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OBJECTIVE: The application of inflammation-regulated therapeutic gene expression in arthritis conditions increases the efficiency of gene therapy by self-limiting the transgene. Incidentally, constitutive overexpression of transgenes typically leads to detrimental effects in disease conditions; therefore, regulation of expression is warranted. We undertook this study to validate a new gene therapy approach using a cell culture-based inflammation model and a novel self-limiting, inflammation-responsive promoter construct. METHODS: We designed a self-limiting promoter construct that expresses an antiinflammatory gene (interleukin-4 [IL-4]) only in the presence of inflammation. Our construct featured a truncated promoter sequence of cyclooxygenase 2 (COX-2) upstream of the IL-4 gene. We triggered inflammation in vitro in articular chondrocytes by applying the inflammatory cytokines IL-1beta and tumor necrosis factor alpha (TNFalpha) together exogenously, and we studied the extent of IL-4 expression and its effect on the inflammatory cascade. RESULTS: Using articular chondrocytes, we showed that our COX-2 promoter construct expressed IL-4 only in the presence of IL-1beta and TNFalpha. IL-4 expressed in the presence of IL-1beta and TNFalpha down-regulated a series of inflammation mediators, prostaglandins, and matrix metalloproteinases. CONCLUSION: The use of this construct for the expression of antiinflammatory genes allows production of a therapeutic gene product that is controlled by the severity of the disease. The effectiveness of this promoter construct for combating inflammation makes it a suitable candidate for the development of a new local gene therapy strategy for the treatment of osteoarthritis, in which IL-1beta and TNFalpha trigger a signal cascade that elevates COX-2 levels. [ABSTRACT FROM AUTHOR]

Background: Chronic inflammatory skin diseases are of great social and medical importance and require effective drug therapy. Phosphodiesterase 4 (PDE4) inhibitors represent a possible therapeutic option by regulating inflammatory processes. PDEs cause the release of proinflammatory cytokines by interfering with signaling pathways. The PDE4 inhibitors apremilast (treatment of psoriasis and Behçet's disease), roflumilast (treatment of chronic obstructive pulmonary disease), and crisaborole (treatment of atopic dermatitis) are currently approved in Europe., Psoriasis: Apremilast is used for second-line treatment of plaque psoriasis and psoriatic arthritis and has a favorable side effect profile. Topical PDE4 inhibitors are currently being researched and have not yet been approved by the European Medicines Agency (EMA)., Atopic Dermatitis: The topical PDE4 inhibitor crisaborole was approved by the EMA in 2020 as a topical treatment alternative to glucocorticoids and calcineurin inhibitors. Although the substance has shown good tolerability in studies and also alleviates the accompanying itching, it did not find its way onto the German market. BEHçET'S DISEASE: Apremilast is approved for the treatment of Behçet's disease in adults with refractory, severe oral ulcers., Outlook: Case studies have also demonstrated the efficacy of systemic PDE4 inhibition in other skin diseases (including blistering autoimmune dermatoses, lichen planus, and acantholytic genodermatoses). The substances are also being researched and used to treat extracutaneous inflammatory diseases., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Background and Objectives: Oral lichen planus (OLP) is a T cell driven disorder that significantly impairs patients' quality of life. Previous reports suggest that both cellular and humoral activities against desmoglein (dsg) 1 and 3 may be involved in OLP pathogenesis. Here, we aim to analyze the frequency of occurrence and pathological significance of anti-dsg antibodies in a large cohort of OLP patients., Materials and Methods: OLP patients were screened for anti-dsg antibodies by enzyme-linked immunosorbent assay in three tertiary referral centers. OLP sera with anti-dsg antibodies were further analyzed by Western blot and dispase-based keratinocyte dissociation assay (DDA) to identify the targeted dsg ectodomains and to assess their pathogenicity., Results: Of 151-screened individuals with OLP, only four patients (2.6%) with erosive OLP showed serum IgG against dsg1/3. Western blot analysis with recombinant dsg3 ectodomains revealed preferential recognition of the extracellular domain 5. By DDA with spontaneously immortalized human keratinocytes, none of the sera from these four patients induced acantholysis., Conclusions: Activation of humoral immunity occurs prevalently in patients with erosive OLP, probably due to epitope spreading. OLP serum antibodies are unable to induce loss of intercellular adhesion in vitro, strongly suggesting that they are not disease causing but rather an epiphenomenon., (© 2024 The Author(s). Journal der Deutschen Dermatologischen Gesellschaft published by John Wiley & Sons Ltd on behalf of Deutsche Dermatologische Gesellschaft.)

