Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

ENDOR Spectroscopy Reveals the "Free" 5'-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe-4S]2+ Cluster.

1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.

Tumor-Associated Macrophages Expand Chemoresistant, Ovarian Cancer Stem-Like Cells.

The persistence of ovarian cancer stem-like cells (OvCSCs) after chemotherapy resistance has been implicated in relapse. However, the ability of these relatively quiescent cells to produce the robust tumor regrowth necessary for relapse remains an enigma. Since normal stem cells exist in a niche, and tumor-associated macrophages (TAMs) are the highest abundance immune cell within ovarian tumors, we hypothesized that TAMs may influence OvCSC proliferation. To test this, we optimized OvCSC enrichment by sphere culture and in vitro polarization of monocytes to a TAM-like M2 phenotype. Using cocultures that permitted the exchange of only soluble factors, we found that M2 macrophages increased the proliferation of sphere cells. Longer-term exposure (5-7 days) to soluble TAM factors led to retention of some stem cell features by OvCSCs but loss of others, suggesting that TAMs may support an intermediate stemness phenotype in OvCSCs. Although TAM coculture decreased the percentage of OvCSCs surviving chemotherapy, it increased the overall number. We therefore sought to determine the influence of this interaction on chemotherapy efficacy in vivo and found that inhibiting macrophages improved chemotherapy response. Comparing the gene expression changes in OvCSCs cocultured with TAMs to publicly available patient data identified 34 genes upregulated in OvCSCs by exposure to soluble TAM factors whose expression correlates with outcome. Overall, these data suggest that TAMs may influence OvCSC proliferation and impact therapeutic response.

Computational Description of Alkylated Iron-Sulfur Organometallic Clusters.

The radical S-adenosyl methionine (SAM) enzyme superfamily has widespread roles in hydrogen atom abstraction reactions of crucial biological importance. In these enzymes, reductive cleavage of SAM bound to a [4Fe-4S]1+ cluster generates the 5'-deoxyadenosyl radical (5'-dAdo•) which ultimately abstracts an H atom from the substrate. However, overwhelming experimental evidence has surprisingly revealed an obligatory organometallic intermediate Ω exhibiting an Fe-C5'-adenosyl bond, whose properties are the target of this theoretical investigation. We report a readily applied, two-configuration version of broken symmetry DFT, denoted 2C-DFT, designed to allow the accurate description of the hyperfine coupling constants and g-tensors of an alkyl group bound to a multimetallic iron-sulfur cluster. This approach has been validated by the excellent agreement of its results both with those of multiconfigurational complete active space self-consistent field computations for a series of model complexes and with the results from electron nuclear double-resonance/electron paramagnetic resonance spectroscopic studies for the crystallographically characterized complex, M-CH3, a [4Fe-4S] cluster with a Fe-CH3 bond. The likewise excellent agreement between spectroscopic results and 2C-DFT computations for Ω confirm its identity as an organometallic complex with a bond between an Fe of the [4Fe-4S] cluster and C5' of the deoxyadenosyl moiety, as first proposed.

Characterization by ENDOR Spectroscopy of the Iron-Alkyl Bond in a Synthetic Counterpart of Organometallic Intermediates in Radical SAM Enzymes.

Members of the radical S-adenosyl-l-methionine (SAM) enzyme superfamily initiate a broad spectrum of radical transformations through reductive cleavage of SAM by a [4Fe-4S]1+ cluster it coordinates to generate the reactive 5'-deoxyadenosyl radical (5'-dAdo•). However, 5'-dAdo• is not directly liberated for reaction and instead binds to the unique Fe of the cluster to create the catalytically competent S = 1/2 organometallic intermediate Ω. An alternative mode of reductive SAM cleavage, especially seen photochemically, instead liberates CH3•, which forms the analogous S = 1/2 organometallic intermediate with an Fe-CH3 bond, ΩM. The presence of a covalent Fe-C bond in both structures was established by the ENDOR observation of 13C and 1H hyperfine couplings to the alkyl groups that show isotropic components indicative of Fe-C bond covalency. The synthetic [Fe4S4]3+-CH3 cluster, M-CH3, is a crystallographically characterized analogue to ΩM that exhibits the same [Fe4S4]3+ cluster state as Ω and ΩM, and thus an analysis of its spectroscopic properties─and comparison with those of Ω and ΩM─can be grounded in its crystal structure. We report cryogenic (2 K) EPR and 13C/1/2H ENDOR measurements on isotopically labeled M-CH3. At low temperatures, the complex exhibits EPR spectra from two distinct conformers/subpopulations. ENDOR shows that at 2 K, one contains a static methyl, but in the other, the methyl undergoes rapid tunneling/hopping rotation about the Fe-CH3 bond. This generates an averaged hyperfine coupling tensor whose analysis requires an extended treatment of rotational averaging. The methyl group 13C/1/2H hyperfine couplings are compared with the corresponding values for Ω and ΩM.

