Impact of engineering the ATP synthase rotor ring on photosynthesis in tobacco chloroplasts.

The chloroplast ATP synthase produces the ATP needed for photosynthesis and plant growth. The trans-membrane flow of protons through the ATP synthase rotates an oligomeric assembly of c subunits, the c-ring. The ion-to-ATP ratio in rotary F1F0-ATP synthases is defined by the number of c-subunits in the rotor c-ring. Engineering the c-ring stoichiometry is, therefore, a possible route to manipulate ATP synthesis by the ATP synthase and hence photosynthetic efficiency in plants. Here, we describe the construction of a tobacco (Nicotiana tabacum) chloroplast atpH (chloroplastic ATP synthase subunit c gene) mutant in which the c-ring stoichiometry was increased from 14 to 15 c-subunits. Although the abundance of the ATP synthase was decreased to 25% of wild-type (WT) levels, the mutant lines grew as well as WT plants and photosynthetic electron transport remained unaffected. To synthesize the necessary ATP for growth, we found that the contribution of the membrane potential to the proton motive force was enhanced to ensure a higher proton flux via the c15-ring without unwanted low pH-induced feedback inhibition of electron transport. Our work opens avenues to manipulate plant ion-to-ATP ratios with potentially beneficial consequences for photosynthesis.

Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology.

We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing a strong membrane curvature of ∼100°. Our structure explains the structural basis of cristae formation in mitochondria, a landmark signature of eukaryotic cell morphology.

Deep vein thrombosis after sclerotherapy and endovenous laser ablation of varicose veins - an observational study.

Background: The risk of developing deep vein thrombosis (DVT) after endovenous ablation of varicose veins varies in the literature. Little is known about the characteristics of this complication and associated factors. This study aimed: 1) to study the occurrence of DVT after ultrasound-guided foam sclerotherapy (UGFS) alone or combined with endovenous laser ablation (EVLA) for lower-limb varicose veins; 2) to identify factors associated with DVT. Patients and methods: The study included all outpatients aged 18 years or older who underwent UGFS and EVLA or UGFS alone at the University Hospital of Zurich between 2011 and 2015. Data were extracted from the hospital electronic medical record. Patients were surveyed about their level of pain after the procedure and their level of satisfaction with the procedure. Duplex ultrasound was used to assess the deep venous system 7-10 days and 6-8 months after the procedure. Regression analysis was used to examine the association of patient and procedure characteristics with the development of DVT. Results: A total of 334 patients (561 procedures performed in 393 different sessions) were included: 73% of the patients underwent combined UGFS and EVLA and 27% underwent UGFS alone. DVT occurred in 24 (7.2%) patients, of whom 88% underwent combined procedures and 17% underwent interventions involving both the great and small saphenous veins on the same session. DVT occurred in 8.2% of patients receiving thromboprophylaxis and in 9.5% of patients not receiving thromboprophylaxis. DVT occurred in 5.2% of women and 11.9% of men. No factors associated with a diagnosis of DVT after intervention were identified. Pain and satisfaction levels did not differ between patients with and without DVT. Conclusions: This study adds to the knowledge of the risk of DVT following UGFS alone or combined with EVLA. Further studies are needed to revise thromboprophylaxis.

Serious Games and Gamification: Health Care Workers' Experience, Attitudes, and Knowledge.

OBJECTIVE: With the rapid advancement of digital technology due to COVID-19, the health care field is embracing the use of digital technologies for learning, which presents an opportunity for teaching methods such as serious games to be developed and improved. Technology offers more options for these educational approaches. The goal of this study was to assess health care workers' experiences, attitudes, and knowledge regarding serious games in training. METHODS: The convenience sample consisted of 223 participants from the specialties of internal medicine and psychiatry who responded to questions regarding sociodemographic data, experience, attitudes, and knowledge regarding serious games. This study used an ordinal regression model to analyze the relationship between knowledge, attitudes, and experiences and the idea or wish to implement serious games. RESULTS: The majority of healthcare workers were not familiar with serious games or gamification. The results show gender and age differences regarding familiarity and willingness to use serious games. With increasing age, the respondents preferred conventional and traditional learning methods to playful teaching elements; younger generations were significantly more motivated than older generations when envisioning using elements of serious games in the future. CONCLUSIONS: The COVID-19 pandemic has encouraged the use of new technologies and digitalization. This study describes positive attitudes toward serious games, mainly in younger people working in health care. Serious games present an opportunity to develop new approaches for postgraduate medical teachings and continuing medical education.

Tailoring the Luminescent Properties of SrS:Ce3+ by Sr-Deficiency and Na+ Doping.

