Riociguat treatment in patients with chronic thromboembolic pulmonary hypertension: Final safety data from the EXPERT registry

Objective: The soluble guanylate cyclase stimulator riociguat is approved for the treatment of adult patients with pulmonary arterial hypertension (PAH) and inoperable or persistent/recurrent chronic thromboembolic pulmonary hypertension (CTEPH) following Phase

Mass Spectrometry Strategies for Characterization of Contact Allergens and their Protein Conjugates in Vitro and in Vivo

Humans are daily exposed to chemicals from various sources, including cosmetics, jewelry, clothes, and hair dyes, which can result in the occurrence of contact allergy and subsequent allergic contact dermatitis (ACD), a type IV delayed hypersensitivity reaction. ACD is characterized by inflammation and eczema at the site of exposure, and no definitive cure for this condition has been identified to date, with only symptomatic treatment options involving corticosteroids being available. The research presented in this thesis is centered around mass spectrometry (MS) strategies aimed at enhancing our comprehension of events that occur during the early stages of the development of contact allergy. Special emphasis is given to characterizing various contact allergens (haptens) and their interactions with endogenous proteins, as these interactions are considered crucial in the initiation of contact allergy. Moreover, the thesis endeavors to explore the activation of prehaptens and prohaptens, which are non-reactive compounds capable of transforming into haptens outside or inside the skin, respectively. In Paper I, a bottom-up proteomics approach was employed to investigate the adductome of two major blood proteins, human serum albumin (HSA) and hemoglobin (Hb). The study aimed to identify the most reactive sites on these proteins upon exposure to different haptens with varying sensitization potencies. Highly susceptible sites on HSA and Hb were identified as the most likely targets for in vivo modification. This study is the first investigation of the Hb adductome in the context of contact allergy and may contribute to the development of improved diagnostic tools using blood samples. With Hb on focus, Paper II evaluated three different MS-based methods, including bottom-up proteomics, detachment of N-terminal adducts by FIRE, and limited proteolysis (LiP), to determine the most suitable approach for assessing exposure through this protein. The three methods showed different strengths and limitations depending on the nature of the hapten. In Paper III, the research conducted revealed the presence of a hapten-protein conjugate in blood samples mice treated with the synthetic hapten tetramethyl rhodamine isothiocyanate (TRITC) topically. The identified protein was the macrophage migration inhibitory factor (MIF), marking the first instance of such a conjugate being detected in blood samples after topical hapten application. The study also indicated that MIF could potentially be modified by other contact allergens, suggesting its potential as a biomarker for the condition. In Paper IV, contact allergy to propolis, a by-product of honey used in biocosmetics, was investigated. Air oxidation experiments with a model peptide and MS detection, revealed that quinones formed from the oxidation of major propolis components are responsible for adduct formation. The identified adducts are likely the cause of contact allergy to propolis, providing valuable insights into the underlying mechanisms of propolis contact allergy and potential implications for clinical diagnosis. In Paper V, the bioactivation of cinnamic alcohol, a common ingredient in many cosmetic products, was investigated using in vitro systems and a targeted MS approach. Two metabolites, namely pOH-cinnamic alcohol and pOH-cinnamic aldehyde, were identified as of particular interest and their sensitizing potency was evaluated, with the latter categorized as a moderate sensitizer. In summary, this doctoral thesis employed MS techniques to characterize contact allergens and their protein conjugates, yielding valuable insights into the molecular mechanisms underlying contact allergy development. The findings have potential implications for improving diagnostic tools and strategies for preventing and treating contact allergy.