Comparison of the Simple Patient-Centric Atopic Dermatitis Scoring System PEST with SCORAD in Young Children Using a Ceramide Dominant Therapeutic Moisturizer.

INTRODUCTION: Patient eczema severity time (PEST) is a new atopic dermatitis (AD) scoring system based on patients' own perception of their disease. Conventional scales such as SCORing of atopic dermatitis (SCORAD) reflect the clinician's observations during the clinic visit. Instead, the PEST score captures eczema severity, relapse and recovery as experienced by the patient or caregiver on a daily basis, promoting patient engagement, compliance with treatment and improved outcomes. This study aims to determine the correlation between carer-assessed PEST and clinician-assessed SCORAD in paediatric AD patients after 12 weeks of treatment using a ceramide-dominant therapeutic moisturizer. METHODS: Prospective, open-label, observational, multi-centre study in which children with AD aged 6 months to 6 years were treated with a ceramide dominant therapeutic moisturizer twice daily for 12 weeks; 58 children with mild-to-moderate AD were included. Correlation between the 7-day averaged PEST and SCORAD scores for assessment of AD severity was measured within a general linear model. PEST and SCORAD were compared in week 4 and week 12. RESULTS: At week 12, a moderate correlation was found between the SCORAD and PEST scores (r = 0.51). The mean change in SCORAD and PEST scores from baseline to week 12 was -11.46 [95% confidence interval (CI) -14.99 to -7.92, p < 0.0001] and -1.33 (95% CI -0.71 to -0.10, p < 0.0001) respectively. PEST demonstrated greater responsiveness to change (33.3% of scale) compared to SCORAD (13.8% of scale). CONCLUSION: The PEST score correlates well with the SCORAD score and may have improved sensitivity when detecting changes in the severity of AD. The ceramide-dominant therapeutic moisturizer used was safe and effective in the management of AD in young children. FUNDING: Hyphens Pharma Pte Ltd. TRIAL REGISTRATION: clinicaltrials.gov identifier, NCT02073591.

Guidelines for the Management of Atopic Dermatitis in Singapore.

INTRODUCTION: Atopic dermatitis is a common, chronic pruritic condition affecting both children and adults, which has a negative impact on the quality of life. These guidelines were developed by an expert workgroup appointed by the Dermatological Society of Singapore, to provide doctors with information to assist in the management of their patients with atopic dermatitis. The workgroup members are experienced dermatologists with interest and expertise in eczemas. MATERIALS AND METHODS: Workgroup members arrived at a consensus on the topics to be included. Relevant studies from the literature were assessed for best evidence, supplemented by the collective experience of the workgroup. RESULTS: For mild atopic dermatitis, emollients, mild potency topical steroids and topical calcineurin inhibitors are recommended. For moderate-to-severe atopic dermatitis, the use of emollients, moderate-to-potent topical steroids, topical calcineurin inhibitors, wet dressings, antimicrobials for secondary skin infection, phototherapy, and systemic therapy (e.g. prednisolone, cyclosporine, azathioprine or methotrexate) may be warranted. Patients with moderate-to-severe atopic dermatitis should be managed in conjunction with a dermatologist. CONCLUSION: Good outcomes can be achieved with an individualised therapeutic approach combined with adequate patient and parental education.

Anxiety and Depression in Patients with Atopic Dermatitis in a Southeast Asian Tertiary Dermatological Centre.

INTRODUCTION: This study aimed to assess the frequency of anxiety and depression in a cohort of adult patients with atopic dermatitis (AD) in a tertiary dermatological centre, using the Hospital Anxiety and Depression Scale (HADS). We looked for any correlation between anxiety and depression with skin disease severity. MATERIALS AND METHODS: Patients with AD were recruited from the National Skin Centre, Singapore, from 2008 to 2009 for a prospective cross-sectional study. The scoring atopic dermatitis (SCORAD) grade was determined and the HADS was administered via interviews. RESULTS: A total of 100 patients (78 males, 22 females) were enrolled (92% Chinese, 4% Malays and 4% Indians). Their average age was 25.7 years. Sixty-five percent used topical steroids, 14% had previously taken oral prednisolone for the control of disease flares, and 20% were on concurrent systemic therapy. The mean SCORAD was 55.0, with 99% of patients having moderate or severe AD. The mean HADS anxiety score was 7.2 and the mean depression score was 5.0. The level of anxiety correlated well with that of depression (Spearman's rank correlation coefficient, ρ = 0.59, P <0.05); 18% were considered as cases of anxiety and 5% as cases of depression. These patients also had higher SCORAD values compared to other patients with lower scores for anxiety or depression (P <0.05). Linear regression demonstrated a statistically significant positive relationship between anxiety and depression scores, and SCORAD scores. CONCLUSION: Our study identified, by means of the HADS, the frequency of anxiety and depression amongst a cohort of Singaporean patients with AD. More severe skin disease correlated to greater psychological burden. The HADS is a useful screening tool that can constitute part of the overall holistic management of patients with AD so as to improve patient care.

