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Achondroplasia is the most common skeletal dysplasia. With the development of new growth-promoting drug treatment for children and adolescents with achondroplasia, multidisciplinary care has become increasingly more important. In addition to orthopedic care, a specialized team comprised of pediatrics/endocrinology, radiology, neurosurgery, otorhinolaryngology, anesthesiology, physiotherapy, psychology and other disciplines is necessary to develop and implement a holistic concept to improve the quality of life for individuals affected by achondroplasia. The orthopedic management of achondroplasia has become even more complex and multidisciplinary due to these new medical and surgical developments. These new developments herald a new era of orthopedic and multidisciplinary care for achondroplasia., Competing Interests: Einhaltung ethischer Richtlinien. Interessenkonflikt: Gemäß den Richtlinien des Springer Medizin Verlags werden Autoren und Wissenschaftliche Leitung im Rahmen der Manuskripterstellung und Manuskriptfreigabe aufgefordert, eine vollständige Erklärung zu ihren finanziellen und nichtfinanziellen Interessen abzugeben. Autoren: G. Mindler: A. Finanzielle Interessen: Kyowa Kirin, BioMarin, Alexion, Newclip. – Bezahlter Berater/Vortragender für Kyowa Kirin, BioMarin, Alexion. – B. Nichtfinanzielle Interessen: Angestellt: Orthopädisches Spital Speising, Niedergelassen: Privatordination Dr. Mindler | Mitgliedschaften: VKO, EPOS, ÖGO, ÖGOuT, GAMMA, GEVR, ISCBH | Vereinstätigkeiten: Medizinischer Beirat XLH Austria. C. Chiari: A. Finanzielle Interessen: Universität für Weiterbildung Krems, Vortragende im Masterstudienlehrgang „Orthopädie und Traumatologie“. – B. Nichtfinanzielle Interessen: Abteilungsleiterin Kinderorthopädie und Fußchirurgie, Orthopädisches Spital Speising, Wien | Mitgliedschaften: ÖGO, ÖGOUT, VKO, VSOU, ICRS, GOTS, EPOS | Past Präsidentin: ÖGO | Vorstandsmitglied: ÖGO, ÖGOUT, EPOS. N. Walik: A. Finanzielle Interessen: N. Walik gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: Klinische Psychologin im Orthopädischen Spital Speising Wien | Mitgliedschaften: Berufsverband österreichischer Psychologen BÖP, Milton Erikson Gesellschaft MEGA. A. Stauffer: A. Finanzielle Interessen: Smith & Nephew – Übernahme von Reise- und Übernachtungskosten sowie bezahlte Kursteilnehmergebühr. – B. Nichtfinanzielle Interessen: Assistenzärztin in Ausbildung zur Fachärztin am Orthopädischen Spital Speising | Mitgliedschaften: GOTS, ÖGU. S. Siegert: A. Finanzielle Interessen: Forschungsförderung zur persönlichen Verfügung: CCP Starter Gran 2021, 1000 €, Medizinische Universität Wien. – PCT Advisory Board AADC Referentinnenhonorar, Biogen Advisory Board, Friedreich Ataxie Referentinnenhonorar. – B. Nichtfinanzielle Interessen: Fachärztin an der Uniklinik für Kinder- und Jugendmedizin der Medizinischen Universität Wien | Mitgliedschaften: Öst. Gesellschaft f. Kinder- und Jugendheilkunde, EPNS. A. Reinprecht: A. Finanzielle Interessen: A. Reinprecht gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: Angestellte Neurochirurgin (Medizinische Universität Wien) | Mitgliedschaften: Österr. Gesellschaft für Neurochirurgie (ÖGNC), European Society for Pediatric Neurosurgery (ESPN). R. Rath-Wacenovsky: A. Finanzielle Interessen: R. Rath–Wacenovsky gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: Leitung und Inhaberin, Kindermedizinisches Zentrum: Kinder und Jugendgesundheit Leuchtturm! Seestadt | Barbara Prammer Allee 9/1, 1220 Wien, Österreich | Mitgliedschaften: ÖGP, ÖGSM, ÖGKJ. J. Patsch: A. Finanzielle Interessen: J. Patsch gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: Angestellte Kinderradiologin (Assoziierte Professorin Medizinische Universität Wien); Niedergelassene Wahlärztin (Minischall.at) | Vorstandsmitglied Gesellschaft für Pädiatrische Radiologie; AG-Leiterin Kinderradiologie Österr. Röntgengesellschaft (ÖRG); Mitglied ÖRG | Mitgliedschaften: Deutsche Gesellschaft für Osteologie (DVO), European Society of Radiology (ESR), European Society of Pediatric Radiology (ESPR), ESPR MSK Taskforce Subcommittee Member. V. Kranebitter: A. Finanzielle Interessen: V. Kranebitter gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: HNO-Fachärztin und Phoniaterin/Oberärztin/Klinische Abteilung für Phoniatrie-Logopädie, Universitätsklinik für Hals‑, Nasen- und Ohrenkrankheiten, Medizinische Universität Wien/Österreich. B. Kraler: A. Finanzielle Interessen: B. Kraler gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: Facharzt Orthopädie und Traumatologie am Orthopädischen Spital Speising Wien | Ordinationstätigkeit Orthopädie und Traumatologie Nest 13, Wien | Mitgliedschaften: European Pediatric Orthopaedic Society EPOS, Swiss Orthopaedics, OeGOuT und ÖGO, Swiss Pediatric Orthopedic Society, European Society for Movement Analysis in Adults and Children (ESMAC). N. Kadrnoska: A. Finanzielle Interessen: N. Kadrnoska gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: Angestellt von Gemeinde Wien, Physiotherapeutin, AKH Wien. G. Hartmann: A. Finanzielle Interessen: Honorare für die Teilnahme an Advisory Boards der Firma BioMarin zum Thema Achondroplasie. – B. Nichtfinanzielle Interessen: Pädiatrische Endokrinologin an der MedUni Wien (Bereichsleitung) | Koordinatorin des national und international designierten Expertisezentrums für Seltene Knochenerkrankungen (Vienna Bone & Growth Center, BOND ERN). J. Falger: A. Finanzielle Interessen: J. Falger gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: Ärztliche Direktorin, REHA Bad Erlach GmbH, kokon Bad Erlach, Reha für junge Menschen, Thermenstraße 1, 2822 Bad Erlach. N. Ahyai: A. Finanzielle Interessen: N. Ahyai gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: Angestellte Anästhesistin, Orthopädisches Spital Speising, Wien. K. Block: A. Finanzielle Interessen: K. Block gibt an, dass kein finanzieller Interessenkonflikt besteht. – B. Nichtfinanzielle Interessen: Obfrau des Bundesverbandes kleinwüchsiger Menschen und ihrer Familien | Der Bkmf-AUSTRIA ist für die Interessenvertretung von Menschen mit Kleinwuchs in allen Bereichen zuständig. Wissenschaftliche Leitung: Die vollständige Erklärung zum Interessenkonflikt der Wissenschaftlichen Leitung finden Sie am Kurs der zertifizierten Fortbildung auf www.springermedizin.de/cme . Der Verlag: erklärt, dass für die Publikation dieser CME-Fortbildung keine Sponsorengelder an den Verlag fließen. Für diesen Beitrag wurden von den Autor/-innen keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien. Für Bildmaterial oder anderweitige Angaben innerhalb des Manuskripts, über die Patient/-innen zu identifizieren sind, liegt von ihnen und/oder ihren gesetzlichen Vertretern/Vertreterinnen eine schriftliche Einwilligung vor., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Background: Biallelic SUFU variants have originally been linked to Joubert syndrome, comprising cerebellar abnormalities, dysmorphism, and polydactyly. In contrast, heterozygous truncating variants have recently been associated with developmental delay and ocular motor apraxia, but only a limited number of patients have been reported. Here, we aim to delineate further the mild end of the phenotypic spectrum related to SUFU haploinsufficiency., Methods: Nine individuals (from three unrelated families) harboring truncating SUFU variants were investigated, including two previously reported individuals (from one family). We provide results from a comprehensive assessment comprising neuroimaging, neuropsychology, video-oculography, and genetic testing., Results: We identified three inherited or de novo truncating variants in SUFU (NM_016169.4): c.895C>T p.(Arg299∗), c.71dup p.(Ala25Glyfs∗23), and c.71del p.(Pro24Argfs∗72). The phenotypic expression showed high variability both between and within families. Clinical features include motor developmental delay (seven of nine), axial hypotonia (five of nine), ocular motor apraxia (three of nine), and cerebellar signs (three of nine). Four of the six reported children had macrocephaly. Neuropsychological and developmental assessments revealed mildly delayed language development in the youngest children, whereas general cognition was normal in all variant carriers. Subtle but characteristic SUFU-related neuroimaging abnormalities (including superior cerebellar dysplasia, abnormalities of the superior cerebellar peduncles, rostrally displaced fastigium, and vermis hypoplasia) were observed in seven of nine individuals., Conclusions: Our data shed further light on the mild but recognizable features of SUFU haploinsufficiency and underline its marked phenotypic variability, even within families. Notably, neurodevelopmental and behavioral abnormalities are mild compared with Joubert syndrome and seem to be well compensated over time., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Narcolepsy is a rare neurological disease caused by dysfunction of hypocretin-producing neurons. Hypocretin concentrations in the cerebrospinal fluid (CSF) of less than 110 pg/ml are considered pathological in adults., Objectives: To compare hypocretin levels of children with narcolepsy type 1, type 2 and disease control groups, in addition to a detailed CSF analysis, clinical and polysomnographic parameters., Methods: In a retrospective, cross-sectional study, children diagnosed with narcolepsy based on clinical and polysomnographic parameters, who received a CSF analysis and hypocretin measurement, in addition to controls, were included. CSF was analyzed for the presence of cells, total protein, lactate, intrathecal synthesis of antibodies against measles, rubella and/or varicella zoster, and oligoclonal bands. All children had a complete sleep study including a multiple sleep latency test (MSLT)., Results: 49 children with narcolepsy type 1, 15 children with type 2 and 37 children with other (suspected) neurological diseases were included. CSF routine analysis did not reveal any differences between the three groups. All children with narcolepsy type 1 had hypocretin levels of less than 110 pg/ml (range: 10-101 pg/ml). Hypocretin levels in type 2 patients ranged from 43 to 436 pg/ml (median 157 pg/ml). The median hypocretin level in the control cohort was 365 pg/ml (range: 153-583 pg/ml). In 4 children with narcolepsy type 2 the diagnosis was changed to narcolepsy level 1 because of a CSF hypocretin level of less than 110 pg/ml according to the recently proposed criteria, which consider the measurement of hypocretin in CSF., Conclusion: Children with narcolepsy type 1 showed significantly lower CSF hypocretin levels than children with narcolepsy type 2 and controls. As suggested by the recently published narcolepsy criteria, hypocretin levels of less than 110 pg/ml should be used as an additional criterion for the presence of narcolepsy type 1 in children., Competing Interests: Declaration of competing interest M.Otto received research support from the EU Joint Programm neurodegenerative diseases networks Genfi-Prox, the EU (MOOD-MARKER), the German Research Foundation/DFG (SFB1279), the foundation of the state Baden-Württemberg D.3830), Boehringer Ingelheim Ulm University BioCenter (D.5009) and the Thierry Latran Foundation. M. Otto gave scientific advice to Roche, Biogen, Axon, and Fujirebio. K.Rostasy received honoraria from UCB, Horizon, and Euroimmun, and serves as Consultant for Roche in the Operetta2 trial. A.Bertolini received travel funds from Octapharma, honoraria for the advisory board through Horizon. E.Wendel received travel funds from UCB. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Aim: This exploratory study evaluates rating scale usage by experts from the European Reference Network for Rare Neurological Diseases (ERN-RND) for paediatric MD, considering factors like diagnosis, intellectual disability, age, and transition to adult care. The aim is to propose a preliminary framework for consistent application., Methods: A multicentre survey among 25 ERN-RND experts from 10 European countries examined rating scale usage in paediatric MD, categorizing MD into acute, non-progressive, and neurodegenerative types. Factors influencing scale choice and the transition to adult care practices were analysed. A comprehensive literature search was conducted to identify the earliest age of application of these scales in paediatric patients., Results: The study identifies various rating scales and establishes their usage frequencies for different MDs. Experts highlighted the need for standardized scales and proposed preliminary evaluation strategies based on clinical contexts. Challenges in applying scales to young, non-cooperative patients were acknowledged., Interpretation: The study recommends developing standardized rating scales for paediatric MDs to improve evaluations and data collection. It suggests potential scales for specific clinical scenarios to better evaluate disease progression. Comprehensive, patient-centred care remains crucial during the transition to adult care, despite the identified challenges. This exploratory approach aims to enhance patient outcomes and care., Competing Interests: Declaration of competing interest We hereby affirm that all authors involved in the preparation of this manuscript declare no conflicts of interest. This includes financial, personal, or professional relationships that could potentially influence the interpretation of the work presented herein., (Copyright © 2024 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Introduction: Multiparametric MRI (mpMRI) is gold standard for the primary diagnostic work-up of clinically significant prostate cancer (csPCa). The aim of this study was to assess the benefit of the perfusion sequence and the non-inferiority of an MRI without contrast administration (bpMRI) compared to mpMRI while taking clinical parameters into account., Methods: In this retrospective, non-interventional study we examined MRI data from 355 biopsy-naïve patients, performed on a 3T MRI system, evaluated by a board-certified radiologist with over 10 years of experience with subsequent mpMRI-TRUS fusion biopsy., Discussion: Only 16/355 (4.5%) patients benefited from dynamic contrast enhanced. In only 3/355 (0.8%) patients, csPCa would have been missed in bpMRI. BpMRI provided sensitivity and specificity (81.4%; 79.4%) comparable to mpMRI (75.2%; 81.8%). Additionally, bpMRI and mpMRI were independent predictors for the presence of csPCa, individually (OR: 15.36; p < 0.001 vs. 12.15; p = 0.006) and after accounting for established influencing factors (OR: 12.81; p < 0.001 vs. 6.50; p = 0.012). When clinical parameters were considered, a more balanced diagnostic performance between sensitivity and specificity was found for mpMRI and bpMRI. Overall, PSA density showed the highest diagnostic performance (area under the curve = 0.81) for the detection of csPCa., Conclusion: The premise of the study was confirmed. Therefore, bpMRI should be adopted as soon as existing limitations have been lifted by prospective multi-reader studies., (© 2024 S. Karger AG, Basel.)

