Stepwise assembly of the active site of [NiFe]-hydrogenase.

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2 into 2e- and 2H+ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN- molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

Actinium-225 as an example for monitoring of internal exposure of occupational intakes of radionuclides in face of new nuclear-medical applications for short-lived alpha emitting particles.

Monitoring of internal exposure to short-lived alpha-emitting radionuclides such as actinium-225 (225Ac), which are becoming increasingly important in nuclear medicine, plays an important role in the radiation protection of occupationally exposed persons. After having tested gamma spectrometry, liquid scintillation counting and alpha spectrometry for monitoring of internal exposure, the focus of the present study was on solid phase extraction of 225Ac from urine in combination with alpha spectrometry. The development of the method was based on recent findings from the literature on this topic. The method was used in a pilot phase to monitor internal exposure of four workers who were directly or indirectly involved in the manufacture and/or use of 225Ac. The monitoring protocol allowed a relatively short 24-hour urine sample analysis with excellent recovery of the internal standard, but it did not allow for a detection limit of less than 1 mBq nor a sufficient yield of 225Ac. Based on these results it is concluded that an in vitro excretion analysis alone is not appropriate for monitoring internal exposure to 225Ac. Instead, different radiation monitoring techniques have to be combined to ensure the radiation protection of employees.

Digital Behavior Change Interventions for the Prevention and Management of Type 2 Diabetes: Systematic Market Analysis.

BACKGROUND: Advancements in technology offer new opportunities for the prevention and management of type 2 diabetes. Venture capital companies have been investing in digital diabetes companies that offer digital behavior change interventions (DBCIs). However, little is known about the scientific evidence underpinning such interventions or the degree to which these interventions leverage novel technology-driven automated developments such as conversational agents (CAs) or just-in-time adaptive intervention (JITAI) approaches. OBJECTIVE: Our objectives were to identify the top-funded companies offering DBCIs for type 2 diabetes management and prevention, review the level of scientific evidence underpinning the DBCIs, identify which DBCIs are recognized as evidence-based programs by quality assurance authorities, and examine the degree to which these DBCIs include novel automated approaches such as CAs and JITAI mechanisms. METHODS: A systematic search was conducted using 2 venture capital databases (Crunchbase Pro and Pitchbook) to identify the top-funded companies offering interventions for type 2 diabetes prevention and management. Scientific publications relating to the identified DBCIs were identified via PubMed, Google Scholar, and the DBCIs' websites, and data regarding intervention effectiveness were extracted. The Diabetes Prevention Recognition Program (DPRP) of the Center for Disease Control and Prevention in the United States was used to identify the recognition status. The DBCIs' publications, websites, and mobile apps were reviewed with regard to the intervention characteristics. RESULTS: The 16 top-funded companies offering DBCIs for type 2 diabetes received a total funding of US $2.4 billion as of June 15, 2021. Only 4 out of the 50 identified publications associated with these DBCIs were fully powered randomized controlled trials (RCTs). Further, 1 of those 4 RCTs showed a significant difference in glycated hemoglobin A1c (HbA1c) outcomes between the intervention and control groups. However, all the studies reported HbA1c improvements ranging from 0.2% to 1.9% over the course of 12 months. In addition, 6 interventions were fully recognized by the DPRP to deliver evidence-based programs, and 2 interventions had a pending recognition status. Health professionals were included in the majority of DBCIs (13/16, 81%,), whereas only 10% (1/10) of accessible apps involved a CA as part of the intervention delivery. Self-reports represented most of the data sources (74/119, 62%) that could be used to tailor JITAIs. CONCLUSIONS: Our findings suggest that the level of funding received by companies offering DBCIs for type 2 diabetes prevention and management does not coincide with the level of evidence on the intervention effectiveness. There is considerable variation in the level of evidence underpinning the different DBCIs and an overall need for more rigorous effectiveness trials and transparent reporting by quality assurance authorities. Currently, very few DBCIs use automated approaches such as CAs and JITAIs, limiting the scalability and reach of these solutions.

Dienamine-Induced Divinylcyclopropane-Cycloheptadiene Rearrangements.

Sigmatropic rearrangements constitute an important group of pericyclic reactions. In contrast to cycloaddition reactions, examples of catalytic variants of electrocyclic reactions and sigmatropic rearrangements are still scarce in the chemical literature. Herein, we report the first organocatalytic Cope rearrangement of in situ-generated divinylcyclopropanes. The reactive motif was generated by condensation of 4-(2-vinylcyclopropyl)but-2-enal derivatives with a secondary amine catalyst to form a transient dienamine. The cycloheptadiene products could be obtained in high yield and excellent diastereoselectivity. Importantly, the reaction was demonstrated to be stereospecific, proceeding under mild conditions, while exhibiting broad functional group tolerance.

O2-Tolerant H2 Activation by an Isolated Large Subunit of a [NiFe] Hydrogenase.

