Immunomodulatory and clinical effects of receptor-interacting protein kinase 1 (RIPK1) inhibitor eclitasertib (SAR443122) in patients with severe COVID-19: a phase 1b, randomized, double-blinded, placebo-controlled study.

BACKGROUND: Targeting receptor-interacting serine/threonine protein kinase 1 could mitigate the devastating sequelae of the hyperinflammatory state observed in severe cases of COVID-19. This study explored the immunomodulatory and clinical effects of the receptor-interacting serine/threonine protein kinase 1 inhibitor SAR443122 (eclitasertib) in patients with severe COVID-19. METHODS: In this Phase 1b, double-blinded, placebo-controlled study (NCT04469621) a total of 82 patients were screened, of whom 68 patients were eligible and randomized (2:1) to receive eclitasertib 600 mg (300 mg twice daily) or placebo up to 14 days. Primary outcome was relative change in C-reactive protein from baseline to Day 7. Time to clinical improvement using 7-point ordinal scale, ventilator/respiratory failure-free days, change in SpO2/FiO2 ratio, and biomarkers of severe COVID-19 were explored. RESULTS: Geometric mean ratio (point estimate [90% confidence interval]) of the relative change from baseline in C-reactive protein with eclitasertib vs. placebo on Day 7 was 0.85 (0.49-1.45; p = 0.30). Median time to 50% decrease in C-reactive protein from baseline was 3 days vs. 5 days (p = 0.056) with eclitasertib vs. placebo. Median time to ≥ 2-point improvement on 7-point clinical symptoms scale was 8 days vs. 10 days with eclitasertib vs. placebo (p = 0.38). Mean ventilator/respiratory failure-free days, change in baseline-adjusted SpO2/FiO2 ratio, and clinical biomarkers showed consistent numerical improvements with eclitasertib vs. placebo. The most frequently reported treatment-emergent adverse events were gastrointestinal disorders and condition aggravated/worsened COVID-19 pneumonia. CONCLUSIONS: Eclitasertib was well tolerated with consistent trends toward more rapid resolution of inflammatory biomarkers and clinical improvement in severe COVID-19 patients than placebo. GOV IDENTIFIER: NCT04469621, first posted on clinicaltrials.gov on July 14, 2020.

Comparison of Different Preparation Techniques of Thermophoretic Swimmers and Their Propulsion Velocity.

The motion of partly gold (Au)-coated Janus particles under laser irradiation is caused by self-thermophoresis. Despite numerous studies addressing this topic, the impact of the preparation method and the degree of coverage of the particle with Au on the resulting thermophoretic velocity has not yet been fully understood. A detailed understanding of the most important tuning parameters during the preparation process is crucial to design Janus particles that are optimized for Au coverage to receive a high thermophoretic velocity. In this study, we explore the influence of the fabrication process, which changes the Au cap size, on the resulting self-propulsion behavior of partly Au-coated polystyrene particles (Au-PS). Additionally, the impact of an underlying adhesion chromium layer is investigated. In addition to the most commonly used qualitative SEM and EDX measurements, we propose a novel and fast technique utilizing AFM studies to quantify the cap size. This non-invasive technique can be used to determine both the size and the maximum thickness of the Au cap. The Au cap size was systematically varied in a range between about 36 and 74% by different preparation strategies. Nevertheless, we showed that the differing Au cap sizes of the Janus particles in this range have no obvious effect on the thermophoretic velocity. This is a surprising result since one would expect an effect of the Au cap size due to different solvent flows around the Janus particles and is attributed to an additional torque near the surface of the measuring cell.

Originalarbeiten / Original Articles. Die Behandlung von Kindern und Jugendlichen mit substanzbezogenen Störungen im deutschen Gesundheitssystem ­ eine Quantitative Studie.

