Key inflammatory markers in bronchoalveolar lavage predict bronchiectasis progression in young children with CF.

INTRODUCTION: Inflammation appears early in cystic fibrosis (CF) pathogenesis, with specific elevated inflammatory markers in bronchoalveolar lavage fluid (BALF) correlating with structural lung disease. Our aim was to identify markers of airway inflammation able to predict bronchiectasis progression over two years with high sensitivity and specificity. METHODS: Children with CF with two chest computed tomography (CT) scans and bronchoscopies at a two-year interval were included (n= 10 at 1 and 3 years and n= 27 at 3 and 5 years). Chest CTs were scored for increase in bronchiectasis (Δ%Bx), using the PRAGMA-CF score. BALF collected with the first CT scan were analyzed for neutrophil% (n= 36), myeloperoxidase (MPO) (n= 25), neutrophil elastase (NE) (n= 26), and with a protein array for inflammatory and fibrotic markers (n= 26). RESULTS: MPO, neutrophil%, and inducible T-cell costimulator ligand (ICOSLG), but not clinical characteristics, correlated significantly with Δ%Bx. Evaluation of neutrophil%, NE, MPO, interleukin-8 (IL-8), ICOSLG, and hepatocyte growth factor (HGF), for predicting an increase of > 0.5% of Δ%Bx in two years, showed that IL-8 had the best sensitivity (82%) and specificity (73%). Neutrophil%, ICOSLG and HGF had sensitivities of 85, 82, and 82% and specificities of 59, 67 and 60%, respectively. The odds ratio for risk of >0.5% Δ%Bx was higher for IL-8 (12.4) than for neutrophil%, ICOSLG, and HGF (5.9, 5.3, and 6.7, respectively). Sensitivity and specificity were lower for NE and MPO). CONCLUSIONS: High levels of IL-8, neutrophil%, ICOSGL and HGF in BALF may be good predictors for progression of bronchiectasis in young children with CF.

The 'omics of obesity in B-cell acute lymphoblastic leukemia.

The obesity pandemic currently affects more than 70 million Americans and more than 650 million individuals worldwide. In addition to increasing susceptibility to pathogenic infections (eg, SARS-CoV-2), obesity promotes the development of many cancer subtypes and increases mortality rates in most cases. We and others have demonstrated that, in the context of B-cell acute lymphoblastic leukemia (B-ALL), adipocytes promote multidrug chemoresistance. Furthermore, others have demonstrated that B-ALL cells exposed to the adipocyte secretome alter their metabolic states to circumvent chemotherapy-mediated cytotoxicity. To better understand how adipocytes impact the function of human B-ALL cells, we used a multi-omic RNA-sequencing (single-cell and bulk transcriptomic) and mass spectroscopy (metabolomic and proteomic) approaches to define adipocyte-induced changes in normal and malignant B cells. These analyses revealed that the adipocyte secretome directly modulates programs in human B-ALL cells associated with metabolism, protection from oxidative stress, increased survival, B-cell development, and drivers of chemoresistance. Single-cell RNA sequencing analysis of mice on low- and high-fat diets revealed that obesity suppresses an immunologically active B-cell subpopulation and that the loss of this transcriptomic signature in patients with B-ALL is associated with poor survival outcomes. Analyses of sera and plasma samples from healthy donors and those with B-ALL revealed that obesity is associated with higher circulating levels of immunoglobulin-associated proteins, which support observations in obese mice of altered immunological homeostasis. In all, our multi-omics approach increases our understanding of pathways that may promote chemoresistance in human B-ALL and highlight a novel B-cell-specific signature in patients associated with survival outcomes.

Development of a Universal Second-Tier Newborn Screening LC-MS/MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids.

First-tier MS-based newborn screening by flow injection analysis can have high presumptive positive rates, often due to isomeric/isobaric compounds or poor biomarker specificity. These presumptive positive samples can be analyzed by second-tier screening assays employing separations such as liquid chromatography-mass spectrometry (LC-MS/MS), which increases test specificity and drastically reduces false positive referrals. The ability to screen for multiple disorders in a single multiplexed test simplifies workflows and maximizes public health laboratories' resources. In this study, we developed and validated a highly multiplexed second-tier method for dried blood spots using a hydrophilic interaction liquid chromatography (HILIC) column coupled to an MS/MS system. The LC-MS/MS method was capable of simultaneously detecting second-tier biomarkers for maple syrup urine disease, homocystinuria, methylmalonic acidemia, propionic acidemia, glutaric acidemia type 1, glutaric acidemia type 2, guanidinoacetate methyltransferase deficiency, short-chain acyl-CoA dehydrogenase deficiency, adrenoleukodystrophy, and Pompe disease.