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease., (© 2023. The Author(s).)

Background and Purpose: Rituximab (RTX) is frequently used off-label in multiple sclerosis. However, studies on the risk-benefit profile of RTX in pediatric-onset multiple sclerosis are scarce., Methods: In this multicenter retrospective cohort study, patients with pediatric-onset multiple sclerosis from Sweden, Austria and Germany, who received RTX treatment were identified by chart review. Annualized relapse rates, Expanded Disability Status Scale scores and magnetic resonance imaging parameters (new T2 lesions and contrast-enhancing lesions) were assessed before and during RTX treatment. The proportion of patients who remained free from clinical and disease activity (NEDA-3) during RTX treatment was calculated. Side effects such as infusion-related reactions, infections and laboratory abnormalities were assessed., Results: Sixty-one patients received RTX during a median (interquartile range) follow-up period of 20.9 (35.6) months. The annualized relapse rate decreased from 0.6 (95% confidence interval [CI] 0.38-0.92) to 0.03 (95% CI 0.02-0.14). The annual rate of new T2 lesions decreased from 1.25 (95% CI 0.70-2.48) to 0.08 (95% CI 0.03-0.25) and annual rates of new contrast-enhancing lesions decreased from 0.86 (95% CI 0.30-3.96) to 0. Overall, 70% of patients displayed no evidence of disease activity (NEDA-3). Adverse events were observed in 67% of patients. Six patients discontinued treatment due to ongoing disease activity or adverse events., Conclusion: Our study provides class IV evidence that RTX reduces clinical and radiological activity in pediatric-onset multiple sclerosis., (© 2024 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Being involved in various physiological and pathophysiological mechanisms (ageing, kidney damage, cardiovascular diseases, etc.), Klotho is a parameter of increasing interest. Studies in veterinary medicine are still rare, but it is exciting to find out whether the findings obtained can be transferred to animals. The aim of this study was therefore to investigate Klotho in cats. This study addressed α-Klotho concentrations in the serum of two groups of cats: one diseased group affected by hypertrophic cardiomyopathy ( n = 27) and one healthy control group ( n = 35). α-Klotho concentrations in serum were measured using an ELISA. The results were evaluated in the context of several echocardiographic measurement parameters in the diseased group. No significant difference between α-Klotho concentrations in the two groups was found. A slight negative correlation was found between α-Klotho concentrations and the relation of left atrium/aorta (La/Ao) in the diseased group. Gaining initial information on α-Klotho in cats, it was not possible to draw definite conclusions concerning cardiomyopathies in this species. The assessment of Klotho should be considered in terms of its broad implications in disease processes, but it is also recommended to focus on specific disease features. Both approaches might be promising as possible applications of Klotho in veterinary medicine.