Mass Spectrometry Strategies for Characterization of Contact Allergens and their Protein Conjugates in Vitro and in Vivo

Humans are daily exposed to chemicals from various sources, including cosmetics, jewelry, clothes, and hair dyes, which can result in the occurrence of contact allergy and subsequent allergic contact dermatitis (ACD), a type IV delayed hypersensitivity reaction. ACD is characterized by inflammation and eczema at the site of exposure, and no definitive cure for this condition has been identified to date, with only symptomatic treatment options involving corticosteroids being available. The research presented in this thesis is centered around mass spectrometry (MS) strategies aimed at enhancing our comprehension of events that occur during the early stages of the development of contact allergy. Special emphasis is given to characterizing various contact allergens (haptens) and their interactions with endogenous proteins, as these interactions are considered crucial in the initiation of contact allergy. Moreover, the thesis endeavors to explore the activation of prehaptens and prohaptens, which are non-reactive compounds capable of transforming into haptens outside or inside the skin, respectively. In Paper I, a bottom-up proteomics approach was employed to investigate the adductome of two major blood proteins, human serum albumin (HSA) and hemoglobin (Hb). The study aimed to identify the most reactive sites on these proteins upon exposure to different haptens with varying sensitization potencies. Highly susceptible sites on HSA and Hb were identified as the most likely targets for in vivo modification. This study is the first investigation of the Hb adductome in the context of contact allergy and may contribute to the development of improved diagnostic tools using blood samples. With Hb on focus, Paper II evaluated three different MS-based methods, including bottom-up proteomics, detachment of N-terminal adducts by FIRE, and limited proteolysis (LiP), to determine the most suitable approach for assessing exposure through this protein. The three methods showed different strengths and limitations depending on the nature of the hapten. In Paper III, the research conducted revealed the presence of a hapten-protein conjugate in blood samples mice treated with the synthetic hapten tetramethyl rhodamine isothiocyanate (TRITC) topically. The identified protein was the macrophage migration inhibitory factor (MIF), marking the first instance of such a conjugate being detected in blood samples after topical hapten application. The study also indicated that MIF could potentially be modified by other contact allergens, suggesting its potential as a biomarker for the condition. In Paper IV, contact allergy to propolis, a by-product of honey used in biocosmetics, was investigated. Air oxidation experiments with a model peptide and MS detection, revealed that quinones formed from the oxidation of major propolis components are responsible for adduct formation. The identified adducts are likely the cause of contact allergy to propolis, providing valuable insights into the underlying mechanisms of propolis contact allergy and potential implications for clinical diagnosis. In Paper V, the bioactivation of cinnamic alcohol, a common ingredient in many cosmetic products, was investigated using in vitro systems and a targeted MS approach. Two metabolites, namely pOH-cinnamic alcohol and pOH-cinnamic aldehyde, were identified as of particular interest and their sensitizing potency was evaluated, with the latter categorized as a moderate sensitizer. In summary, this doctoral thesis employed MS techniques to characterize contact allergens and their protein conjugates, yielding valuable insights into the molecular mechanisms underlying contact allergy development. The findings have potential implications for improving diagnostic tools and strategies for preventing and treating contact allergy.

Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders

AbstractGlycosylation is one of the most common and important post-translational modifications, influencing both the chemical and the biological properties of proteins. Studying the glycosylation of the entire protein population of a sample can be challenging because variations in the concentrations of certain proteins can enhance or obscure changes in glycosylation. Furthermore, alterations in the glycosylation pattern of individual proteins, exhibiting larger variability in disease states, have been suggested as biomarkers for different types of cancer, as well as inflammatory and neurodegenerative diseases. In this paper, we present a rapid and efficient method for glycosylation analysis of individual proteins focusing on changes in the degree of fucosylation or other alterations to the core structure of the glycans, such as the presence of bisecting N-acetylglucosamines and a modified degree of branching. Streptavidin-coated magnetic beads are used in combination with genetically engineered immunoaffinity binders, called VHH antibody fragments. A major advantage of the VHHs is that they are nonglycosylated; thus, enzymatic release of glycans from the targeted protein can be performed directly on the beads. After deglycosylation, the glycans are analyzed by MALDI-TOF-MS. The developed method was evaluated concerning its specificity, and thereafter implemented for studying the glycosylation pattern of two different proteins, alpha-1-antitrypsin and transferrin, in human serum and cerebrospinal fluid. To our knowledge, this is the first example of a protein array-type experiment that employs bead-based immunoaffinity purification in combination with mass spectrometry analysis for fast and efficient glycan analysis of individual proteins in biological fluid

