Numerical and Experimental Analysis of Strength Loss of 1.2709 Maraging Steel Produced by Selective Laser Melting (SLM) under Thermo-Mechanical Fatigue Conditions.

The result of the development of additive manufacturing (AM) methods is the increasing use of the selective laser melting (SLM) method as a technique for producing tooling for injection moulds and die casting pressure moulds from maraging steel powders. The mould components are subjected to varying thermo-mechanical loads during these operations. This paper presents a numerical model that is used to predict the fatigue life of a material that is loaded with a time-varying temperature field according to the classic and modified Coffin test.

Abrasive Wear Resistance of Ultrafine Ausferritic Ductile Iron Intended for the Manufacture of Gears for Mining Machinery.

The purpose of this study was to experimentally determine the abrasion wear properties of ausferritic ductile iron austempered at 250 °C in order to obtain cast iron of class EN-GJS-1400-1. It has been found that such a cast iron grade makes it possible to create structures for material conveyors used for short-distance transport purposes, required to perform in terms of abrasion resistance under extreme conditions. The wear tests addressed in the paper were conducted at a ring-on-ring type of test rig.

Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.

A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar.

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes.

A bacteriophage capsid protein provides a general amyloid interaction motif (GAIM) that binds and remodels misfolded protein assemblies.

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold.

Biophysical fragment screening of the β1-adrenergic receptor: identification of high affinity arylpiperazine leads using structure-based drug design.

Biophysical fragment screening of a thermostabilized β1-adrenergic receptor (β1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein-ligand crystal structures of the β1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized β1AR complexed with 19 and 20 were determined at resolutions of 2.

CCR5 is a receptor for Staphylococcus aureus leukotoxin ED.

Pore-forming toxins are critical virulence factors for many bacterial pathogens and are central to Staphylococcus aureus-mediated killing of host cells. S. aureus encodes pore-forming bi-component leukotoxins that are toxic towards neutrophils, but also specifically target other immune cells.

Interactions of the human LIP5 regulatory protein with endosomal sorting complexes required for transport.

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions.

Structure of a proteasome Pba1-Pba2 complex: implications for proteasome assembly, activation, and biological function.

The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly.

Femtomolar Fab binding affinities to a protein target by alternative CDR residue co-optimization strategies without phage or cell surface display.

In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position.

Survey of the 2009 commercial optical biosensor literature.

We took a different approach to reviewing the commercial biosensor literature this year by inviting 22 biosensor users to serve as a review committee. They set the criteria for what to expect in a publication and ultimately decided to use a pass/fail system for selecting which papers to include in this year's reference list. Of the 1514 publications in 2009 that reported using commercially available optical biosensor technology, only 20% passed their cutoff.

Fragment screening of stabilized G-protein-coupled receptors using biophysical methods.

Biophysical studies with G-protein-coupled receptors (GPCRs) are typically very challenging due to the poor stability of these receptors when solubilized from the cell membrane into detergent solutions. However, the stability of a GPCR can be greatly improved by introducing a number of point mutations into the protein sequence to give a stabilized receptor or StaR®. Here, we present the utility of StaRs for biophysical studies and the screening of fragment libraries.

Biacore analysis with stabilized G-protein-coupled receptors.

Using stabilized forms of β₁ adrenergic and A₂(A) adenosine G-protein-coupled receptors, we applied Biacore to monitor receptor activity and characterize binding constants of small-molecule antagonists spanning more than 20,000-fold in affinity. We also illustrate an improved method for tethering His-tagged receptors on NTA (carboxymethylated dextran preimmobilized with nitrilotriacetic acid) chips to yield stable, high-capacity, high-activity surfaces as well as a novel approach to regenerate receptor binding sites. Based on our success with this approach, we expect that the combination of stabilized receptors with biosensor technology will become a common method for characterizing members of this receptor family.

Two independent histidines, one in human prolactin and one in its receptor, are critical for pH-dependent receptor recognition and activation.

Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition.

Biosensor-based fragment screening using FastStep injections.

We have developed a novel analyte injection method for the SensíQ Pioneer surface plasmon resonance-based biosensor referred to as "FastStep." By merging buffer and sample streams immediately prior to the reaction flow cells, the instrument is capable of automatically generating a two- or threefold dilution series (of seven or five concentrations, respectively) from a single analyte sample. Using sucrose injections, we demonstrate that the production of each concentration within the step gradient is highly reproducible.

Direct DNA methylation profiling using methyl binding domain proteins.

Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG) and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay.

Exploring minimal biotinylation conditions for biosensor analysis using capture chips.

Using Biacore's new regenerateable streptavidin capture (CAP) sensor chips, we investigated a number of biotinylation conditions for target ligands. We explored standard amine as well as the less commonly used carboxyl biotinylation methods. We illustrate the time scales required for efficient biotinylation as well as the hazards of overbiotinylation.

Kinetic analysis and fragment screening with Fujifilm AP-3000.

We evaluated the performance of Fujifilm's new AP-3000 surface plasmon resonance biosensor for kinetic analysis and fragment screening. Using carbonic anhydrase II as a model system, we characterized a set of 10 sulfonamide-based inhibitors that range in molecular mass from 98 to 341Da and approximately 10,000-fold in affinity (0.4mM to 20nM).

Grading the commercial optical biosensor literature-Class of 2008: 'The Mighty Binders'.

Optical biosensor technology continues to be the method of choice for label-free, real-time interaction analysis. But when it comes to improving the quality of the biosensor literature, education should be fundamental. Of the 1413 articles published in 2008, less than 30% would pass the requirements for high-school chemistry.

Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors.

Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa.

Structural models for interactions between the 20S proteasome and its PAN/19S activators.

Proteasome activity is regulated by sequestration of its proteolytic centers in a barrel-shaped structure that limits substrate access. Substrates enter the proteasome by means of activator complexes that bind to the end rings of proteasome alpha subunits and induce opening of an axial entrance/exit pore. The PA26 activator binds in a pocket on the proteasome surface using main chain contacts of its C-terminal residues and uses an internal activation loop to trigger gate opening by repositioning the proteasome Pro-17 reverse turn.

A medical-legal partnership as a component of a palliative care model.

Introduction: A medical-legal partnership (MLP) incorporated as part of a comprehensive palliative care model addresses unmet social and material needs for patients. This study retrospectively reviews the experience of one MLP and quantifies the benefits of the program for both patients and the host health care institution.

Methods: The Legal Services Program, an MLP, reviewed their program referral and outcomes from April 1, 2004 to December 31, 2007 to document legal needs resolved.

Structural basis for ESCRT-III protein autoinhibition.

Endosomal sorting complexes required for transport-III (ESCRT-III) subunits cycle between two states: soluble monomers and higher-order assemblies that bind and remodel membranes during endosomal vesicle formation, midbody abscission and enveloped virus budding. Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member. IST1 and its ESCRT-III binding partner, CHMP1B, both form higher-order helical structures in vitro, and IST1-CHMP1 interactions are required for abscission.

E2 interaction and dimerization in the crystal structure of TRAF6.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-kappaB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure.

Inhibition and binding kinetics of the hepatitis C virus NS3 protease inhibitor ITMN-191 reveals tight binding and slow dissociative behavior.

The protease activity of hepatitis C virus nonstructural protein 3 (NS3) is essential for viral replication. ITMN-191, a macrocyclic inhibitor of the NS3 protease active site, promotes rapid, multilog viral load reductions in chronic HCV patients. Here, ITMN-191 is shown to be a potent inhibitor of NS3 with a two-step binding mechanism.