Purpose: One-capsule, once-daily valbenazine is approved for tardive dyskinesia and under evaluation for chorea associated with Huntington's disease, conditions in which patients often experience dysphagia. In vitro studies were conducted to assess the suitability of crushing the contents of valbenazine capsules (40 and 80 mg) for mixing with soft foods or liquids or administration via a gastrostomy tube (G-tube)., Methods: In study 1, the dissolution of whole valbenazine capsules and crushed capsule contents were measured serially for 1 hour. In study 2, valbenazine recovery was evaluated after crushed contents were mixed with soft foods, buffer solutions (pH range, 1.2-6.8), and fed-state simulated gastric fluid. In study 3, valbenazine recovery was evaluated after crushed contents were dispersed in water and delivered via a G-tube. In studies 2 and 3, acceptable valbenazine recovery was 90% to 110%., Findings: Study 1 indicated rapid and complete drug release for whole valbenazine capsules and crushed capsule contents, with similar release at 10 minutes (whole, 94%-99%; crushed, 98%-100%) and 60 minutes (whole, 101%-103%; crushed, 101%-102%). Study 2 found acceptable valbenazine recovery within 2 hours of adding crushed capsule contents to tested foods, buffers, or fed-state simulated gastric fluid (recovery, 92%-102%). Study 3 found acceptable valbenazine recovery when crushed contents were added to cold or hot water and delivered via G-tube, with a water cup rinse to capture residual contents (recovery, 91%-97%)., Implications: These studies indicate the potential viability of valbenazine formulation(s) that can be added to soft foods or liquids or delivered via G-tube. Such formulations will be important for individuals who require treatment with a vesicular monoamine transporter 2 inhibitor but cannot swallow whole pills., Competing Interests: Declaration of Competing Interest The three studies included in this report were supported by Neurocrine Biosciences, Inc. MS has received the following: research grants within past 3 years from International Society for Bipolar Disorders (ISBD), National Institutes of Health (NIH), Centers for Disease Control and Prevention (CDC), Patient-Centered Outcomes Research Institute (PCORI), Merck, Otsuka, Alkermes; consultant fees in the past 1 year from Alkermes, Otsuka, Janssen, Lundbeck, Teva, Neurelis; royalties in the past 1 year from Springer Press, Johns Hopkins University Press, Oxford Press, UpToDate; and compensation in the past 1 year for CME preparation/participation in CME activities: American Physician's Institute (CMEtoGo), Psychopharmacology Institute, American Epilepsy Society, Clinical Care Options, American Academy of Child and Adolescent Psychiatry, Neurocrine. AP has received consulting fees from Neurocrine Biosciences, Inc. All remaining authors are full-time employees of Neurocrine Biosciences, Inc., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA methylation classifiers ("episignatures") help to determine the pathogenicity of variants of uncertain significance (VUS). However, their sensitivity is limited due to their training on unambiguous cases with strong-effect variants so that the classification of variants with reduced effect size or in mosaic state may fail. Moreover, episignature evaluation of mosaics as a function of their degree of mosaicism has not been developed so far. We improved episignatures with respect to three categories. Applying (i) minimum-redundancy-maximum-relevance feature selection we reduced their length by up to one order of magnitude without loss of accuracy. Performing (ii) repeated re-training of a support vector machine classifier by step-wise inclusion of cases in the training set that reached probability scores larger than 0.5, we increased the sensitivity of the episignature-classifiers by 30%. In the newly diagnosed patients we confirmed the association between DNA methylation aberration and age at onset of KMT2B-deficient dystonia. Moreover, we found evidence for allelic series, including KMT2B-variants with moderate effects and comparatively mild phenotypes such as late-onset focal dystonia. Retrained classifiers also can detect mosaics that previously remained below the 0.5-threshold, as we showed for KMT2D-associated Kabuki syndrome. Conversely, episignature-classifiers are able to revoke erroneous exome calls of mosaicism, as we demonstrated by (iii) comparing presumed mosaic cases with a distribution of artificial in silico-mosaics that represented all the possible variation in degree of mosaicism, variant read sampling and methylation analysis., (© 2023. The Author(s).)

Mitochondrial networks remodel their connectivity, content, and subcellular localization to support optimized energy production in conditions of increased environmental or cellular stress. Microglia rely on mitochondria to respond to these stressors, however our knowledge about mitochondrial networks and their adaptations in microglia in vivo is limited. Here, we generate a mouse model that selectively labels mitochondria in microglia. We identify that mitochondrial networks are more fragmented with increased content and perinuclear localization in vitro vs. in vivo . Mitochondrial networks adapt similarly in microglia closest to the injury site after optic nerve crush. Preventing microglial UCP2 increase after injury by selective knockout induces cellular stress. This results in mitochondrial hyperfusion in male microglia, a phenotype absent in females due to circulating estrogens. Our results establish the foundation for mitochondrial network analysis of microglia in vivo , emphasizing the importance of mitochondrial-based sex effects of microglia in other pathologies., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Mastitis is one of the most common diseases of dairy cows. Procalcitonin (PCT) has been described as an endogenous inflammatory biomarker for bacterial infections. The aim of this study was to find possible correlations between PCT concentrations in the serum and milk of cows with mastitis and their clinical signs and disease progression. In total, 88 dairy cows were examined, of which 30 animals were diagnosed with clinical mastitis, 30 had subclinical mastitis, and 28 were designated as a healthy control group. The diseased animals were re-examined after 12 days. All PCT levels in this study were determined by a species-specific ELISA. All three groups could be differentiated from each other based on serum and milk PCT levels. The animals with clinical mastitis showed the highest mean concentrations of PCT (serum: 2641 pg/mL; milk: 1326 pg/mL), and the lowest PCT concentrations were found in the healthy control group (serum: 1166 pg/mL; milk: 176 pg/m). Over the course of the disease, results from the kinetics study showed that PCT levels remained high for the entire observation period. The results from this study showed that the PCT concentration could be used to differentiate between clinical mastitis, subclinical mastitis, and healthy cows.

