Determining KRAS4B-Targeting Compound Specificity by Top-Down Mass Spectrometry.

We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform.

Partitioning to ordered membrane domains regulates the kinetics of secretory traffic.

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants.

Creating safety amidst chronic contamination: A mixed-method analysis of residents' experiences in a Southern Italian steel town.

This study analyses how residents create safety in Taranto, Italy, a city located next to one of the largest steel plants in Europe. Combining long-term ethnographic research with an online-based survey, our study shows that most respondents recognise and criticise the presence of environmental risks in their daily lives but encounter such risks in complex ways. Contrary to previous scholarship suggesting that pollution can result in alienating residents from their lived environment, this research shows that acute awareness of environmental risks does not necessarily undermine attachment to place but rather can co-exist with or even strengthen it.

FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines.

A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement.

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors.

Partitioning to ordered membrane domains regulates the kinetics of secretory traffic.

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants.

Improved Sequence Analysis of Intact Proteins by Parallel Ion Parking during Electron Transfer Dissociation.

Electron transfer dissociation (ETD) is an analytically useful tool for primary structure interrogation of intact proteins, but its utility is limited by higher-order reactions with the products. To inhibit these higher-order reactions, first-generation fragment ions are kinetically excited by applying an experimentally tailored parallel ion parking waveform during ETD (ETD-PIP). In combination with subsequent ion/ion proton transfer reactions, precursor-to-product conversion was maximized as evidenced by the consumption of more than 90% of the 21 kDa Protein G precursor to form ETD product ions.

Hypertrophic cardiomyopathy in infant newborns of diabetic mother: a heterogeneous condition, the importance of anamnesis, physical examination and follow-up.

Background: Hypertrophic cardiomyopathy (HCM) in neonates is a rare and heterogeneous disorder. HCM accounts for 25 to 40% of all pediatric cardiomyopathy cases and the highest incidence in pediatric population is reported in children < 1 year.

Case Presentation: we report two clinical cases of neonates, born to mothers respectively with a pre-pregnancy insulin-dependent diabetic mellitus type 2 and a suspected diabetes, with inadequate prenatal glycemic control for the first and underestimated glycemic control for the second case, with a different evolution.

Refining the N-Termini of the SARS-CoV-2 Spike Protein and Its Discrete Receptor-Binding Domain.

Previous work employing five SARS-CoV-2 spike protein receptor-binding domain (RBD) constructs, comprising versions originally developed by Mt. Sinai or the Ragon Institute and later optimized in-house, revealed potential heterogeneity which led to questions regarding variable seropositivity assay performance. Each construct was subjected to N-deglycosylation and subsequent intact mass analysis, revealing significant deviations from predicted theoretical mass for all five proteins.

What is new in anaphylaxis?

Available information suggests that anaphylaxis must be promptly recognized keeping in mind the airway patency, breathing (ventilation and respiration), circulation and mental status and treated. The first treatment is adrenaline. After successful treatment of an anaphylactic episode, attention must be paid to the prevention of early recurrences (biphasic anaphylaxis) and assessment of causes.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications.

Sequencing a Bispecific Antibody by Controlling Chain Concentration Effects When Using an Immobilized Nonspecific Protease.

Complete sequence coverage of monospecific antibodies was previously achieved using immobilized aspergillopepsin I in a single LC-MS/MS analysis. Bispecific antibodies are asymmetrical compared to their monospecific antibody counterparts, resulting in a decrease in the concentration of individual subunits. Four standard proteins were used to characterize the effect of a decrease in concentration when using this immobilized enzyme reactor.

Myelin-Associated MAL and PLP Are Unusual among Multipass Transmembrane Proteins in Preferring Ordered Membrane Domains.

Eukaryotic membranes can be partitioned into lipid-driven membrane microdomains called lipid rafts, which function to sort lipids and proteins in the plane of the membrane. As protein selectivity underlies all functions of lipid rafts, there has been significant interest in understanding the structural and molecular determinants of raft affinity. Such determinants have been described for lipids and single-spanning transmembrane proteins; however, how multipass transmembrane proteins (TMPs) partition between ordered and disordered phases has not been widely explored.

Clinical variability in children with dolichoarteriopathies of the internal carotid artery.

Introduction: Dolichoarteriopathies of the internal carotid artery (DICA) are frequent non-atheromatous anatomical changes in the general population. The etiology of DICA is still controversial: several hypotheses have been suggested, including an anomaly of embryological development, or a degenerative loss of elasticity of the vessel wall. DICA have been related to a wide spectrum of clinical presentations in adults, varying from asymptomatic forms to acute cerebrovascular events.

