Piperazate-Guided Isolation of Caveamides A and B, Cyclohexenylalanine-Containing Nonribosomal Peptides from a Cave Actinomycete.

Hybrid genome-mining/15N-NMR was used to target compounds containing piperazate (Piz) residues, leading to the discovery of caveamides A (1) and B (2) from Streptomyces sp. strain BE230, isolated from New Rankin Cave (Missouri). Caveamides are highly dynamic molecules containing an unprecedented ß-ketoamide polyketide fragment, two Piz residues, and a new N-methyl-cyclohexenylalanine residue. Caveamide B (2) exhibited nanomolar cytotoxicity against several cancer cell lines and nanomolar antimicrobial activity against MRSA and E. coli.

New reactions by pyridoxal phosphate-dependent enzymes.

Pyridoxal phosphate (PLP) is a cofactor that is widely employed in enzymology. This pyridine-containing cofactor can be used for reactions ranging from transaminations to oxidations. The catalytic versatility can be understood by considering the chemical features of this cofactor. In recent years, exciting new reactions involving PLP have been discovered in natural products biosynthesis, upending our understanding of what this cofactor is capable of. Here we review some of the most exciting PLP-dependent reactions from the last five years.

Two Iron(II), α-Ketoglutarate-Dependent Enzymes Encoded by the PPZ Gene Cluster of Metarhizium majus Enable Production of 8-Hydroxyperamine.

Entomopathogenic fungus Metarhizium majus contains the nine-gene PPZ cluster, with ppzA, encoding a peramine-producing nonribosomal peptide synthetase, as the central component. In this work, the roles of two α-ketoglutarate, iron-dependent oxygenases encoded by the PPZ genes ppzC and ppzD were elucidated. PpzD was found to produce both trans-4-hydroxy-l-proline and trans-3-hydroxy-l-proline in a 13.1:1 ratio, yielding a key precursor for peramine biosynthesis. PpzC was found to act directly on peramine, yielding the novel analogue 8-hydroxyperamine.

Childhood, adolescent, and adult primary brain and central nervous system tumor statistics for practicing healthcare providers in neuro-oncology, CBTRUS 2015-2019.

Background: The Central Brain Tumor Registry of the United States (CBTRUS), in collaboration with the Centers for Disease Control and Prevention (CDC) and National Cancer Institute (NCI), is the largest aggregation of histopathology-specific population-based data for primary brain and other central nervous system (CNS) in the US. CBTRUS publishes an annual statistical report which provides critical reference data for the broad neuro-oncology community. Here, we summarize the key findings from the 2022 CBTRUS annual statistical report for healthcare providers. Methods: Incidence data were obtained from the CDC's National Program of Cancer Registries (NPCR) and NCI's Surveillance, Epidemiology, and End Results Program for 52 central cancer registries (CCRs). Survival data were obtained from 42 NPCR CCRs. All rates are per 100 000 and age-adjusted using the 2000 US standard population. Overall median survival was estimated using Kaplan-Meier models. Survival data for selected molecularly defined histopathologies are from the National Cancer Database. Mortality data are from the National Vital Statistics System. Results: The average annual age-adjusted incidence rate of all primary brain and other CNS tumors was 24.25/100 000. Incidence was higher in females and non-Hispanics. The most commonly occurring malignant and predominately non-malignant tumors was glioblastoma (14% of all primary brain tumors) and meningioma (39% of all primary brain tumors), respectively. Mortality rates and overall median survival varied by age, sex, and histopathology. Conclusions: This summary describes the most up-to-date population-based incidence, mortality, and survival, of primary brain and other CNS tumors in the US and aims to serve as a concise resource for neuro-oncology providers.

An imine reductase that captures reactive intermediates in the biosynthesis of the indolocarbazole reductasporine.

Imine reductases (IREDs) and reductive aminases have been used in the synthesis of chiral amine products for drug manufacturing; however, little is known about their biological contexts. Here we employ structural studies and site-directed mutagenesis to interrogate the mechanism of the IRED RedE from the biosynthetic pathway to the indolocarbazole natural product reductasporine. Cocrystal structures with the substrate-mimic arcyriaflavin A reveal an extended active site cleft capable of binding two indolocarbazole molecules. Site-directed mutagenesis of a conserved aspartate in the primary binding site reveals a new role for this residue in anchoring the substrate above the NADPH cofactor. Variants targeting the secondary binding site greatly reduce catalytic efficiency, while accumulating oxidized side-products. As indolocarbazole biosynthetic intermediates are susceptible to spontaneous oxidation, we propose the secondary site acts to protect against autooxidation, and the primary site drives catalysis through precise substrate orientation and desolvation effects. The structure of RedE with its extended active site can be the starting point as a new scaffold for engineering IREDs and reductive aminases to intercept large substrates relevant to industrial applications.