Objectives: To evaluate eravacycline (ERV) activity against Gram-negative and Gram-positive bacteria collected between 2017 and 2020 from worldwide locations., Methods: MIC determinations were performed using Clinical and Laboratory Standards Institute (CLSI) broth microdilution methodology. ERV and tigecycline susceptibility was interpreted using United States Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. Comparator susceptibility was interpreted using CLSI and EUCAST breakpoints., Results: ERV MIC 90 was 0.5 µg/mL against 12 436 Enterobacteriaceae isolates, which only increased to 1 µg/mL against multidrug-resistant (MDR) isolates (n = 2931) (23.6%). Similar activity was shown against 1893 Acinetobacter baumannii (MIC 90 1 µg/mL) and 356 Stenotrophomonas maltophilia (MIC 90 2 µg/mL). ERV was more active against Gram-positive bacteria: 415 Streptococcus pneumoniae (MIC 90 0.008 µg/mL), 273 S. anginosus group (MIC 90 0.015 µg/mL), 1876 Enterococcus faecalis and 1724 E. faecium (MIC 90 2 µg/mL), 2158 Staphylococcus aureus and 575 S. saprophyticus (MIC 90 0.12 µg/mL), 1143 S. epidermidis and 423 S. haemolyticus (MIC 90 0.25 µg/mL). ERV MIC 90 against methicillin-resistant staphylococci and vancomycin-resistant enterococci was similar to susceptible strains. However, ERV susceptibility varied between EUCAST or FDA against staphylococci, especially S. epidermidis (91.5% vs. 47.2%), and vancomycin-resistant E. faecalis (98.3% vs. 76.5%)., Conclusion: This study reaffirms ERV's consistent broad-spectrum activity, which has been evaluated since 2003. ERV remains a key agent for the treatment of bacterial infections, including resistant isolates, but urgent reassessment of clinical breakpoints is required for staphylococci and enterococci., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Currently, there are no data available on SARS-CoV-2 vaccine responses in pediatric-onset multiple sclerosis (POMS), and little is known about the course of SARS-CoV-2 infection in this age group. We therefore investigated humoral immune responses after COVID-19 vaccination and/or infection in POMS., Methods: We retrospectively analyzed seroconversion rates and SARS-CoV-2-specific antibody levels in 30 POMS and one pediatric CIS patient treated with no disease-modifying therapy (no DMT), immunomodulatory DMT (IM-DMT), or immunosuppressive DMT (IS-DMT) from two Austrian MS centers., Results: The median age at MS onset was 15.39 years (interquartile range [IQR]: 1.97). The median age at the first COVID-19 vaccination was 17.43 years (IQR: 2.76). After two vaccine doses, seroconversion (≥0.8 BAU/ml) was reached in 25 of 28 patients (89.3%). All patients with no DMT or IM-DMT generated robust immune responses to vaccination (seroconversion: no DMT: 6/6, IM-DMT: 7/7 [100%]; median titers: no DMT: 2075 BAU [IQR: 1268.50], IM-DMT: 2500 BAU [IQR: 0]). In the IS-DMT group, seroconversion was achieved in 12 of 14 patients (80%), and median titers were 50.8 BAU (IQR 254.63). Titers were significantly higher in no DMT versus IS-DMT (P = 0.012) and in IM-DMT versus IS-DMT (P = 0.001). Infection with SARS-CoV-2 occurred in 11 of 31 patients, and symptoms were mild in all cases. One relapse occurred after infection, but no relapses were documented after vaccination., Conclusions: Generally, mRNA vaccinations were well tolerated in POMS patients with and without DMT. Immune response was significantly reduced in patients treated with IS-DMT. No unexpected adverse events or relapses related to vaccinations were observed., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Defensins are peptides with antimicrobial and immunomodulatory effects. Their concentration could be altered during infections and thus provide information on the prognosis and course of the disease. The aim of the present study was to investigate the defensin concentration in cows with mastitis in order to find correlations between clinical expression and course of the disease and the defensin concentration in milk and blood., Methods: A total of 85 dairy cows were examined. Of these, 30 animals suffered from acute clinical mastitis, 25 animals were diagnosed with subclinical mastitis and 30 animals were considered a healthy comparison group. Beta-Defensin-4 (DEFB-4) was determined by a species-specific enzyme-linked immunosorbent assay (ELISA) (Bovine Defensin Beta 4 ELISA Kit, MyBioSource)., Results: The highest concentrations of DEFB-4 were detected in the animals with acute clinical mastitis. Values of 0 to 895 pg/mL (median: 115 pg/mL) were measured in milk and 40-1,016 pg/mL (median: 245 pg/mL) in serum. The concentrations of this group differed significantly from those of the animals with subclinical mastitis ( p < 0.0001 serum; p = 0.015 milk). In this group, concentrations of 15-211 pg/mL (median: 46 pg/mL) were recorded in milk and 20-271 pg/mL (median: 85 pg/mL) in serum., Discussion: Our results also show that in cases of acute mastitis after 12 days of treatment there is still an active inflammatory process in the tissue, because no significant reduction of somatic cells and defensin could be found after re-examination. Since the DEFB-4 concentrations of animals with clinical mastitis that had to be treated with antibiotics differed significantly from those of animals with subclinical mastitis that did not require antibiotic treatment, it can be assumed that bovine DEFB-4 is an important endogenous parameter for the defense against bacterial infections of the udder., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Neumann, Siegert and Fischer.)

To understand how potential gene manipulations affect in vitro microglia, we provide a set of short protocols to evaluate microglia identity and function. We detail steps for immunostaining to determine microglia identity. We describe three functional assays for microglia: phagocytosis, calcium response following ATP stimulation, and cytokine expression upon inflammatory stimuli. We apply these protocols to human induced-pluripotent-stem-cell (hiPSC)-derived microglia, but they can be also applied to other in vitro microglial models including primary mouse microglia. For complete details on the use and execution of this protocol, please refer to Bartalska et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Environmental cues influence the highly dynamic morphology of microglia. Strategies to characterize these changes usually involve user-selected morphometric features, which preclude the identification of a spectrum of context-dependent morphological phenotypes. Here we develop MorphOMICs, a topological data analysis approach, which enables semiautomatic mapping of microglial morphology into an atlas of cue-dependent phenotypes and overcomes feature-selection biases and biological variability. We extract spatially heterogeneous and sexually dimorphic morphological phenotypes for seven adult mouse brain regions. This sex-specific phenotype declines with maturation but increases over the disease trajectories in two neurodegeneration mouse models, with females showing a faster morphological shift in affected brain regions. Remarkably, microglia morphologies reflect an adaptation upon repeated exposure to ketamine anesthesia and do not recover to control morphologies. Finally, we demonstrate that both long primary processes and short terminal processes provide distinct insights to morphological phenotypes. MorphOMICs opens a new perspective to characterize microglial morphology., (© 2022. The Author(s).)

G protein-coupled receptors (GPCRs) regulate processes ranging from immune responses to neuronal signaling. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additionally, dissecting cell type-specific responses is challenging when the same GPCR is expressed on different cells within a tissue. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that bind clozapine-N-oxide and mimic a GPCR-of-interest. We show that chimeric DREADD-β2AR triggers responses comparable to β2AR on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate β2AR-mediated filopodia formation in microglia, an immune cell capable of driving central nervous system inflammation. When dissecting microglial inflammation, we included two additional DREADD-based chimeras mimicking microglia-enriched GPR65 and GPR109A. DREADD-β2AR and DREADD-GPR65 modulate the inflammatory response with high similarity to endogenous β2AR, while DREADD-GPR109A shows no impact. Our DREADD-based approach allows investigation of cell type-dependent pathways without known endogenous ligands., (© 2022. The Author(s).)

The alveolar hydatid disease, also known as alveolar echinococcosis, of humans is certainly one of the most dangerous zoonoses worldwide. The disease is caused by Echinococcus multilocularis - the fox tapeworm. Red foxes ( Vulpes vulpes ) are currently counted as the most important carriers (reservoirs) of E. multilocularis in the Northern Hemisphere. The possible routes of infection of E. multilocularis to humans are complex and still require research. Until now, it has been unknown whether E. multilocularis eggs can be moved by wind at all. This analysis shows, based on calculations, that E. multilocularis eggs can be transported by wind. Using a mathematical model, flight distances depending on wind speed and take-off heights are calculated for dense and less dense (coniferous) forest areas. The results - differentiated for seasons and as overall average - are based on mean values of wind speeds which were measured over a ten-year period in an experimental forest stand in the Solling (Germany). Due to their rate of descent, wind-related spreading of E. multilocularis eggs is possible. The average flight distance covered by E. multilocularis eggs in forest areas, depending on their starting altitude and wind speed, is between approximately 1.3 m and approximately 17 m. From the mathematical point of view, the wind factor can definitely be seen as one of the multiple vectors associated with environmental contamination by E. multilocularis eggs. Consequently, the possible wind-borne spread of E. multilocularis eggs poses an infection risk to humans that should be considered and requires further research.

