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Neurological symptoms, including cognitive impairment and fatigue, can occur in both the acute infection phase of coronavirus disease 2019 (COVID-19) and at later stages, yet the mechanisms that contribute to this remain unclear. Here we profiled single-nucleus transcriptomes and proteomes of brainstem tissue from deceased individuals at various stages of COVID-19. We detected an inflammatory type I interferon response in acute COVID-19 cases, which resolves in the late disease phase. Integrating single-nucleus RNA sequencing and spatial transcriptomics, we could localize two patterns of reaction to severe systemic inflammation, one neuronal with a direct focus on cranial nerve nuclei and a separate diffuse pattern affecting the whole brainstem. The latter reflects a bystander effect of the respiratory infection that spreads throughout the vascular unit and alters the transcriptional state of mainly oligodendrocytes, microglia and astrocytes, while alterations of the brainstem nuclei could reflect the connection of the immune system and the central nervous system via, for example, the vagus nerve. Our results indicate that even without persistence of severe acute respiratory syndrome coronavirus 2 in the central nervous system, local immune reactions are prevailing, potentially causing functional disturbances that contribute to neurological complications of COVID-19., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling., (© 2023. The Author(s).)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive., Methods: Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results., Results: We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection., Conclusions: Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment., Competing Interests: Conflict of interest: J-C. Rückert and H. Radbruch report support from DFG RA 2491/1-1, BMBF (Defeat Pandemics). A.E. Hauser reports support from Charité – Universitätsmedizin Berlin and Deutsches Rheuma-Forschungszentrum Berlin, and grants from Deutsche Forschungsgemeinschaft (HA5354/10-1, TRR130,P17 and C01, HA5354/8-1). T. Wolff reports support from Federal Ministry of Education and Research (BMBF) grant 01K12006F. M. Kessler reports grants from BMBF Organo-Strat, Einstein 3R. M. Dohmen reports contracts with Max-Delbrück Center, Berlin; grants from Gender Equality Fund, Berlin Institute of Health. F. Klauschen reports consulting fees, lecture honoraria, travel support and participation on advisory boards with BMS, Novartis, Roche and Lilly, and is a co-founder of AI-BIH/Charité-Spinoff Aignostics GmbH. F. Heppner reports consulting fees, lecture honoraria, payment for expert testimony and leadership roles at Novartis, AstraZeneca and ThinkHealth Hygiene Solutions. V.M. Corman reports the following patents: 20210190797 (Methods and reagents for diagnosis of SARS-CoV-2 infection); 9841834 (Human recombinant monoclonal antibody against SARS-CoV-2 spike glycoprotein); 9909654 (A pharmaceutical combination comprising an anti-viral protonophore and a serine protease inhibitor). D. Niemeyer reports that Technische Universität Berlin, Freie Universität Berlin and Charité – Universitätsmedizin have filed a patent application for siRNAs inhibiting SARS-CoV-2 replication with D. Niemeyer as coauthor. M.A. Müller reports the following patents: 20210190797 (Methods and reagents for diagnosis of SARS-CoV-2 infection); 9841834 (Human recombinant monoclonal antibody against SARS-CoV-2 spike glycoprotein); 9909654 (A pharmaceutical combination comprising an anti-viral protonophore and a serine protease inhibitor); and has participated on an advisory board for ECDC/WHO. S. Ludwig reports consulting fees from Atriva Therapeutics GmbH, Biontec SE; and has patent PCT/EP2021/063485 pending. M. Witzenrath reports grants from Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, Deutsche Gesellschaft für Pneumologie, European Respiratory Society, Marie Curie Foundation, Else Kröner Fresenius Stiftung, Capnetz Stiftung, International Max Planck Research School, Quark Pharma, Takeda Pharma, Noxxon, Pantherna, Silence Therapeutics, Vaxxilon, Actelion, Bayer Health Care, Biotest and Boehringer Ingelheim; consulting fees from Noxxon, Pantherna, Silence Therapeutics, Vaxxilon, Aptarion, GlaxoSmithKline, Sinoxa and Biotest; lecture honoraria from AstraZeneca, Berlin Chemie, Chiesi, Novartis, Teva, Actelion, Boehringer Ingelheim, GlaxoSmithKline, Biotest, Bayer Health Care; and has the following patents issued: EPO 12181535.1 (IL-27 for modulation of immune response in acute lung injury), WO/2010/094491 (Means for inhibiting the expression of Ang-2), DE 102020116249.9 (Camostat/Niclosamide cotreatment in SARS-CoV-2 infected human lung cells). All other authors have nothing to disclose., (Copyright ©The authors 2022.)