Ce3+-doped SrS phosphors with a charge-compensating Na+ addition were successfully synthesized via a solid-state reaction method, and the related X-ray diffraction patterns can be indexed to the rock-salt-like crystal structure of the Fm3̅m space group. SrS:(Ce3+)x (0.005 ≤ x ≤ 0.05) and SrS:(Ce3+)0.01,(Na+)y (0.005 ≤ y ≤ 0.030) phosphors were excited by 430 nm UV-Vis light, targeted to the 5d1 → 4f1 transition of Ce3+. The composition-optimized SrS:(Ce3+)0.01, (Na+)0.015 phosphors showed an intense broad emission band at λ = 430-700 nm. The doping of Na+ was probed by solid-state nuclear magnetic resonance. The 430 nm pumped white light-emitting diode structure fabricated with a combination of SrS:(Ce3+)0.01,(Na+)0.015 and Sr2Si5N8:Eu2+ phosphors shows a color-rendering index (Ra) of 89.7. The proposed strategy provides new avenues for the design and realization of novel high color quality solid-state LEDs.

Dimers of mitochondrial ATP synthase induce membrane curvature and self-assemble into rows.

Mitochondrial ATP synthases form dimers, which assemble into long ribbons at the rims of the inner membrane cristae. We reconstituted detergent-purified mitochondrial ATP synthase dimers from the green algae Polytomella sp. and the yeast Yarrowia lipolytica into liposomes and examined them by electron cryotomography. Tomographic volumes revealed that ATP synthase dimers from both species self-assemble into rows and bend the lipid bilayer locally. The dimer rows and the induced degree of membrane curvature closely resemble those in the inner membrane cristae. Monomers of mitochondrial ATP synthase reconstituted into liposomes do not bend membrane visibly and do not form rows. No specific lipids or proteins other than ATP synthase dimers are required for row formation and membrane remodelling. Long rows of ATP synthase dimers are a conserved feature of mitochondrial inner membranes. They are required for cristae formation and a main factor in mitochondrial morphogenesis.

SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activation.

We have previously shown that overexpression of SKI, an endogenous TGF-ß1 repressor, deactivates the pro-fibrotic myofibroblast phenotype in the heart. We now show that SKI also functions independently of SMAD/TGF-ß signaling, by activating the Hippo tumor-suppressor pathway and inhibiting the Transcriptional co-Activator with PDZ-binding motif (TAZ or WWTR1). The mechanism(s) by which SKI targets TAZ to inhibit cardiac fibroblast activation and fibrogenesis remain undefined. A rat model of post-myocardial infarction was used to examine the expression of TAZ during acute fibrogenesis and chronic heart failure. Results were then corroborated with primary rat cardiac fibroblast cell culture performed both on plastic and on inert elastic substrates, along with the use of siRNA and adenoviral expression vectors for active forms of SKI, YAP, and TAZ. Gene expression was examined by qPCR and luciferase assays, while protein expression was examined by immunoblotting and fluorescence microscopy. Cell phenotype was further assessed by functional assays. Finally, to elucidate SKI's effects on Hippo signaling, the SKI and TAZ interactomes were captured in human cardiac fibroblasts using BioID2 and mass spectrometry. Potential interactors were investigated in vitro to reveal novel mechanisms of action for SKI. In vitro assays on elastic substrates revealed the ability of TAZ to overcome environmental stimuli and induce the activation of hypersynthetic cardiac myofibroblasts. Further cell-based assays demonstrated that SKI causes specific proteasomal degradation of TAZ, but not YAP, and shifts actin cytoskeleton dynamics to inhibit myofibroblast activation. These findings were supported by identifying the bi-phasic expression of TAZ in vivo during post-MI remodeling and fibrosis. BioID2-based interactomics in human cardiac fibroblasts suggest that SKI interacts with actin-modifying proteins and with LIM Domain-containing protein 1 (LIMD1), a negative regulator of Hippo signaling. Furthermore, we found that LATS2 interacts with TAZ, whereas LATS1 does not, and that LATS2 knockdown prevented TAZ downregulation with SKI overexpression. Our findings indicate that SKI's capacity to regulate cardiac fibroblast activation is mediated, in part, by Hippo signaling. We postulate that the interaction between SKI and TAZ in cardiac fibroblasts is arbitrated by LIMD1, an important intermediary in focal adhesion-associated signaling pathways. This study contributes to the understanding of the unique physiology of cardiac fibroblasts, and of the relationship between SKI expression and cell phenotype.

Rotor subunits adaptations in ATP synthases from photosynthetic organisms.