Cerebral organoids differentiated from human-induced pluripotent stem cells (hiPSC) provide a unique opportunity to investigate brain development. However, organoids usually lack microglia, brain-resident immune cells, which are present in the early embryonic brain and participate in neuronal circuit development. Here, we find IBA1 + microglia-like cells alongside retinal cups between week 3 and 4 in 2.5D culture with an unguided retinal organoid differentiation protocol. Microglia do not infiltrate the neuroectoderm and instead enrich within non-pigmented, 3D-cystic compartments that develop in parallel to the 3D-retinal organoids. When we guide the retinal organoid differentiation with low-dosed BMP4, we prevent cup development and enhance microglia and 3D-cysts formation. Mass spectrometry identifies these 3D-cysts to express mesenchymal and epithelial markers. We confirmed this microglia-preferred environment also within the unguided protocol, providing insight into microglial behavior and migration and offer a model to study how they enter and distribute within the human brain., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Stem and progenitor cells residing in the intestinal crypts drive the majority of colorectal cancers (CRCs), yet vascular contribution to this niche remains largely unexplored. VEGFA is a key driver of physiological and tumor angiogenesis. Accordingly, current anti-angiogenic cancer therapies target the VEGFA pathway. Here we report that in CRC expansion of the stem/progenitor pool in intestinal crypts requires VEGFA-independent growth and remodeling of blood vessels. Epithelial transformation induced expression of the endothelial peptide apelin, directs migration of distant venous endothelial cells towards progenitor niche vessels ensuring optimal perfusion. In the absence of apelin, loss of injury-inducible PROX1+ epithelial progenitors inhibited both incipient and advanced intestinal tumor growth. Our results establish fundamental principles for the reciprocal communication between vasculature and the intestinal progenitor niche and provide a mechanism for resistance to VEGFA-targeting drugs in CRCs., Competing Interests: Competing interests TVP receives research funding from Roche for an unrelated project. All other authors declare no conflict of interest.

In the last decade, health-related quality of life (HrQoL) has become an increasingly important outcome parameter in children and adolescents with chronic health conditions; among them are pediatric patients with inborn metabolic diseases (IMDs). Hence, knowledge on this topic is increasing, but findings on non-medical influences on the HrQoL of IMD patients are still scarce. In the present study, we retrospectively evaluated the self-reported generic HrQoL of a cohort of pediatric patients (ages 7 to 17 years) with diverse IMDs (n = 204) and explored associations between HrQoL and psychosocial and medical characteristics of the patients. We aimed to identify risk factors for impaired HrQoL to improve and tailor support for the patients and economize resources. Generic HrQoL was assessed with the KINDL-R questionnaire. We compared the HrQoL scores to published German normative data and analyzed the impact of demographic variables and intellectual and psychosocial functioning on the HrQoL. Moreover, we examined the influence of the diagnostic category and the health impairment (as judged by the physicians) on our patients' HrQoL. Overall, the HrQoL of the adolescent patients was comparable to the HrQoL of the norm group. Disorders of intellectual development, impaired psychosocial functioning, and a severe health impairment were associated with lower HrQoL scores.Conclusion: We recommend evaluating these factors in children and adolescents with IMDs to identify patients at risk for impaired HrQoL. What is Known: • Studies on HrQoL in pediatric patients with IMDs mainly focused on subgroups with specific diagnoses and found normal HrQoL in some of those subgroups. • In healthy children and adolescents as well as in pediatric patients with various chronic diseases, associations between psychosocial factors and HrQoL are well known. What is New: • Impaired psychosocial functioning, disorders of intellectual development, and a significant disease and/or treatment burden are risk factors for impaired HrQoL in pediatric patients with IMDs. • Evaluating these factors in children and adolescents with IMDs can help identify patients and families in need of enhanced psychological support., (© 2021. The Author(s).)

Pyroglutamate amyloid beta3-42 (pGlu-Abeta3-42), a highly amyloidogenic and neurotoxic form of Abeta, is N-terminally truncated to form a pyroglutamate and has recently been proposed as a key target for immunotherapy. Optimized ACI-24, a vaccine in development for the treatment and prevention of Alzheimer's disease, focuses the antibody response on the first 15 N-terminal amino acids of Abeta (Abeta1-15). Importantly, clinical data with an initial version of ACI-24 incorporating Abeta1-15, established the vaccine's safety and tolerability with evidence of immunogenicity. To explore optimized ACI-24's capacity to generate antibodies to pGlu-Abeta3-42, pre-clinical studies were carried out. Vaccinating mice and non-human primates demonstrated that optimized ACI-24 was well-tolerated and induced an antibody response against Abeta1-42 as expected, as well as high titres of IgG reactive with pyroGlu-Abeta. Epitope mapping of the polyclonal response confirmed these findings revealing broad coverage of epitopes particularly for Abeta peptides mimicking where cleavage occurs to form pGlu-Abeta3-42. These data are in striking contrast to results obtained with other clinically tested Abeta targeting vaccines which generated restricted and limited antibody diversity. Taken together, our findings demonstrate that optimized ACI-24 vaccination represents a breakthrough to provide a safe immune response with a broader Abeta sequence recognition compared to previously tested vaccines, creating binders to pathogenic forms of Abeta important in pathogenesis including pGlu-Abeta3-42., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Enzymatic digestion of the extracellular matrix with chondroitinase-ABC reinstates juvenile-like plasticity in the adult cortex as it also disassembles the perineuronal nets (PNNs). The disadvantage of the enzyme is that it must be applied intracerebrally and it degrades the ECM for several weeks. Here, we provide two minimally invasive and transient protocols for microglia-enabled PNN disassembly in mouse cortex: repeated treatment with ketamine-xylazine-acepromazine (KXA) anesthesia and 60-Hz light entrainment. We also discuss how to analyze PNNs within microglial endosomes-lysosomes. For complete details on the use and execution of this protocol, please refer to Venturino et al. (2021)., Competing Interests: The authors declare no competing interests. A.V. and S.S. disclose an international patent application (PCT/EP2020/079365)., (© 2021 The Author(s).)

Biallelic truncating FAM149B1 variants result in cilia dysfunction and have been reported in four infants with Joubert syndrome and orofaciodigital syndrome type VI, respectively. We report here on three adult siblings, 18 to 40 years of age, homozygous for the known FAM149B1 c.354&#95;357delinsCACTC (p.Gln118Hisfs*20) variant. Detailed clinical examinations were performed including ocular and gait analyses, skeletal- and neuroimaging. All three patients presented with neurological and oculomotor symptoms since birth and mild skeletal dysplasia in infancy resulting in characteristic gait abnormalities. We document mild skeletal dysplasia, abnormal gait with increased hip rotation and increased external foot rotation, ataxia, variable polydactyly, ocular Duane syndrome, progressive ophthalmoplegia, nystagmus, situs inversus of the retinal vessels, olfactory bulb aplasia, and corpus callosal dysgenesis as novel features in FAM149B1 -ciliopathy. We show that intellectual disability is mild to moderate and retinal, renal and liver function is normal in these affected adults. Our study thus expands the FAM149B1 -related Joubert syndrome to a mainly neurological and skeletal ciliopathy phenotype with predominant oculomotor dysfunction but otherwise stable outcome in adults. Diagnosis of FAM149B1 -related disorder was impeded by segregation of multiple neurogenetic disorders in the same family, highlighting the importance of extended clinical and genetic studies in families with complex phenotypes.

Background: Recent research suggested an hippocalcin (HPCA)-related form of DYT2-like autosomal recessive dystonia. Two reports highlight a broad spectrum of the clinical phenotype. Here, we describe a novel HPCA gene variant in a pediatric patient and two affected relatives., Methods: Whole exome sequencing was applied after a thorough clinical and neurological examination of the index patient and her family members. Results of neuropsychological testing were analyzed., Results: Whole exome sequencing revealed a novel homozygous missense variant in the HPCA gene [c.182C>T p.(Ala61Val)] in our pediatric patient and the two affected family members. Clinically, the cases presented with dystonia, dysarthria, and jerky movements. We observed a particular cognitive profile with executive dysfunctions in our patient, which corresponds to the cognitive deficits that have been observed in the patients previously described., Conclusion: We present a novel genetic variant of the HPCA gene associated with autosomal recessive dystonia in a child with childhood-onset dystonia supporting its clinical features. Furthermore, we propose specific HPCA -related cognitive changes in homozygous carriers, underlining the importance of undertaking a systematic assessment of cognition in HPCA -related dystonia., Competing Interests: None declared., (Thieme. All rights reserved.)

Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells' heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1 CreERT2 mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Background: Individuals with tardive dyskinesia (TD) who completed a long-term study (KINECT 3 or KINECT 4) of valbenazine (40 or 80 mg/day, once-daily for up to 48 weeks followed by 4-week washout) were enrolled in a subsequent study (NCT02736955) that was primarily designed to further evaluate the long-term safety of valbenazine., Methods: Participants were initiated at 40 mg/day (following prior valbenazine washout). At week 4, dosing was escalated to 80 mg/day based on tolerability and clinical assessment of TD; reduction to 40 mg/day was allowed for tolerability. The study was planned for 72 weeks or until termination due to commercial availability of valbenazine. Assessments included the Clinical Global Impression of Severity-TD (CGIS-TD), Patient Satisfaction Questionnaire (PSQ), and treatment-emergent adverse events (TEAEs)., Results: At study termination, 85.7% (138/161) of participants were still active. Four participants had reached week 60, and none reached week 72. The percentage of participants with a CGIS-TD score ≤2 (normal/not ill or borderline ill) increased from study baseline (14.5% [23/159]) to week 48 (64.3% [36/56]). At baseline, 98.8% (158/160) of participants rated their prior valbenazine experience with a PSQ score ≤2 (very satisfied or somewhat satisfied). At week 48, 98.2% (55/56) remained satisfied. Before week 4 (dose escalation), 9.4% of participants had ≥1 TEAE. After week 4, the TEAE incidence was 49.0%. No TEAE occurred in ≥5% of participants during treatment (before or after week 4)., Conclusions: Valbenazine was well-tolerated and persistent improvements in TD were found in adults who received once-daily treatment for >1 year.

Perineuronal nets (PNNs), components of the extracellular matrix, preferentially coat parvalbumin-positive interneurons and constrain critical-period plasticity in the adult cerebral cortex. Current strategies to remove PNN are long-lasting, invasive, and trigger neuropsychiatric symptoms. Here, we apply repeated anesthetic ketamine as a method with minimal behavioral effect. We find that this paradigm strongly reduces PNN coating in the healthy adult brain and promotes juvenile-like plasticity. Microglia are critically involved in PNN loss because they engage with parvalbumin-positive neurons in their defined cortical layer. We identify external 60-Hz light-flickering entrainment to recapitulate microglia-mediated PNN removal. Importantly, 40-Hz frequency, which is known to remove amyloid plaques, does not induce PNN loss, suggesting microglia might functionally tune to distinct brain frequencies. Thus, our 60-Hz light-entrainment strategy provides an alternative form of PNN intervention in the healthy adult brain., Competing Interests: Declaration of interests The authors declare no competing interests. A.V. and S.S. disclose an international patent application (PCT/EP2020/079365)., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Primary glandular bladder tumours (bladder adenocarcinoma [BAC], urachal adenocarcinoma [UAC], urothelial carcinoma with glandular differentiation [UCg]) are rare malignancies with histological resemblance to colorectal adenocarcinoma (CORAD) in the majority of this subgroup. Definite case numbers are very low, molecular data are limited and the pathogenesis remains poorly understood. Therefore, this study was designed to complement current knowledge by in depth analysis of BAC (n = 12), UAC (n = 13), UCg (n = 11) and non-invasive glandular lesions (n = 19). In BAC, in addition to known alterations in TP53, Wnt, MAP kinase and MTOR pathway, mutations in SMAD4, ARID1A and BRAF were identified. Compared to published data on muscle invasive bladder cancer (BLCA) and CORAD, UCg exhibited frequent "urothelial" like alterations while BAC and UAC were characterised by a more "colorectal" like mutational pattern. Immunohistochemically, there was no evidence of DNA mismatch repair deficiency or PD-L1 tumour cell positivity in any sample. Depending on the used antibody 0-45% of BAC, 0-30% of UCg and 0% UAC cases exhibited PD-L1 expressing tumour associated immune cells. A single BAC (9%, 1/11) showed evidence of ARID1A protein loss, and two cases of UCg (20%, 2/10) showed loss of SMARCA1 and PBRM1, respectively. Taken together, our data suggest at least in part involvement of similar pathways driving tumourigenesis of adenocarcinomas like BAC, UAC and CORAD independent of their tissue origin. Alterations of TERT and FBXW7 in single cases of intestinal metaplasia further point towards a possible precancerous character in line with previous reports.

Stored red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation. RBC storage lesions are often assessed by microscopic examination or biochemical and biophysical assays, which are complex, time-consuming, and destructive to fragile cells. Here we demonstrate the use of label-free imaging flow cytometry and deep learning to characterize RBC lesions. Using brightfield images, a trained neural network achieved 76.7% agreement with experts in classifying seven clinically relevant RBC morphologies associated with storage lesions, comparable to 82.5% agreement between different experts. Given that human observation and classification may not optimally discern RBC quality, we went further and eliminated subjective human annotation in the training step by training a weakly supervised neural network using only storage duration times. The feature space extracted by this network revealed a chronological progression of morphological changes that better predicted blood quality, as measured by physiological hemolytic assay readouts, than the conventional expert-assessed morphology classification system. With further training and clinical testing across multiple sites, protocols, and instruments, deep learning and label-free imaging flow cytometry might be used to routinely and objectively assess RBC storage lesions. This would automate a complex protocol, minimize laboratory sample handling and preparation, and reduce the impact of procedural errors and discrepancies between facilities and blood donors. The chronology-based machine-learning approach may also improve upon humans' assessment of morphological changes in other biomedically important progressions, such as differentiation and metastasis., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Several studies have demonstrated high work related stress and burnout symptoms in physicians. The study examined the longitudinal relationships of burnout and gratification crisis during 6 years of postgraduate medical education, controlled for gender and parental status. Data acquisition was carried out within the KarMed-study. Administered were the Maslach Burnout Inventory (MBI-HSS (MP)) (MBI) and the Effort-Reward-Imbalance (ERI). The analyses focused on general linear models and general linear mixed models with repeated measure designs. The amount of burnout perceived at the beginning of the postgraduate medical education is highly associated with the perceived amount of burnout at the end. Gratification crisis were significant for one subscale of the MBI (emotional exhaustion), yet not for personal accomplishment nor depersonalization. Significant time effects were found on all 3 subscales of the MBI, with gender effects on the subscales emotional exaustion and depersonalization. Having kids did not have significant effects, controlled for all measurement points of burnout as well as for gratification crisis. Theoretical implications and practical consequences for doctors, as well as the limitations of the study, are discussed., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (© Georg Thieme Verlag KG Stuttgart · New York.)

Objective: To evaluate the effects of once-daily valbenazine (40 or 80 mg/d) in older and younger adults with tardive dyskinesia (TD)., Methods: Data were pooled from three 6-week, randomized, double-blind, placebo-controlled (DBPC) studies (KINECT [NCT01688037], KINECT 2 [NCT01733121], and KINECT 3 [NCT02274558]) and two long-term studies (KINECT 3 extension and KINECT 4 [NCT02405091]). Outcomes analyzed in older and younger participants (55 years or older and younger than 55 years, respectively) included Abnormal Involuntary Movement Scale (AIMS) response (threshold of greater than or equal to 50% improvement from baseline in total score [items 1 to 7]) and Clinical Global Impression of Change-Tardive Dyskinesia (CGI-TD) response (score 2 or less ["very much improved" or "much improved"]). Safety assessments included treatment-emergent adverse events (TEAEs)., Results: At week 6 (end of DBPC treatment), the percentage of participants who met the AIMS response threshold was higher with valbenazine versus placebo in both subgroups: 55 years or older (80 mg/d, 39.7% [P < .001]; 40 mg/d, 28.6% [P < .01]; placebo, 9.7%); younger than 55 years (80 mg/d, 39.5% [P < .001]; 40 mg/d, 20.0% [P > .05]; placebo, 10.8%). The percentage of participants with CGI-TD response was also higher with valbenazine versus placebo: 55 years or older (80 mg/d, 41.3% [P < .01]; 40 mg/d, 30.2% [P > .05]; placebo, 19.4%); younger than 55 years (80 mg/d, 39.5% [P < .05]; 40 mg/d, 35.3% [P < .05]; placebo, 18.5%). Responses at week 48 (end of long-term treatment, combined doses) were as follows: 55 years or older (AIMS, 70.7%; CGI-TD, 82.8%); younger than 55 years (AIMS, 58.7%; CGI-TD, 72.3%). No significant differences between older and younger subgroups were found for AIMS or CGI-TD response. No new safety signals or TEAEs of clinical concern were found in older participants who received long-term treatment., Conclusions: Valbenazine improved TD and was generally well tolerated in older and younger adults., (© 2019 The Authors. International Journal of Geriatric Psychiatry published by John Wiley & Sons Ltd.)