Neurological symptoms in SARS-CoV-2-infected patients have been reported, but their cause remains unclear. In theory, the neurological symptoms observed after SARS-CoV-2 infection could be (1) directly caused by the virus infecting brain cells, (2) indirectly by our body's local or systemic immune response toward the virus, (3) by coincidental phenomena, or (4) a combination of these factors. As indisputable evidence of intact and replicating SARS-CoV-2 particles in the central nervous system (CNS) is currently lacking, we suggest focusing on the host's immune reaction when trying to understand the neurocognitive symptoms associated with SARS-CoV-2 infection. In this perspective, we discuss the possible immune-mediated mechanisms causing functional or structural CNS alterations during acute infection as well as in the post-infectious context. We also review the available literature on CNS affection in the context of COVID-19 infection, as well as observations from animal studies on the molecular pathways involved in sickness behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-β, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-β. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-β, and is distracted from itself.

Innate lymphoid cells (ILCs) emerge in the last few years as important regulators of immune responses and biological processes. Although ILCs are mainly known as tissue-resident cells, their precise localization and interactions with the microenvironment are still unclear. Here we combine a multiplexed immunofluorescence technique and a customized computational, open-source analysis pipeline to unambiguously identify CD127 + ILCs in situ and characterize these cells and their microenvironments. Moreover, we reveal the transcription factor IRF4 as a marker for tonsillar ILC3, and identify conserved stromal landmarks characteristic for ILC localization. We also show that CD127 + ILCs share tissue niches with plasma cells in the tonsil. Our works thus provide a platform for multiparametric histological analysis of ILCs to improve our understanding of ILC biology.

The newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic respiratory disease. Moreover, thromboembolic events throughout the body, including in the CNS, have been described. Given the neurological symptoms observed in a large majority of individuals with COVID-19, SARS-CoV-2 penetrance of the CNS is likely. By various means, we demonstrate the presence of SARS-CoV-2 RNA and protein in anatomically distinct regions of the nasopharynx and brain. Furthermore, we describe the morphological changes associated with infection such as thromboembolic ischemic infarction of the CNS and present evidence of SARS-CoV-2 neurotropism. SARS-CoV-2 can enter the nervous system by crossing the neural-mucosal interface in olfactory mucosa, exploiting the close vicinity of olfactory mucosal, endothelial and nervous tissue, including delicate olfactory and sensory nerve endings. Subsequently, SARS-CoV-2 appears to follow neuroanatomical structures, penetrating defined neuroanatomical areas including the primary respiratory and cardiovascular control center in the medulla oblongata.

The multiple sclerosis therapeutic teriflunomide is known to block the de novo synthesis of pyrimidine in mitochondria by inhibiting the enzyme dihydroorotate-dehydrogenase (DHODH). The metabolic processes of oxidative phosphorylation and glycolysis are further possible downstream targets. In healthy adult mice, high levels of dihydroorotate-dehydrogenase (DHODH) activity are measured in the central nervous system (CNS), and DHODH inhibition may cause indirect effects on reactive oxygen species production and NADPH oxidase (NOX) mediated oxidative stress, known to be key aspects of the inflammatory response of the CNS. However, little is known about the effect of teriflunomide on the dynamics of NOX activation in CNS cells and subsequent alterations of neuronal function in vivo . In this study, we employed fluorescence lifetime imaging (FLIM) and phasor analysis of the endogeneous fluorescence of NAD(P)H (nicotinamide adenine dinucleotide phosphate) in the brain stem of mice to visualize the effect of teriflunomide on cellular metabolism. Furthermore, we simultaneously studied neuronal Ca 2+ signals in transgenic mice with a FRET-based Troponin C Ca 2+ sensor based (CerTN L15) quantified using FRET-FLIM. Hence, we directly correlated neuronal (dys-)function indicated by steadily elevated calcium levels with metabolic activity in neurons and surrounding CNS tissue. Employing our intravital co-registered imaging approach, we could not detect any significant alteration of NOX activation after incubation of the tissue with teriflunomide. Furthermore, we could not detect any changes of the inflammatory induced neuronal dysfunction due to local treatment with teriflunomide. Concerning drug safety, we can confirm that teriflunomide has no metabolic effects on neuronal function in the CNS tissue during neuroinflammation at concentrations expected in orally treated patients. The combined endogenous FLIM and calcium imaging approach developed by us and employed here uniquely meets the need to monitor cellular metabolism as a basic mechanism of tissue functions in vivo ., (Copyright © 2020 Mothes, Ulbricht, Leben, Günther, Hauser, Radbruch and Niesner.)