The Treatment of Children and Adolescents with Substance-Related Disorders in the German Health Care System - a Quantitative Study This study evaluates the minimum characteristics of OPS code 9-694 for specific treatment in special settings for substance-related disorders in children and adolescents. The research questions to be answered are the extent to which given minimum characteristics and potentials are assessed as practical, up-to-date, needs-oriented and necessary. The survey was conducted using a nationwide online survey in special wards of child and adolescent psychiatric clinics. Given minimum characteristics reflect everyday clinical practice and are assessed as practical, up-to-date and relevant. Nevertheless, setting- and method-specific potential for further development was identified, most of which has already been implemented in the clinics but has not yet been incorporated into the minimum characteristics. The topic has received little research attention to date. The findings of the original study can provide a basis for the optimization and adaptation of minimum characteristics and treatment offers for children and adolescents with substance-related disorders and lead to the further development of the OPS code. A theory-practice transfer tailored to the target group requires the networking of individual players in the help system as well as the health policy discussion and gradual adaptation of the content of given minimum characteristics and potential for change.

[The Specific Treatment of Children and Adolescents with Substance Use Disorders in a Special Setting - A Qualitative Study of the Minimum Characteristics of OPS Code 9-694]. / Die spezifische Behandlung von substanzbedingten Störungen bei Kindern und Jugendlichen im besonderen Setting.

The Specific Treatment of Children and Adolescents with Substance Use Disorders in a Special Setting - A Qualitative Study of the Minimum Characteristics of OPS Code 9-694 Abstract: Objective: The minimum characteristics for the treatment of children and adolescents with substance use disorders are shown in the additional code 9-694 of the Operation and Procedure Code (OPS). The OPS acts as an instrument of the PEPP remuneration system as well as for external quality assurance. In the presented study, we present the assessments of practitioners with regard to the practical relevance and feasibility of the OPS code as well as possible further development potentials. Methods: We used a qualitative research design to evaluate the topicality and practical feasibility of applicable minimum characteristics. A draft of a treatment concept prepared by the author according to the minimum characteristics of OPS code 9-694 served as the starting material for the study. The results of the expert interviews were evaluated using qualitative content analysis. Results: The minimum characteristics are assessed as being practical, with additional work becoming a burden, while resources remain the same. There is a need for adapting and defining disorder- and age-specific peculiarities as well as for designing the treatment setting and therapeutic methods. A clear setting, transparency, and the possibility of individual procedures enable practical implementation. Conclusions: This study reveals the need for specialized treatment concepts that map needs and specifications equally. Accordingly, the results of the study represent an important reference for the design of future treatment concepts. The implementation of further development potentials as well as professional transfer of theory to practice require further evaluation involving all those involved in the treatment process.

Lichtgesteuerte Translation von mRNA in Eukaryoten.

Messenger RNA (mRNA) shows great potential for medical applications, as recently demonstrated by the mRNA-based vaccines against the coronavirus. In addition, it has long been used for ectopic gene expression in cells and model organisms. While numerous methodologies are available for controlling gene expression at the level of transcription, approaches to control translation are scarce. Here we review strategies for direct light-mediated activation of mRNA translation via photocleavable groups and their potential to achieve spatial and temporal control of protein production.

Translational drug discovery and development with the use of tissue-relevant biomarkers: Towards more physiological relevance and better prediction of clinical efficacy.

Due to the clinical development of drugs such as secukinumab, ustekinumab and dupilumab, major changes have been achieved in the treatment of patients diagnosed with psoriasis and atopic dermatitis. In academia and the pharmaceutical industry, research is increasingly moving towards the development of bispecific antibodies and multi-specific nanobodies, as there is a compelling need for new treatment modalities for patients suffering from autoimmune or malignant disease. The purpose of this review is to discuss aspects of translational drug development with a particular emphasis on indications such as psoriasis and atopic dermatitis. The identification of biomarkers, the assessment of target organ pharmacokinetic and pharmacodynamics interactions and a wide range of in vitro, ex vivo and in vivo models should contribute to an appropriate prediction of a biological effect in the clinical setting. As human biology may not be perfectly reflected by approaches such as skin equivalents or animal models, novel approaches such as the use of human skin and dermal microperfusion assays in healthy volunteers and patients appear both reasonable and mandatory. These models may indeed generate highly translationally relevant data that have the potential to reduce the failure rate of drugs currently undergoing clinical development.

LC/MS/MS analyses of open-flow microperfusion samples quantify eicosanoids in a rat model of skin inflammation.