Structure of methyltransferase RedM that forms the dimethylpyrrolinium of the bisindole reductasporine.

Bisindoles are biologically active natural products that arise from the oxidative dimerization of two molecules of l-tryptophan. In bacterial bisindole pathways, a core set of transformations is followed by the action of diverse tailoring enzymes that catalyze reactions that lead to diverse bisindole products. Among bisindoles, reductasporine is distinct due to its dimethylpyrrolinium structure. Its previously reported biosynthetic gene cluster encodes two unique tailoring enzymes, the imine reductase RedE and the dimethyltransferase RedM, which were shown to produce reductasporine from a common bisindole intermediate in recombinant E. coli. To gain more insight into the unique tailoring enzymes in reductasporine assembly, we reconstituted the biosynthetic pathway to reductasporine in vitro and then solved the 1.7 Å resolution structure of RedM. Our work reveals RedM adopts a variety of conformational changes with distinct open and closed conformations, and site-directed mutagenesis alongside sequence analysis identifies important active site residues. Finally, our work sets the stage for understanding how RedM evolved to react with a pyrrolinium scaffold and may enable the development of new dimethyltransferase catalysts.

Idiopathic granulomatous mastitis: a 5-year retrospective review of cases in a tertiary centre in Dublin, Ireland.

AIMS: Idiopathic granulomatous mastitis (IGM) is a rare, benign, inflammatory breast disorder of unknown aetiology usually affecting women of reproductive age. It classically presents as a unilateral painful breast mass. It is frequently mistaken for carcinoma or other inflammatory breast diseases. Diagnostic investigations include clinical examination, appropriate imaging and tissue sampling. A link between IGM and infection with the Corynebacterium species in particular Corynebacterium kroppenstedtii has been described. METHODS: A retrospective single-centre cohort study was conducted over a 5-year period (2017-2022); all cases of IGM were identified. RESULTS: Forty-one patients were diagnosed with IGM. Breast lump was the most common presenting complaint (n=29). The average age was 45 years. Eighteen patients had samples sent for culture and sensitivity, 11 of which had positive microbiology results indicative of Corynebacterium spp infection.An 82% resolution rate (27 of 33) was recorded in those who received either a short-antibiotic course or none at all. Eight patients reported persistent disease at 3 months, five of which had evidence of Corynebacterium spp. DISCUSSION: This 5-year review highlights the impact of IGM in a tertiary centre in Dublin, Ireland. Although no treatment guidelines exist, options include antibiotics, immunomodulators and surgery. Due to risk of fistulae and unfavourable cosmetic outcomes, surgery should be reserved for refractory IGM. We suspect that there may be a subset of patients where prolonged antibiotic therapy should be considered. Defining this subgroup requires further study, but likely includes those with cystic neutrophilic granulomatous mastitis, relapsing disease and in whom Corynebacterium spp is recovered.

Parental Support and Adolescents' Coping with Academic Stressors: A Longitudinal Study of Parents' Influence Beyond Academic Pressure and Achievement.

Adolescents face many academic pressures that require good coping skills, but coping skills can also depend on social resources, such as parental support and fewer negative interactions. The aim of this study was to determine if parental support and parental negative interactions concurrently and longitudinally relate to adolescents' ways of academic coping, above and beyond the impact of three types of academic stress, students' achievement at school (i.e., grades in school), and age. Survey data were collected from 839 Australian students in grades 5 to 10 (Mage = 12.2, SD = 1.72; 50% girls). Students completed measures of support and negative interactions with parents; academic stress from workload, external pressure (teachers/parents) to achieve, and intrapsychic pressure for high achievement; and ways of academic coping that were grouped into two positive and two negative types. Hypothesized associations were tested concurrently and from one year to the next using path modeling. Beyond the numerous significant influences of academic stress and achievement on coping, and control for age and COVID-19 timing, adolescents with more parental support reported more use of engagement coping (e.g., strategizing) and comfort-seeking, whereas those who reported more negative interactions with parents reported more use of disengagement coping (e.g., concealment) and escape. In the longitudinal model, parental support predicted an increase in engagement and comfort-seeking and a decrease in disengagement coping, whereas negative interaction with parents predicted an increase in disengagement coping. Overall, the findings support the view that coping with academic stressors will continue to depend on parent-adolescent relationships even into the teen years.

Structure of the Oxygen, Pyridoxal Phosphate-Dependent Capuramycin Biosynthetic Protein Cap15.