Objective: To investigate whether low-density granulocytes (LDGs) are an immunophenotypic feature of patients with multiple sclerosis (MS) or neuromyelitis optica spectrum disorder (NMOSD). Methods: Blood samples were collected from 20 patients with NMOSD and 17 patients with MS, as well as from 15 patients with Systemic Lupus Erythematosus (SLE) and 23 Healthy Donors (HD). We isolated peripheral blood mononuclear cells (PBMCs) with density gradient separation and stained the cells with antibodies against CD14, CD15, CD16, and CD45, and analyzed the cells by flow cytometry or imaging flow cytometry. We defined LDGs as CD14 - CD15 high and calculated their share in total PBMC leukocytes (CD45 + ) as well as the share of CD16 hi LDGs. Clinical data on disease course, medication, and antibody status were obtained. Results: LDGs were significantly more common in MS and NMOSD than in HDs, comparable to SLE samples (median values HD 0.2%, MS 0.9%, NMOSD 2.1%, SLE 4.3%). 0/23 of the HDs, but 17/20 NMOSD and 11/17 MS samples as well as 13/15 SLE samples had at least 0.7 % LDGs. NMOSD patients without continuous immunosuppressive treatment had significantly more LDGs compared to their treated counterparts. LDG nuclear morphology ranged from segmented to rounded, suggesting a heterogeneity within the group. Conclusion: LDGs are a feature of the immunophenotype in some patients with MS and NMOSD., (Copyright © 2019 Ostendorf, Mothes, van Koppen, Lindquist, Bellmann-Strobl, Asseyer, Ruprecht, Alexander, Niesner, Hauser, Paul and Radbruch.)

Objective: To investigate the relevance of dysfunctional T cells in immune-mediated myopathies. We analyzed T-cell exhaustion and senescence, in the context of programmed cell death protein 1 (PD1)-related immunity in skeletal muscle biopsies from patients with immune-mediated necrotizing myopathy (IMNM), sporadic inclusion body myositis (sIBM), and myositis induced by immune checkpoint inhibitors (irMyositis)., Methods: Skeletal muscle biopsies from 12 patients with IMNM, 7 patients with sIBM, and 8 patients with irMyositis were analyzed by immunostaining and immunofluorescence as well as by quantitative PCR. Eight biopsies from nondisease participants served as controls., Results: CD3 + CD8 + T cells in biopsies from IMNM, sIBM, and irMyositis were largely PD1-positive, while CD68 + macrophages were sparsely positive to the ligand of programmed cell death protein 1 (PD-L1). The sarcolemma of myofibers was PD-L2 + and was colocalized with major histocompatibility complex (MHC) class I. CD68 + macrophages were colocalized with PD-L2. Senescent T cells were strongly enriched in skeletal muscle of sIBM, revealing a distinct immunologic signature. Biopsies from patients with irMyositis showed mild signs of senescence and exhaustion., Conclusion: Persistent exposure to antigens in IMNMs and sIBM may lead to T-cell exhaustion, a process controlled by the PD1 receptor and its cognate ligands PD-L1/PD-L2. To our knowledge, these data are the first evidence of presence of dysfunctional T cells and relevance of the PD1 pathway in IMNM, sIBM, and irMyositis. These findings may guide the way to a novel understanding of the immune pathogenesis of immune-mediated myopathies.