Fluorine-19 ( 19 F) magnetic resonance imaging (MRI) of injected perfluorocarbons (PFCs) can be used for the quantification and monitoring of inflammation in diseases such as atherosclerosis. To advance the translation of this technique to the clinical setting, we aimed to 1) demonstrate the feasibility of quantitative 19 F MRI in small inflammation foci on a clinical scanner, and 2) to characterize the PFC-incorporating leukocyte populations and plaques. To this end, thirteen atherosclerotic apolipoprotein-E-knockout mice received 2 × 200 µL PFC, and were scanned on a 3 T clinical MR system. 19 F MR signal was detected in the aortic arch and its branches in all mice, with a signal-to-noise ratio of 11.1 (interquartile range IQR = 9.5-13.1) and a PFC concentration of 1.15 mM (IQR = 0.79-1.28). Imaging flow cytometry was used on another ten animals and indicated that PFC-labeled leukocytes in the aortic arch and it branches were mainly dendritic cells, macrophages and neutrophils (ratio 9:1:1). Finally, immunohistochemistry analysis confirmed the presence of those cells in the plaques. We thus successfully used 19 F MRI for the noninvasive quantification of PFC in atherosclerotic plaque in mice on a clinical scanner, demonstrating the feasibility of detecting very small inflammation foci at 3 T, and advancing the translation of 19 F MRI to the human setting.

Background: Data on pediatric DBS is still limited because of small numbers in single center series and lack of systematic multi-center trials., Objectives: We evaluate short- and long-term adverse events (AEs) of patients undergoing deep brain stimulation (DBS) during childhood and adolescence., Methods: Data collected by the German registry on pediatric DBS (GEPESTIM) were analyzed according to reversible and irreversible AEs and time of occurrence with relation to DBS-surgery: Intraoperative, perioperative (<4 weeks), postoperative (4 weeks < 6 months) and long term AEs (>6 months)., Results: 72 patients with childhood-onset dystonia from 10 DBS-centers, who received 173 DBS electrodes and 141 implantable pulse generators (IPG), were included in the registry. Mean time of postoperative follow-up was 4.6 ± 4 years. In total, 184 AEs were documented in 53 patients (73.6%). 52 DBS-related AEs in 26 patients (36.1%) required 45 subsequent surgical interventions 4.7 ± 4.1 years (range 3 months-15 years) after initial implantation. The total risk of an AE requiring surgical intervention was 7.9% per electrode-year. Hardware-related AEs were the most common reason for surgery. There was a tendency of a higher rate of AEs in patients aged 7-9 years beyond 6 months after implantation., Discussion: The intraoperative risk of AEs in pediatric patients with dystonia undergoing DBS is very low, whereas the rate of postoperative hardware-related AEs is a prominent feature with a higher occurrence compared to adults, especially on long-term follow-up., Conclusion: Factors leading to such AEs must be identified and patient management has to be focused on risk minimization strategies in order to improve DBS therapy and maximize outcome in pediatric patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In neuroscience, recombinant viruses such as lentiviruses and adeno-associated viruses (AAVs) have been successfully used to target various cell types in the brain, although effective transduction of microglia is rare. In this review, we provide a short background of lentiviruses and AAVs, and strategies for designing recombinant viral vectors. Then, we will summarize recent literature on successful microglial transductions in vitro and in vivo, and discuss the current challenges. Finally, we provide guidelines for reporting the efficiency and specificity of viral targeting in microglia, which will enable the microglial research community to assess and improve methodologies for future studies., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Neurodegeneration with Brain Iron Accumulation type I (NBIA-I) is a rare hereditary neurodegenerative disorder with pallidal degeneration leading to disabling generalized dystonia and parkinsonism. Pallidal or subthalamic deep brain stimulation can partially alleviate motor symptoms. Disease-specific patterns of abnormally enhanced oscillatory neuronal activity recorded from the basal ganglia have been described in patients with movement disorders undergoing deep brain stimulation (DBS). Here we studied oscillatory activity recorded from the internal globus pallidus (GPi) and the subthalamic nucleus (STN) to characterize neuronal activity patterns in NBIA-I., Methods: We recorded local field potentials (LFP) from DBS electrodes in 6 juvenile patients with NBIA-I who underwent functional neurosurgery. Four patients were implanted in the STN and two patients in the GPi. Recordings were performed during wakeful rest. An FFT-based approach was used to analyze the power spectrum in the target area., Results: In all patients we found distinct peaks in the low frequency (7-12 Hz) and in 5 out 6 also in the beta frequency range (15-30 Hz) with the largest beta peak in the patient that presented with the most prominent bradykinesia. No distinct peaks occurred in the gamma frequency range (35-100 Hz). The oscillatory pattern did not differ between STN and GPi., Conclusions: Here we show for the first time the oscillatory activity pattern in the STN and the GPi in juvenile patients with dystonia plus syndrome due to NBIA-I. The low frequency peak we found is in line with previous studies in patients with isolated idiopathic dystonia. In our cohort, the pallidal beta band activity may be related to more severe motor slowing in dystonia plus syndrome such as NBIA-I., Significance: Our results further support the link between hyperkinetic motor symptoms such as dystonia and enhanced basal ganglia low frequency activity irrespective of the underlying etiology of dystonia., (Copyright © 2019 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

Background: Few studies have assessed the treatment of tardive dyskinesia (TD) in patients with primary mood disorders who are managed with antipsychotics. The effects of once-daily valbenazine on TD were evaluated in adults with a bipolar or depressive disorder., Methods: Data were pooled from two 6-week double-blind placebo-controlled trials (KINECT 2 and KINECT 3; 114 mood participants) and a long-term blinded extension study (KINECT 3 extension; 77 mood participants) of valbenazine in adults with TD. Efficacy assessments included Abnormal Involuntary Movement Scale (AIMS) total score (sum of items 1-7), Clinical Global Impression of Change-Tardive Dyskinesia (CGI-TD), and Patient Global Impression of Change (PGIC). Safety assessments included treatment-emergent adverse events (TEAEs), Young Mania Rating Scale, and Montgomery-Åsberg Depression Rating Scale., Results: At Week 6, mean improvements in AIMS total score were significantly greater with valbenazine versus placebo (40 mg/day, -3.1 [P < 0.01]; 80 mg/day, -3.5 [P < 0.001]; placebo, -0.9). Significant differences between valbenazine (80 mg/day) and placebo were also found for Week 6 AIMS response (≥50% total score improvement) and CGI-TD response ("much improved" or "very much improved"), but not PGIC response. Sustained improvements in AIMS, CGI-TD, and PGIC were found through 48 weeks. Valbenazine was generally well tolerated, with no unexpected TEAEs, worsening in psychiatric symptoms, or emergence of suicidality., Limitations: Pooled analyses were conducted post hoc, and neither study was designed to focus solely on mood disorder patients., Conclusions: In participants with primary mood disorders, once-daily treatment with valbenazine was generally well tolerated and resulted in 6-week and sustained TD improvements., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: This study examines the real length of postgraduate medical education in Germany. The regulations define minimal time periods in months for each discipline. We especially analyze the differences between minimum and real length, as well as the percentages of interrupting or dropping out physicians by gender., Methods: Within the KarMed study, annual postal surveys were sent to graduates of seven medical faculties in Germany from their last year of undergraduate education until after six years of postgraduate training. The return rate at baseline was 48 % (n = 1012) and the 5 surveys after baseline reached rates of 85 % and more. Descriptive statistics were used for analyses., Results: Half of the residents did not finish within the minimum time period. This proportion was higher among women (65 %) then men (43 %); among women with child/children this proportion reached 91 %. Female residents expecting a prolongation estimated an average time period of 19.6 months (SD = 14.7), male residents of 11.5 months (SD = 8.6).Female residents named problems in relation to children as the most important factor. Half of the reasons given by male residents (and 31 % of those given by females) are related to organizational and curricular problems of postgraduate education., Conclusion: Effective measures are needed to reduce the problem of continuation of postgraduate education in the course of getting/having children and/or to promote the return to training in the hospital. For this, curricular and organizational measures, e. g. rotation plans and better possibilities for part-time training, should be offered., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (© Georg Thieme Verlag KG Stuttgart · New York.)

