A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t1/2 of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of "lost" [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper et al. (Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344-10356) regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35% of the muscarinic receptors from cells treated for 30 min with 100 microM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a "light vesicle" form occurred with a t1/2 approximately 10 min, and reversed with a t1/2 approximately 20 min. In contrast to previous results in this cell line regarding beta-adrenergic receptors (Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441-443), agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein

New Molecular Reporters for Rapid Protein Folding Assays

The GFP folding reporter assay [1] uses a C-terminal GFP fusion to report on the folding success of upstream fused polypeptides. The GFP folding assay is widely-used for screening protein variants with improved folding and solubility [2]–[8], but truncation artifacts may arise during evolution, i.e. from de novo internal ribosome entry sites [9]. One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants. Circular permutants of fluorescent proteins often misfold and are non-fluorescent, and do not readily tolerate fused polypeptides within the fluorescent protein scaffolding [10]–[12]. To overcome these limitations, and to increase the dynamic range for reporting on protein misfolding, we have created eight GFP insertion reporters with different sensitivities to protein misfolding using chimeras of two previously described GFP variants, the GFP folding reporter [1] and the robustly-folding “superfolder” GFP [13]. We applied this technology to engineer soluble variants of Rv0113, a protein from Mycobacterium tuberculosis initially expressed as inclusion bodies in Escherichia coli. Using GFP insertion reporters with increasing stringency for each cycle of mutagenesis and selection led to a variant that produced large amounts of soluble protein at 37°C in Escherichia coli. The new reporter constructs discriminate against truncation artifacts previously isolated during directed evolution of Rv0113 using the original C-terminal GFP folding reporter. Using GFP insertion reporters with variable stringency should prove useful for engineering protein variants with improved folding and solubility, while reducing the number of artifacts arising from internal cryptic ribosome initiation sites

Quantitative mineral resource assessment of undiscovered porphyry copper resources in South America

A quantitative resource assessment to be published this fall identifies and evaluates 26 tracts in South America where the geology isconsidered permissive for the occurrence of porphyry copper deposits. For each tract, information is provided on: (1) the rationale fordelineating the tract, (2) examples of important deposits in the tract, (3) the rationale for choosing the mineral deposit model used forthe assessment, (4) exploration history, and (5) expected spatial distribution of undiscovered deposits in the tract. The scale used toevaluate geologic information and define tracts is 1:1,000,000. There are about 600 million tonnes of copper in known porphyry copper deposits in South America. This study estimates there areapproximately 720 million tonnes of additional copper in undiscovered porphyry copper deposits, yielding a combined endowment ofabout 1.3 billion tonnes of copper. There are about 69 known porphyry copper deposits using the criteria adopted here to define a well-explored deposit. This study estimates that a mean of about 140 deposits remain to be found. In other words, about twice as manynew deposits might be found as have already been found. Overall, deposit densities in South America are comparable to those in therest of the world but differ in important details that are reflected in mapped distributions of deposits, metal densities, and percentagesof undiscovered deposits in each tract. The deposits in the tracts that include Chuquicamata and El Teniente are significantly larger intonnage and grade and are reported in a separate model that is more representative of their characteristics. The results of theassessment afirm that not all porphyry copper deposits in South America are located in the Andes. Geologic indications support thepresence of undiscovered deposits in Patagonia as well as the Amazon of Brazil. A preliminary 1:4,000,000 map of the 26 tracts will bedisplayed

STXBP2-R190C Variant in a Patient With Neonatal Hemophagocytic Lymphohistiocytosis (HLH) and G6PD Deficiency Reveals a Critical Role of STXBP2 Domain 2 on Granule Exocytosis

