Generation of immunocompetent somatic glioblastoma mouse models through in situ transformation of subventricular zone neural stem cells

Disease-relevant in vivo tumor models are essential tools for both discovery and translational research. Here, we describe a highly genetically tractable technique for generating immunocompetent somatic glioblastoma (GBM) mouse models using piggyBac transposition and CRISPR-Cas9-mediated gene editing in wild-type mice. We describe steps to deliver plasmids into subventricular zone endogenous neural stem cells by injection and electroporation, leading to the development of adult tumors that closely recapitulate the histopathological, molecular, and cellular features of human GBM. For complete details on the use and execution of this protocol, please refer to Garcia-Diaz et al.1

Emerging regulatory paradigms in glutathione metabolism

One of the hallmarks of cancer is the ability to generate and withstand unusual levels of oxidative stress. In part, this property of tumor cells is conferred by elevation of the cellular redox buffer glutathione. Though enzymes of the glutathione synthesis and salvage pathways have been characterized for several decades, we still lack a comprehensive understanding of their independent and coordinate regulatory mechanisms. Recent studies have further revealed that overall central metabolic pathways are frequently altered in various tumor types, resulting in significant increases in biosynthetic capacity, and feeding into glutathione synthesis. In this review, we will discuss the enzymes and pathways affecting glutathione flux in cancer, and summarize current models for regulating cellular glutathione through both de novo synthesis and efficient salvage. In addition, we examine the integration of glutathione metabolism with other altered fates of intermediary metabolites, and highlight remaining questions about molecular details of the accepted regulatory modes

Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insights into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wildtype or mutant GCLC and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to wild-type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using an S. cerevisiae model system. Mutant GCLC alone could not complement a glutathione-deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency

Monitoring bioaerosol and odour emissions from composting facilities - WR1121

Government policy requires that valuable resources should be recovered and recycled from biodegradable waste. A successful and growing organics recycling industry delivers this policy with composting being one of the principal technologies deployed to process suitable feedstock such as garden and food waste. Composting inevitably generates bioaerosols – particulate matter comprising cells or cellular components that are released into the air as a result of disturbance of composting feedstock or the processing of final product. Exposure to bioaerosols has the potential to be harmful to human and animal health. The Environment Agency adopts a precautionary and risk-based approach to the regulation of composting facilities which was developed on the basis of research by Wheeler et al. (2001) and which has been updated as new evidence has become available. The Environment Agency also requires site operators to monitor bioaerosols around their facilities using methods specified in a standard protocol which relies upon classical microbiology methods which are tried and tested but which are labour-intensive, slow and offer only a snapshot view of a highly dynamic system. A recent IOM review commissioned by Defra (Searl, 2009) on exposure-response relationships for bioaerosol emissions from waste treatment processes identified significant gaps in knowledge of exposure to bioaerosols and recommended that more research was needed into alternatives to viable microbial monitoring such as priority biomarkers (notably endotoxin) and potential surrogates such as particulate matter. The IOM review also concluded that there is a lack of information to support the development of appropriate stand-off distances. The overall aim of this project was to provide evidence on bioaerosol production, dispersion and potential exposures from composting facilities in support of future developments in policy and regulation of biowaste facilities. The objectives were: (i) to undertake a comprehensive set of standard and novel bioaerosol measurements at representative composting sites to assess comparability between different methods and also to measure spatial and temporal variations; and (ii) to determine the odour emissions and then compare these with bioaerosol emissions to see if odour is a marker of significant bioaerosol exposure. Standard (AfOR, 2009) and novel (CEN filter method, endotoxin, glucan, qPCR, real-time particulates) bioaerosols measurements were taken on a minimum of three to a maximum of six occasions over a twelve month period at four different composting facilities in England. The composting facilities were selected to represent sites of varying sizes (tonnages) and to allow a comparison of bioaerosol concentrations at standard open windrow sites versus a fully-contained site. Additional supporting information was collected including meteorological data at the time of sampling, observation of site operations and measurements of odour at one of the sites. Supporting bioaerosol and odour dispersion modelling was conducted at the site where the odour measurements were made. The spatial trend of bioaerosol concentrations described by Wheeler et al., (1991) and upon which EA regulatory policy is based was broadly corroborated by this dataset. Excursions above the EA acceptable levels at or beyond 250m from source were rare. Bioaerosol concentrations at the enclosed site were generally lower than at the open windrow sites. There was no evidence of a seasonal pattern in bioaerosol concentrations at any of the sites whereas between-sampling day variations were apparent. The cause(s) of these variations were not identified. No consistent relationship was observed between the concentration of bioaerosols measured by the two AfOR standard methods. The two methods displayed certain strengths and weakness in different situations. The IOM sampling device proved to be better suited to situations where high bioaerosol concentrations were encountered (close to source); the Andersen proving to be more effective in the lower concentration range typically found upwind of a site or at distance downwind from source. The higher volume filtration device tested in this project (referred to as the CEN method) produced data that did not consistently match either of the AfOR standard methods. This device demonstrated greater sensitivity than the IOM filter method but suffered drawbacks associated with its weight and a lack of ease of use in the field. Endotoxin concentrations were normally below the level recommended by the Dutch Expert Committee on Occupational Safety but occasional exceedances of this standard were detected at the larger open windrow sites. The majority of glucan measurements were below a widely referred to 10ng/m3 threshold. Significantly elevated concentrations were detected at one of the larger open windrow sites. The dynamic range of the qPCR method is wider (4-5-log) than either of the AfOR and the CEN methods. It is also quicker to carry out and has the potential for automation. The results from the qPCR method are mainly higher than standard AfOR methods, as the method does not distinguish viable and non-viable spores. The spatial distribution of Aspergillus fumigatus spores (by qPCR) along sampling transects, gives similar results compared to AfOR (and CEN) methods. Real time particle detection showed that both TSP and PM10 are correlated to Aspergillus fumigatus spore concentration. No consistent relationship was observed between odour and bioaerosol concentrations (although this was a limited dataset). The envelope of modelled (back-extrapolated) bioaerosol emission rates straddles several orders of magnitude. Distinguishing the influences of meteorological conditions on this variability was not possible. It was not possible to predict bioaerosol or odour emission rates with confidence. This continues to hamper confidence in modelling of odours and bioaerosols from open windrow facilities. The findings of this research have implications for the current standard monitoring protocol which should be reviewed accordingly. The findings of this multi-site survey accord with existing regulatory policy and are supportive of the general trend towards enclosed facilities. Notwithstanding this, continuing research is needed to enhance the database on emission from bioaerosol and odour abatement technologies (e.g. biofilters); to determine the cause(s) of occasional bioaerosol peaks from open facilities; to improve exposure assessments through better modelling protocols; and to link enhanced exposure information to future health impact studies