Driven by transmembrane electrochemical ion gradients, F-type ATP synthases are the primary source of the universal energy currency, adenosine triphosphate (ATP), throughout all domains of life. The ATP synthase found in the thylakoid membranes of photosynthetic organisms has some unique features not present in other bacterial or mitochondrial systems. Among these is a larger-than-average transmembrane rotor ring and a redox-regulated switch capable of inhibiting ATP hydrolysis activity in the dark by uniquely adapted rotor subunit modifications. Here, we review recent insights into the structure and mechanism of ATP synthases specifically involved in photosynthesis and explore the cellular physiological consequences of these adaptations at short and long time scales.

High reproducibility of the interferon-gamma release assay T-SPOT.TB in serial testing.

Longitudinal studies regarding the reproducibility of Interferon-gamma release assay (IGRA) T-SPOT.TB for the diagnosis of Mycobacterium tuberculosis (M. tb) infection in serial testing are limited. We retrospectively analysed results of serially tested subjects in a medical laboratory in Germany over a time period of 14 years. From October 2004 to December 2018, a total of 5440 subjects were identified with a second T-SPOT.TB test after a median time interval of 258 days (interquartile range [IQR] 62-665). Consistently negative (n = 4520) or positive results (n = 682) were observed in 5202 (95.6%) subjects, indicating a high degree of concordance in serial testing (κ = 0.83). Test conversions occurred in 101 of 4621 (2.2%) subjects with initially negative tests. Of 819 subjects with initially positive test results, 137 (16.7%) had a test reversion which was associated with low spot numbers of the first test. Of 529 subjects retested within 1 year, only 60 (11.3%) displayed a test reversion. In subjects retested after more than 1 year, 77 of 290 (26.6%) tests reverted. This significantly higher rate of test reversions after more than 1 year was age-dependent and only observed in subjects above the age of 40 years. In the medical laboratory, the T-SPOT.TB test demonstrates a high reproducibility in serial testing.

Molecular architecture of the N-type ATPase rotor ring from Burkholderia pseudomallei.

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N1No-type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c17 ring is H+ specific, demonstrating that the ATPase is proton-coupled. The c17 ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes.

The aspartimide problem persists: Fluorenylmethyloxycarbonyl-solid-phase peptide synthesis (Fmoc-SPPS) chain termination due to formation of N-terminal piperazine-2,5-diones.

Aspartimide (Asi) formation is a notorious side reaction in peptide synthesis that is well characterized and described in literature. In this context, we observed significant amounts of chain termination in Fmoc-SPPS while synthesizing the N-terminal Xaa-Asp-Yaa motif. This termination was caused by the formation of piperazine-2,5-diones. We investigated this side reaction using a linear model peptide and independently synthesizing its piperazine-2,5-dione derivative. Nuclear magnetic resonance (NMR) data of the side product present in the crude linear peptide proves that exclusively the six-membered ring is formed whereas the theoretically conceivable seven-membered 1,4-diazepine-2,5-dione is not found. We propose a mechanism where nucleophilic attack of the N-terminal amino function takes place at the α-carbon of the carbonyl group of the corresponding Asi intermediate. In addition, we systematically investigated the impact of (a) different adjacent amino acid residues, (b) backbone protection, and (c) side chain protection of flanking amino acids. The side reaction is directly related to the Asi intermediate. Hence, hindering or avoiding Asi formation reduces or completely suppresses this side reaction.

Origin and Manipulation of Stable Vortex Ground States in Permalloy Nanotubes.

We present a detailed study on the static magnetic properties of individual permalloy nanotubes (NTs) with hexagonal cross-sections. Anisotropic magnetoresistance (AMR) measurements and scanning transmission X-ray microscopy (STXM) are used to investigate their magnetic ground states and its stability. We find that the magnetization in zero applied magnetic field is in a very stable vortex state. Its origin is attributed to a strong growth-induced anisotropy with easy axis perpendicular to the long axis of the tubes. AMR measurements of individual NTs in combination with micromagnetic simulations allow the determination of the magnitude of the growth-induced anisotropy for different types of NT coatings. We show that the strength of the anisotropy can be controlled by introducing a buffer layer underneath the magnetic layer. The magnetic ground states depend on the external magnetic field history and are directly imaged using STXM. Stable vortex domains can be introduced by external magnetic fields and can be erased by radio-frequency magnetic fields applied at the center of the tubes via a strip line antenna.

How to engineer glucose oxidase for mediated electron transfer.