Background/Objectives: Retinal hyperreflective foci, 25-50 µm in diameter, that can be imaged by noninvasive optical coherence tomography (OCT) may represent microglial activity related to inflammation. This study aimed to detect hyperreflective foci in the OCT-hyporeflective avascular outer nuclear layer of the retina in relapsing-remitting MS (RRMS) patients without ongoing eye or optic nerve disease. Methods: A cohort of 13 RRMS patients (8 eyes with and 18 eyes without prior optic neuritis) underwent retinal OCT at baseline, after 1 month, after 6 months, and then every 6 months for 3 years. The data were compared with single-examination data from 106 eyes in 53 age-matched healthy subjects. Results: The prevalence of hyperreflective foci at baseline was higher in RRMS patients than in healthy subjects (46.2% vs. 1.8%, p < 0.005). Patients with optic neuritis had much more foci than those without ( p < 0.001). Hyperreflective foci recurred in 23.1% of RRMS patients, bilaterally in one with prior optic neuritis and unilaterally in two without. Conclusions: Patients with RRMS, notably those with prior optic neuritis, had elevated rates of retinal infiltration in the absence of retinal disease, suggesting that the phenomenon may represent elevated activity of an immune surveillance or housekeeping mechanism rather than retinal disease.

This study tested if a high-resolution, multi-modal, multi-scale retinal imaging instrument can provide novel information about structural abnormalities in vivo. The study examined 11 patients with very mild to moderate non-proliferative diabetic retinopathy (NPDR) and 10 healthy subjects using fundus photography, optical coherence tomography (OCT), OCT angiography (OCTA), adaptive optics scanning laser ophthalmoscopy (AO-SLO), adaptive optics OCT and OCTA (AO-OCT(A)). Of 21 eyes of 11 patients, 11 had very mild NPDR, 8 had mild NPDR, 2 had moderate NPDR, and 1 had no retinopathy. Using AO-SLO, capillary looping, inflections and dilations were detected in 8 patients with very mild or mild NPDR, and microaneurysms containing hyperreflective granular elements were visible in 9 patients with mild or moderate NPDR. Most of the abnormalities were seen to be perfused in the corresponding OCTA scans while a few capillary loops appeared to be occluded or perfused at a non-detectable flow rate, possibly because of hypoperfusion. In one patient with moderate NPDR, non-perfused capillaries, also called ghost vessels, were identified by alignment of corresponding en face AO-OCT and AO-OCTA images. The combination of multiple non-invasive imaging methods could identify prominent microscopic abnormalities in diabetic retinopathy earlier and more detailed than conventional fundus imaging devices., (© 2024. The Author(s).)

Motor performance is monitored continuously by specialized brain circuits and used adaptively to modify behavior on a moment-to-moment basis and over longer time periods. During vocal behaviors, such as singing in songbirds, internal evaluation of motor performance relies on sensory input from the auditory and vocal-respiratory systems. Sensory input from the auditory system to the motor system, often referred to as auditory feedback, has been well studied in singing zebra finches ( Taeniopygia guttata ), but little is known about how and where nonauditory sensory feedback is evaluated. Here we show that brief perturbations in air sac pressure cause short-latency neural responses in the higher-order song control nucleus HVC (used as proper name), an area necessary for song learning and song production. Air sacs were briefly pressurized through a cannula in anesthetized or sedated adult male zebra finches, and neural responses were recorded in both nucleus parambigualis (PAm), a brainstem inspiratory center, and HVC, a cortical premotor nucleus. These findings show that song control nuclei in the avian song system are sensitive to perturbations directly targeted to vocal-respiratory, or viscerosensory, afferents and support a role for multimodal sensory feedback integration in modifying and controlling vocal control circuits. NEW & NOTEWORTHY This study presents the first evidence of sensory input from the vocal-respiratory periphery directly activating neurons in a motor circuit for vocal production in songbirds. It was previously thought that this circuit relies exclusively on sensory input from the auditory system, but we provide groundbreaking evidence for nonauditory sensory input reaching the higher-order premotor nucleus HVC, expanding our understanding of what sensory feedback may be available for vocal control.

Reduced expression of the plasma membrane citrate transporter SLC13A5 , also known as INDY, has been linked to increased longevity and mitigated age-related cardiovascular and metabolic diseases. Citrate, a vital component of the tricarboxylic acid cycle, constitutes 1-5% of bone weight, binding to mineral apatite surfaces. Our previous research highlighted osteoblasts' specialized metabolic pathway facilitated by SLC13A5 regulating citrate uptake, production, and deposition within bones. Disrupting this pathway impairs bone mineralization in young mice. New Mendelian randomization analysis using UK Biobank data indicated that SNPs linked to reduced SLC13A5 function lowered osteoporosis risk. Comparative studies of young (10 weeks) and middle-aged (52 weeks) osteocalcin-cre-driven osteoblast-specific Slc13a5 knockout mice ( Slc13a5 cKO ) showed a sexual dimorphism: while middle-aged females exhibited improved elasticity, middle-aged males demonstrated enhanced bone strength due to reduced SLC13A5 function. These findings suggest reduced SLC13A5 function could attenuate age-related bone fragility, advocating for SLC13A5 inhibition as a potential osteoporosis treatment.