Eicosanoids are lipid-mediator molecules with key roles in inflammatory skin diseases, such as psoriasis. Eicosanoids are released close to the source of inflammation, where they elicit local pleiotropic effects and dysregulations. Monitoring inflammatory mediators directly in skin lesions could provide new insights and therapeutic possibilities. Here, we analyzed dermal interstitial fluid samples obtained by dermal open-flow microperfusion in a rat model of skin inflammation. We developed a solid-phase extraction ultra-HPLC/MS/MS method to reliably and precisely analyze small-volume samples and quantified 11 eicosanoids [thromboxane B2, prostaglandin (PG) E2, PGD2, PGF2α, leukotriene B4, 15-HETE, 12-HETE, 5-HETE, 12-hydroxyeicosapentaenoic acid, 13-HODE, and 17-hydroxydocosahexaenoic acid]. Our method achieved a median intraday precision of approximately 5% and interday precision of approximately 8%. All calibration curves showed excellent linearity between 0.01 and 50 ng/ml (R2 > 0.980). In the rat model, eicosanoids were significantly increased in imiquimod-treated inflamed skin sites compared with untreated control sites. Oral treatment with an anti-inflammatory glucocorticoid decreased eicosanoid concentrations. These results show that a combination of tissue-specific sampling with LC/MS analytics is well suited for analyzing small sample volumes from minimally invasive sampling methods such as open-flow microperfusion or microdialysis to study local inflammation and the effect of treatments in skin diseases.

Anti-GPVI Fab SAR264565 effectively blocks GPVI function in ex vivo human platelets under arterial shear in a perfusion chamber.

INTRODUCTION: Glycoprotein VI (GPVI) is the major platelet receptor for collagen-mediated platelet adhesion and activation. SAR264565 is an anti-GPVI-Fab, binds to GPVI with high affinity, and blocks GPVI function in human platelets in vitro. METHODS: The effect of SAR26456 on platelet responsiveness in the blood of 21 healthy male subjects was investigated using Sakariassen's ex vivo thrombogenesis perfusion chamber model on a collagen-coated surface under conditions mimicking arterial flow. Ex vivo effects of SAR264565 (10 and 100 µg/mL) were investigated before administration of aspirin or clopidogrel to study subjects (baseline), after aspirin (2× 300 mg) administration alone, and after combined aspirin (2× 300 mg)/clopidogrel (600 mg) administration. Additional ex vivo and in vitro platelet tests were also performed. RESULTS: Addition of SAR264565 to the perfusion chamber dose-dependently reduced platelet and fibrin deposition, reaching statistical significance at 100 µg/mL (415 ± 67 compared to 137 ± 36 platelets/cm2, [p < 0.01] and fibrin 0.095 ± 0.014 compared to 0.032 ± 0.008 µg/cm2, [p < 0.001]). Aspirin administration caused an additive and dose-dependent reduction of SAR264565-induced platelet and fibrin deposition. Combined aspirin/clopidogrel administration did not lead to additional SAR264565-induced inhibition of platelet or fibrin deposition. CONCLUSION: GPVI antagonism by the anti-GPVI-Fab fragment SAR264565 dose-dependently inhibits platelet adhesion and fibrin formation on a collagen surface under arterial shear. Additive inhibition is observed after prior aspirin administration with no further amplification on top of a combination of aspirin with clopidogrel. Ex vivo antiplatelet tests confirmed a selective inhibiting effect of SAR264565 on collagen-induced platelet activation.

Severe Neonatal Interstitial Lung Disease Caused by a Rare Surfactant Protein C Mutation.