The catalytic properties of hydrogenases are nature's answer to the seemingly simple reaction H2 ⇌ 2H+ + 2e-. Members of the phylogenetically diverse subgroup of [NiFe] hydrogenases generally consist of at least two subunits, where the large subunit harbors the H2-activating [NiFe] site and the small subunit contains iron-sulfur clusters mediating e- transfer. Typically, [NiFe] hydrogenases are susceptible to inhibition by O2. Here, we conducted system minimization by isolating and analyzing the large subunit of one of the rare members of the group of O2-tolerant [NiFe] hydrogenases, namely the preHoxG protein of the membrane-bound hydrogenase from Ralstonia eutropha. Unlike previous assumptions, preHoxG was able to activate H2 as it clearly performed catalytic hydrogen/deuterium exchange. However, it did not execute the entire catalytic cycle described for [NiFe] hydrogenases. Remarkably, H2 activation was performed by preHoxG even in the presence of O2, although the unique [4Fe-3S] cluster located in the small subunit and described to be crucial for tolerance toward O2 was absent. These findings challenge the current understanding of O2 tolerance of [NiFe] hydrogenases. The applicability of this minimal hydrogenase in basic and applied research is discussed.

Kreon® (Creon®) vs. Lipancrea®: In Vitro Comparison of Two Encapsulated Pancreatin Preparations.

Kreon® (Creon®) and Lipancrea® are pancreatic enzyme supplements indicated in the treatment of exocrine pancreatic insufficiency. In order to determine their interchangeability, an in vitro comparison of their physical properties and enzymatic activity was carried out. Capsule fill weight and particle size were also determined in order to establish their physical properties. Amylase, lipase and protease activities, lipase release at different pHs and the dissolution time of pellets were assessed for enzymatic analysis. The length range of Kreon® and Lipancrea® pellets was 1.1-2.2 mm and 1.5-2.8 mm, respectively. Protease activity was below the label claim for Lipancrea® and above for Kreon® presentations. Lipase and amylase activity were equal to or higher than the label claim in both preparations. In dissolution experiments simulating the stomach passage, significant release of lipase activity was observed for Lipancrea® (% actual activity: 41% for Lipancrea® 8000; 21% for Lipancrea® 16000) after 60 min at pH 5.0. No release of lipase activity was observed for Kreon® at that particular pH. Enzyme release for Lipancrea® at pH 6.0 was generally slower than for Kreon® and seemed to be influenced by the preceding incubation at lower pH. More than 85% of Kreon® and Lipancrea® dissolved in a pH 6.0 phosphate buffer within 20 min. Despite the similarities of the enzyme content on the respective labels, Kreon® and Lipancrea® differ in pellet size, enzymatic activity and release. This may impact their therapeutic efficacy and, therefore, may limit their interchangeability.

A membrane-bound [NiFe]-hydrogenase large subunit precursor whose C-terminal extension is not essential for cofactor incorporation but guarantees optimal maturation.

[NiFe]-hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)2 (CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo-protein carries usually a C-terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron-transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C-terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C-terminal extension from the apo-form of the large subunit of the membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H2 -dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of the R. eutropha MBH, the C-terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator.

Somatodendritic surface expression of epitope-tagged and KChIP binding-deficient Kv4.2 channels in hippocampal neurons.

Kv4.2 channels mediate a subthreshold-activating somatodendritic A-type current (ISA) in hippocampal neurons. We examined the role of accessory Kv channel interacting protein (KChIP) binding in somatodendritic surface expression and activity-dependent decrease in the availability of Kv4.2 channels. For this purpose we transfected cultured hippocampal neurons with cDNA coding for Kv4.2 wild-type (wt) or KChIP binding-deficient Kv4.2 mutants. All channels were equipped with an externally accessible hemagglutinin (HA)-tag and an EGFP-tag, which was attached to the C-terminal end. Combined analyses of EGFP self-fluorescence, surface HA immunostaining and patch-clamp recordings demonstrated similar dendritic trafficking and functional surface expression for Kv4.2[wt]HA,EGFP and the KChIP binding-deficient Kv4.2[A14K]HA,EGFP. Coexpression of exogenous KChIP2 augmented the surface expression of Kv4.2[wt]HA,EGFP but not Kv4.2[A14K]HA,EGFP. Notably, activity-dependent decrease in availability was more pronounced in Kv4.2[wt]HA,EGFP + KChIP2 coexpressing than in Kv4.2[A14K]HA,EGFP + KChIP2 coexpressing neurons. Our results do not support the notion that accessory KChIP binding is a prerequisite for dendritic trafficking and functional surface expression of Kv4.2 channels, however, accessory KChIP binding may play a potential role in Kv4.2 modulation during intrinsic plasticity processes.

[Planning for brain surgery in brain tumors]. / Zur Berechnung von Hirnarealverschiebungen bei Hirntumoren.

