This paper describes the current update on macromolecular model validation services that are provided at the MolProbity website, emphasizing changes and additions since the previous review in 2010. ...There have been many infrastructure improvements, including rewrite of previous Java utilities to now use existing or newly written Python utilities in the open‐source CCTBX portion of the Phenix software system. This improves long‐term maintainability and enhances the thorough integration of MolProbity‐style validation within Phenix. There is now a complete MolProbity mirror site at http://molprobity.manchester.ac.uk. GitHub serves our open‐source code, reference datasets, and the resulting multi‐dimensional distributions that define most validation criteria. Coordinate output after Asn/Gln/His “flip” correction is now more idealized, since the post‐refinement step has apparently often been skipped in the past. Two distinct sets of heavy‐atom‐to‐hydrogen distances and accompanying van der Waals radii have been researched and improved in accuracy, one for the electron‐cloud‐center positions suitable for X‐ray crystallography and one for nuclear positions. New validations include messages at input about problem‐causing format irregularities, updates of Ramachandran and rotamer criteria from the million quality‐filtered residues in a new reference dataset, the CaBLAM Cα‐CO virtual‐angle analysis of backbone and secondary structure for cryoEM or low‐resolution X‐ray, and flagging of the very rare cis‐nonProline and twisted peptides which have recently been greatly overused. Due to wide application of MolProbity validation and corrections by the research community, in Phenix, and at the worldwide Protein Data Bank, newly deposited structures have continued to improve greatly as measured by MolProbity's unique all‐atom clashscore.
MolProbity is a structure‐validation web service that provides broad‐spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It ...relies heavily on the power and sensitivity provided by optimized hydrogen placement and all‐atom contact analysis, complemented by updated versions of covalent‐geometry and torsion‐angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X‐ray crystallography provides a wealth of biologically important molecular data in the form of atomic three‐dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystallization to data collection to phasing to model building to refinement, have made solving a structure using crystallography easier than ever. However, despite these improvements, local errors that can affect biological interpretation are widespread at low resolution and even high‐resolution structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is critical both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.
Systematic studies of cancer genomes have provided unprecedented insights into the molecular nature of cancer. Using this information to guide the development and application of therapies in the ...clinic is challenging. Here, we report how cancer-driven alterations identified in 11,289 tumors from 29 tissues (integrating somatic mutations, copy number alterations, DNA methylation, and gene expression) can be mapped onto 1,001 molecularly annotated human cancer cell lines and correlated with sensitivity to 265 drugs. We find that cell lines faithfully recapitulate oncogenic alterations identified in tumors, find that many of these associate with drug sensitivity/resistance, and highlight the importance of tissue lineage in mediating drug response. Logic-based modeling uncovers combinations of alterations that sensitize to drugs, while machine learning demonstrates the relative importance of different data types in predicting drug response. Our analysis and datasets are rich resources to link genotypes with cellular phenotypes and to identify therapeutic options for selected cancer sub-populations.
Display omitted
•We integrate heterogeneous molecular data of 11,289 tumors and 1,001 cell lines•We measure the response of 1,001 cancer cell lines to 265 anti-cancer drugs•We uncover numerous oncogenic aberrations that sensitize to an anti-cancer drug•Our study forms a resource to identify therapeutic options for cancer sub-populations
A look at the pharmacogenomic landscape of 1,001 human cancer cell lines points to new treatment applications for hundreds of known anti-cancer drugs.
OBJECTIVE:We sought to characterize the effect of postoperative complications on long-term survival after colorectal cancer (CRC) resection.
BACKGROUND:The impact of early morbidity on long-term ...survival after curative-intent CRC surgery remains controversial.
METHODS:The Veterans Affairs Surgical Quality Improvement Program and Central Cancer Registry databases were linked to acquire perioperative and cancer-specific data for 12,075 patients undergoing resection for nonmetastatic CRC (1999–2009). Patients were categorized by presence of any complication within 30 days and by type of complication (noninfectious vs infectious). Univariate and multivariate survival analyses adjusted for patient, disease, and treatment factors were performed, excluding early deaths (<90 days). Subset analysis was performed to determine the specific impact of severe postoperative infections.
RESULTS:The overall morbidity and infectious complication rates were 27.8% and 22.5%, respectively. Patients with noninfectious postoperative complications were older, had lower preoperative serum albumin, had worse functional status, and had higher American Society of Anesthesiologists scores than patients with infectious complications and without complications (all P < 0.001). The presence of any complication was independently associated with decreased long-term survival hazard ratio, 1.24; 95% confidence interval (1.15–1.34). Multivariate analysis by complication type demonstrated increased risk only with infectious complications hazard ratio, 1.31; 95% confidence interval (1.21–1.42). Subset analysis demonstrated this effect predominantly in patients with severe infections hazard ratio, 1.41; 95% confidence interval (1.15–1.73).
CONCLUSIONS:The presence of postoperative complications after CRC resection is associated with decreased long-term survival, independent of patient, disease, and treatment factors. The impact on long-term outcome is primarily driven by infectious complications, particularly severe postoperative infections.
