Rationalizing mAb Candidate Screening Using a Single Holistic Developability Parameter.

A framework for the rational selection of a minimal suite of nondegenerate developability assays (DAs) that maximize insight into candidate developability or storage stability is lacking. To address this, we subjected nine formulation:mAbs to 12 mechanistically distinct DAs together with measurement of their accelerated and long-term storage stability. We show that it is possible to identify a reduced set of key variables from this suite of DAs by using orthogonal statistical methods.

Differential Protease Specificity by Collagenase as a Novel Approach to Serum Proteomics That Includes Identification of Extracellular Matrix Proteins without Enrichment.

Circulating extracellular matrix (ECM) proteins are serological biomarkers of interest due to their association with pathologies involving disease processes such as fibrosis and cancers. In this study, we investigate the potential for serum biomarker research using differential protease specificity (DPS), leveraging alternate protease specificity as a targeting mechanism to selectively digest circulating ECM protein serum proteins. A proof-of-concept study is presented using serum from patients with cirrhotic liver or hepatocellular carcinoma.

Stochastic assembly of biomacromolecular complexes: impact and implications on charge interpretation in native mass spectrometry.

Native mass spectrometry is a potent method for characterizing biomacromolecular assemblies. A critical aspect to extracting accurate mass information is the correct inference of the ion ensemble charge states. While a variety of experimental strategies and algorithms have been developed to facilitate this, virtually all approaches rely on the implicit assumption that any peaks in a native mass spectrum can be directly attributed to an underlying charge state distribution.

Methodology-related pseudohyperkalemia associated with marked muscle enzyme leakage in a dog.

Background: As hyperkalemia may be life-threatening, it is critical to recognize artifactually increased potassium concentrations. Pseudohyperkalemia may occur in myopathies when using the VetScan2 analyzer (VS2), but the degree of pseudohyperkalemia and relationships relative to creatine kinase activity (CK) are unknown.

Objectives: We aimed to determine what degree of muscle enzyme leakage, as reflected by increased serum CK activity, results in cases with falsely elevated potassium concentrations when measured by the VS2.

Perspectives on pediatric congenital aortic valve stenosis: Extracellular matrix proteins, post translational modifications, and proteomic strategies.

In heart valve biology, organization of the extracellular matrix structure is directly correlated to valve function. This is especially true in cases of pediatric congenital aortic valve stenosis (pCAVS), in which extracellular matrix (ECM) dysregulation is a hallmark of the disease, eventually leading to left ventricular hypertrophy and heart failure. Therapeutic strategies are limited, especially in pediatric cases in which mechanical and tissue engineered valve replacements may not be a suitable option.

Defining the Tumor Microenvironment by Integration of Immunohistochemistry and Extracellular Matrix Targeted Imaging Mass Spectrometry.

Breast stroma plays a significant role in breast cancer risk and progression yet remains poorly understood. In breast stroma, collagen is the most abundantly expressed protein and its increased deposition and alignment contributes to progression and poor prognosis. Collagen post-translation modifications such as hydroxylated-proline (HYP) control deposition and stromal organization.

Methemoglobin concentrations in three salmonid species following exposure to benzocaine or tricaine methanesulfonate.

Methemoglobin is hemoglobin containing ferric iron rather than ferrous iron which renders it incapable of binding to oxygen. Blood sampling of fish is done under sedation or general anesthesia. Tricaine methanesulfonate (TMS) or benzocaine is commonly used but both can cause oxidation of hemoglobin to methemoglobin.

An in vivo platform to select and evolve aggregation-resistant proteins.

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E.

A Rapid Array-Based Approach to -Glycan Profiling of Cultured Cells.

Typically, N-glycosylation studies done on cultured cells require up to millions of cells followed by lengthy preparation to release, isolate, and profile -glycans. To overcome these limitations, we report a rapid array-based workflow for profiling -glycan signatures from cells, adapted from imaging mass spectrometry used for on-tissue -glycan profiling. Using this approach, -glycan profiles from a low-density array of eight cell chambers could be reported within 4 h of completing cell culture.