Lichen nitidus presenting with nail changes--case report and review of the literature.

Lichen nitidus of the nail is rare and can precede the onset of skin lesions. Delayed diagnosis is common. We present an unusual case of lichen nitidus-associated nail changes that preceded the onset of skin lesions in a 4-year-old Indian girl. We also conduct a review of six other cases of lichen nitidus with nail involvement from the English-language literature. Clues to the diagnosis of lichen nitidus include violaceous or pigmentary changes of the nail fold and subtle lichenoid papules on the affected digits. Lichen nitidus of the nails appears to be less severe than nail changes of lichen planus and is generally self-limiting. Understanding the natural history of lichen nitidus of the nails will help physicians better counsel patients and their families.

Cutaneous protothecosis in a patient with previously undiagnosed HIV infection.

Protothecosis is an uncommon condition resulting from infection by achlorophyllous algae of the Prototheca species. Immunocompromised individuals are generally most susceptible to protothecal infection and tend to develop severe and disseminated disease. However, the association between protothecosis and HIV-induced immunosuppression is not clear, with only a handful of cases having been described to date. Here we report a case of cutaneous protothecosis in a Chinese man with previously undiagnosed HIV infection that responded well to oral itraconazole.

Atopic dirty neck or acquired atopic hyperpigmentation? An epidemiological and clinical study from the National Skin Centre in Singapore.

BACKGROUND: 'Atopic dirty neck' is a poorly understood acquired hyperpigmentation in patients with atopic dermatitis (AD). OBJECTIVE: To report a single-centre experience with synthesis of this entity's features. METHODS: All patients with AD with dirty neck seen over a 5-month period at the National Skin Centre were invited to participate. RESULTS: Out of 544 AD patients examined, 78 (14.3%) had acquired pigmentation of the neck. The majority had moderate-to-severe underlying eczema. Histopathology showed increased epidermal melanin and dermal melanophages, a thickened basement membrane and a dense superficial perivascular infiltrate. CONCLUSION: Acquired atopic hyperpigmentation has a high prevalence, particularly in adolescent Asian males. Clinico-pathological correlation suggests it results from both frictional melanosis and post-inflammatory hyperpigmentation. The rippled appearance and the onset in adolescence are probably due to accentuation of the juxta-clavicular beaded lines. Optimal control of eczema may improve and potentially prevent the development, which is of importance considering the psychosocial impact of the condition.

An 11-year review of dermatomyositis in Asian patients.

INTRODUCTION: Dermatomyositis (DM) is a multisystem inflammatory disease with a strong association with malignancy. We aimed to describe a series of Asian patients with DM and identify any significant clinical factors associated with malignancy. MATERIALS AND METHODS: This was a retrospective review of a multi-racial cohort of 69 Asian patients diagnosed with DM over an 11-year period from 1996 to 2006. RESULTS: Malignancy was detected in 15 out of 68 patients (22%), the most common of which was nasopharyngeal carcinoma (7 cases). Compared to the non-malignancy group, the malignancy-associated group was older and had more male patients. There were no statistically significant clinical, serological or laboratory factors associated with a higher risk of malignancy. CONCLUSION: This study highlights the importance of ongoing malignancy screening especially for nasopharyngeal carcinoma in Asian patients with DM.

Contact dermatitis: a common adverse reaction to topical traditional Chinese medicine.

BACKGROUND: Over the past decade, there has been an increase in the use of topical traditional Chinese medicine (TTCM). The most common adverse event seen in the use of TTCM is contact dermatitis. This retrospective study was designed to determine the types of commonly used TTCM in Singapore causing contact dermatitis. METHODS: We retrospectively reviewed the case notes of 10 patients seen at the National Skin Centre over a 4-month period with a diagnosis of adverse cutaneous reactions to TTCMs. RESULTS: A total of 10 patients were diagnosed with contact dermatitis to TTCM at our institution over a 4-month period. The clinical manifestation ranged from eczematous lesions to urticarial plaques and blisters. Five of these patients were patch-tested to our standard series, our in-house TTCM series, as well as to the TTCM they had used. The other five patients declined patch testing. The results of patch testing confirmed the diagnosis of allergic contact dermatitis in three of the tested patients. Negative patch testing results inferred the diagnosis of irritant contact dermatitis to TTCM in the other two patients. Two of the patients with positive patch tests also had a positive reaction to balsam of peru and fragrance mix. The three positive patch tests were all caused by medicated oils. CONCLUSIONS: These results suggest it is likely that essential oils present as constituents of the medicated oils could be the possible allergen. The study also confirms that contact dermatitis is a common adverse event associated with the use of TTCM.