Glucose oxidase (GOx) is of high industrial interest for glucose sensing because of its high ß-d-glucose specificity. The efficient and specific electrochemical communication between the redox center and electrodes is crucial to ensure accurate glucose determination. The efficiency of the electron transfer rates (ETR) with GOx, together with quinone diamine based mediators, is low and differs even among mediator derivatives. To design optimized enzyme-mediator couples and to describe a mediator binding model, a joint experimental and computational study was performed based on an oxygen-independent GOx variant V7 and two quinone diimine based electron mediators (QDM-1 and QDM-2), which differ in polarity and size, and ferrocenemethanol (FM). A site saturation library at position 414 was screened with all three mediators and yielded four beneficial substitutions Tyr, Met, Leu, and Val. The variants showed increased mediator activity for the more polar QDM-2 with a simultaneously decreased activity for the less polar and smaller QDM-1 and for FM. The variant GOx V7-I414Y exhibited the biggest change for the quinone diimine derivatives compared with V7 (QDM-1: 55.9 U/mg V7, 33.2 U/mg V7-I414Y; QDM-2: 2.7 U/mg V7, 12.9 U/mg V7-I414Y). Theoretical ETR calculated based on the Marcus theory were in good agreement with the experimental results. Molecular docking studies revealed a preferable binding of the two QD mediators directly in the active site, 3.5 Å away from the N5 atom of the flavin adenine dinucleotide (FAD) and in direct vicinity to position 414. In summary, position 414 in the active site was identified to modulate the electron shuttling from the FAD of the GOx to small water-soluble mediators dependent on the polarity and size of residue 414 and on the polarity and size of the mediator. The presented mediator binding model offers a promising possibility for the design of optimized enzyme-mediator couples.

On the principle of ion selectivity in Na+/H+-coupled membrane proteins: experimental and theoretical studies of an ATP synthase rotor.

Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na(+) or H(+), thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na(+)/H(+) selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H(+) selective, although not sufficiently to be functionally coupled to H(+), owing to the large excess of Na(+) in physiological settings. The functional Na(+) specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H(+) selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na(+)- and H(+)-coupled systems in other families of membrane transporters and enzymes.

[Leg ulcers (ulcus cruris): The frequent macrovascular causes]. / Ulcus cruris: Die häufigen, makrovaskulären Ursachen.

Leg ulcers (ulcus cruris): The frequent macrovascular causes Abstract. Four pathologies make up the macrovascular etiologies of leg uclers: Venous leg ulcers (50 %), mixed venous-arterial leg ulcers (20 %), arterial leg ulcers (5 %), and Martorell hypertensive ischemic leg ulcer (5 %). The remaining 20 % concern a large array of other etiologies. Every leg ulcer requires vascular (arterial and venous) work-up, that can be completed with microbiology, biopsy, and more in-depth internal diagnostics, as indicated. Venous leg ulcers are treated with compression therapy. Incompetent saphenous veins and tributaries are abolished if the deep venous system is patent. Occluded iliac veins are recanalised and stented, as possible. Refractory venous leg ulcers are grafted with split skin or punch grafts, depending on their surface. Extensive dermatolipofasciosclerosis may be tangentially removed by shave therapy or fasciectomy, that can be combined with negative pressure wound treatment (NPWT). Skin equivalents are an alternative to treat superficial venous leg ulcers that fail to epithelialise. Their indication in the treatment of more complex leg ulcers still needs to be better investigated and understood. The use of dermal matrices leads to more stable scars. Mixed venous-arterial leg ulcers heal slower and recur more frequently. Compression needs to be reduced. Refractory cases require arterial revascularisation, to transform the mixed venous-arterial into a venous leg ulcer. Arterial leg ulcers require arterial revascularization and split skin graft. Martorell hypertensive ischemic leg ulcer is still underrecognised and often confounded with with pyoderma gangrenosum, which leads therapy into a wrong direction. Necrosectomy, antibiotic treatment in the presence of relevant bacterial superinfection, and repeated split skin grafts eventually heal the vast majority of these extremely painful and potentially mortal wounds.

Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin.

We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-ß-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.

Substitution determination of Fmoc-substituted resins at different wavelengths.

In solid-phase peptide synthesis, the nominal batch size is calculated using the starting resin substitution and the mass of the starting resin. The starting resin substitution constitutes the basis for the calculation of a whole set of important process parameters, such as the number of amino acid derivative equivalents. For Fmoc-substituted resins, substitution determination is often performed by suspending the Fmoc-protected starting resin in 20% (v/v) piperidine in DMF to generate the dibenzofulvene-piperidine adduct that is quantified by ultraviolet-visible spectroscopy. The spectrometric measurement is performed at the maximum absorption wavelength of the dibenzofulvene-piperidine adduct, that is, at 301.0 nm. The recorded absorption value, the resin weight and the volume are entered into an equation derived from Lambert-Beer's law, together with the substance-specific molar absorption coefficient at 301.0 nm, in order to calculate the nominal substitution. To our knowledge, molar absorption coefficients between 7100 l mol-1  cm-1 and 8100 l mol-1  cm-1 have been reported for the dibenzofulvene-piperidine adduct at 301.0 nm. Depending on the applied value, the nominal batch size may differ up to 14%. In this publication, a determination of the molar absorption coefficients at 301.0 and 289.8 nm is reported. Furthermore, proof is given that by measuring the absorption at 289.8 nm the impact of wavelength accuracy is reduced. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Percutaneous sclerotherapy for spongiform venous malformations - analysis of patient-evaluated outcome and satisfaction.