Aim: Undergraduate medical education is currently in a fundamental transition towards competency-based programs around the globe. A major curriculum reform implies a dual challenge: the change of the curriculum and the delivering organization. Both are closely interwoven. In this article, we provide practical insights into our approach of managing such a fundamental reform of the large undergraduate medical program at the Charité - Universitätsmedizin Berlin., Methods: Members of the project management team summarized the key features of the process with reference to the literature., Results: Starting point was a traditional, discipline-based curriculum that was reformed into a fully integrated, competency-based program. This change process went through three phases: initiation, curriculum development and implementation, and sustainability. We describe from a change management perspective, their main characteristics, and the approaches that were employed to manage them successfully., Conclusions: Our report is intended to provide practical insights and guidance for those institutions which are yet considering or have already started to undergo a major reform of their undergraduate programs towards competency medical education.

Although oligoclonal bands in the cerebrospinal fluid have been a hallmark of multiple sclerosis diagnosis for over three decades, the role of antibody-secreting cells in multiple sclerosis remains unclear. T and B cells are critical for multiple sclerosis pathogenesis, but increasing evidence suggests that plasma cells also contribute, through secretion of autoantibodies. Long-lived plasma cells are known to drive various chronic inflammatory conditions as e.g. systemic lupus erythematosus, however, to what extent they are present in autoimmune central nervous system inflammation has not yet been investigated. In brain biopsies from multiple sclerosis patients and other neurological diseases, we could detect non-proliferating plasma cells (CD138 + Ki67 - ) in the parenchyma. Based on this finding, we hypothesized that long-lived plasma cells can persist in the central nervous system (CNS). In order to test this hypothesis, we adapted the multiple sclerosis mouse model experimental autoimmune encephalomyelitis to generate a B cell memory response. Plasma cells were found in the meninges and the parenchyma of the inflamed spinal cord, surrounded by tissue areas resembling survival niches for these cells, characterized by an up-regulation of chemokines (CXCL12), adhesion molecules (VCAM-1) and survival factors (APRIL and BAFF). In order to determine the lifetime of plasma cells in the chronically inflamed CNS, we labeled the DNA of proliferating cells with 5-ethynyl-2'-deoxyuridine (EdU). Up to five weeks later, we could detect EdU + long-lived plasma cells in the murine CNS. To our knowledge, this is the first study describing non-proliferating plasma cells directly in the target tissue of a chronic inflammation in humans, as well as the first evidence demonstrating the ability of plasma cells to persist in the CNS, and the ability of the chronically inflamed CNS tissue to promote this persistence. Hence, our results suggest that the CNS provides survival niches for long-lived plasma cells, similar to the niches found in other organs. Targeting these cells in the CNS offers new perspectives for treatment of chronic autoimmune neuroinflammatory diseases, especially in patients who do not respond to conventional therapies.

In aging individuals, both protective as well as regulatory immune functions are declining, resulting in an increased susceptibility to infections as well as to autoimmunity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2-deficiency in immune cell subsets has been shown to be associated with aging. Using intravital marker-free NAD(P)H-fluorescence lifetime imaging, we have previously identified microglia/myeloid cells and astrocytes as main cellular sources of NADPH oxidase (NOX) activity in the CNS during neuroinflammation, due to an overactivation of NOX. The overactivated NOX enzymes catalyze the massive production of the highly reactive [Formula: see text] which initiates in a chain reaction the overproduction of diverse reactive oxygen species (ROS). Age-dependent oxidative distress levels in the brain and their cellular sources are not known. Furthermore, it is unclear whether in age-dependent diseases oxidative distress is initiated by overproduction of ROS or by a decrease in antioxidant capacity, subsequently leading to neurodegeneration in the CNS. Here, we compare the activation level of NOX enzymes in the cerebral cortex of young and aged mice as well as in a model of vascular amyloid pathology. Despite the fact that a striking change in the morphology of microglia can be detected between young and aged individuals, we find comparable low-level NOX activation both in young and old mice. In contrast, aged mice with the human APP E693Q mutation, a model for cerebral amyloid angiopathy (CAA), displayed increased focal NOX overactivation in the brain cortex, especially in tissue areas around the vessels. Despite activated morphology in microglia, NOX overactivation was detected only in a small fraction of these cells, in contrast to other pathologies with overt inflammation as experimental autoimmune encephalomyelitis (EAE) or glioblastoma. Similar to these pathologies, the astrocytes majorly contribute to the NOX overactivation in the brain cortex during CAA. Together, these findings emphasize the role of other cellular sources of activated NOX than phagocytes not only during EAE but also in models of amyloid pathology. Moreover, they may strengthen the hypothesis that microglia/monocytes show a diminished potential for clearance of amyloid beta protein.

Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

A hallmark of autoimmune retinal inflammation is the infiltration of the retina with cells of the innate and adaptive immune system, leading to detachment of the retinal layers and even to complete loss of the retinal photoreceptor layer. As the only optical system in the organism, the eye enables non-invasive longitudinal imaging studies of these local autoimmune processes and of their effects on the target tissue. Moreover, as a window to the central nervous system (CNS), the eye also reflects general neuroinflammatory processes taking place at various sites within the CNS. Histological studies in murine neuroinflammatory models, such as experimental autoimmune uveoretinitis (EAU) and experimental autoimmune encephalomyelitis, indicate that immune infiltration is initialized by effector CD4 + T cells, with the innate compartment (neutrophils, macrophages, and monocytes) contributing crucially to tissue degeneration that occurs at later phases of the disease. However, how the immune attack is orchestrated by various immune cell subsets in the retina and how the latter interact with the target tissue under in vivo conditions is still poorly understood. Our study addresses this gap with a novel approach for intravital two-photon microscopy, which enabled us to repeatedly track CD4 + T cells and LysM phagocytes during the entire course of EAU and to identify a specific radial infiltration pattern of these cells within the inflamed retina, starting from the optic nerve head. In contrast, highly motile [Formula: see text] cells display an opposite radial motility pattern, toward the optic nerve head. These inflammatory processes induce modifications of the microglial network toward an activated morphology, especially around the optic nerve head and main retinal blood vessels, but do not affect the neurons within the ganglion cell layer. Thanks to the new technology, non-invasive correlation of clinical scores of CNS-related pathologies with immune infiltrate behavior and subsequent tissue dysfunction is now possible. Hence, the new approach paves the way for deeper insights into the pathology of neuroinflammatory processes on a cellular basis, over the entire disease course.

Successive treatment of the diethyl ester of 3,4-bis(oxamate)phenylene benzoic acid (3,4-bopbH2Et2OH; ) with [Bu4N]OH and MCl2·nH2O (M = Cu, Ni; n = 2, 6) gave, upon extraction into CH2Cl2, binuclear complexes [Bu4N]4[{M(3,4-bopbO)}2CH2] (, M = Cu, Ni) as novel symmetric methylene diesters. In contrast, subsequent addition of [Bu4N]OH and anhydrous NiCl2 to afforded the very hygroscopic [Bu4N]3[Ni(3,4-bopbO)] () instead. In order to verify reaction conditions to achieve the corresponding carboxamides to - as a crucial step for the synthesis of higher generation dendrimers - was shown to react with RNH2 (R = Me (), Pr ()) to [RNH3][tdqc] (R = Me (), Pr (); tdqc = 1,2,3,4-tetrahydro-2,3-dioxo-6-quinoxaline carboxylate) by rearrangement reactions. Alternatively, if was converted first to 3,4-bopbH2EtF () and treated next with PrNH2, the corresponding carboxamide 3,4-bopbH2Et2NPrH () could be obtained in high yields. The reaction of with [Bu4N]OH and MCl2·nH2O (M = Cu, Ni; n = 2, 6) gave conveniently mononuclear [Bu4N]2[M(3,4-bopbNPrH)] (, M = Cu, Ni). After thus optimizing reaction conditions, for the synthesis of higher-branched bis(oxamato) type complexes the (polyamido)amines (en(II)H2), (den(III)H3), (den(V)H5) and (den(X)H10), possessing two, three, five or ten terminal amino groups, were reacted with appropriate equiv. of to give the corresponding carboxamides en(II)(3,4-bopbH2Et2)2 (), den(III)(3,4-bopbH2Et2)3 (), den(V)(3,4-bopbH2Et2)5 () and den(X)(3,4-bopbH2Et2)10 (). Compounds were converted to the corresponding Cu(II)-containing complexes , which were treated with the corresponding equiv. of [Cu(pmdta)][BF4]2 to afford . Complexes of the series and possess two, three, five or ten end-grafted mononuclear {Cu(3,4-bopb)}(2-) and trinuclear {Cu3(3,4-bopb)(pmdta)2}(2+) complex fragments, respectively. The identities of were established by NMR spectroscopy and ESI-MS measurements. For and , ESI-MS, UV/Vis and ESR studies were applied to confirm the identities of these species. The magnetic properties of the {Cu3(3,4-bopb)(pmdta)2}(2+) end-grafted poly(amidoamine) dendrimers were studied by susceptibility measurements vs. temperature to give J values between -112 () and -118 cm(-1) (), which are typically observed for discrete trinuclear Cu(II)-containing bis(oxamato) type complexes.