Pyridoxal phosphate-dependent enzymes able to use oxygen as a co-substrate have emerged in multiple protein families. Here, we use crystallography to solve the 2.40 Å resolution crystal structure of Cap15, a nucleoside biosynthetic enzyme that catalyzes the oxidative decarboxylation of glycyl uridine. Our structural study captures the internal aldimine, pinpointing the active site lysine as K230 and showing the site of phosphate binding. Our docking studies reveal how Cap15 is able to catalyze a stereoselective deprotonation reaction, and bioinformatic analysis reveals active site residues that distinguish Cap15 from the structurally related d-glucosaminate-6-phosphate ammonia lyase and l-seryl-tRNA(Sec) selenium transferase (SelA). Our work provides the structural basis for further mechanistic investigation of a unique biosynthetic enzyme and provides a blueprint for understanding how oxygen reactivity emerged in the SelA-like protein family.

Reductases Produce Nitric Oxide in an Alternative Pathway to Form the Diazeniumdiolate Group of l-Alanosine.

l-Alanosine is a diazeniumdiolate (N-nitrosohydroxylamine) antibiotic that inhibits MTAP-deficient tumor cells by blocking de novo adenine biosynthesis. Previous work revealed the early steps in the biosynthesis of l-alanosine. In the present study, we used genome mining to discover two new l-alanosine-producing strains that lack the aspartate-nitrosuccinate pathway genes found in the original l-alanosine producer. Instead, nitrate is reduced with a unique set of nitrate-nitrite reductases. These enzymes are typically used as part of the nitrogen cycle for denitrification or assimilation, and our report here shows how enzymes from the nitrogen cycle can be repurposed for the biosynthesis of specialized metabolites. The widespread distribution of nitric-oxide-producing reductases also indicates a potential for the discovery of new nitric-oxide-derived natural products.

Development of a Model to Identify Febrile Children at Low Risk for Multisystem Inflammatory Syndrome.

OBJECTIVES: The case definition for multisystem inflammatory syndrome in children (MIS-C) is broad and encompasses symptoms and signs commonly seen in children with fever. Our aim was to identify clinical predictors that, independently or in combination, identify febrile children presenting to the emergency department (ED) as low risk for MIS-C. METHODS: We conducted a retrospective single-center study of otherwise healthy children 2 months to 20 years of age presenting to the ED with fever and who had a laboratory evaluation for MIS-C between April 15, 2020, and October 31, 2020. We excluded children with a diagnosis of Kawasaki disease. Our outcome was an MIS-C diagnosis defined by the Centers for Disease Control and Prevention criteria. We conducted multivariable logistic regression analyses to identify variables independently associated with MIS-C. RESULTS: Thirty-three patients with and 128 patients without MIS-C were analyzed. Of those with MIS-C, 16 of 33 (48.5%) had hypotension for age, signs of hypoperfusion, or required ionotropic support. Four variables were independently associated with the presence of MIS-C; known or suspected SARS CoV-2 exposure (adjusted odds ratio [aOR], 4.0; 95% confidence interval [CI], 1.4-11.9) and the following 3 symptoms and signs: abdominal pain on history (aOR, 4.8; 95% CI, 1.7-15.0), conjunctival injection (aOR, 15.2; 95% CI, 5.4-48.1), and rash involving the palms or soles (aOR, 12.2; 95% CI, 2.4-69.4). Children were at low risk of MIS-C if none of the 3 symptoms or signs were present (sensitivity 87.9% [95% CI, 71.8-96.6]; specificity 62.5% [53.5-70.9], negative predictive value 95.2% [88.3-98.7]). Of the 4 MIS-C patients without any of these 3 factors, 2 were ill-appearing in the ED and the other 2 had no cardiovascular involvement during their clinical course. CONCLUSIONS: A combination of 3 clinical symptoms and signs had moderate to high sensitivity and high negative predictive value for identifying febrile children at low risk of MIS-C. If validated, these factors could aid clinicians in determining the need to obtain or forego an MIS-C laboratory evaluation during SARS-CoV-2 prevalent periods in febrile children.

Approximating exposure therapy in the lab: Replacing the CS+ with a similar versus a different stimulus and including additional stimuli resembling the CS+ during extinction.