Childhood interstitial lung disease (chILD) is a collective term for a group of rare lung disorders of heterogeneous origin. Surfactant dysfunction disorders are a cause of chILD with onset during the neonatal period and infancy. Clinical signs of tachypnea and hypoxemia are nonspecific and usually caused by common conditions like lower respiratory tract infections. We report on a full-term male newborn who was readmitted to the hospital at 7 days of age with marked tachypnea and poor feeding during the respiratory syncytial virus season. After exclusion of infection and other, more common congenital disorders, chILD was diagnosed using chest computed tomography and genetic analysis. A likely pathogenic heterozygous variant of SFTPC (c.163C>T, L55F) was detected by whole exome sequencing. The patient received supplemental oxygen and noninvasive respiratory support and was treated with intravenous methylprednisolone pulses and hydroxychloroquine. Despite the treatment, his respiratory situation deteriorated continuously, leading to several hospitalizations and continuous escalation of noninvasive ventilatory support. At 6 months of age, the patient was listed for lung transplant and transplanted successfully aged 7 months.

Target occupancy biomarker assay development using a conformation-selective antibody against small-molecule-bound TNF.

Background: An antibody specific to small-molecule inhibitor-bound TNF has enabled the development of target occupancy biomarker assays to support the development of novel treatments for autoimmune disorders. Materials & methods: ELISAs were developed for inhibitor-bound and total TNF to determine the percentage of TNF occupancy in samples from stimulated blood. Inhibitor-saturated samples allowed measurement of total and inhibitor-bound TNF in a single electrochemiluminescence immunoassay. Results: TNF occupancy was proportional to inhibitor concentration in plasma samples. An electrochemiluminescence method for inhibitor-bound TNF was validated for use as a potential clinical occupancy biomarker assay. Conclusion: Development of these assays has allowed measurement of a target occupancy biomarker, which has supported progression of the first small-molecule inhibitors of TNF.

Experimental evidence of a size-dependent sign change of the Seebeck coefficient of Bi nanowire arrays.

The electrical transport in bismuth nanowires is strongly influenced by both sample geometry and crystallinity. Compared to bulk bismuth, the electrical transport in nanowires is dominated by size effects and influenced by surface states, which gain increasing relevance with increasing surface-to-volume ratios, i.e. with decreasing wire diameter. Bismuth nanowires with tailored diameter and crystallinity constitute, therefore, excellent model systems, allowing to study the interplay of the different transport phenomena. Here, we present temperature-dependent Seebeck coefficient and relative electrical resistance measurements of parallel bismuth nanowire arrays with diameters between 40 and 400 nm synthesized by pulsed electroplating in polymer templates. Both electrical resistance and Seebeck coefficient exhibit a non-monotonic temperature dependence, with the sign of the Seebeck coefficient changing from negative to positive with decreasing temperature. The observed behavior is size-dependent and is attributed to limitations of the mean free path of the charge carriers within the nanowires. The observed size-dependent Seebeck coefficient and in particular the size-dependent sign change opens a promising avenue for single-material thermocouples with p- and n-legs made from nanowires with different diameters.

Selective non-lipid modulator of LPA5 activity in human platelets.

Lysophosphatidic acid (LPA) is a potent activator of human platelets in vitro. Recently, the G protein-coupled receptor LPA5/GPR92 has been identified to be the relevant LPA receptor responsible for the activation of human platelets by LPA. In a high-throughput screening campaign we identified a diphenyl pyrazole carboxylic acid as a small-molecule inhibitor for LPA5. Confirmation for the specificity of this small molecule was achieved in human platelets as the relevant cellular in vitro model. We could confirm using antagonists for alternative LPA receptors that we identified in our work the first non-lipid, small-molecule inhibitor for LPA5/GPR92 specifically inhibiting LPA-mediated platelet activation in vitro.

Interstitial lung disease in infancy and early childhood: a clinicopathological primer.

Children's interstitial lung disease (chILD) encompasses a wide and heterogeneous spectrum of diseases substantially different from that of adults. Established classification systems divide chILD into conditions more prevalent in infancy and other conditions occurring at any age. This categorisation is based on a multidisciplinary approach including clinical, radiological, genetic and histological findings. The diagnostic evaluation may include lung biopsies if other diagnostic approaches failed to identify a precise chILD entity, or if severe or refractory respiratory distress of unknown cause is present. As the majority of children will be evaluated and diagnosed outside of specialist centres, this review summarises relevant clinical, genetic and histological findings of chILD to provide assistance in clinical assessment and rational diagnostics.

An orally available small molecule that targets soluble TNF to deliver anti-TNF biologic-like efficacy in rheumatoid arthritis.