A precise knowledge of the localization of an intracerebral mass is a basic requirement for the planning of neurosurgical operations. Stereotactic atlases offer the possibility to adapt pre-operative imaging data onto normal anatomical conditions in the CNS. These atlases, however, reflect the standard variants of the CNS and do not allow to draw conclusions on local and secondary changes of the anatomy caused by the presence of pathological processes. The physical model proposed in this paper provides an estimate of the displacement of brain areas by an intracerebral mass. The modeling of brain parenchyma deformation is based on the mechanics of deformed media. The implementation of the model is successful in the group of primary brain tumors and meningiomas, and uses empirically-obtained data of a prospectively-selected patient population. The aim of the proposed model is, as further step, the integration and adaptation in apposite software solutions for the stereotactic orientation in the CNS.

A chronic model of arthritis supported by a strain-specific periarticular lymph node in BALB/c mice.

Current animal models of arthritis only partially reflect the complexity of rheumatoid arthritis and typically lack either chronicity or autoantibody formation. Here we describe a model that combines features of antigen-induced arthritis and collagen-induced arthritis, which can be efficiently induced in BALB/c and C57BL/6 mice. However, BALB/c mice generate significantly higher titres of anticollagen and anticitrullinated peptide antibodies, show a stronger progressive joint destruction, and in the chronic phase the disease spreads between joints. Concomitant to the observation of a more severe pathology, we discovered a previously undescribed small periarticular lymph node in close proximity to the knee joint of BALB/c mice, which acts as the primary draining lymph node for the synovial cavity. Our model more closely reflects the pathology of rheumatoid arthritis than classical models of arthritis and is hence particularly suitable for further studies of disease pathogenesis.

CXCR5- and CCR7-dependent lymphoid neogenesis in a murine model of chronic antigen-induced arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease with unknown etiology and only partially defined pathogenesis. The aim of this study was to establish a murine model of chronic arthritis in which the development of tertiary lymphoid tissue, a hallmark of human RA, is locally induced, and to characterize the roles of the homeostatic chemokine receptors CXCR5 and CCR7 in this process. METHODS: We developed a modified model of chronic antigen-induced arthritis (AIA) in mice with a strong bias toward inflammation. Disease pathology was assessed up to 9 months in wild-type, CXCR5-deficient, and CCR7-deficient mice by determination of knee joint swelling and cellular and humoral immune responses, as well as by histologic analysis of arthritic knee joints. RESULTS: In this novel model of AIA, mice developed organized ectopic lymphoid follicles with topologically segregated B cell and T cell areas, high endothelial venules, and germinal center formation within the chronically inflamed synovial tissue. Analysis of the initiation and progression of AIA in wild-type, CXCR5-/-, and CCR7-/- mice revealed a reduction of acute inflammatory parameters in both knockout strains as well as significantly reduced joint destruction in CXCR5-/- mice. Most importantly, the development and organization of tertiary lymphoid tissue were significantly impaired in CXCR5-deficient and CCR7-deficient mice. CONCLUSION: Our results suggest that an inflammatory microenvironment efficiently triggers lymphoid neogenesis in autoimmune diseases such as RA. Moreover, the generation of autoreactive tertiary lymphoid tissues, which is entirely dependent on homeostatic chemokines, may in turn maintain local aberrant chronic immune responses.

Selectin ligand-independent priming and maintenance of T cell immunity during airborne tuberculosis.

Immunity to Mycobacterium tuberculosis infection is critically dependent on the timely priming of T effector lymphocytes and their efficient recruitment to the site of mycobacterial implantation in the lung. E-, P-, and L-selectin counterreceptors control lymphocyte homing to lymph nodes and leukocyte trafficking to peripheral sites of acute inflammation, their adhesive function depending on fucosylation by fucosyltransferases (FucT) IV and VII. To address the relative importance of differentially glycosylated selectin counterreceptors for priming of T cell effector functions in a model of mycobacteria-induced granulomatous pulmonary inflammation, we used aerosol-borne M. tuberculosis to infect FucT-IV-/-, FucT-VII-/-, FucT-IV-/-/FucT-VII-/-, or wild-type control mice. In lymph nodes, infected FucT-IV-/-/FucT-VII-/- and, to a lesser extent, FucT-VII-/- mice had severely reduced numbers of T cells and reduced Ag-specific effector responses. By contrast, recruitment of activated T cells into the lungs was similar in all four groups of mice during infection and expression of T cell, and macrophage effector functions were only delayed in lungs of FucT-IV-/-/FucT-VII-/- mice. Importantly, lungs from all groups expressed CXCL13, CCL21, and CCL19 and displayed organized follicular neolymphoid structures after infection with M. tuberculosis, which suggests that the lung served as a selectin ligand-independent priming site for immune responses to mycobacterial infection. All FucT-deficient strains were fully capable of restricting M. tuberculosis growth in infected organs until at least 150 days postinfection. Our observations indicate that leukocyte recruitment functions dictated by FucT-IV and FucT-VII-dependent selectin ligand activities are not critical for inducing or maintaining T cell effector responses at levels necessary to control pulmonary tuberculosis.