Despite recent development of vaccines to prevent SARS-CoV-2 infection, treatment of critically ill COVID-19 patients remains an important goal. In principle, genome-wide association studies (GWASs) provide a shortcut to the clinical evidence needed to repurpose existing drugs; however, genes identified frequently lack a causal disease link. We report an alternative method for finding drug repurposing targets, focusing on disease-causing traits beyond immediate disease genetics. Sixty blood cell types and biochemistries, and body mass index, were screened on a cohort of critically ill COVID-19 cases and controls that exhibited mild symptoms after infection, yielding high neutrophil cell count as a possible causal trait for critical illness. Our methodology identified CDK6 and janus kinase (JAK) inhibitors as treatment targets that were validated in an ex vivo neutrophil extracellular trap (NET) formation assay. Our methodology demonstrates the increased power for drug target identification by leveraging large disease-causing trait datasets., Competing Interests: H.A.B., J.L.C., C.N.J.R., M.R.J.L., J.E.K., and M.F.S. are employees of biotx.ai GmbH. A.R.E.C.S. was an employee of biotx.ai GmbH., (© 2023 The Author(s).)

Pemphigus and pemphigoid are two potentially life-threatening groups of autoimmune diseases, characterized by autoantibodies targeting structural components of desmosomes or hemidesmosomes, respectively. Affected patients typically show itchy/painful plaques or blistering skin lesions and/or impairing mucosal blistering and erosions, which may strongly impact their quality of life. Since the milestone work of Walter Lever in 1953, who differentiated these two groups of diseases by histopathological analysis of the level of antibody-mediated skin cleavage, enormous progresses occurred. Achievements made in laboratory diagnostics now allow to identify antigen specific structural proteins of the skin that are targeted by pathogenic autoantibodies. These progresses were accompanied by an increased understanding of the pathogenesis of these diseases thanks to the establishment of animal models reproducing disease and on studies on skin and blood of affected individuals, which have been leading to novel and disease-specific treatments. Yet, given their phenotypical overlap with more common dermatological diseases, correct diagnosis and appropriate treatment are often delayed, in some cases leading to irreversible sequelae, including organ dysfunction (i.e., loss of vision in mucous membrane pemphigoid). Here, we provide a concise overview of the clinical appearance, diagnosis and therapeutic management of pemphigus and pemphigoid diseases., (© 2023 The Authors. Journal der Deutschen Dermatologischen Gesellschaft published by John Wiley & Sons Ltd on behalf of Deutsche Dermatologische Gesellschaft.)

A scar develops following the appearance of a deep tissue defect as part of the physiological wound healing process. The initial inflammatory response is followed by proliferation of connective tissue cells, which form fibrosis as a final tissue substitute. Disorders can occur at all stages of the process and are most commonly manifested as impaired wound healing or the formation of atrophic and hypertrophic scars or keloids. The focus of this article is on the treatment of pathologic scars, which are an indication for therapy due to functional limitations, complaints, and stigmatization, among other reasons. Conservative medical, physical, surgical and laser therapeutic approaches are pursued. The basis for this is an understanding of the pathophysiological mechanisms and factors influencing the choice of therapy, as well as an interdisciplinary and interprofessional therapeutic approach., (© 2023 The Authors. Journal der Deutschen Dermatologischen Gesellschaft published by John Wiley & Sons Ltd on behalf of Deutsche Dermatologische Gesellschaft.)

