Practical Spectrophotometric Assay for the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Succinyl-L,L-Diaminopimelic Acid Desuccinylase, a Potential Antibiotic Target

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics

Indoline‐6‐Sulfonamide Inhibitors of the Bacterial Enzyme DapE

Inhibitors of the bacterial enzyme dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18) hold promise as antibiotics with a new mechanism of action. Herein we describe the discovery of a new series of indoline sulfonamide DapE inhibitors from a high-throughput screen and the synthesis of a series of analogs. Inhibitory potency was measured by a ninhydrin-based DapE assay recently developed by our group. Molecular docking experiments suggest active site binding with the sulfonamide acting as a zinc-binding group (ZBG)

Definitions, Criteria and Global Classification of Mast Cell Disorders with Special Reference to Mast Cell Activation Syndromes: A Consensus Proposal

Activation of tissue mast cells (MCs) and their abnormal growth and accumulation in various organs are typically found in primary MC disorders also referred to as mastocytosis. However, increasing numbers of patients are now being informed that their clinical findings are due to MC activation (MCA) that is neither associated with mastocytosis nor with a defined allergic or inflammatory reaction. In other patients with MCA, MCs appear to be clonal cells, but criteria for diagnosing mastocytosis are not met. A working conference was organized in 2010 with the aim to define criteria for diagnosing MCA and related disorders, and to propose a global unifying classification of all MC disorders and pathologic MC reactions. This classification includes three types of `MCA syndromes' (MCASs), namely primary MCAS, secondary MCAS and idiopathic MCAS. MCA is now defined by robust and generally applicable criteria, including (1) typical clinical symptoms, (2) a substantial transient increase in serum total tryptase level or an increase in other MC-derived mediators, such as histamine or prostaglandin D 2, or their urinary metabolites, and (3) a response of clinical symptoms to agents that attenuate the production or activities of MC mediators. These criteria should assist in the identification and diagnosis of patients with MCAS, and in avoiding misdiagnoses or overinterpretation of clinical symptoms in daily practice. Moreover, the MCAS concept should stimulate research in order to identify and exploit new molecular mechanisms and therapeutic targets. Copyright (C) 2011 S. Karger AG, Base

Standards of genetic testing in the diagnosis and prognostication of systemic mastocytosis in 2022: Recommendations of the EU-US cooperative group

Mastocytosis comprises rare heterogeneous diseases characterized by an increased accumulation of abnormal mast cells in various organs/tissues. The pathogenesis of mastocytosis is strongly linked to the presence of KIT-activating mutations. In systemic mastocytosis (SM), the most frequent mutation encountered is KIT p.D816V, whose presence constitutes one of the minor diagnostic criteria. Different techniques are used to search and quantify the KIT p.D816V mutant; however, allele-specific quantitative PCR and droplet digital PCR are today the most sensitive. The analysis of the KIT p.D816V allele burden has undeniable interest for diagnostic, prognostic, and therapeutic monitoring. The analysis of non–mast cell hematological compartments in SM is similarly important because KIT p.D816V multilineage involvement is associated with a worse prognosis. In addition, in advanced forms of SM, mutations in genes other than KIT are frequently identified and affect negatively disease outcome and response to therapy. Thus, combined quantitative and sensitive analysis of KIT mutations and next-generation sequencing of other recurrently involved myeloid genes make it possible to better characterize the extent of the affected cellular compartments and additional molecular aberrations, providing a more detailed overview of the complex mutational landscape of SM, in relation with the clinical heterogeneity of the disease. In this article, we report the latest recommendations of the EU-US Cooperative Group presented in September 2020 in Vienna during an international working conference, on the techniques we consider standard to detect and quantify the KIT p.D816V mutant in SM and additional myeloid mutations found in SM subtypes.D.D.M., J.J.L., and M.C.C. were supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. P.V. was supported by the Austrian Science Fund (FWF) (grant nos. F4704-B20 and P32470-B)

Standards of pathology in the diagnosis of systemic mastocytosis: recommendations of the EU-US cooperative group

Pathology plays a central role in the diagnosis of systemic mastocytosis (SM), its delineation from other neoplasms and reactive conditions, and in monitoring of SM under therapy. The morphologic hallmark of SM is the accumulation of spindle-shaped, hypogranulated mast cells (MCs) in bone marrow (BM) and other extracutaneous tissues. Four of the 5 World Health Organization–defined diagnostic criteria (ie, compact MC aggregates [=major criterion]; atypical MC morphology; activating KIT point mutations; aberrant expression of CD25 and/or CD2 and/or CD30 in MCs [=minor criteria]) can be addressed by the pathologist. The final classification of SM variants as either BM mastocytosis, indolent SM, smoldering SM, aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN), or MC leukemia (MCL) has important prognostic significance and requires the integration of certain morphological, clinical, radiological, and biochemical data, referred to as B- and C-findings. Substantial diagnostic challenges may be posed to the pathologist and clinician especially in the so-called advanced SM variants, that is, ASM, MCL, and SM-AHN. In this article, updated recommendations of the EU-US Cooperative Group regarding standards of pathology in the diagnosis of SM, presented during the year 2020 Working Conference held in September in Vienna, are reported.T. I. George was supported by the ARUP Institute for Clinical and Experimental Pathology. K. Hartmann was supported by the Swiss National Science Foundation, grant number 310030_207705. D. D. Metcalfe, J. J. Lyons, and M. Carter were supported by the Division of Intramural Research, National Institutes of Allergic and Infectious Diseases, National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not represent the official views of the NIH. P. Valent was supported by the Austrian Science Funds (FWF), projects F4701-B20 and F4704-B20

Genomic Characterization of Methanomicrobiales Reveals Three Classes of Methanogens

BACKGROUND:Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. METHODOLOGY/PRINCIPAL FINDINGS:In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. CONCLUSIONS/SIGNIFICANCE:Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III)

Narcissism and the strategic pursuit of short-term mating : universal links across 11 world regions of the International Sexuality Description Project-2.

Previous studies have documented links between sub-clinical narcissism and the active pursuit of short-term mating strategies (e.g., unrestricted sociosexuality, marital infidelity, mate poaching). Nearly all of these investigations have relied solely on samples from Western cultures. In the current study, responses from a cross-cultural survey of 30,470 people across 53 nations spanning 11 world regions (North America, Central/South America, Northern Europe, Western Europe, Eastern Europe, Southern Europe, Middle East, Africa, Oceania, Southeast Asia, and East Asia) were used to evaluate whether narcissism (as measured by the Narcissistic Personality Inventory; NPI) was universally associated with short-term mating. Results revealed narcissism scores (including two broad factors and seven traditional facets as measured by the NPI) were functionally equivalent across cultures, reliably associating with key sexual outcomes (e.g., more active pursuit of short-term mating, intimate partner violence, and sexual aggression) and sex-related personality traits (e.g., higher extraversion and openness to experience). Whereas some features of personality (e.g., subjective well-being) were universally associated with socially adaptive facets of Narcissism (e.g., self-sufficiency), most indicators of short-term mating (e.g., unrestricted sociosexuality and marital infidelity) were universally associated with the socially maladaptive facets of narcissism (e.g., exploitativeness). Discussion addresses limitations of these cross-culturally universal findings and presents suggestions for future research into revealing the precise psychological features of narcissism that facilitate the strategic pursuit of short-term mating