Tumor necrosis factor (TNF) is a pleiotropic cytokine belonging to a family of trimeric proteins with both proinflammatory and immunoregulatory functions. TNF is a key mediator in autoimmune diseases and during the last couple of decades several biologic drugs have delivered new therapeutic options for patients suffering from chronic autoimmune diseases such as rheumatoid arthritis and chronic inflammatory bowel disease. Attempts to design small molecule therapies directed to this cytokine have not led to approved products yet. Here we report the discovery and development of a potent small molecule inhibitor of TNF that was recently moved into phase 1 clinical trials. The molecule, SAR441566, stabilizes an asymmetrical form of the soluble TNF trimer, compromises downstream signaling and inhibits the functions of TNF in vitro and in vivo. With SAR441566 being studied in healthy volunteers we hope to deliver a more convenient orally bioavailable and effective treatment option for patients suffering with chronic autoimmune diseases compared to established biologic drugs targeting TNF.

Novel inhibitor ZED3197 as potential drug candidate in anticoagulation targeting coagulation FXIIIa (F13a).

BACKGROUND: Factor XIII (FXIII) is the final enzyme of the coagulation cascade. While the other enzymatic coagulation factors are proteases, FXIII belongs to the transglutaminase family. FXIIIa covalently crosslinks the fibrin clot and represents a promising target for drug development to facilitate fibrinolysis. However, no FXIII-inhibiting compound has entered clinical trials. Here, we introduce the features of a peptidomimetic inhibitor of FXIIIa (ZED3197) as a potential drug candidate. METHODS: The potency of ZED3197 against FXIIIa and the selectivity against other human transglutaminases were characterized using transamidation and isopeptidase assays. The inhibition of fibrin crosslinking was evaluated by biochemical methods and thromboelastometry. Further, the pharmacology of the compound was explored in a rabbit model of venous stasis and reperfusion. RESULTS: ZED3197 proved to be a potent and selective inhibitor of human FXIIIa. Further, the compound showed broad inhibitory activity against cellular FXIIIA from various animal species. Rotational thromboelastometry in whole human blood indicated that the inhibitor, in a dose-dependent manner, prolonged clot formation, reduced clot firmness, and facilitated clot lysis without affecting the clotting time, indicating minimal impact on hemostasis. In vivo, the novel FXIIIa inhibitor effectively decreased the weight of clots and facilitated flow restoration without prolongation of the bleeding time. CONCLUSIONS: ZED3197 is the first drug-like potent compound targeting FXIIIa, a yet untapped target in anticoagulation. Due to the function of FXIII downstream of thrombin the approach provides minimal impact on hemostasis. In vivo data imply that the inhibitor dissociates an antithrombotic effect from increased bleeding tendency.

Chronic Granulomatous Disease First Diagnosed in Adulthood Presenting With Spinal Cord Infection.

Chronic granulomatous disease (CGD) is a rare genetic immunodeficiency, which is characterized by recurrent severe bacterial and fungal infections caused by a defect in phagocytic cells due to loss of superoxide production. The disease usually manifests within the first years of life. Early diagnosis allows therapeutic intervention to improve the limited life expectancy. Nevertheless, only half of the patients exceed the age of 25. Here, we present the case of a 41-year old female patient who presented with an extensive spinal cord infection and atypical pneumonia mimicking tuberculosis. The medical history with recurrent granulomatous infections and microbiological findings with multiple unusual opportunistic pathogens was the key to the diagnosis of CGD, which is exceptionally rare first diagnosed in patients in the fifth decade of life. The late diagnosis in this case was likely due to the lack of knowledge of the disease by the treating teams before but not because the patient did not have typical CGD infections along her life. The extensive progressive developing granulomas in our patient with fatal outcome raise the question of early immunosuppressive therapy in addition to anti-infectious treatment. We recommend appropriate CGD diagnostics in adult patients with unclear granulomatous diseases of the nervous system.

Fluorescence In Situ Hybridization for Diagnosis of Whipple's Disease in Formalin-Fixed Paraffin-Embedded Tissue.