Dual oxidase 1 promotes antiviral innate immunity.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

Airway profile of bioactive lipids predicts early progression of lung disease in cystic fibrosis.

BACKGROUND: Previously, we showed that abnormal levels of bioactive lipids in bronchoalveolar lavage fluid (BALF) from infants with cystic fibrosis (CF) correlated with early structural lung damage. METHOD: To extend these studies, BALF bioactive lipid measurement by mass spectrometry and chest computed tomography (CT, combined with the sensitive PRAGMA-CF scoring method) were performed longitudinally at 2-year intervals in a new cohort of CF children (n = 21, aged 1-5 yrs). RESULTS: PRAGMA-CF, neutrophil elastase activity, and myeloperoxidase correlated with BALF lysolipids and isoprostanes, markers of oxidative stress, as well as prostaglandin E2 and combined ceramide precursors (Spearman's Rho > 0.5; P < 0.01 for all). Multiple protein agonists of inflammation and tissue remodeling, measured by Olink protein array, correlated positively (r = 0.44-0.79, p < 0.05) with PRAGMA-CF scores and bioactive lipid levels. Notably, levels of lysolipids, prostaglandin E2 and isoprostanes at first BALF predicted the evolution of PRAGMA-CF scores 2 years later. In wild-type differentiated primary bronchial epithelial cells, and in CFTR-inducible iCFBE cells, treatment with a lysolipid receptor agonist (VPC3114) enhanced shedding of pro-inflammatory and pro-fibrotic proteins. CONCLUSIONS: Together, our findings suggest that bioactive lipids in BALF correlate with and possibly predict structural lung disease in CF children, which supports their use as biomarkers of disease progression and treatment efficacy. Furthermore, our data suggest a causative role of airway lysolipids and oxidative stress in the progression of early CF lung disease, unveiling potential therapeutic targets.

The thiocyanate analog selenocyanate is a more potent antimicrobial pro-drug that also is selectively detoxified by the host.

A hallmark of cystic fibrosis (CF) lung pathology is an increased susceptibility to pulmonary infections. Thiocyanate (-SCN) is an endogenous component of the innate immunity's peroxidase system that converts -SCN to the antimicrobial agent hypothiocyanite (HOSCN). We have previously shown that the host thioredoxin reductase (TrxR), but not the pathogen's TrxR, can selectively detoxify HOSCN thereby decreasing inflammation and oxidative stress. We tested whether the -SCN analog selenocyanate (-SeCN) shares these properties against several clinical CF bacterial isolates. We examined oxidant production from a lactoperoxidase (LPO) system using -SeCN as a potential substrate. The LPO system generated an oxidant similar in nature to HOSCN and consistent with being HOSeCN. The rate of oxidant generation using -SeCN was significantly less than seen for -SCN. An LPO system was used to generate HOSCN or HOSeCN and compared for antimicrobial activity during in situ exposure of clinical CF isolates of P. aeruginosa (PA), B. cepacia complex (BCC), and methicillin-resistant S. aureus (MRSA) obtained from CF sputum samples. Bacterial viability was assessed by colony forming units. Selective detoxification of HOSeCN was determined by comparing its metabolism by mammalian thioredoxin reductase (TrxR) to bacterial TrxR following the consumption of NADPH. We also assessed potential toxicity of equivalent HOSeCN generation, which demonstrated in situ antimicrobial activity, in human bronchial epithelial cells with a cell viability assay. The -SeCN/HOSeCN system was much more potent than -SCN/HOSCN system at killing PA, BCC and MRSA isolates. The -SeCN/HOSeCN system was more effective at killing -SCN/HOSCN resistant isolates. Mammalian TrxR selectively detoxified HOSeCN whereas the bacterial TrxR enzyme showed little activity. Human bronchial epithelial cells exposed to equivalent flux of HOSeCN that killed several CF pathogens showed no decrease in viability. -SeCN may be an effective therapeutic for the treatment of CF lung pathogens that are difficult to treat with current antibiotics.

