Uncoupling of p97 ATPase activity has a dominant negative effect on protein extraction

p97 is a highly abundant, homohexameric AAA+ ATPase that performs a variety of essential cellular functions. Characterized as a ubiquitin-selective chaperone, p97 recognizes proteins conjugated to K48-linked polyubiquitin chains and promotes their removal from chromatin and other molecular complexes. Changes in p97 expression or activity are associated with the development of cancer and several related neurodegenerative disorders. Although pathogenic p97 mutations cluster in and around p97's ATPase domains, mutant proteins display normal or elevated ATPase activity. Here, we show that one of the most common p97 mutations (R155C) retains ATPase activity, but is functionally defective. p97-R155C can be recruited to ubiquitinated substrates on chromatin, but is unable to promote substrate removal. As a result, p97-R155C acts as a dominant negative, blocking protein extraction by a similar mechanism to that observed when p97's ATPase activity is inhibited or inactivated. However, unlike ATPase-deficient proteins, p97-R155C consumes excess ATP, which can hinder high-energy processes. Together, our results shed new insight into how pathogenic mutations in p97 alter its cellular function, with implications for understanding the etiology and treatment of p97-associated diseases

Lesinurad, a novel, oral compound for gout, acts to decrease serum uric acid through inhibition of urate transporters in the kidney.

BackgroundExcess body burden of uric acid promotes gout. Diminished renal clearance of uric acid causes hyperuricemia in most patients with gout, and the renal urate transporter (URAT)1 is important for regulation of serum uric acid (sUA) levels. The URAT1 inhibitors probenecid and benzbromarone are used as gout therapies; however, their use is limited by drug-drug interactions and off-target toxicity, respectively. Here, we define the mechanism of action of lesinurad (Zurampic®; RDEA594), a novel URAT1 inhibitor, recently approved in the USA and Europe for treatment of chronic gout.MethodssUA levels, fractional excretion of uric acid (FEUA), lesinurad plasma levels, and urinary excretion of lesinurad were measured in healthy volunteers treated with lesinurad. In addition, lesinurad, probenecid, and benzbromarone were compared in vitro for effects on urate transporters and the organic anion transporters (OAT)1 and OAT3, changes in mitochondrial membrane potential, and human peroxisome proliferator-activated receptor gamma (PPARγ) activity.ResultsAfter 6 hours, a single 200-mg dose of lesinurad elevated FEUA 3.6-fold (p < 0.001) and reduced sUA levels by 33 % (p < 0.001). At concentrations achieved in the clinic, lesinurad inhibited activity of URAT1 and OAT4 in vitro, did not inhibit GLUT9, and had no effect on ABCG2. Lesinurad also showed a low risk for mitochondrial toxicity and PPARγ induction compared to benzbromarone. Unlike probenecid, lesinurad did not inhibit OAT1 or OAT3 in the clinical setting.ConclusionThe pharmacodynamic effects and in vitro activity of lesinurad are consistent with inhibition of URAT1 and OAT4, major apical transporters for uric acid. Lesinurad also has a favorable selectivity and safety profile, consistent with an important role in sUA-lowering therapy for patients with gout

Well-differentiated papillary mesothelioma of the peritoneum is genetically defined by mutually exclusive mutations in TRAF7 and CDC42.

Well-differentiated papillary mesothelioma is an uncommon mesothelial neoplasm that most frequently arises in the peritoneal cavity of women of reproductive age. Whereas malignant mesothelioma is an aggressive tumor associated with poor outcome, well-differentiated papillary mesothelioma typically exhibits indolent behavior. However, histologically differentiating between these two entities can be challenging, necessitating the development of distinguishing biomarkers. While the genetic alterations that drive malignant mesothelioma have recently been determined, the molecular pathogenesis of well-differentiated papillary mesothelioma is unknown. Here we performed genomic profiling on a cohort of ten well-differentiated papillary mesothelioma of the peritoneum. We identified that all tumors harbored somatic missense mutations in either the TRAF7 or CDC42 genes, and lacked alterations involving BAP1, NF2, CDKN2A, DDX3X, SETD2, and ALK that are frequent in malignant mesothelioma. We recently identified that another mesothelial neoplasm, adenomatoid tumor of the genital tract, is genetically defined by somatic missense mutations in the TRAF7 gene, indicating a shared molecular pathogenesis between well-differentiated papillary mesothelioma and adenomatoid tumors. To the best of our knowledge, well-differentiated papillary mesothelioma is the first human tumor type found to harbor recurrent mutations in the CDC42 gene, which encodes a Rho family GTPase. Immunohistochemistry demonstrated intact BAP1 expression in all cases of well-differentiated papillary mesothelioma, indicating that this is a reliable marker for distinguishing well-differentiated papillary mesothelioma from malignant mesotheliomas that frequently display loss of expression. Additionally, all well-differentiated papillary mesothelioma demonstrated robust expression of L1 cell adhesion molecule (L1CAM), a marker of NF-kB pathway activation, similar to that observed in adenomatoid tumors. In contrast, we have previously shown that L1CAM staining is not observed in normal mesothelial cells and malignant mesotheliomas of the peritoneum. Together, these studies demonstrate that well-differentiated papillary mesothelioma is genetically defined by mutually exclusive mutations in TRAF7 and CDC42 that molecularly distinguish this entity from malignant mesothelioma

