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Paul Ritsche,1 Ralf Roth,1 Thomas Bernhard,2 Lukas Nebiker,1 Eric Lichtenstein,1 Martino Franchi,3 Jörg Spörri,4,5 Oliver Faude1 1Department of Sport, Exercise and Health, University of Basel, Basel, Switzerland; 2FC Basel 1893, Basel, Switzerland; 3Human Neuromuscular Physiology Lab, Department of Biomedical Sciences, University of Padua, Padova, Italy; 4Sports Medical Research Group, Department of Orthopaedics, Balgrist University Hospital, University of Zurich, Zurich, Switzerland; 5University Centre for Prevention and Sports Medicine, Department of Orthopaedics, Balgrist University Hospital, University of Zurich, Zurich, SwitzerlandCorrespondence: Paul Ritsche, University of Basel, Department of Sport, Exercise and Health, Grosse Allee 6, Basel, 4052, Switzerland, Email Paul.ritsche@unibas.chPurpose: Adolescent soccer players experience distinct physiological changes due to chronological and biological maturation, impacting their soccer performance. Here, we explored age-related variations and associations between quadriceps geometry and strength in male national-level adolescent soccer players.Patients and Methods: We used ultrasonography to examine the regional architecture and morphology of the rectus femoris (RF) and vastus lateralis (VL) muscles, and we assessed knee extension strength by isometric and isokinetic dynamometry. Players were categorized into four age groups: under (U) 15 (n=18, age=13.7± 0.5 years), U16 (n=15, age=14.7± 0.5), U17 (n=19, age=15.7± 0.5), U18 (n=18, age=16.7± 0.5) and U21 (n=25, age=18.5± 0.5).Results: The absolute and relative strengths were higher in the U16 compared to U15 by 12– 15% and 6– 8%, 11– 12% and 6– 7% in the U17 compared to U16, 5– 7% and − 1– 2% in the U18 compared to U17 and 0– 15% and − 1– 11% in the U21 compared to U18 age groups, respectively. VL architecture did not change relevantly between the age groups. The muscle anatomical cross-sectional area (ACSA) of the VL and RF differed non-uniformly and muscle region-specific by 10– 36%, with highest values in the U21 age group. Moderate correlations between the VL architecture and knee extension strength in both legs were observed only in the U16 age group. The quadriceps ACSA showed age-specific correlations with knee extension strength.Conclusion: Our findings highlight non-uniform differences in quadriceps muscle morphology and absolute and relative strength among male national-level adolescent soccer players in different age groups. The correlations observed between muscle morphology or architecture and strength were muscle, muscle region, leg and age dependent.Keywords: strength, youth, muscle architecture, vastus lateralis, rectus femoris

An ab initio (i.e., from first principles) theoretical framework capable of providing a unified description of the structure and low-energy reaction properties of light nuclei is desirable to further our understanding of the fundamental interactions among nucleons, and provide accurate predictions of crucial reaction rates for nuclear astrophysics, fusion-energy research, and other applications. In this contribution we review ab initio calculations for nucleon and deuterium scattering on light nuclei starting from chiral two- and three-body Hamiltonians, obtained within the framework of the ab initio no-core shell model with continuum. This is a unified approach to nuclear bound and scattering states, in which square-integrable energy eigenstates of the A-nucleon system are coupled to (A−a)+a target-plus-projectile wave functions in the spirit of the resonating group method to obtain an efficient description of the many-body nuclear dynamics both at short and medium distances and at long ranges.

We present recent results for 4He and 6Li obtained with improved NN interactions derived from chiral effective field theory up to N4LO. The many-body calculations are performed order-by-order in the chiral expansion. At N3LO and N4LO additional renormalization using the Similarity Renormalization Group is adopted to improve numerical convergence of the many-body calculations. We discuss results for the ground state energies, as well as the magnetic moment and the low-lying spectrum of 6Li.

We present a cross-section of recent advances in ab initio nuclear structure theory, which have changed the horizons of this field. Starting from chiral effective field theory to construct the nuclear Hamiltonian and the similarity renormalization group to further soften it, we address several many-body approaches that have seen major developments over the past few years. We show that the domain of ab initio nuclear structure theory has been pushed well beyond the p-shell and that quantitative QCD-based predictions are becoming possible all the way from the proton to the neutron drip line up into the medium-mass regime.

Although fish removal for biomanipulation is often highly size-selective, our understanding of the nutrient cycling effects of this size-selection is poor. To better understand these effects, we measured nutrient excretion by gizzard shad (Dorosoma cepedianum) of differing sizes from four central Florida (USA) lakes and combined these measures with gillnet biomass and size-structure data to compare lake-wide effects among lakes and years. Direct removal of P in fish tissue ranged from 0.16−1.00 kg·P·ha-1·yr-1. The estimated reduction in P excretion due to harvest ranged from 30.8−202.5 g·P·ha-1·month-1, with effects strongly tied to the biomass and size structure harvested. The amount of P release prevented per kg of fish removed was lower in previously unharvested lakes, due to the initial removal of larger fish with lower mass-specific excretion rates. Gill net mesh size impacted the size distribution of harvested fish, with smaller fish that excrete more P per gram being more vulnerable to smaller mesh sizes. In Lake Apopka, decreasing the mesh size by 1.3 cm yielded P excretion reductions that were 10.7−15.1% larger. Fish harvesting to reduce internal nutrient cycling can be most effective by increasing total harvest and by harvesting smaller size classes over multiple years.