Background: Hyperreflective granular elements with a transient presence in the retina can be detected non-invasively by optical coherence tomography (OCT). Such foci or dots may represent aggregates of activated microglia. However, in multiple sclerosis an increased number of hyperreflective foci has so far not been demonstrated in the intrinsically hyporeflective and avascular outer nuclear layer of the retina where there are no fixed elements in healthy eyes. Therefore, the present study intended to investigate the presence of hyperreflective foci in the outer nuclear layer in patients with relapsing- remitting multiple sclerosis (RRMS) by using a high-resolution OCT scanning protocol., Methods: This cross-sectional exploratory study examined 88 eyes in 44 RRMS patients and 106 eyes in 53 age- and sex-matched healthy subjects. None of the patients had any sign of retinal disease. All patients and healthy subjects each underwent one session of spectral domain OCT imaging. A total of 23,200 B-scans extracted from 8 × 8 mm blocks of linear B-scans at 60 μm intervals were analysed for hyperreflective foci in the outer nuclear layer of the retina. Analyses were made of the total block scan and a circular 6-mm diameter fovea-centered field in each eye. Multivariate logistic regression analysis was used to assess associations between parameters., Results: Hyperreflective foci were observed in 31 out of 44 (70.5 %) multiple sclerosis patients compared to 1 out of 53 (1.8%) healthy subjects (p < 0.0001). From analyses of the total block scans, the median number of hyperreflective foci in the outer nuclear layer was 1 (range 0-13) in patients and 0 (range 0-2) in healthy subjects (p < 0.0001). In total, 66.2% of all hyperreflective foci were located within 6 mm of the center of the macula. There was no detectable association between the presence of hyperreflective foci and retinal nerve fiber layer or ganglion cell layer thickness., Conclusion: Hyperreflective granular foci in the avascular outer nuclear layer of the retina seen by OCT were almost completely absent in healthy subjects, whereas they were found, albeit at low density, in the majority of patients with RRMS. Hyperreflective foci can be repeatedly examined by non-invasive means and without pupil dilation, which opens a new field of investigation of infiltrating elements in an unmyelinated part of the central nervous system., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Hyperreflective foci are poorly understood transient elements seen on optical coherence tomography (OCT) of the retina in both healthy and diseased eyes. Systematic studies may benefit from the development of automated tools that can map and track such foci. The outer nuclear layer (ONL) of the retina is an attractive layer in which to study hyperreflective foci as it has no fixed hyperreflective elements in healthy eyes. In this study, we intended to evaluate whether automated image analysis can identify, quantify and visualize hyperreflective foci in the ONL of the retina., Methods: This longitudinal exploratory study investigated 14 eyes of seven patients including six patients with optic neuropathy and one with mild non-proliferative diabetic retinopathy. In total, 2596 OCT B-scan were obtained. An image analysis blob detector algorithm was used to detect candidate foci, and a convolutional neural network (CNN) trained on a manually labelled subset of data was then used to select those candidate foci in the ONL that fitted the characteristics of the reference foci best., Results: In the manually labelled data set, the blob detector found 2548 candidate foci, correctly detecting 350 (89%) out of 391 manually labelled reference foci. The accuracy of CNN classifier was assessed by manually splitting the 2548 candidate foci into a training and validation set. On the validation set, the classifier obtained an accuracy of 96.3%, a sensitivity of 88.4% and a specificity of 97.5% (AUC 0.989)., Conclusion: This study demonstrated that automated image analysis and machine learning methods can be used to successfully identify, quantify and visualize hyperreflective foci in the ONL of the retina on OCT scans., (© 2022 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Healthy human skin is constantly exposed to sterile and microbial agents. The skin immune system plays an important role in immune surveillance between tolerance and immune activation. This is mainly mediated by neutrophils, macrophages and most importantly lymphocytes. Keratinocytes, which form the outer skin barrier (epidermis) are also critical for cutaneous homeostasis. Being a non-professional immune cell, recognition of danger signals in keratinocytes is mediated by innate immune receptors (pattern recognition receptors, PRR). While Toll-like receptors are located on the cell membrane or the endosomes, nucleotide-binding domain and leucine-rich repeat containing gene family receptors (NLR) are intracellular PRRs. Some of these, once activated, trigger the formation of inflammasomes. Inflammasomes are multiprotein complexes and serve as platforms that mediate the release of innate cytokines after successful recognition, thereby attracting immune cells. Moreover, they mediate the pro-inflammatory cell death pyroptosis. Best characterized is the NLRP3 inflammasome. The function of inflammasomes differs significantly between different cell types (keratinocytes versus immune cells) and between different species (human versus mouse). In recent years, great progress has been made in deciphering the activation mechanisms. Dysregulation of inflammasomes can lead to diseases with varying degrees of severity. Here we focus on the structure, function, and associated pathologies of the NLRP1 inflammasome, which is the most relevant inflammasome in keratinocytes., 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 Burian, Schmidt and Yazdi.)