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation.

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo-ratiometrically and time-resolved by fluorescence lifetime imaging-and show their concrete application in the context of neuroinflammation in adult mice.

Background: Despite considerable progress made in the past decade through salt iodization programs, over 2 billion people worldwide still have inadequate iodine intake, with devastating consequences for brain development and intellectual capacity. To optimize these programs with regard to salt iodine content, careful monitoring of salt iodine content is essential, but few methods are available to quantitatively measure iodine concentration in a simple, fast, and safe way., Objective: We have validated a newly developed device that quantitatively measures the content of potassium iodate in salt in a simple, safe, and rapid way., Methods: The linearity, determination and detection limit, and inter- and intra-assay variability of this colorimetric method were assessed and the method was compared with iodometric titration, using salt samples from several countries., Results: Linearity of analysis ranged from 5 to 75 mg/kg iodine, with 1 mg/kg being the determination limit; the intra- and interassay imprecision was 0.9%, 0.5%, and 0.7% and 1.5%, 1.7%, and 2.5% for salt samples with iodine contents of 17, 30, and 55 mg/kg, respectively; the interoperator imprecision for the same samples was 1.2%, 4.9%, and 4.7%, respectively. Comparison with the iodometric method showed high agreement between the methods (R2 = 0.978; limits of agreement, -10.5 to 10.0 mg/kg)., Conclusions: The device offers a field- and user-friendly solution to quantifying potassium iodate salt content reliably. For countries that use potassium iodide in salt iodization programs, further validation is required.

Background: β-Carotene is an important precursor of vitamin A, and is associated with bovine fertility. β-Carotene concentrations in plasma are used to optimize β-carotene supplementation in cattle, but measurement requires specialized equipment to separate plasma and extract and measure β-carotene, either using spectrophotometry or high performance liquid chromatography (HPLC)., Objective: The objective of this study was to validate a new 2-step point-of-care (POC) assay for measuring β-carotene in whole blood and plasma., Methods: β-carotene concentrations in plasma from 166 cows were measured using HPLC and compared with results obtained using a POC assay, the iCheck-iEx-Carotene test kit. Whole blood samples from 23 of these cattle were also evaluated using the POC assay and compared with HPLC-plasma results from the same 23 animals. The POC assay includes an extraction vial (iEx Carotene) and hand-held photometer (iCheck Carotene)., Results: Concentrations of β-carotene in plasma measured using the POC assay ranged from 0.40 to 15.84 mg/L (n = 166). No differences were observed between methods for assay of plasma (mean ± SD; n = 166): HPLC-plasma 4.23 ± 2.35 mg/L; POC-plasma 4.49 ± 2.36 mg/L. Similar good agreement was found when plasma analyzed using HPLC was compared with whole blood analyzed using the POC system (n = 23): HPLC-plasma 3.46 ± 2.12 mg/L; POC-whole blood 3.67 ± 2.29 mg/L., Conclusions: Concentrations of β-carotene can be measured in blood and plasma from cattle easily and rapidly using a POC assay, and results are comparable to those obtained by the highly sophisticated HPLC method. Immediate feedback regarding β-carotene deficiency facilitates rapid and appropriate optimization of β-carotene supplementation in feed., (© 2012 American Society for Veterinary Clinical Pathology.)

Well-dispersed Ag@Pd supported on magnetite nanoparticles have been obtained through a simple colloidal impregnation method. The as-synthesised nanocomposite exhibits greatly enhanced catalytic reactivity and reusability towards 4-nitrophenol hydrogenation., (This journal is © The Royal Society of Chemistry 2011)

Vitamin A deficiency continues to be a global public health problem. Fortification of oil with vitamin A is considered a cost-effective, feasible strategy to prevent this problem but quality control poses a challenge to program implementation. To overcome this, we have validated a newly developed device that quantitatively measures the content of retinyl palmitate in refined palm oil, is simple to use, and yields immediate results.Linearity of analysis ranged from 2.5 - 30 mg retinol equivalents (RE)/ kg of palm oil, with 2.5 mg RE/kg being the determination limit; inter- and intra-assay precision ranged from 1.4 - 7.1 %. Comparison with a high-performance liquid chromatography method showed high agreement between the methods (R(2) = 0.92; Limits of Agreement: -1.24 mg to 2.53 mg RE/kg), and further comparisons illustrate that the new device is useful in low-resource settings. This device offers a field- and user-friendly solution to quantifying the vitamin A content in refined palm oil.

