025-01-LIB)John D. Proctor Foundation (Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship)United States. Army Medical Research and Materiel Command. Prostate Cancer Research Program (Grant

Using Electronic Noses to Detect Tumors During Neurosurgery

It has been proposed to develop special-purpose electronic noses and algorithms for processing the digitized outputs of the electronic noses for determining whether tissue exposed during neurosurgery is cancerous. At present, visual inspection by a surgeon is the only available intraoperative technique for detecting cancerous tissue. Implementation of the proposal would help to satisfy a desire, expressed by some neurosurgeons, for an intraoperative technique for determining whether all of a brain tumor has been removed. The electronic-nose technique could complement multimodal imaging techniques, which have also been proposed as means of detecting cancerous tissue. There are also other potential applications of the electronic-nose technique in general diagnosis of abnormal tissue. In preliminary experiments performed to assess the viability of the proposal, the problem of distinguishing between different types of cultured cells was substituted for the problem of distinguishing between normal and abnormal specimens of the same type of tissue. The figure presents data from one experiment, illustrating differences between patterns that could be used to distinguish between two types of cultured cancer cells. Further development can be expected to include studies directed toward answering questions concerning not only the possibility of distinguishing among various types of normal and abnormal tissue but also distinguishing between tissues of interest and other odorous substances that may be present in medical settings

Cross-Study Projections of Genomic Biomarkers: An Evaluation in Cancer Genomics

Human disease studies using DNA microarrays in both clinical/observational and experimental/controlled studies are having increasing impact on our understanding of the complexity of human diseases. A fundamental concept is the use of gene expression as a “common currency” that links the results of in vitro controlled experiments to in vivo observational human studies. Many studies – in cancer and other diseases – have shown promise in using in vitro cell manipulations to improve understanding of in vivo biology, but experiments often simply fail to reflect the enormous phenotypic variation seen in human diseases. We address this with a framework and methods to dissect, enhance and extend the in vivo utility of in vitro derived gene expression signatures. From an experimentally defined gene expression signature we use statistical factor analysis to generate multiple quantitative factors in human cancer gene expression data. These factors retain their relationship to the original, one-dimensional in vitro signature but better describe the diversity of in vivo biology. In a breast cancer analysis, we show that factors can reflect fundamentally different biological processes linked to molecular and clinical features of human cancers, and that in combination they can improve prediction of clinical outcomes