Conservation of mechanism in flavoprotein-catalyzed amine oxidation.

The goals of this presentation are to summarize the present understanding of the mechanism of amine oxidation by flavoproteins and to examine the possibility that a member of the monoamine oxidase family catalyzes oxidation of a carbon-carbon bond. In the discussion of mechanism, the emphasis is on the protonation state of the amine substrate, since the once-controversial mechanism of oxidation appears to be resolved. The argument will be made that flavoproteins catalyzing amine oxidation preferentially bind the form of the substrate in which the reacting nitrogen is uncharged.

Biochemical and biophysical approaches to characterization of the aromatic amino acid hydroxylases.

The aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase utilize a non-heme iron to catalyze the hydroxylation of the aromatic rings of their amino acid substrates, with a tetrahydropterin serving as the source of the electrons necessary for the monooxygenation reaction. These enzymes have been subjected to a variety of biochemical and biophysical approaches, resulting in a detailed understanding of their structures and mechanism. We summarize here the experimental approaches that have led to this understanding.

A novel in vitro high-content imaging assay for the prediction of drug-induced lung toxicity.

The development of inhaled drugs for respiratory diseases is frequently impacted by lung pathology in non-clinical safety studies. To enable design of novel candidate drugs with the right safety profile, predictive in vitro lung toxicity assays are required that can be applied during drug discovery for early hazard identification and mitigation. Here, we describe a novel high-content imaging-based screening assay that allows for quantification of the tight junction protein occludin in A549 cells, as a model for lung epithelial barrier integrity.

Mechanistic safety assessment via multi-omic characterisation of systemic pathway perturbations following in vivo MAT2A inhibition.

The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis.

The aromatic amino acid hydroxylases: Structures, catalysis, and regulation of phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase.

The aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase are non-heme iron enzymes that catalyze key physiological reactions. This review discusses the present understanding of the common catalytic mechanism of these enzymes and recent advances in understanding the relationship between their structures and their regulation.

An International Comparison of Presentation, Outcomes and CORONET Predictive Score Performance in Patients with Cancer Presenting with COVID-19 across Different Pandemic Waves.

Patients with cancer have been shown to have increased risk of COVID-19 severity. We previously built and validated the COVID-19 Risk in Oncology Evaluation Tool (CORONET) to predict the likely severity of COVID-19 in patients with active cancer who present to hospital. We assessed the differences in presentation and outcomes of patients with cancer and COVID-19, depending on the wave of the pandemic.

Thermodynamics of iron, tetrahydrobiopterin, and phenylalanine binding to phenylalanine hydroxylase from Chromobacterium violaceum.

Phenylalanine hydroxylase (PheH) is a pterin-dependent, mononuclear nonheme iron(II) oxygenase that uses the oxidative power of O to hydroxylate phenylalanine to form tyrosine. PheH is a member of a superfamily of O-activating enzymes that utilizes a common metal binding motif: the 2-His-1-carboxylate facial triad. Like most members of this superfamily, binding of substrates to PheH results in a reorganization of its active site to allow O activation.

Establishment of CORONET, COVID-19 Risk in Oncology Evaluation Tool, to Identify Patients With Cancer at Low Versus High Risk of Severe Complications of COVID-19 Disease On Presentation to Hospital.

Purpose: Patients with cancer are at increased risk of severe COVID-19 disease, but have heterogeneous presentations and outcomes. Decision-making tools for hospital admission, severity prediction, and increased monitoring for early intervention are critical. We sought to identify features of COVID-19 disease in patients with cancer predicting severe disease and build a decision support online tool, COVID-19 Risk in Oncology Evaluation Tool (CORONET).

Biomarker identification using dynamic time warping analysis: a longitudinal cohort study of patients with COVID-19 in a UK tertiary hospital.