The formation of central CD8 T cell memory in response to infection depends on the transcription factor Tcf1 (Tcf7). Tcf1 is expressed at high levels in naive CD8 T cells but downregulated in most CD8 T cells during effector differentiation. The relevance of Tcf1 downregulation for effector differentiation and the signals controlling Tcf1 expression have not been elucidated. Here, we show that systemic inflammatory signals downregulated Tcf1 in CD8 T cells during dendritic cell vaccination and bacterial infections. The suppressive effect was mediated by the inflammatory cytokine interleukin 12 (IL-12), which acted via STAT4 in CD8 T cells. IL-12-induced Tcf1 downregulation required cell cycling, occurred at the transcriptional level, and was prevented in part by inhibiting DNA methyltransferases. Absence of Tcf1 during T cell priming circumvented the need of systemic inflammation for effector differentiation. We conclude that silencing of Tcf1 by systemic inflammation facilitates effector CD8 T cell differentiation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gamma synchronization increases during movement and scales with kinematic parameters. Here, disease-specific characteristics of this synchronization and the dopamine-dependence of its scaling in Parkinson's disease are investigated. In 16 patients undergoing deep brain stimulation surgery, movements of different velocities revealed that subthalamic gamma power peaked in the sensorimotor part of the subthalamic nucleus, correlated positively with maximal velocity and negatively with symptom severity. These effects relied on movement-related bursts of transient synchrony in the gamma band. The gamma burst rate highly correlated with averaged power, increased gradually with larger movements and correlated with symptom severity. In the dopamine-depleted state, gamma power and burst rate significantly decreased, particularly when peak velocity was slower than ON medication. Burst amplitude and duration were unaffected by the medication state. We propose that insufficient recruitment of fast gamma bursts during movement may underlie bradykinesia as one of the cardinal symptoms in Parkinson's disease., Competing Interests: RL, WN, AB, AH, JH, SS, GS, JK, AK No competing interests declared, (© 2018, Lofredi et al.)

Background/objectives: Fatty liver disease (FLD) is an important intermediate trait along the cardiometabolic disease spectrum and strongly associates with type 2 diabetes. Knowledge of biological pathways implicated in FLD is limited. An untargeted metabolomic approach might unravel novel pathways related to FLD., Subjects/methods: In a population-based sample (n=555) from Northern Germany, liver fat content was quantified as liver signal intensity using magnetic resonance imaging. Serum metabolites were determined using a non-targeted approach. Partial least squares regression was applied to derive a metabolomic score, explaining variation in serum metabolites and liver signal intensity. Associations of the metabolomic score with liver signal intensity and FLD were investigated in multivariable-adjusted robust linear and logistic regression models, respectively. Metabolites with a variable importance in the projection >1 were entered in in silico overrepresentation and pathway analyses., Results: In univariate analysis, the metabolomics score explained 23.9% variation in liver signal intensity. A 1-unit increment in the metabolomic score was positively associated with FLD (n=219; odds ratio: 1.36; 95% confidence interval: 1.27-1.45) adjusting for age, sex, education, smoking and physical activity. A simplified score based on the 15 metabolites with highest variable importance in the projection statistic showed similar associations. Overrepresentation and pathway analyses highlighted branched-chain amino acids and derived gamma-glutamyl dipeptides as significant correlates of FLD., Conclusions: A serum metabolomic profile was associated with FLD and liver fat content. We identified a simplified metabolomics score, which should be evaluated in prospective studies.

Information for patients with rare diseases has to adhere to strict quality criteria in order to support individual treatment decisions or coping strategies. However, developers are facing specific challenges: For example, the evidence is often insufficient or of very low quality. In the context of the National Action League for People with Rare Diseases (NAMSE), criteria have been developed that assure high-quality information on rare diseases. Core criteria comprise the involvement of patients or their advocates in all stages of the development process, the systematic search and assessment of the evidence, systematic collection of patient experience, transparency in terms of people involved and funding, and nondirective and neutral formulation of content and documentation of the process. In a joint project between the Alliance for Chronic Rare Diseases (ACHSE e. V.) and the German Agency for Quality in Medicine (ÄZQ), ten short information leaflets on different rare diseases have been developed in the past three years, conceived to show the applicability of these criteria. First experiences with this format show that the criteria are adaptive to a broad range of diverse rare diseases and settings. Involving patients and their advocates throughout the whole development process - from prioritization to development of methods and provision of patient experience and coping strategies - is crucial. Insufficient evidence remains a challenge. The examples show that in the absence of proven findings, information that matters to patients and reflects this uncertainty is feasible.

Th17 cells play critical roles in host defense and autoimmunity. Emerging data support a role for Notch signaling in Th17 cell differentiation but whether it is a positive or negative regulator remains unclear. We report here that T cell-specific deletion of Notch receptors enhances Th17 cell differentiation in the gut, with a corresponding increase in IL-17 secretion. An increase in Th17 cell frequency was similarly observed following immunization of T cell specific Notch mutant mice with OVA/CFA. However, in this setting, Th17 cytokine secretion was impaired, and increased intracellular retention of IL-17 was observed. Intracellular IL-17 co-localized with the CD71 iron transporter in the draining lymph node of both control and Notch-deficient Th17 cells. Immunization induced CD71 surface expression in control, but not in Notch-deficient Th17 cells, revealing defective CD71 intracellular transport in absence of Notch signaling. Moreover, Notch receptor deficient Th17 cells had impaired mTORC2 activity. These data reveal a context-dependent impact of Notch on vesicular transport during high metabolic demand suggesting a role for Notch signaling in the bridging of T cell metabolic demands and effector functions. Collectively, our findings indicate a prominent regulatory role for Notch signaling in the fine-tuning of Th17 cell differentiation and effector function.

Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1β. The nature of endogenous stress signals that activate inflammasomes remains unclear. Here we show that an inhibitor of the HIV aspartyl protease, Nelfinavir, triggers inflammasome formation and elicits an IL-1R-dependent inflammation in mice. We found that Nelfinavir impaired the maturation of lamin A, a structural component of the nuclear envelope, thereby promoting the release of DNA in the cytosol. Moreover, deficiency of the cytosolic DNA-sensor AIM2 impaired Nelfinavir-mediated inflammasome activation. These findings identify a pharmacologic activator of inflammasome and demonstrate the role of AIM2 in detecting endogenous DNA release upon perturbation of nuclear envelope integrity.

Dupuytren´s disease, a fibromatosis of the connective tissue in the palm, is a common complex disease with a strong genetic component. Up to date nine genetic loci have been found to be associated with the disease. Six of these loci contain genes that code for Wnt signalling proteins. In spite of this striking first insight into the genetic factors in Dupuytren´s disease, much of the inherited risk in Dupuytren´s disease still needs to be discovered. The already identified loci jointly explain ~1% of the heritability in this disease. To further elucidate the genetic basis of Dupuytren´s disease, we performed a genome-wide meta-analysis combining three genome-wide association study (GWAS) data sets, comprising 1,580 cases and 4,480 controls. We corroborated all nine previously identified loci, six of these with genome-wide significance (p-value < 5x10-8). In addition, we identified 14 new suggestive loci (p-value < 10-5). Intriguingly, several of these new loci contain genes associated with Wnt signalling and therefore represent excellent candidates for replication. Next, we compared whole-transcriptome data between patient- and control-derived tissue samples and found the Wnt/β-catenin pathway to be the top deregulated pathway in patient samples. We then conducted network and pathway analyses in order to identify protein networks that are enriched for genes highlighted in the GWAS meta-analysis and expression data sets. We found further evidence that the Wnt signalling pathways in conjunction with other pathways may play a critical role in Dupuytren´s disease.

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here, we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes, where they trap T cells by releasing the CCR7 ligand, CCL19, and to inflamed central nervous system, where they locally limit immunopathogenesis through interleukin-10 production, thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment, where proB cells develop, is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition, these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases.

In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer's disease (AD) have been failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas9 will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD., (Copyright © 2015 Elsevier Inc. All rights reserved.)