BACKGROUND: Congenital venous malformations are frequently treated with sclerotherapy. Primary treatment goal is to control the often size-related symptoms. Functional impairment and aesthetical aspects as well as satisfaction have rarely been evaluated. PATIENTS AND METHODS: Medical records of patients who underwent sclerotherapy of spongiform venous malformations were reviewed and included in this retrospective study. The outcome of sclerotherapy as self-reported by patients was assessed in a 21 item questionnaire. RESULTS: Questionnaires were sent to 166 patients with a total of 327 procedures. Seventy-seven patients (48 %) with a total of 159 procedures (50 %) responded to the survey. Fifty-seven percent of patients were male. The age ranged from 1 to 38.1 years with a median age of 16.4 years. The lower extremities were the most common treated area. Limitations caused by the venous malformation improved in the majority of patients (e.g. pain improvement 87 %, improvement of swelling 83 %) but also worsening of symptoms occurred in a minority of cases. Seventy-seven per cent would undergo sclerotherapy again. CONCLUSIONS: Sclerotherapy for treatment of venous malformations results in significant reduction of symptoms. Multiple treatments are often needed, but patients are willing to undergo them.

Efficacy of idebenone on respiratory function in patients with Duchenne muscular dystrophy not using glucocorticoids (DELOS): a double-blind randomised placebo-controlled phase 3 trial.

BACKGROUND: Cardiorespiratory failure is the leading cause of death in Duchenne muscular dystrophy. Based on preclinical and phase 2 evidence, we assessed the efficacy and safety of idebenone in young patients with Duchenne muscular dystrophy who were not taking concomitant glucocorticoids. METHODS: In a multicentre phase 3 trial in Belgium, Germany, the Netherlands, Switzerland, France, Sweden, Austria, Italy, Spain, and the USA, patients (age 10-18 years old) with Duchenne muscular dystrophy were randomly assigned in a one-to-one ratio with a central interactive web response system with a permuted block design with four patients per block to receive idebenone (300 mg three times a day) or matching placebo orally for 52 weeks. Study personnel and patients were masked to treatment assignment. The primary endpoint was change in peak expiratory flow (PEF) as percentage predicted (PEF%p) from baseline to week 52, measured with spirometry. Analysis was by intention to treat (ITT) and a modified ITT (mITT), which was prospectively defined to exclude patients with at least 20% difference in the yearly change in PEF%p, measured with hospital-based and weekly home-based spirometry. This study is registered with ClinicalTrials.gov, number NCT01027884. FINDINGS: 31 patients in the idebenone group and 33 in the placebo group comprised the ITT population, and 30 and 27 comprised the mITT population. Idebenone significantly attenuated the fall in PEF%p from baseline to week 52 in the mITT (-3·05%p [95% CI -7·08 to 0·97], p=0·134, vs placebo -9·01%p [-13·18 to -4·84], p=0·0001; difference 5·96%p [0·16 to 11·76], p=0·044) and ITT populations (-2·57%p [-6·68 to 1·54], p=0·215, vs -8·84%p [-12·73 to -4·95], p<0·0001; difference 6·27%p [0·61 to 11·93], p=0·031). Idebenone also had a significant effect on PEF (L/min), weekly home-based PEF, FVC, and FEV1. The effect of idebenone on respiratory function outcomes was similar between patients with previous corticosteroid use and steroid-naive patients. Treatment with idebenone was safe and well tolerated with adverse event rates were similar in both groups. Nasopharyngitis and headache were the most common adverse events (idebenone, eight [25%] and six [19%] of 32 patients; placebo, nine [26%] and seven [21%] of 34 patients). Transient and mild diarrhoea was more common in the idebenone group than in the placebo group (eight [25%] vs four [12%] patients). INTERPRETATION: Idebenone reduced the loss of respiratory function and represents a new treatment option for patients with Duchenne muscular dystrophy. FUNDING: Santhera Pharmaceuticals.

A new type of Na(+)-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1F0-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Šresolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.