Behavioural weight loss treatment preferences of college students with overweight and obesity.

The college environment increases risk of weight gain in young adults with overweight/obesity. Behavioural weight loss interventions are proven effective, however, young adults' adherence to such programs is poor. The purpose of the study was to determine weight loss treatment preferences of 2- and 4-year college students for the development of population-specific interventions.

Unambiguous Sequence Characterization of a Monoclonal Antibody in a Single Analysis Using a Nonspecific Immobilized Enzyme Reactor.

Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is a methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1 to 20 kDa.

Protamines from liverwort are produced by post-translational cleavage and C-terminal di-aminopropanelation of several male germ-specific H1 histones.

Protamines are small, highly-specialized, arginine-rich, and intrinsically-disordered chromosomal proteins that replace histones during spermiogenesis in many organisms. Previous evidence supports the notion that, in the animal kingdom, these proteins have evolved from a primitive replication-independent histone H1 involved in terminal cell differentiation. Nevertheless, a direct connection between the two families of chromatin proteins is missing.

High-throughput doping control analysis of 28 amphetamine-type stimulants in equine plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

A hydrophilic interaction liquid chromatography-tandem mass spectrometry method (HILIC-MS/MS) was developed for the simultaneous determination of 28 amphetamine-type stimulants (ATSs) in equine plasma for doping control analysis. In this method, stimulants were recovered from equine plasma by liquid-liquid extraction (LLE) at pH 9.5 using methyl tert-butyl ether and detected on a Thermo Finnigan triple quadrupole mass spectrometer operating in positive-ion mode electrospray ionization.

Pre-hospital lung ultrasound for cardiac heart failure and COPD: is it worthwhile?

Background: Pre-hospital ultrasound is a new challenge and lung ultrasound could be an interesting opportunity in the pre-hospital medical service. The aim of our study was to evaluate the efficacy of lung ultrasound in out-of-hospital non-traumatic respiratory insufficiency.

Methods: We planned a case-controlled study in the ULSS 5 ovest vicentino area (Vicenza-Italy) enrolling subjects with severe dyspnea caused by cardiac heart failure or acute exacerbation of chronic obstructive pulmonary disease.

Distinct metaplastic and inflammatory phenotypes in autoimmune and adenocarcinoma-associated chronic atrophic gastritis.

Background: Autoimmune gastritis (AIG) and adenocarcinoma-associated chronic atrophic gastritis (CAG) are both associated with oxyntic atrophy, but AIG patients demonstrate an increased risk of carcinoid tumors rather than the elevated risk of adenocarcinoma observed with CAG. We therefore sought to compare the characteristics of the metaplastic mucosa in AIG and CAG patients.

Methods: We examined markers for metaplasia (spasmolytic polypeptide expressing metaplasia (SPEM) and intestinal metaplasia) as well as proliferation (Ki67) and immune cell populations (neutrophils, macrophages, and eosinophils) in gastric sections from 16 female patients with autoimmune thyroiditis and AIG and 17 patients with CAG associated with gastric adenocarcinoma.

Recombinant Human Thyrotropin Improves Endothelial Coronary Flow Reserve in Thyroidectomized Patients with Differentiated Thyroid Cancer.

Background: The role of thyrotropin (TSH) on the cardiovascular system has been poorly investigated. It is unknown whether the changes in the vasculature associated with thyroid diseases result from altered thyroid hormone action or whether they are a consequence of a direct effect of TSH on endothelial cells. The present study was designed to evaluate the endothelial response of coronary flow to TSH in patients with differentiated thyroid cancer (DTC) without cardiovascular risk factors.

Risk factors for silent myocardial ischemia in patients with well-controlled essential hypertension.

Silent myocardial ischemia (SMI) is frequently observed in patients with essential hypertension (EH). The major risk factor for SMI is uncontrolled blood pressure (BP), but SMI is also observed in patients with well-controlled BP. To evaluate the prevalence of SMI and the factors associated with SMI in EH patients with well-controlled BP.

Cyclosporin promotes neurorestoration and cell replacement therapy in pre-clinical models of Parkinson's disease.

Background: The early clinical trials using fetal ventral mesencephalic (VM) allografts in Parkinson's disease (PD) patients have shown efficacy (albeit not in all cases) and have paved the way for further development of cell replacement therapy strategies in PD. The preclinical work that led to these clinical trials used allografts of fetal VM tissue placed into 6-OHDA lesioned rats, while the patients received similar allografts under cover of immunosuppression in an α-synuclein disease state. Thus developing models that more faithfully replicate the clinical scenario would be a useful tool for the translation of such cell-based therapies to the clinic.