Stakeholder Requirements for an Ethical Framework to Sustain Multiple Research Projects in an Emerging Living Lab Involving Older Adults.

Living Lab (LL) research should follow clear ethical guidelines and principles. While these exist in specific disciplinary contexts, there is a lack of tailored and specific ethical guidelines for the design, development, and implementation of LL projects. As well as the complexity of these dynamic and multi-faceted contexts, the engagement of older adults, and adults with reducing cognitive and physical capacity in LL research, poses additional ethical challenges.

An exploratory study of barriers to inclusion in the European workplace.

Background: The European Disability Strategy (2010-2020) seeks to significantly raise the proportion of people with disabilities working in the open labour market. The ERGO WORK project is a collaboration of academic and industrial partners in six European countries, focused on understanding and tackling barriers to workplace inclusion for workers with disabilities.

Methods: This study sought to explore the perceptions and needs of stakeholders in terms of workplace adaptation to the needs of employees with disabilities.

An in vivo platform for identifying inhibitors of protein aggregation.

Protein aggregation underlies an array of human diseases, yet only one small-molecule therapeutic targeting this process has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, that is capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains.

Insights into the consequences of co-polymerisation in the early stages of IAPP and Aβ peptide assembly from mass spectrometry.

The precise molecular mechanisms by which different peptides and proteins assemble into highly ordered amyloid deposits remain elusive. The fibrillation of human amylin (also known as islet amyloid polypeptide, hIAPP) and the amyloid-beta peptide (Aβ-40) are thought to be pathogenic factors in Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), respectively. Amyloid diseases may involve co-aggregation of different protein species, in addition to the self-assembly of single precursor sequences.

ESI-IMS-MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules.

Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is a powerful method for the study of conformational changes in protein complexes, including oligomeric species populated during protein self-aggregation into amyloid fibrils. Information on the mass, stability, cross-sectional area and ligand binding capability of each transiently populated intermediate, present in the heterogeneous mixture of assembling species, can be determined individually in a single experiment in real-time. Determining the structural characterisation of oligomeric species and alterations in self-assembly pathways observed in the presence of small molecule inhibitors is of great importance, given the urgent demand for effective therapeutics.

Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry.

The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition.

Characterization of functionally distinct mitochondrial subpopulations.

Mitochondrial stress results in changes in mitochondrial function, morphology and homeostasis (biogenesis, fission/fusion, mitophagy) and may lead to changes in mitochondrial subpopulations. While flow cytometric techniques have been developed to quantify features of individual mitochondria related to volume, Ca(2+) concentration, mtDNA content, respiratory capacity and oxidative damage, less information is available concerning the identification and characterization of mitochondrial subpopulations, particularly in epithelial cells. Mitochondria from rabbit kidneys were stained with molecular probes for cardiolipin content (nonyl acridine orange, NAO) and membrane potential (tetramethylrhodamine, TMRM) and analyzed using flow cytometry.

Comparison of NADH-dependent cytochrome b5 reductase activity and in vitro methemoglobin induction by sodium nitrite in Oncorhynchus mykiss, Salmo salar, and Salvelinus fontinalis.

Methemoglobin is hemoglobin containing ferric iron. Methemoglobin cannot bind to oxygen and at high concentrations causes tissue hypoxia. Brook trout (Salvelinus fontinalis) develop significantly greater methemoglobinemia than Atlantic salmon (Salmo salar) or rainbow trout (Oncorhynchus mykiss) following general anesthesia with benzocaine or tricaine methanesulfonate.

Evaluation of co-oximetry for the measurement of methemoglobin in rainbow trout (Oncorhynchus mykiss) and values in 3 salmonid species.

Background: Methemoglobin (metHb) is oxidized hemoglobin that cannot reversibly bind oxygen, and concentrations in healthy fish have been reported to be 0.6-24.8% compared with 0-3% in healthy mammals.