Genomic profiling of malignant peritoneal mesothelioma reveals recurrent alterations in epigenetic regulatory genes BAP1, SETD2, and DDX3X.

Malignant mesothelioma is a rare cancer that arises from the mesothelial cells that line the pleural cavity and less commonly from the peritoneal lining of the abdomen and pelvis. Most pleural mesotheliomas arise in patients with a history of asbestos exposure, whereas the association of peritoneal mesotheliomas with exposure to asbestos and other potential carcinogens is less clear, suggesting that the genetic alterations that drive malignant peritoneal mesothelioma may be unique from those in pleural mesothelioma. Treatment options for all malignant mesotheliomas are currently limited, with no known targeted therapies available. To better understand the molecular pathogenesis of malignant peritoneal mesothelioma, we sequenced 510 cancer-related genes in 13 patients with malignant mesothelioma arising in the peritoneal cavity. The most frequent genetic alteration was biallelic inactivation of the BAP1 gene, which occurred in 9/13 cases, with an additional two cases demonstrating monoallelic loss of BAP1. All 11 of these cases demonstrated loss of BAP1 nuclear staining by immunohistochemistry, whereas two tumors without BAP1 alteration and all 42 cases of histologic mimics in peritoneum (8 multilocular peritoneal inclusion cyst, 6 well-differentiated papillary mesothelioma of the peritoneum, 16 adenomatoid tumor, and 12 low-grade serous carcinoma of the ovary) demonstrated intact BAP1 nuclear staining. Additional recurrently mutated genes in this cohort of malignant peritoneal mesotheliomas included NF2 (3/13), SETD2 (2/13), and DDX3X (2/13). While these genes are known to be recurrently mutated in pleural mesotheliomas, the frequencies are distinct in peritoneal mesotheliomas, with nearly 85% of peritoneal tumors harboring BAP1 alterations versus only 20-30% of pleural tumors. Together, these findings demonstrate the importance of epigenetic modifiers including BAP1, SETD2, and DDX3X in mesothelial tumorigenesis and suggest opportunities for targeted therapies

Adenomatoid tumors of the male and female genital tract are defined by TRAF7 mutations that drive aberrant NF-kB pathway activation.

Adenomatoid tumors are the most common neoplasm of the epididymis, and histologically similar adenomatoid tumors also commonly arise in the uterus and fallopian tube. To investigate the molecular pathogenesis of these tumors, we performed genomic profiling on a cohort of 31 adenomatoid tumors of the male and female genital tracts. We identified that all tumors harbored somatic missense mutations in the TRAF7 gene, which encodes an E3 ubiquitin ligase belonging to the family of tumor necrosis factor receptor-associated factors (TRAFs). These mutations all clustered into one of five recurrent hotspots within the WD40 repeat domains at the C-terminus of the protein. Functional studies in vitro revealed that expression of mutant but not wild-type TRAF7 led to increased phosphorylation of nuclear factor-kappa B (NF-kB) and increased expression of L1 cell adhesion molecule (L1CAM), a marker of NF-kB pathway activation. Immunohistochemistry demonstrated robust L1CAM expression in adenomatoid tumors that was absent in normal mesothelial cells, malignant peritoneal mesotheliomas and multilocular peritoneal inclusion cysts. Together, these studies demonstrate that adenomatoid tumors of the male and female genital tract are genetically defined by TRAF7 mutation that drives aberrant NF-kB pathway activation

Nano-Scale Strain-Induced Giant Pseudo-Magnetic Fields and Charging Effects in CVD-Grown Graphene on Copper