Metal-organics [((RO)(3)P)(m)CuO(2)CCF(3)] (R = CH(3): 11a, m = 1; 11b, m = 2; 11c, m = 3. R = CH(2)CH(3): 12a, m = 1; 12b, m = 2; 12c, m = 3. R = CH(2)CF(3): 13a, m = 1; 13b, m = 2; 13c, m = 3) are either accessible by the reaction of [((RO)(3)P)(m)CuCl] (R = CH(3): 5a, m = 1; 5b, m = 2; 5c, m = 3. R = CH(2)CH(3): 6a, m = 1; 6b, m = 2; 6c, m = 3) with [KO(2)CCF(3)] (7), or treatment of [Cu(2)O] (8) with HO(2)CCF(3) (9) and P(OR)(3) (2, R = CH(3); 3, R = CH(2)CH(3); 4, R = CH(2)CF(3)). (31)P{(1)H} NMR spectra [((CH(3)O)(3)P)(m)CuO(2)CCF(3)] (m = 1, 1.5, 2, 2.5, 3, 3.5, and 4) have been studied at 25 and -80 °C showing phosphite ligand exchange in solution. The molecular structures of 11a and 13a-13c in the solid state are reported. Complexes 11a and 13a are tetramers featuring μ-η(2)(1κO:2κO')- and μ(3)-η(2)(1κO:2κO':3κO')-(11a) or μ(3)-η(2)(1κO:2κO':3κO')-bonded O(2)CCF(3) ligands (13a) with the Cu(I) ions being part of CuPO(2) and CuPO(3) units (11a), while in 13a solely a CuPO(3) moiety is present. Skeletal isomerism of 11a vs. 13a is discussed. Compound 13b is dimeric ({CuP(2)O(2)}(2)) with pseudo-tetrahedral Cu environments and μ-η(2)(1κO:2κO')O(2)CCF(3) functionalities. In monomeric 13c the O(2)CCF(3) ligand is η(1)(κO)-bonded to a tetra-coordinated Cu(i) ion. The thermal solid state properties of 11, 12 and 13 were studied by Thermo Gravimetry (TG). These complexes decompose by phosphite elimination, decarboxylation and dealkylation. Hot-wall Chemical Vapour Deposition (CVD) experiments were carried out at 380 °C using 11c as precursor for the deposition of copper onto pieces of TiN-coated oxidized silicon substrates. Copper layers of high purity were obtained with grain sizes between 200-1200 nm.

The effect of progressive phosphorylation by phosphorous oxychloride upon the conformation of the 300 kDa storage protein (cruciferin) from rapeseed has been studied using chemical analysis, SDS-PAGE, HPLC, analytical ultracentrifugation, viscometry, fluorescence spectroscopy, and hydrophobicity measurement. The amount of phosphorous in the protein increased with the excess of phosphorous oxychloride and the pH of reaction. The bulk of phosphorus was only loosely bound to the protein and was removed by washing with cold perchloric acid. The more stably bound phosphorus groups after reaction at pH 8 were found to be nearly equally attached to amino and hydroxyl groups, whereas phosphorylation at pH 10-11 led to predominant O-phosphorylation as detected by studying the acid- and alkali-lability of the protein-phosphorous bonds. A 50 kDa component appeared as a product of covalent cross-linking of the constituent alpha- and beta-polypeptide chains. A 2.5S fraction appeared as the main product of dissociation, which takes place after a critical step of modification. The higher the extent of phosphorylation, the larger was the percentage of higher molecular weight products, the percentage of which was most significant after modification under strongly alkaline conditions. They may be attributed both to products of chemical cross-linking and to noncovalently linked aggregates formed by interactions of partially unfolded derivatives exhibiting an increased surface hydrophobicity.