What conformations do protein molecules populate in solution? Crystallography provides a high-resolution description of protein structure in the crystal environment, while NMR describes structure in ...solution but using less data. NMR structures display more variability, but is this because crystal contacts are absent or because of fewer data constraints? Here we report unexpected insight into this issue obtained through analysis of detailed protein energy landscapes generated by large-scale, native-enhanced sampling of conformational space with Rosetta@home for 111 protein domains. In the absence of tightly associating binding partners or ligands, the lowest-energy Rosetta models were nearly all <2.5 Å CαRMSD from the experimental structure; this result demonstrates that structure prediction accuracy for globular proteins is limited mainly by the ability to sample close to the native structure. While the lowest-energy models are similar to deposited structures, they are not identical; the largest deviations are most often in regions involved in ligand, quaternary, or crystal contacts. For ligand binding proteins, the low energy models may resemble the apo structures, and for oligomeric proteins, the monomeric assembly intermediates. The deviations between the low energy models and crystal structures largely disappear when landscapes are computed in the context of the crystal lattice or multimer. The computed low-energy ensembles, with tight crystal-structure-like packing in the core, but more NMR-structure-like variability in loops, may in some cases resemble the native state ensembles of proteins better than individual crystal or NMR structures, and can suggest experimentally testable hypotheses relating alternative states and structural heterogeneity to function.
Display omitted
► Near-native Rosetta sampling shows native states as energy minima. ► Some computed energy minima differ from the target experimental structure. ► Many computed alternate states are demonstrated to be biologically relevant. ► Simulations in artificial crystals enhance native recapitulation. ► Structure prediction is limited more by sampling than by the energy function.
Pancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby ...preventing Ca
-dependent cell death. The ATP-generating pyruvate kinase-M2 (PKM2) is oncogenic and overexpressed in PDAC. This study investigated the PKM2-derived ATP supply to the PMCA as a potential therapeutic locus.
PDAC cell growth, migration and death were assessed by using sulforhodamine-B/tetrazolium-based assays, gap closure assay and poly-ADP ribose polymerase (PARP1) cleavage, respectively. Cellular ATP and metabolism were assessed using luciferase/fluorescent-based assays and the Seahorse XFe96 analyzer, respectively. Cell surface biotinylation identified membrane-associated proteins. Fura-2 imaging was used to assess cytosolic Ca
overload and in situ Ca
clearance. PKM2 knockdown was achieved using siRNA.
The PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca
overload. PKM2 associates with plasma membrane proteins providing a privileged ATP supply to the PMCA. PKM2 knockdown reduced PMCA activity and reduced the sensitivity of shikonin-induced cell death.
Cutting off the PKM2-derived ATP supply to the PMCA represents a novel therapeutic strategy for the treatment of PDAC.
We have developed a suite of protein redesign algorithms that improves realistic in silico modeling of proteins. These algorithms are based on three characteristics that make them unique: (1) ...improved flexibility of the protein backbone, protein side-chains, and ligand to accurately capture the conformational changes that are induced by mutations to the protein sequence; (2) modeling of proteins and ligands as ensembles of low-energy structures to better approximate binding affinity; and (3) a globally optimal protein design search, guaranteeing that the computational predictions are optimal with respect to the input model. Here, we illustrate the importance of these three characteristics. We then describe OSPREY, a protein redesign suite that implements our protein design algorithms. OSPREY has been used prospectively, with experimental validation, in several biomedically relevant settings. We show in detail how OSPREY has been used to predict resistance mutations and explain why improved flexibility, ensembles, and provability are essential for this application.
OSPREY is free and open source under a Lesser GPL license. The latest version is OSPREY 2.0. The program, user manual, and source code are available at www.cs.duke.edu/donaldlab/software.php.
osprey@cs.duke.edu.
The presence of CD8
T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the ...mechanism of CD8
T cell invasion into BEC. CD8
T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8
T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8
T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin
CD8
T cells formed β-catenin-associated interactions with BEC, were larger than E-cadherin
CD8
T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin
CD103
CD69
CD8
T cells into BEC.
This paper describes the design and performance of a photo-acoustic aerosol absorption spectrometer (PAS) built for operation on a research aircraft platform. The PAS instrument is capable of ...measuring dry absorption at 659 nm, 532 nm, and 404 nm, and absorption enhancement due to coatings at 532 nm and 404 nm. The measurement accuracy for all channels is < = 10% and in flight 1 Hz sensitivities lie within the range of 0.5-1.5 Mm
−1
. PAS measurements of calibrated absorbing aerosol samples are shown to be consistent with measurements made by a previous generation single channel photo-acoustic instrument. Aircraft data collected during a recent field campaign in California are used to demonstrate the capabilities of the PAS. In combination with an aircraft cavity ring down aerosol extinction spectrometer described in a companion paper, the new PAS instrument provides a sensitive airborne in-situ characterization of aerosol optics.
Copyright 2012 American Association for Aerosol Research
Linear infrastructure such as pipelines and power lines is ubiquitous and responsible for loss of habitats and disruption of landscape connectivity. We reviewed published research to answer the ...following questions: (1) Which organisms are commonly used to indicate impacts of pipelines and power lines to biodiversity? (2) How do pipelines and power lines impact biodiversity? and (3) How are these impacts mitigated? Studies of pipelines most often used mammals and plants as bioindicators, whereas studies of power lines focused largely on birds and plants. A myriad of impacts were identified, including the mortality of plants during construction, changes to the structure and composition of plant and animal communities that resulted from construction, the creation of open and shrubby corridors within intact forests, and collisions and electrocutions of birds with power lines. However, in most studies baseline data were not collected, so magnitudes of the impacts are often unknown. Mitigation in many studies was mentioned only in the discussion as a way to reduce impacts, but mitigation techniques were rarely tested directly. We outline considerations when selecting bioindicators—research that takes a community- or ecosystem-level approach will more fully determine the scope of impacts of linear infrastructure than the historical approach of focusing on populations of select bioindicators. Mitigation strategies must ultimately result from appropriate baseline studies, scientific data collection and analyses, and be implemented within an adaptive management strategy.
PDF