Scanning tunneling microscopic and spectroscopic (STM/STS) studies of graphene grown by chemical vapor deposition (CVD) on copper reveal that the monolayer carbon structures remaining on copper are strongly strained and rippled, with different regions exhibiting different lattice structures and local electronic density of states (LDOS). The large and non-uniform strain induces pseudo-magnetic field up to ∼ 50 Tesla, as manifested by the integer and fractional pseudo-magnetic field quantum Hall effects (IQHE and FQHE) in the LDOS of graphene. Additionally, ridges appear along the boundaries of different lattice structures, which exhibit excess charging effects. For graphene transferred from copper to SiO_2 substrates after the CVD growth, the average strain and the corresponding charging effects and pseudo-magnetic fields become much reduced. These findings suggest the feasibility of strain-engineering of graphene-based nano-electronics

Confirmation of a recent bipolar ejection in the very young hierarchical multiple system IRAS 16293-2422

We present and analyze two new high-resolution (approx 0.3 arcsec), high-sensitivity (approx 50 uJy beam-1) Very Large Array 3.6 cm observations of IRAS 16293-2422 obtained in 2007 August and 2008 December. The components A2alpha and A2beta recently detected in this system are still present, and have moved roughly symmetrically away from source A2 at a projected velocity of 30-80 km s-1. This confirms that A2alpha and A2beta were formed as a consequence of a very recent bipolar ejection from A2. Powerful bipolar ejections have long been known to occur in low-mass young stars, but this is -to our knowledge-- the first time that such a dramatic one is observed from its very beginning. Under the reasonable assumption that the flux detected at radio wavelengths is optically thin free-free emission, one can estimate the mass of each ejecta to be of the order of 10^-8 Msun. If the ejecta were created as a consequence of an episode of enhanced mass loss accompanied by an increase in accretion onto the protostar, then the total luminosity of IRAS 16293-2422 ought to have increased by 10-60% over the course of at least several months. Between A2alpha and A2beta, component A2 has reappeared, and the relative position angle between A2 and A1 is found to have increased significantly since 2003-2005. This strongly suggests that A1 is a protostar rather than a shock feature, and that the A1/A2 pair is a tight binary system. Including component B, IRAS 16293-2422 therefore appears to be a very young hierarchical multiple system.Comment: Accepted for publication in The Astrophysical Journa

Navier-Stokes Analysis of a High Wing Transport High-Lift Configuration with Externally Blown Flaps

Insights and lessons learned from the aerodynamic analysis of the High Wing Transport (HWT) high-lift configuration are presented. Three-dimensional Navier-Stokes CFD simulations using the OVERFLOW flow solver are compared with high Reynolds test data obtained in the NASA Ames 12 Foot Pressure Wind Tunnel (PWT) facility. Computational analysis of the baseline HWT high-lift configuration with and without Externally Blown Flap (EBF) jet effects is highlighted. Several additional aerodynamic investigations, such as nacelle strake effectiveness and wake vortex studies, are presented. Technical capabilities and shortcomings of the computational method are discussed and summarized

An endoscopie imaging system based on a two-dimensional CMUT array: real-time imaging results

Real-time catheter-based ultrasound imaging tools are needed for diagnosis and image-guided procedures. The continued development of these tools is partially limited by the difficulty of fabricating two-dimensional array geometries of piezoelectric transducers. Using capacitive micromachined ultrasonic transducer (CMUT) technology, transducer arrays with widely varying geometries, high frequencies, and wide bandwidths can be fabricated. A volumetric ultrasound imaging system based on a two-dimensional, 16×l6-element, CMUT array is presented. Transducer arrays with operating frequencies ranging from 3 MHz to 7.5 MHz were fabricated for this system. The transducer array including DC bias pads measures 4 mm by 4.7 mm. The transducer elements are connected to flip-chip bond pads on the array back side with 400-μm long through-wafer interconnects. The array is flip-chip bonded to a custom-designed integrated circuit (IC) that comprises the front-end electronics. Integrating the front-end electronics with the transducer array reduces the effects of cable capacitance on the transducer's performance and provides a compact means of connecting to the transducer elements. The front-end IC provides a 27-V pulser and 10-MHz bandwidth amplifier for each element of the array. An FPGA-based data acquisition system is used for control and data acquisition. Output pressure of 230 kPa was measured for the integrated device. A receive sensitivity of 125 mV/kPa was measured at the output of the amplifier. Amplifier output noise at 5 Mhz is 112 nV/√Hz. Volumetric images of a wire phantom and vessel phantom are presented. Volumetric data for a wire phantom was acquired in real-time at 30 frames per second.Publisher's Versio