Whipple's disease (WD) is a rare chronic systemic infection with a wide range of clinical symptoms, routinely diagnosed in biopsies from the small intestine and other tissues by periodic acid-Schiff (PAS) diastase staining and immunohistological analysis with specific antibodies. The aim of our study was to improve the pathological diagnosis of WD. Therefore, we analyzed the potential of fluorescence in situ hybridization (FISH) for diagnosing WD, using a Tropheryma (T.) whipplei-specific probe. 19 formalin-fixed paraffin-embedded (FFPE) duodenal biopsy specimens of 12 patients with treated (6/12) and untreated (6/12) WD were retrospectively examined using PAS diastase staining, immunohistochemistry, and FISH. 20 biopsy specimens with normal intestinal mucosa, Helicobacter pylori, or mycobacterial infection, respectively, served as controls. We successfully detected T. whipplei in tissue biopsies with a sensitivity of 83% in untreated (5/6) and 40% in treated (4/10) cases of WD. In our study, we show that FISH-based diagnosis of individual vital T. whipplei in FFPE specimens is feasible and can be considered as ancillary diagnostic tool for the diagnosis of WD in FFPE material. We show that FISH not only detect active WD but also be helpful as an indicator for the efficiency of antibiotic treatment and for detection of recurrence of disease when the signal of PAS diastase and immunohistochemistry lags behind the recurrence of disease, especially if the clinical course of the patient and antimicrobial treatment is considered.

In vivo visualization of osteoarthritic hypertrophic lesions.

Osteoarthritis (OA) is one of the most common diseases in the aging population. While disease progress in humans is monitored indirectly by X-ray or MRI, small animal OA lesions detection always requires surgical intervention and histology. Here we introduce bimodal MR/NIR probes based on cartilage-targeting 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid amide (DOTAM) that are directly administered to the joint cavity. We demonstrate applications in healthy and diseased rat joints by MRI in vivo. The same joints are inspected post-mortem by fluorescence microscopy, showing not only the precise location of the reagents but also revealing details such as focal cartilage damage and chondrophyte or osteophyte formation. This allows for determining the distinct pathological state of the disease and the regeneration capability of the animal model and will help to correctly assess the effect of potential disease modifying OA drugs (DMOADs) in the future.

Supernatants of HIV-infected immune cells affect the barrier function of human HT-29/B6 intestinal epithelial cells.

OBJECTIVES: Characterization of the diarrhoea-inducing effect of altered cytokine production in HIV infection. METHODS: Monocyte-derived macrophages (MDM) were infected with macrophagetropic (SF162) and lymphocytotropic (IIIB) HIV-1 strains and cocultured with autologous peripheral blood mononuclear cells (PBMC). After 24 h the supernatants were collected and tested for their immunoreactive levels of cytokines by enzyme-linked immunosorbent assay. The effects of the supernatants and the respective recombinant human cytokines on barrier function of HT-29/B6 cells were determined. RESULTS: Infection of MDM with HIV-1 SF162 or IIIB led to increased production of tumour necrosis factor-alpha (TNFalpha), interleukin-1-beta, interferon-alpha and interferon-gamma after cell-cell contact with PBMC. Supernatants of infected cells decreased transepithelial resistance (R(t)), with higher effects on R(t) in HIV IIIB infection, which was due to higher cytokine concentrations. The effect was not due to cytotoxicity (negative LDH assay) or epithelial monolayer disruption [zonula occludens protein-1 (ZO-1) immunofluorescence staining]. The effect of HIV-1 IIIB coculture supernatants could be mimicked by the respective recombinant human cytokines. TNFalpha is an effector cytokine, because inhibition of TNFalpha by its soluble receptor decreased the effect of the supernatants on transepithelial resistance. Conductance scanning indicated the cytokine-induced barrier defect to be due to both, induction of epithelial apoptoses and tight junction alterations. CONCLUSIONS: Cell-cell interaction of HIV-infected macrophages with PBMC leads to a release of cytokines sufficient to alter intestinal epithelial barrier function. The main effect was mediated by TNFalpha inducing a leak-flux which may contribute to the diarrhoea by HIV per se (HIV-enteropathy).

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.