Neonatal hemophagocytic lymphohistiocytosis (HLH) is a medical emergency that can be associated with significant morbidity and mortality. Often these patients present with familial HLH (f-HLH), which is caused by gene mutations interfering with the cytolytic pathway of cytotoxic T-lymphocytes (CTLs) and natural killer cells. Here we describe a male newborn who met the HLH diagnostic criteria, presented with profound cholestasis, and carried a maternally inherited heterozygous mutation in syntaxin-binding protein-2 [STXBP2, c.568C\u3eT (p.Arg190Cys)] in addition to a severe pathogenic variant in glucose 6-phosphate dehydrogenase [G6PD, hemizygous c.1153T\u3eC (Cys385Arg)]. Although mutations in STXBP2 gene are associated with f-HLH type 5, the clinical and biological relevance of the p.Arg190Cys mutation identified in this patient was uncertain. To assess its role in disease pathogenesis, we performed functional assays and biochemical and microscopic studies. We found that p.Arg190Cys mutation did not alter the expression or subcellular localization of STXBP2 or STX11, neither impaired the STXBP2/STX11 interaction. In contrast, forced expression of the mutated protein into normal CTLs strongly inhibited degranulation and reduced the cytolytic activity outcompeting the effect of endogenous wild-type STXBP2. Interestingly, arginine 190 is located in a structurally conserved region of STXBP2 where other f-HLH-5 mutations have been identified. Collectively, data strongly suggest that STXBP2-R190C is a deleterious variant that may act in a dominant-negative manner by probably stabilizing non-productive interactions between STXBP2/STX11 complex and other still unknown factors such as the membrane surface or Munc13-4 protein and thus impairing the release of cytolytic granules. In addition to the contribution of STXBP2-R190C to f-HLH, the accompanied G6PD mutation may have compounded the clinical symptoms; however, the extent by which G6PD deficiency has contributed to HLH in our patient remains unclear

North Tropical Atlantic influence on western Amazon fire season variability

The prevailing wet climate in the western Amazon is not favorable to the natural occurrence of fires. Nevertheless, the current process of clearing of humid forests for agriculture and cattle ranching has increased the vulnerability of the region to the spread of fires. Using meteorological stations precipitation and the Moderate Resolution Spectroradiometer (MODIS) Active-Fires (AF) during 2000-2009, we show that fire anomalies vary closely with July-August-September (JAS) precipitation variability as measured by the Standardized Precipitation Index (SPI). The precipitation variability is, in turn, greatly determined by sea surface temperature (SST) anomalies in the North Tropical Atlantic (NTA). We develop a linear regression model to relate local fire activity to an index of the NTA-SST. By using seasonal forecasts of SST from a coupled model, we are able to predict anomalous JAS fire activity as early as April. We applied the method to predict the severe 2010 JAS season, which indicated strongly positive seasonal fire anomalies within the 95% prediction confidence intervals in most western Amazon. The spatial distribution of predicted SPI was also in accordance with observed precipitation anomalies. This three months lead time precipitation and fire prediction product in the western Amazon could help local decision makers to establish an early warning systems or other appropriate course of action before the fire season begins

URBAN TERRAIN CLIMATOLOGY AND REMOTE SENSING *

. Urban areas have been conceived of as monolithic heat islands because traditional ground observation techniques do not lend themselves to more specific analyses. Observations of urban energy-exchange obtained from calibrated electro-optical scanners combined with energy budget simulation techniques provide tools to relate the urban land use mosaic to the heat island phenomenon. Maps of surface energy-related phenomena were made from airborne scanner outputs for selected flightpaths across the city of Baltimore, Maryland. Conditions for the flight time were simulated according to the various types of land use using an energy budget simulation model which lends itself to extrapolation of simulated grid-point conditions into a map form. Maps made by simulation compare sufficiently well with those made by aerial observation to encourage further refinement of the simulation approach.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72392/1/j.1467-8306.1976.tb01110.x.pd

A preliminary quantitative mineral resource assessment of undiscovered porphyry copper resources in the Andes mountains of South America

Quantitative information on the probable locations and amounts of undiscovered porphyry copper resources of the world is important to international exploration managers, land-use and environmental planners, economists, and policy makers. The U.S. Geological Survey is organizing and facilitating a cooperative assessment, in collaboration with interested geological surveys and geological organizations, of the likely global distribution, quantity, and quality of selected undiscovered nonfuel mineral resources. This report on undiscovered porphyry copper deposits of the Andes Mountains was produced jointly with the geological surveys of Argentina, Chile, Colombia, and Peru, and is a summary of the more extensive report (>300 pages) that will be published soon. Reports on undiscovered copper resources of other regions of the world are being prepared and will be followed by assessments of the global undiscovered resources of platinum-group minerals and potash