Myeloperoxidase oxidation of methionine associates with early cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease progressively worsens from infancy to adulthood. Disease-driven changes in early CF airway fluid metabolites may identify therapeutic targets to curb progression.CF patients aged 12-38 months (n=24; three out of 24 later denoted as CF screen positive, inconclusive diagnosis) received chest computed tomography scans, scored by the Perth-Rotterdam Annotated Grid Morphometric Analysis for CF (PRAGMA-CF) method to quantify total lung disease (PRAGMA-%Dis) and components such as bronchiectasis (PRAGMA-%Bx). Small molecules in bronchoalveolar lavage fluid (BALF) were measured with high-resolution accurate-mass metabolomics. Myeloperoxidase (MPO) was quantified by ELISA and activity assays.Increased PRAGMA-%Dis was driven by bronchiectasis and correlated with airway neutrophils. PRAGMA-%Dis correlated with 104 metabolomic features (p<0.05, q<0.25). The most significant annotated feature was methionine sulfoxide (MetO), a product of methionine oxidation by MPO-derived oxidants. We confirmed the identity of MetO in BALF and used reference calibration to confirm correlation with PRAGMA-%Dis (Spearman's ρ=0.582, p=0.0029), extending to bronchiectasis (PRAGMA-%Bx; ρ=0.698, p=1.5×10-4), airway neutrophils (ρ=0.569, p=0.0046) and BALF MPO (ρ=0.803, p=3.9×10-6).BALF MetO associates with structural lung damage, airway neutrophils and MPO in early CF. Further studies are needed to establish whether methionine oxidation directly contributes to early CF lung disease and explore potential therapeutic targets indicated by these findings.

Exploiting the Catalytic Diversity of Short-Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope.

Numerous short-chain dehydrogenases/reductases (SDRs) have found biocatalytic applications in C=O and C=C (enone) reduction. For NADPH-dependent C=N reduction, imine reductases (IREDs) have primarily been investigated for extension of the substrate range. Here, we show that SDRs are also suitable for a broad range of imine reductions. The SDR noroxomaritidine reductase (NR) is involved in Amaryllidaceae alkaloid biosynthesis, serving as an enone reductase. We have characterized NR by using a set of typical imine substrates and established that the enzyme is active with all four tested imine compounds (up to 99 % conversion, up to 92 % ee). Remarkably, NR reduced two keto compounds as well, thus highlighting this enzyme family's versatility. Using NR as a template, we have identified an as yet unexplored SDR from the Amaryllidacea Zephyranthes treatiae with imine-reducing activity (≤95 % ee). Our results encourage the future characterization of SDR family members as a means of discovering new imine-reducing enzymes.

The Amaryllidaceae alkaloids: biosynthesis and methods for enzyme discovery.

Amaryllidaceae alkaloids are an example of the vast diversity of secondary metabolites with great therapeutic promise. The identification of novel compounds in this group with over 300 known structures continues to be an area of active study. The recent identification of norbelladine 4'-O-methyltransferase (N4OMT), an Amaryllidaceae alkaloid biosynthetic enzyme, and the assembly of transcriptomes for Narcissus sp. aff. pseudonarcissus and Lycoris aurea highlight the potential for discovery of Amaryllidaceae alkaloid biosynthetic genes with new technologies. Recent technical advances of interest include those in enzymology, next generation sequencing, genetic modification, nuclear magnetic resonance spectroscopy (NMR), and mass spectrometry (MS).

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities.

Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.

CYP96T1 of Narcissus sp. aff. pseudonarcissus Catalyzes Formation of the Para-Para' C-C Phenol Couple in the Amaryllidaceae Alkaloids.

The Amaryllidaceae alkaloids are a family of amino acid derived alkaloids with many biological activities; examples include haemanthamine, haemanthidine, galanthamine, lycorine, and maritidine. Central to the biosynthesis of the majority of these alkaloids is a C-C phenol-coupling reaction that can have para-para', para-ortho', or ortho-para' regiospecificity. Through comparative transcriptomics of Narcissus sp. aff. pseudonarcissus, Galanthus sp., and Galanthus elwesii we have identified a para-para' C-C phenol coupling cytochrome P450, CYP96T1, capable of forming the products (10bR,4aS)-noroxomaritidine and (10bS,4aR)-noroxomaritidine from 4'-O-methylnorbelladine. CYP96T1 was also shown to catalyzed formation of the para-ortho' phenol coupled product, N-demethylnarwedine, as less than 1% of the total product. CYP96T1 co-expresses with the previously characterized norbelladine 4'-O-methyltransferase. The discovery of CYP96T1 is of special interest because it catalyzes the first major branch in Amaryllidaceae alkaloid biosynthesis. CYP96T1 is also the first phenol-coupling enzyme characterized from a monocot.

Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.