A conserved intracellular allosteric binding site (IABS) has recently been identified at several G protein-coupled receptors (GPCRs). Ligands targeting the IABS, so-called intracellular allosteric antagonists, are highly promising compounds for pharmaceutical intervention and currently evaluated in several clinical trials. Beside co-crystal structures that laid the foundation for the structure-based development of intracellular allosteric GPCR antagonists, small molecule tools that enable an unambiguous identification and characterization of intracellular allosteric GPCR ligands are of utmost importance for drug discovery campaigns in this field. Herein, we discuss recent approaches that leverage cellular target engagement studies for the IABS and thus play a critical role in the evaluation of IABS-targeted ligands as potential therapeutic agents., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Photoswitchable ligands as biological tools provide an opportunity to explore the kinetics and dynamics of the clinically relevant μ-opioid receptor. These ligands can potentially activate or deactivate the receptor when desired by using light. Spatial and temporal control of biological activity allows for application in a diverse range of biological investigations. Photoswitchable ligands have been developed in this work, modelled on the known agonist fentanyl, with the aim of expanding the current "toolbox" of fentanyl photoswitchable ligands. In doing so, ligands have been developed that change geometry (isomerize) upon exposure to light, with varying photophysical and biochemical properties. This variation in properties could be valuable in further studying the functional significance of the μ-opioid receptor., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Young age at school entry (ASE) is related to attention-deficit/hyperactivity disorder in higher grades. The reason for this association is unclear, but medical oversupply and stress-related factors are discussed. We aimed to investigate whether ASE is associated with reported symptoms of attention-deficit/hyperactivity (ADH) already in first grade. Data of a population-based prospective cohort study (N = 2003; Mainz-Bingen region; Rhineland-Palatinate; Germany) with baseline assessments prior to school entry and two follow-ups during first grade were analysed. ADH symptoms were assessed by parent and teacher versions of the Strengths and Difficulties Questionnaire. Associations between ASE and scores of the hyperactivity/inattention subscale (range 0-10) were investigated by regression analysis and adjusted for potential confounders and baseline symptoms prior to school entry. In total, 1633 children (52% boys, mean ASE 6.5 years) were included. There were no relationships between ASE and parent-reported scores of the hyperactivity/inattention subscale prior to school entry and 3 months thereafter. However, at the end of first grade, ASE was negatively associated with the hyperactivity/inattention subscale in parent (- 0.7 subscale points per year ASE, standard error = 0.16, p < 0.0001) and teacher reports (- 1.2 subscale points per year ASE, standard error = 0.25, p < 0.0001). This ASE effect appeared more pronounced in girls than in boys. Young ASE is related to more reported symptoms of ADH at the end of first grade, but not before. The evolvement of this effect during first grade may be a clue to ASE-related stress factors., (© 2021. The Author(s).)

Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α 2A -adrenergic receptor (α 2A AR), seeking new α 2A AR agonists chemotypes that lack the sedation conferred by known α 2A AR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential G i and G o signaling. Experimental structures of α 2A AR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit '9087 [mean effective concentration (EC 50 ) of 52 nanomolar] and two analogs, '7075 and PS75 (EC 50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.

Fluorescently labeled ligands are versatile molecular tools to study G protein-coupled receptors (GPCRs) and can be used for a range of different applications, including bioluminescence resonance energy transfer (BRET) assays. Here, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a drug target in oncology and inflammation. Starting from previously reported intracellular CCR2 antagonists, several tetramethylrhodamine (TAMRA)-labeled CCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CCR2. By means of these studies, we developed 14 as a fluorescent CCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a non-isotopic and high-throughput manner. Further, we show that 14 can be used as a tool for fragment-based screening approaches. Thus, our small-molecule-based fluorescent CCR2 ligand 14 represents a promising tool for future studies of CCR2 pharmacology.

Molecular mechanisms underlying auto-antibody-induced acantholysis in pemphigus vulgaris are subject of current research to date. To decipher the discrepancy between ubiquitous antibody binding to the epidermal desmosomes, but discontinuous disease manifestation, we were able to identify Ultraviolet A (UVA) as a cofactor for acantholysis. UVA induces interleukin (IL)-1 secretion in keratinocytes, mirroring innate immune system activation. In an in vitro keratinocyte dissociation assay increased fragmentation was observed when UVA was added to anti-Desmoglein 3 Immunoglobulins (anti-Dsg3 IgG). These results were confirmed in skin explants where UVA enhanced anti-Dsg3-mediated loss of epidermal adhesion. The UVA-mediated effect was blocked in vitro by the pan-caspase-inhibitor zVAD-fmk. Thus, we introduce UVA as a caspase-dependent exogenous cofactor for acantholysis which suggests that local innate immune responses largely contribute to overt clinical blister formation upon autoantibody binding to epidermal cells in pemphigus vulgaris., 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 © 2022 Eichkorn, Schmidt, Walter, Hertl, Baron, Waschke and Yazdi.)