Recent studies have shown that extinction training including the conditional stimulus (CS+) and stimuli that are similar to the CS + enhances extinction retention and generalisation to novel stimuli. However, in a clinical setting, the CS+ is rarely available for use during exposure therapy. The aim of the present study was to determine if replacing the CS+ with a similar versus different stimulus, and including other similar stimuli during extinction, could reduce fear at test on par with extinction using the original CS+ with and without other similar stimuli. In an experiment conducted in a single session, participants completed a habituation phase followed by an acquisition phase using two dog images presented with (CS+) and without (CS-) an acoustic unconditional stimulus (US). Participants were randomly allocated to four extinction conditions: similar CS + dog with novel dog images (Similar replacement extinction condition); different CS + dog with novel dog images (Different replacement extinction condition); original CS + dog with novel dog images (Multiple extinction control condition); and original CS + without novel dog images (Standard extinction control condition). All participants completed a test phase with the original CSs followed by a generalisation test with another two novel dog images. All groups acquired, and then extinguished differential skin conductance responses (SCRs) with no differences observed between groups. Whereas the Similar replacement extinction group and the Multiple and Standard extinction control groups did not exhibit significant differential SCRs when re-exposed to the original CS + relative to the CS- at test, differential responding to the CSs was significant at test in the Different replacement extinction group. There were no significant differences between groups in SCRs to the two novel dog images during the generalisation phase and in between-phase subjective ratings. Findings suggest that replacement stimuli used during extinction should be as similar as possible to the CS + to reduce physiological arousal to the original CS+.

Identification of the Azaserine Biosynthetic Gene Cluster Implicates Hydrazine as an Intermediate to the Diazo Moiety.

Azaserine (1) is a natural product and nonproteinogenic amino acid containing a diazo group. Here we report the biosynthetic gene cluster for 1 from Glycomyces harbinensis. We then use isotopic feeding, gene deletion, and biochemical experiments to support a pathway whereby hydrazinoacetic acid (2) and a peptidyl carrier protein-loaded serine (3) are intermediates on route to the final natural product 1.

Fear learning and extinction predicts anxiety in daily life: a study of Pavlovian conditioning and ecological momentary assessment.

BACKGROUND: The association between anxious mood and aberrant fear learning mechanisms has not been fully elucidated. Studying how fear conditioning and extinction constructs relate to anxiety symptoms and reactivity to stressful and benign moments in everyday life provides a powerful addition to experimental paradigms. METHOD: Fifty-one young adults completed laboratory-based differential conditioning and extinction tasks with (CS + ) and without (CS-) an aversive unconditional stimulus (US). Electrodermal skin conductance responses were measured during each phase, followed by ecological momentary assessment (EMA) tapping anxiety and stressors six times daily for seven days (2, 142 moments). RESULTS: Conditioned electrodermal reactivity to the CS + and overgeneralisation to the CS- were associated with greater change in anxiety (measured via EMA), across non-stressful situations, remaining the same across stressful situations. Likewise, during extinction when the CS + is now safe, more electrodermal reactivity to the CS + was associated with more anxiety change across non-stressful situations and remained the same across stressful situations. Also, during extinction when threat is absent, more electrodermal reactivity at the late stage of the CS- was associated with less momentary anxiety change in response to stressful situations; more electrodermal activity at the late stage of the CS + was associated with more anxiety change across non-stressful situations and remained the same across stressful situations. CONCLUSIONS: Sampling 'in vivo' emotion and stress experiences, study findings revealed links between conditioned electrodermal reactivity and overgeneralisation to safe stimuli and heightened anxious reactivity during non-stressful (i.e. safe) moments in daily life, coupled with less change in response to actual stressors.

CBTRUS Statistical Report: Pediatric Brain Tumor Foundation Childhood and Adolescent Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2014-2018.

The CBTRUS Statistical Report: Pediatric Brain Tumor Foundation Childhood and Adolescent Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2014-2018 comprehensively describes the current population-based incidence of primary malignant and non-malignant brain and other CNS tumors in children and adolescents ages 0-19 years, collected and reported by central cancer registries covering approximately 100% of the United States population. Overall, brain and other CNS tumors are the most common solid tumor, the most common cancer, and the most common cause of cancer death in children and adolescents ages 0-19 years. This report aims to serve as a useful resource for researchers, clinicians, patients, and families.

Clinical insights from Wolman disease: Evaluating infantile hepatosplenomegaly.

There is a broad differential diagnosis of infantile hepatosplenomegaly, with some etiologies being debilitating and treatable. A structured approach to history, examination, and laboratory and radiographic findings is important in diagnosis. Herein, we present a case of Wolman disease presenting as hepatosplenomegaly in an infant. This case details important learning points to help distinguish the diagnosis of Wolman disease from other conditions with overlapping clinical features, such as hemophagocytic lymphohistiocytosis (HLH). The advent of enzyme replacement therapy has dramatically changed the natural history of Wolman disease, and this child showed remarkable improvement with treatment. This child was later found to have extensive adenopathy with retroperitoneal lymph node biopsy demonstrating diffuse infiltration by lipid-laden macrophages, fatty deposits, cholesterol crystals, and calcifications. Similar to the collection of characteristic cells in other lysosomal storage disorders, we postulate that this is characteristic of underlying Wolman disease. We conclude with a summary of learning points from this presentation on infantile hepatosplenomegaly, pertinent to the geneticist, pediatrician, and pediatric subspecialists.

