A method for detergent-free isolation of membrane proteins in their local lipid environment.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins

Recurrent hotspot mutations in HRAS Q61 and PI3K-AKT pathway genes as drivers of breast adenomyoepitheliomas

Adenomyoepithelioma of the breast is a rare tumor characterized by epithelial-myoepithelial differentiation, of which a subset will progress to invasive or metastatic cancer. We sought to define the genomic landscape of adenomyoepitheliomas. Massively parallel sequencing revealed highly recurrent somatic mutations in HRAS and PI3K-AKT pathway-related genes. Strikingly, HRAS mutations were restricted to estrogen receptor (ER)-negative tumors, all affected codon 61, and all but one co-occurred with PIK3CA or PIK3R1 mutations. To interrogate the functional significance of HRAS Q61 mutations in adenomyoepithelial differentiation, we expressed HRASQ61R alone or in combination with PIK3CAH1047R in non-transformed ER-negative breast epithelial cells. HRASQ61R induced characteristic phenotypes of adenomyoepitheliomas such as the expression of myoepithelial markers and loss of e-cadherin, hyperactivation of AKT signaling, and transformative properties that were arrested by combination therapy with AKT and MEK inhibitors. Our results indicate that breast adenomyoepitheliomas often manifest a unique transformation program featuring HRAS activation

Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis

Most tumors exhibit increased glucose metabolism to lactate, however, the extent to which glucose-derived metabolic fluxes are used for alternative processes is poorly understood [1, 2]. Using a metabolomics approach with isotope labeling, we found that in some cancer cells a relatively large amount of glycolytic carbon is diverted into serine and glycine metabolism through phosphoglycerate dehydrogenase (PHGDH). An analysis of human cancers showed that PHGDH is recurrently amplified in a genomic region of focal copy number gain most commonly found in melanoma. Decreasing PHGDH expression impaired proliferation in amplified cell lines. Increased expression was also associated with breast cancer subtypes, and ectopic expression of PHGDH in mammary epithelial cells disrupted acinar morphogenesis and induced other phenotypic alterations that may predispose cells to transformation. Our findings show that the diversion of glycolytic flux into a specific alternate pathway can be selected during tumor development and may contribute to the pathogenesis of human cancer.National Institutes of Health (U.S.)National Cancer Institute (U.S.)Smith Family FoundationDamon Runyon Cancer Research FoundationBurroughs Wellcome Fun

Morphology and resistivity of Al thin films grown on Si(111) by molecular beam epitaxy

Thin films of aluminium metal with varying thickness between 10 and 200nm were grown on (I 1 1) Si substrates at 250 degrees C under UHV conditions using molecular beam epitaxy (MBE). Grown thin films were characterized by in situ Xray photoelectron spectroscopy, and ex situ X-ray diffraction, atomic force microscopy and temperature-dependent electrical resisitivity measurements. The results showed that (i) films grow via 3D-island Volmer-Weber growth mechanism, (ii) with increasing film thickness the average grain size increases and the coalescence takes place for thickness > 60 nm, and (iii) independent of the thickness, films grow with (111) orientation. The room-temperature value of resistivity contrary to the predictions of existing theoretical models is found to increase monotonically up to a thickness of 40 nm. This anomalous feature was understood in terms of the film morphology, whereby charge transport takes place via variable range hopping (VRH). For film thickness = 60 mn the resistivity decreased sharply and the M-I transition disappeared. The bulk value of resistivity (2.59 mu Omega cm) was obtained for thickness > 200 urn. (c) 200

Bias and temperature dependent charge transport in high mobility cobalt-phthalocyanine thin films

The temperature dependent current-voltage (J-V) characteristics of highly-oriented cobalt phthalocyanine films (rocking-curve width=0.11 degrees) deposited on (001) LaAlO(3) substrates are investigated. In the temperature range 300-100 K, charge transport is governed by bulk-limited processes with a bias dependent crossover from Ohmic (J similar to V) to trap-free space-charge-limited conduction (J similar to V(2)). The mobility (mu) at 300 K has a value of similar to 7 cm(2) V(-1) s(-1) and obeys Arrhenius-type (ln mu similar to 1/T) behavior. However, at temperatures < 100 K, the charge transport is electrode-limited, which undergoes a bias dependent transition from Schottky (ln J similar to V(1/2)) to multistep-tunneling (conductivity varying exponentially on the inverse of the square-root of electric field)

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Not AvailableCatla (Catla catla) is a one of the most harvested Indian major carps and is widely cultured fish species in Indian subcontinent. In the present study, genetic variability between hatchery and wild stocks of Catla was surveyed using sequence data of mitochondrial DNA of partial 307 bp of cytochrome b region. A total of 174 Catla individuals were examined from three different river basins and hatcheries. Significant genetic heterogeneity was observed for the sequence data (FST = 0.308, p ≤ 0.001). However, analysis of molecular variance (AMOVA) resulted in insignificant genetic differentiation among the samples of three rivers and culture zones (FCT = −0.10, p = 0.44). The result suggested a significant genetic variation within different riverine system, low genetic differentiation among samples from river basins and a lack of genetic variation in hatchery populations.Not Availabl

Not Available

Not AvailableCatla (Catla catla) is a one of the most harvested Indian major carps and is widely cultured fish species in Indian subcontinent. In the present study, genetic variability between hatchery and wild stocks of Catla was surveyed using sequence data of mitochondrial DNA of partial 307 bp of cytochrome b region. A total of 174 Catla individuals were examined from three different river basins and hatcheries. Significant genetic heterogeneity was observed for the sequence data (FST = 0.308, p ≤ 0.001). However, analysis of molecular variance (AMOVA) resulted in insignificant genetic differentiation among the samples of three rivers and culture zones (FCT = -0.10, p = 0.44). The result suggested a significant genetic variation within different riverine system, low genetic differentiation among samples from river basins and a lack of genetic variation in hatchery populations.Not Availabl

High thermoelectric performance of (AgCrSe2)(0.5)(CuCrSe2)(0.5) nano-composites having all-scale natural hierarchical architectures

Recent studies have shown that thermoelectric materials exhibit a high figure-of-merit if it consists of hierarchically organized microstructures that significantly lower the lattice thermal conductivity without any appreciable change in the power factor. Here, we report a new class of thermoelectric (AgCrSe2)(0.5)(CuCrSe2)(0.5) nano-composites synthesized via the vacuum hot pressing of a mixture of the constituents, which naturally consists of phonon scattering centers in a multiscale hierarchical fashion, i.e. atomic scale disorder, nanoscale amorphous structure, natural grain boundaries due to layered structure and mesoscale grain boundaries/interfaces. The presence of a natural hierarchical architecture of different length scales in the composite samples is confirmed by scanning electron and transmission electron microscopy. Detailed characterization reveals that in the composite samples there is a slight migration of Cu into the Ag site. Composite samples exhibit extremely low thermal conductivity similar to 2 mW cm(-1) K-1 at 773 K, which is nearly one third of the pure AgCrSe2 and CuCrSe2. The composite samples exhibit a high ZT similar to 1.4 at 773 K, which is attributed to the scattering of heat carrying phonons of all wavelengths via the natural hierarchical architecture of the material. The ease of synthesis of such high performance (AgCrSe2)(0.5)(CuCrSe2)(0.5) nanocomposites with a natural hierarchical architecture offers a promise for replacing conventional tellurides