In many vertebrates, courtship occurs through the performance of elaborate behavioral displays that are as spectacular as they are complex. The question of how sexual selection acts upon these animals' neuromuscular systems to transform a repertoire of pre-existing movements into such remarkable (if not unusual) display routines has received relatively little research attention. This is a surprising gap in knowledge, given that unraveling this extraordinary process is central to understanding the evolution of behavioral diversity and its neural control. In many vertebrates, courtship displays often push the limits of neuromuscular performance, and often in a ritualized manner. These displays can range from songs that require rapid switching between two independently controlled 'voice boxes' to precisely choreographed acrobatics. Here, we propose a framework for thinking about how the brain might not only control these displays, but also shape their evolution. Our framework focuses specifically on a major midbrain area, which we view as a likely important node in the orchestration of the complex neural control of behavior used in the courtship process. This area is the periaqueductal grey (PAG), as studies suggest that it is both necessary and sufficient for the production of many instinctive survival behaviors, including courtship vocalizations. Thus, we speculate about why the PAG, as well as its key inputs, might serve as targets of sexual selection for display behavior. In doing so, we attempt to combine core ideas about the neural control of behavior with principles of display evolution. Our intent is to spur research in this area and bring together neurobiologists and behavioral ecologists to more fully understand the role that the brain might play in behavioral innovation and diversification., Competing Interests: RS, MF, MS No competing interests declared, (© 2022, Schwark et al.)

GPR88 is an orphan class A G-protein-coupled receptor that is highly expressed in the striatum and regulates diverse brain and behavioral functions. Here we present cryo-EM structures of the human GPR88-Gi1 signaling complex with or without a synthetic agonist (1R, 2R)-2-PCCA. We show that (1R, 2R)-2-PCCA is an allosteric modulator binding to a herein identified pocket formed by the cytoplasmic ends of transmembrane segments 5, 6, and the extreme C terminus of the α5 helix of Gi1. We also identify an electron density in the extracellular orthosteric site that may represent a putative endogenous ligand of GPR88. These structures, together with mutagenesis studies and an inactive state model obtained from metadynamics simulations, reveal a unique activation mechanism for GPR88 with a set of distinctive structure features and a water-mediated polar network. Overall, our results provide a structural framework for understanding the ligand binding, activation and signaling mechanism of GPR88, and will facilitate the innovative drug discovery for neuropsychiatric disorders and for deorphanization of this receptor., (© 2022. The Author(s).)

A broadly applicable synthesis of peptides incorporating mixed disulfides between cysteine and homocysteine and cysteamine was developed. The method was established using pharmacologically relevant G protein-coupled receptor (GPCR) ligands including the μ-receptor agonist Dmt-DALDA and extended to the orexin derivative Oxa(17-33) and NT(8-13), the C-terminal hexapeptide of neurotensin. The newly developed NT(8-13) analog 6b incorporating an S-functionalized homocysteine revealed covalent binding of the neurotensin receptor 1 (NTSR1) in a radioligand depletion study., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A conserved intracellular allosteric binding site (IABS) has recently been identified at several G protein-coupled receptors (GPCRs). Starting from vercirnon, an intracellular C-C chemokine receptor type 9 (CCR9) antagonist and previous phase III clinical candidate for the treatment of Crohn's disease, we developed a chemical biology toolbox targeting the IABS of CCR9. We first synthesized a fluorescent ligand enabling equilibrium and kinetic binding studies via NanoBRET as well as fluorescence microscopy. Applying this molecular tool in a membrane-based setup and in living cells, we discovered a 4-aminopyrimidine analogue as a new intracellular CCR9 antagonist with improved affinity. To chemically induce CCR9 degradation, we then developed the first PROTAC targeting the IABS of GPCRs. In a proof-of-principle study, we succeeded in showing that our CCR9-PROTAC is able to reduce CCR9 levels, thereby offering an unprecedented approach to modulate GPCR activity., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Pemphigus vulgaris (PV) is a chronic, life-altering autoimmune disease due to the production of anti-desmoglein antibodies causing the loss of cell-cell adhesion in keratinocytes (acantholysis) and blister formation in both skin and mucous membranes. The dispase-based keratinocyte dissociation assay (DDA) is the method of choice to examine the pathogenic effect of antibodies and additional co-stimuli on cell adhesion in vitro. Despite its widespread use, there is a high variability of experimental conditions, leading to inconsistent results. In this paper, we identify and discuss pitfalls in the application of DDA, including generation of a monolayer with optimized density, appropriate culturing conditions to obtain said monolayer, application of mechanical stress in a standardized manner, and performing consistent data processing. Importantly, we describe a detailed protocol for a successful and reliable DDA and the respective ideal conditions for three different types of human keratinocytes: (1) primary keratinocytes, (2) the HaCaT spontaneously immortalized keratinocyte cell line, and (3) the recently characterized HaSKpw spontaneously immortalized keratinocyte cell line. Our study provides detailed protocols which guarantee intra- and inter-experimental comparability of DDA.