The inseparability of sampling and time and its influence on attempts to unify the molecular and fossil records

The two major approaches to studying macroevolution in deep time are the fossil record and reconstructed relationships among extant taxa from molecular data. Results based on one approach sometimes conflict with those based on the other, with inconsistencies often attributed to inherent flaws of one (or the other) data source. What is unquestionable is that both the molecular and fossil records are limited reflections of the same evolutionary history, and any contradiction between them represents a failure of our existing models to explain the patterns we observe. Fortunately, the different limitations of each record provide an opportunity to test or calibrate the other, and new methodological developments leverage both records simultaneously. However, we must reckon with the distinct relationships between sampling and time in the fossil record and molecular phylogenies. These differences impact our recognition of baselines, and the analytical incorporation of age estimate uncertainty. These differences in perspective also influence how different practitioners view the past and evolutionary time itself, bearing important implications for the generality of methodological advancements, and differences in the philosophical approach to macroevolutionary theory across fields.Comment: 29 pages, 1 figure. All others contributed equally to this wor

Pharmacokinetic and Biodistribution Assessment of a Near Infrared-Labeled PSMA-Specific Small Molecule in Tumor-Bearing Mice

Prostate cancer is themost frequently diagnosed cancer in men and often requires surgery. Use of near infrared (NIR) technologies to perform image-guided surgery may improve accurate delineation of tumor margins. To facilitate preclinical testing of such outcomes, here we developed and characterized a PSMA-targeted small molecule, YC-27. IRDye 800CW was conjugated to YC-27 or an anti-PSMA antibody used for reference. Human 22Rv1, PC3M-LN4, and/or LNCaP prostate tumor cells were exposed to the labeled compounds. In vivo targeting and clearance properties were determined in tumor-bearing mice. Organs and tumors were excised and imaged to assess probe localization. YC-27 exhibited a dose dependent increase in signal upon binding. Binding specificity and internalization were visualized by microscopy. In vitro and in vivo blocking studies confirmed YC-27 specificity. In vivo, YC-27 showed good tumor delineation and tissue contrast at doses as low as 0.25 nmole. YC-27 was cleared via the kidneys but bound the proximal tubules of the renal cortex and epididymis. Since PSMA is also broadly expressed on the neovasculature of most tumors, we expect YC-27 will have clinical utility for image-guided surgery and tumor resections