Investigation into Propolis Components Responsible for Inducing Skin Allergy : Air Oxidation of Caffeic Acid and Its Esters Contribute to Hapten Formation

Propolis is a resin-like material produced by bees from the buds of poplar and cone-bearing trees and is used in beehive construction. Propolis is a common additive in various biocosmetics and health-related products, despite the fact that it is a well-known cause of contact allergy. Caffeic acid and its esters have been the primary suspects behind the sensitization potency of propolis-induced contact allergy. However, the chemical structures of the protein adducts formed between these haptens and skin proteins during the process of skin sensitization remain unknown. In this study, the reactivity of three main contact allergens found in propolis, namely, caffeic acid (CA), caffeic acid 1,1-dimethylallyl ester (CAAE), and caffeic acid phenethyl ester (CAPE), was investigated. These compounds were initially subjected to the kinetic direct peptide reactivity assay to categorize the sensitization potency of CA, CAAE, and CAPE, but the data obtained was deemed too unreliable to confidently classify their skin sensitization potential based on this assay alone. To further investigate the chemistry involved in generating possible skin allergy-inducing protein adducts, model peptide reactions with CA, CAAE, and CAPE were conducted and analyzed via liquid chromatography-high-resolution mass spectrometry. Reactions between CA, CAAE, and CAPE and a cysteine-containing peptide in the presence of oxygen, both in closed and open systems, were monitored at specific time points. These studies revealed the formation of two different adducts, one corresponding to thiol addition to the α,β-unsaturated carbonyl region of the caffeic structure and the second corresponding to thiol addition to the catechol, after air oxidation to o-quinone. Observation of these peptide adducts classifies these compounds as prehaptens. Interestingly, no adduct formation was observed when the same reactions were performed under oxygen-free conditions, highlighting the importance of air oxidation processes in CA, CAAE, and CAPE adduct formation. Additionally, through NMR analysis, we found that thiol addition occurs at the C-2 position in the aromatic ring of the CA derivatives. Our results emphasize the importance of air oxidation in the sensitization potency of propolis and shed light on the chemical structures of the resultant haptens which could trigger allergic reactions in vivo

HARPA: Tackling physically induced performance variability

Continuously increasing application demands on both High Performance Computing (HPC) and Embedded Systems (ES) are driving the IC manufacturing industry on an everlasting scaling of devices in silicon. Nevertheless, integration and miniaturization of transistors comes with an important and non-negligible trade-off: time-zero and time-dependent performance variability. Increasing guard-bands to battle variability is not scalable, since worst-case design margins are prohibitive for downscaled technology nodes. This paper discusses the FP7-612069-HARPA project of the European Commission which aims to enable next-generation embedded and high-performance heterogeneous many-cores to cost-effectively confront variations by providing Dependable-Performance: correct functionality and timing guarantees throughout the expected lifetime of a platform under thermal, power, and energy constraints. The HARPA novelty is in seeking synergies in techniques that have been considered virtually exclusively in the ES or HPC domains (worst-case guaranteed partly proactive techniques in embedded, and dynamic best-effort reactive techniques in high-performance)

The HARPA Approach to Ensure Dependable Performance

The goal of the HARPA solution is to overcome the performance variability (PV) by enabling next-generation embedded and high-performance platforms using heterogeneous many-core processors to provide cost-effectively dependable performance: the correct functionality and (where needed) timing guarantees throughout the expected lifetime of a platform. This must be accomplished in the presence of cycle-by-cycle performance variability due to time-dependent variations in silicon devices and wires under thermal, power, and energy constraints. The common challenge for both embedded and high-performance systems is to harness the unsustainable increases in design and operational margins and yet provide dependable performance. For example, resources that are statically determined based on worst-case execution time for real-time applications or lower clock frequency to satisfy excessive timing margins in high-performance processors

Riociguat treatment in patients with chronic thromboembolic pulmonary hypertension: Final safety data from the EXPERT registry

International audienc

Riociguat treatment in patients with pulmonary arterial hypertension: Final safety data from the EXPERT registry

International audienc