Objective: To investigate clinical outcomes in a real-world setting in the complete population-based cohort of alemtuzumab-treated MS patients in Denmark., Methods: Data were retrieved from The Danish Multiple Sclerosis Registry between 2009 and 2019. Demographic and disease-specific patient parameters related to treatment history, efficacy, and safety outcomes were assessed at baseline and during follow-up visits., Results: A total of 209 patients (78% female) started treatment with alemtuzumab during the study period with 3.1 ± 1.4 years follow-up. After 2 years, 75% of patients were relapse-free compared to 48% the year before alemtuzumab ( p  < 0.001). The annual number of relapses was reduced by 69% in year 4 compared with the year prior alemtuzumab. More active disease before alemtuzumab increased the annual hazard rate for relapse (HR: 2.88, p  < 0.001). The Expanded Disability Status Scale (EDSS) score remained stable or improved in 81% of patients after 2 years. The need for an additional treatment course was associated with higher number of relapses in the year before alemtuzumab (odds ratio (OR) = 1.95, p  = 0.001)., Conclusion: In a country with primarily escalation strategy, relapse rate reduction was maintained for 5 years, and EDSS stabilized/improved in majority of patients. Higher relapse rate 1 year before alemtuzumab increased the odds for additional courses. Novel serious AEs were not observed.

The family of neuropeptide Y (NPY) receptors comprises four subtypes (Y 1 R, Y 2 R, Y 4 R, Y 5 R), which are addressed by at least three endogenous peptides, i.e., NPY, peptide YY, and pancreatic polypeptide (PP), the latter showing a preference for Y 4 R. A series of cyclic oligopeptidic Y 4 R ligands were prepared by applying a novel approach, i.e., N-terminus to arginine side-chain cyclization. Most peptides acted as Y 4 R partial agonists, showing up to 60-fold higher Y 4 R affinity compared to the linear precursor peptides. Two cyclic hexapeptides ( 18 , 24 ) showed higher Y 4 R potency (Ca 2+ aequorin assay) and, with p K i values >10, also higher Y 4 R affinity compared to human pancreatic polypeptide (hPP). Compounds such as 18 and 24 , exhibiting considerably lower molecular weight and considerably more pronounced Y 4 R selectivity than PP and previously described dimeric peptidic ligands with high Y 4 R affinity, represent promising leads for the preparation of labeled tool compounds and might support the development of drug-like Y 4 R ligands.

Polygenic risk scores (PRS) aggregating results from genome-wide association studies are the state of the art in the prediction of susceptibility to complex traits or diseases, yet their predictive performance is limited for various reasons, not least of which is their failure to incorporate the effects of gene-gene interactions. Novel machine learning algorithms that use large amounts of data promise to find gene-gene interactions in order to build models with better predictive performance than PRS. Here, we present a data preprocessing step by using data-mining of contextual information to reduce the number of features, enabling machine learning algorithms to identify gene-gene interactions. We applied our approach to the Parkinson's Progression Markers Initiative (PPMI) dataset, an observational clinical study of 471 genotyped subjects (368 cases and 152 controls). With an AUC of 0.85 (95% CI = [0.72; 0.96]), the interaction-based prediction model outperforms the PRS (AUC of 0.58 (95% CI = [0.42; 0.81])). Furthermore, feature importance analysis of the model provided insights into the mechanism of Parkinson's disease. For instance, the model revealed an interaction of previously described drug target candidate genes TMEM175 and GAPDHP25 . These results demonstrate that interaction-based machine learning models can improve genetic prediction models and might provide an answer to the missing heritability problem., Competing Interests: JC, HB, JK, CR, and MS are employed by biotx.ai GmbH. The remaining 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 © 2021 Cope, Baukmann, Klinger, Ravarani, Böttinger, Konigorski and Schmidt.)

Objectives: Long-term survivors of childhood cancer are at increased risk for sequelae such as poor mental health (MH) or impaired health-related quality of life (HrQoL). We aimed to evaluate early adverse effects on MH and HrQoL in young childhood cancer survivors (YCCS) before school entry., Methods: In a nationwide prospective cohort study, children with cancer other than brain tumors diagnosed at preschool age and completed cancer treatments were identified from the German Childhood Cancer Registry. The comparison group was children of the same age without a cancer diagnosis who participated in the prospective population-based health survey ikidS. MH problems and HrQoL were assessed by parental versions of the Strengths and Difficulties Questionnaire (SDQ) and the questionnaire for health-related quality of life in children (KINDL), respectively. Associations between cancer and MH as well as HrQoL were analyzed by multivariable linear regression., Results: Of 382 YCCS contacted, 145 were enrolled (mean age 6.6 years) and 124 analyzed. Compared to children without a cancer diagnosis (3683 contacted, 2003 enrolled, 1422 analyzed), YCCS had more MH problems (13% vs. 3%) and slightly worse HrQoL (median 78.7 vs. 80.2 points). In the adjusted analysis, YCCS had higher SDQ scores (2.2 points, 95% confidence interval [CI] 1.3, 3.0) and lower KINDL scores (-2.4 points, 95% CI -3.7, -1.1) compared to children without cancer diagnosis., Conclusion: Already at preschool age, YCCS may be at increased risk of MH problems and impaired HrQoL. This could have impacts on subsequent school performance and educational attainment. Follow-up health care for YCCS may include early screening for MH problems and reasons for HrQoL deficits., (© 2021 The Authors. Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