Objectives: COVID-19 is a heterogeneous disease, and many reports have described variations in demographic, biochemical and clinical features at presentation influencing overall hospital mortality. However, there is little information regarding longitudinal changes in laboratory prognostic variables in relation to disease progression in hospitalised patients with COVID-19.

Design And Setting: This retrospective observational report describes disease progression from symptom onset, to admission to hospital, clinical response and discharge/death among patients with COVID-19 at a tertiary centre in South East England.

Pharmacological inhibition of MERTK induces in vivo retinal degeneration: a multimodal imaging ocular safety assessment.

The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical.

Wave comparisons of clinical characteristics and outcomes of COVID-19 admissions - Exploring the impact of treatment and strain dynamics.

Objectives: Dexamethasone has now been incorporated into the standard of care for COVID-19 hospital patients. However, larger intensive care unit studies have failed to show discernible improvements in mortality in the recent wave. We aimed to investigate the impacts of these factors on disease outcomes in a UK hospital study.

Patient-derived scaffolds influence secretion profiles in cancer cells mirroring clinical features and breast cancer subtypes.

Background: Breast cancer is a common malignancy with varying clinical behaviors and for the more aggressive subtypes, novel and more efficient therapeutic approaches are needed. Qualities of the tumor microenvironment as well as cancer cell secretion have independently been associated with malignant clinical behaviors and a better understanding of the interplay between these two features could potentially reveal novel targetable key events linked to cancer progression.

Methods: A newly developed human derived in vivo-like growth system, consisting of decellularized patient-derived scaffolds (PDSs) recellularized with standardized breast cancer cell lines (MCF7 and MDA-MB-231), were used to analyze how 63 individual patient specific microenvironments influenced secretion determined by proximity extension assays including 184 proteins and how these relate to clinical outcome.

Research Evaluation Alongside Clinical Treatment in COVID-19 (REACT COVID-19): an observational and biobanking study.

Introduction: The COVID-19 pandemic caused by SARS-CoV-2 places immense worldwide demand on healthcare services. Earlier identification of patients at risk of severe disease may allow intervention with experimental targeted treatments, mitigating the course of their disease and reducing critical care service demand.

Methods And Analysis: This prospective observational study of patients tested or treated for SARS-CoV-2, who are under the care of the tertiary University Hospital Southampton NHS Foundation Trust (UHSFT), captured data from admission to discharge; data collection commenced on 7 March 2020.

Characterization of cell-free breast cancer patient-derived scaffolds using liquid chromatography-mass spectrometry/mass spectrometry data and RNA sequencing data.

Patient-derived scaffolds (PDSs) generated from primary breast cancer tumors can be used to model the tumor microenvironment . Patient-derived scaffolds are generated by repeated detergent washing, removing all cells. Here, we analyzed the protein composition of 15 decellularized PDSs using liquid chromatography-mass spectrometry/mass spectrometry.

Biosimilar infliximab administration for the management of acute graft-versus-host disease.

Introduction: Acute graft-versus-host disease (aGVHD) is a significant immune-mediated complication of allogeneic hematopoietic stem cell transplant (HSCT). Despite prophylactic immunosuppression, the incidence of grades II-IV aGVHD post-HSCT varies from 20 to 80%. Tumor necrosis factor (TNF) is an important cytokine involved in the pathogenesis of GVHD, and medications such as infliximab (Remicade®) have been utilized as second-line treatment options in patients with steroid-refractory GHVD.

Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment.

Tumor cells interact with the microenvironment that specifically supports and promotes tumor development. Key components in the tumor environment have been linked to various aggressive cancer features and can further influence the presence of subpopulations of cancer cells with specific functions, including cancer stem cells and migratory cells. To model and further understand the influence of specific microenvironments we have developed an experimental platform using cell-free patient-derived scaffolds (PDSs) from primary breast cancers infiltrated with standardized breast cancer cell lines.

Translational studies of intravenous and intracerebroventricular routes of administration for CNS cellular biodistribution for BMN 250, an enzyme replacement therapy for the treatment of Sanfilippo type B.