Free Piperazic Acid as a Precursor to Nonribosomal Peptides.

Piperazic acid (Piz) is a nonproteinogenic amino acid possessing a rare nitrogen-nitrogen bond. However, little is known about how Piz is incorporated into nonribosomal peptides, including whether adenylation domains specific to Piz exist. In this study, we show that free piperazic acid is directly adenylated and then incorporated into the incarnatapeptin nonribosomal peptides through isotopic incorporation studies. We also use in vitro reconstitution to demonstrate adenylation of free piperazic acid with a three-domain nonribosomal peptide synthetase from the incarnatapeptin gene cluster. We furthermore use bioinformatics and site-directed mutagenesis to outline consensus sequences for the adenylation of piperazic acid, which can now be used for the prediction of gene clusters linked to piperazic-acid-containing peptides. Finally, we discover a fusion protein of a piperazate synthase and an adenylation domain, highlighting the close biosynthetic relationship of piperazic acid formation and its adenylation. Altogether, our work demonstrates the evolution of biosynthetic systems for the activation of free piperazic acid through adenylation, a pathway we suggest is likely to be employed in the majority of pathways to piperazic-acid-containing peptides.

Natural Products Produced in Culture by Biosynthetically Talented Salinispora arenicola Strains Isolated from Northeastern and South Pacific Marine Sediments.

Laboratory cultures of two 'biosynthetically talented' bacterial strains harvested from tropical and temperate Pacific Ocean sediment habitats were examined for the production of new natural products. Cultures of the tropical Salinispora arenicola strain RJA3005, harvested from a PNG marine sediment, produced salinorcinol (3) and salinacetamide (4), which had previously been reported as products of engineered and mutated strains of Amycolatopsis mediterranei, but had not been found before as natural products. An S. arenicola strain RJA4486, harvested from marine sediment collected in the temperate ocean waters off British Columbia, produced the new aminoquinone polyketide salinisporamine (5). Natural products 3, 4, and 5 are putative shunt products of the widely distributed rifamycin biosynthetic pathway.

Synthetic and biosynthetic routes to nitrogen-nitrogen bonds.

The nitrogen-nitrogen bond is a core feature of diverse functional groups like hydrazines, nitrosamines, diazos, and pyrazoles. Such functional groups are found in >300 known natural products. Such N-N bond-containing functional groups are also found in significant percentage of clinical drugs. Therefore, there is wide interest in synthetic and enzymatic methods to form nitrogen-nitrogen bonds. In this review, we summarize synthetic and biosynthetic approaches to diverse nitrogen-nitrogen-bond-containing functional groups, with a focus on biosynthetic pathways and enzymes.

Examining the Process of Implementing a Three-Step Mental Health and Wellbeing System of Care for Children and Adolescents Across Multiple Community Settings.

Mental health problems affect large numbers of young people. Integrated systems are required that can be applied in diverse settings to reach youth 'where they are'. We evaluated the process of implementing a three-step youth mental health and wellbeing system in diverse community settings according to three implementation outcomes: feasibility, penetration and acceptability. The study describes 49 applications of the 'Life-Fit-Learning system' designed to assess the mental health and wellbeing of youth (Assess step), provide feedback on assessment results (Reflect step), and connect them to resources and services proportionate to their needs (Connect step). Within a participatory research approach, 3798 administrations were conducted with youth between 9 and 18 years and 90 administrations were conducted with adults. Implementation was based on the four phases of the Quality Implementation Framework and was staged to integrate stakeholder and consumer feedback and experience gained from focus groups and two pilot phases before full implementation. Feasibility ratings of successful implementation ranged from 86.7 to 96.4% across applications and settings. High penetration rates were achieved. The Life-Fit-Learning system successfully reached 91.9% to 96% of youth with the Assess and Reflect steps and low intensity Connect step resources. Of those, 14.7% to 23% were identified at-risk for mental health problems and 93% to 97% of those at-risk youth additionally received Connect step co-delivered group-based programs (moderate intensity care) and/or individual treatment (high intensity care). Youth and parents reported high satisfaction across all steps and delivery modes. With strong collaboration, an integrated model of care can be delivered feasibly, effectively and satisfactorily to reach large numbers of young people across settings.