Background: Hyperplasia-associated cutaneous paraneoplasia is an important differential diagnosis in everyday clinical practice. An early diagnosis of the underlying tumor disease can significantly improve the patient's prognosis., Pathogenesis: Hyperplasia is probably mainly cytokine-mediated. The primary tumor and/or the metastases release growth factors and transcription factors which, via epidermal growth factors, lead to hyperproliferation of keratinocytes., Acanthosis Nigricans Maligna: Symmetrical mainly intertriginous hyperpigmentation with partially verrucous hyperplasia and lichenification mostly in association with gastric adenocarcinoma. Special forms are florid cutaneous papillomatosis and tripe palms. Pseudoacanthosis nigricans is to be distinguished (metabolic and hormonal disorders). LESER-TRéLAT SYNDROME: Eruptive occurrence of seborrheic keratosis associated with visceral tumors., Acrokeratosis Bazex: Erythema and scaling initially at the bridge of the nose, ear helix and acra with later spread, associated with tumors of the upper aerodigestive system. It should be clinically differentiated from psoriasis., Therapy: The treatment of the primary tumor is decisive, which also leads to a decrease of cutaneous symptoms. Reappreance suggests tumor recurrence.

The family of human muscarinic acetylcholine receptors (MRs) is characterized by a high sequence homology among the five subtypes (M 1 R-M 5 R), being the reason for a lack of subtype selective MR ligands. In continuation of our work on dualsteric dibenzodiazepinone-type M 2 R antagonists, a series of M 2 R ligands containing a dibenzodiazepinone pharmacophore linked to small basic peptides was synthesized (64 compounds). The linker moiety was varied with respect to length, number of basic nitrogens (0-2) and flexibility. Besides proteinogenic basic amino acids (Lys, Arg), shorter homologues of Lys and Arg, containing three and two methylene groups, respectively, as well as D-configured amino acids were incorporated. The type of linker had a marked impact on M 2 R affinity and also effected M 2 R selectivity. In contrast, the structure of the basic peptide rather determined M 2 R selectivity than M 2 R affinity. For example, the most M 2 R selective compound (UR-CG188, 89) with picomolar M 2 R affinity (pK i 9.60), exhibited a higher M 2 R selectivity (ratio of K i M 1 R/M 2 R/M 3 R/M 4 R/M 5 R: 110:1:5200:55:2300) compared to the vast majority of reported M 2 R preferring MR ligands. For selected ligands, M 2 R antagonism was confirmed in a M 2 R miniG protein recruitment assay., 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 © 2021 Elsevier Masson SAS. All rights reserved.)

Purpose of the Study: Developmental delays at school enrollment can affect early educational achievement. Thus, diagnosis and treatment prior to school entry are important. In Germany, SOPESS - a pediatric developmental screening instrument for preschool health examinations (PHE) - was introduced in several federal states. We investigated the relationships between the results of the language-related domain of the SOPESS and later academic language competencies at the end of first grade., Procedures: Data of the population-based cohort study ikidS (Rhineland Palatinate; Germany) and the PHE provided by public health authorities were linked at individual level. The relationship between the SOPESS-language risk score (range: 0 to 6, higher values indicate more developmental delay) and class teacher-based ratings of academic language competencies (range: -4 to+4, higher values indicate better competencies) was investigated with linear regression models and adjusted for several socio-economic and medical language predictors (e. g. age at school entry, gender, parental education, migration background, hearing disorder). The additional benefits of the SOPESS-language risk score was analyzed using analysis of variance., Results: In total, 1357 children (48% girls, age at PHE 4.9-7.2 years) were included. A clear negative relationship between the SOPESS-language risk score and competencies in spoken and written language was found. Mean academic language competencies decreased with increasing SOPESS-language risk level from 0.8 (SD=1.7) at a risk score of 0 to-3.2 (SD=0.9) at a risk score of 6. The relationship persisted after adjusting for other language predictors. Analysis of variance showed significant benefits of the SOPESS-language risk score, even when further language predictors were considered., Conclusions: There was a clear relationship between results of the SOPESS-language screening and later academic language competencies. Hence, the language-related domain of the SOPESS may be useful for further recommendations of tailored special educational and medical support services, especially if additional socio-economic and medical factors are considered., Competing Interests: Diese Studie wurde durch Forschungsmittel des Bundesministerium für Bildung und Foschung gefördert (BMBF Projekt Nr. 01ER1302 und 01ER1702), (Thieme. All rights reserved.)