BMN 250 is being developed as enzyme replacement therapy for Sanfilippo type B, a primarily neurological rare disease, in which patients have deficient lysosomal alpha-N-acetylglucosaminidase (NAGLU) enzyme activity. BMN 250 is taken up in target cells by the cation-independent mannose 6-phosphate receptor (CI-MPR, insulin-like growth factor 2 receptor), which then facilitates transit to the lysosome. BMN 250 is dosed directly into the central nervous system via the intracerebroventricular (ICV) route, and the objective of this work was to compare systemic intravenous (IV) and ICV delivery of BMN 250 to confirm the value of ICV dosing.

pH and deuterium isotope effects on the reaction of trimethylamine dehydrogenase with dimethylamine.

The flavoprotein trimethylamine dehydrogenase is a member of a small class of flavoproteins that catalyze amine oxidation and transfer the electrons through an Fe/S center to an external oxidant. The mechanism of amine oxidation by this family of enzymes has not been established. Here, we describe the use of pH and kinetic isotope effects with the slow substrate dimethylamine to study the mechanism.

ICU Patient Family Stress Recovery During Breaks in a Hospital Garden and Indoor Environments.

Objectives: Measure the immediate change in intensive care unit (ICU) family members' state stress levels from the beginning to the end of a person's visit to a hospital garden and compare the changes produced by the garden with those associated with spending time in indoor hospital environments intended for respite and relaxation.

Background: No previous research has compared the efficacy of different physical environments as interventions to foster stress reduction in family members of ICU patients, a group of hospital visitors known to experience high levels of distress.

Method: A convenience sample of 42 ICU patient family (from 42 different families) completed the Present Functioning Visual Analogue Scales (PFVAS) before and after each visit (128 total visits) to a garden, an atrium/café, or ICU waiting room.

Hypoxia-induced secretion stimulates breast cancer stem cell regulatory signalling pathways.

It is well known that tumour cells are dependent on communication with the tumour microenvironment. Previously, it has been shown that hypoxia (HX) induces pronounced, diverse and direct effects on cancer stem cell (CSC) qualities in different breast cancer subtypes. Here, we describe the mechanism by which HX-induced secretion influences the spreading of CSCs.

Mechanism of the Flavoprotein d-6-Hydroxynicotine Oxidase: Substrate Specificity, pH and Solvent Isotope Effects, and Roles of Key Active-Site Residues.

The flavoprotein d-6-hydroxynicotine oxidase catalyzes an early step in the oxidation of ( R)-nicotine, the oxidation of a carbon-nitrogen bond in the pyrrolidine ring of ( R)-6-hydroxynicotine. The enzyme is a member of the vanillyl alcohol oxidase/ p-cresol methylhydroxylase family of flavoproteins. The effects of substrate modifications on the steady-state and rapid-reaction kinetic parameters are not consistent with the quinone-methide mechanism of p-cresol methylhydroxylase.

The phenylketonuria-associated substitution R68S converts phenylalanine hydroxylase to a constitutively active enzyme but reduces its stability.

The naturally occurring R68S substitution of phenylalanine hydroxylase (PheH) causes phenylketonuria (PKU). However, the molecular basis for how the R68S variant leads to PKU remains unclear. Kinetic characterization of R68S PheH establishes that the enzyme is fully active in the absence of allosteric binding of phenylalanine, in contrast to the WT enzyme.

Sortilin inhibition limits secretion-induced progranulin-dependent breast cancer progression and cancer stem cell expansion.

Background: Cancer progression is influenced by genetic aberrations in the cancer cell population as well as by other factors including the microenvironment present within a tumour. Direct interactions between various cell types as well as cellular signalling via secreted cytokines can drive key tumourigenic properties associated with disease progression and treatment resistance. Also, cancer stem cell functions are influenced by the microenvironment.