Abstract Background The kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes. Results A number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile. Conclusions The "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety.

The exact treatment of nuclei starting from the constituent nucleons and the fundamental interactions among them has been a long-standing goal in nuclear physics. Above all nuclear scattering and reactions, which require the solution of the many-body quantum-mechanical problem in the continuum, represent an extraordinary theoretical as well as computational challenge for ab initio approaches. We present a new ab initio many-body approach which derives from the combination of the ab initio no-core shell model with the resonating-group method [4]. By complementing a microscopic cluster technique with the use of realistic interactions, and a microscopic and consistent description of the nucleon clusters, this approach is capable of describing simultaneously both bound and scattering states in light nuclei. We will discuss applications to neutron and proton scattering on s- and light p-shell nuclei using realistic nucleon-nucleon potentials, and outline the progress toward the treatment of more complex reactions.

Abstract Background Kulldorff's spatial scan statistic and its software implementation – SaTScan – are widely used for detecting and evaluating geographic clusters. However, two issues make using the method and interpreting its results non-trivial: (1) the method lacks cartographic support for understanding the clusters in geographic context and (2) results from the method are sensitive to parameter choices related to cluster scaling (abbreviated as scaling parameters), but the system provides no direct support for making these choices. We employ both established and novel geovisual analytics methods to address these issues and to enhance the interpretation of SaTScan results. We demonstrate our geovisual analytics approach in a case study analysis of cervical cancer mortality in the U.S. Results We address the first issue by providing an interactive visual interface to support the interpretation of SaTScan results. Our research to address the second issue prompted a broader discussion about the sensitivity of SaTScan results to parameter choices. Sensitivity has two components: (1) the method can identify clusters that, while being statistically significant, have heterogeneous contents comprised of both high-risk and low-risk locations and (2) the method can identify clusters that are unstable in location and size as the spatial scan scaling parameter is varied. To investigate cluster result stability, we conducted multiple SaTScan runs with systematically selected parameters. The results, when scanning a large spatial dataset (e.g., U.S. data aggregated by county), demonstrate that no single spatial scan scaling value is known to be optimal to identify clusters that exist at different scales; instead, multiple scans that vary the parameters are necessary. We introduce a novel method of measuring and visualizing reliability that facilitates identification of homogeneous clusters that are stable across analysis scales. Finally, we propose a logical approach to proceed through the analysis of SaTScan results. Conclusion The geovisual analytics approach described in this manuscript facilitates the interpretation of spatial cluster detection methods by providing cartographic representation of SaTScan results and by providing visualization methods and tools that support selection of SaTScan parameters. Our methods distinguish between heterogeneous and homogeneous clusters and assess the stability of clusters across analytic scales. Method We analyzed the cervical cancer mortality data for the United States aggregated by county between 2000 and 2004. We ran SaTScan on the dataset fifty times with different parameter choices. Our geovisual analytics approach couples SaTScan with our visual analytic platform, allowing users to interactively explore and compare SaTScan results produced by different parameter choices. The Standardized Mortality Ratio and reliability scores are visualized for all the counties to identify stable, homogeneous clusters. We evaluated our analysis result by comparing it to that produced by other independent techniques including the Empirical Bayes Smoothing and Kafadar spatial smoother methods. The geovisual analytics approach introduced here is developed and implemented in our Java-based Visual Inquiry Toolkit.

Abstract Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the cost-effective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem®. In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed.

Abstract Background Childhood obesity is the result of a long lasting imbalance between energy intake and energy expenditure. A major contributing factor is physical inactivity which is closely linked to bone health, cardiovascular disease risk, fitness and psychological factors. The school seems to provide an excellent setting to enhance levels of physical activity (PA). However, there is insufficient data from previous school-based intervention trials on how to enhance overall PA. It is also unknown whether an intervention aimed at increasing PA is effective in improving the children's health. The purpose of this paper is to outline the design of a school-based randomized, controlled trial (RCT) aiming to increase overall PA and to improve fitness and health in 6- to 13-year-old children. Methods/Design 15 schools were randomized to the intervention (n = 9) or the control (n = 6) group, stratified by geographic region (urban vs. rural) and by age (1st and 5th grade). Participation was given for all children in the intervention group since in this group the intervention was part of the normal school curriculum. The intervention during one academic year consisted of: 1. two additional physical education classes per week given by trained physical education teachers adding up to a total of five PA classes per week, 2. short PA breaks (2–5 min each) during academic lessons, 3. PA home work, and 4. adaptation of recreational areas around the school. All children underwent anthropometric measurements, blood pressure assessment, fitness testing, measurement of PA and they filled out questionnaires. At least 70% of all children agreed to blood sampling and measurements of body composition and bone mineral measurements by dual energy x-ray absorptiometry. The primary endpoints of the study after one year were an increase in total PA by accelerometry, an increase in aerobic fitness measured by the 20 m shuttle run, a decrease in percent body fat derived from skinfold measurements and an increase in quality of life as assessed by the child health questionnaire in the intervention group compared to the control group. Secondary outcomes were overall fitness, differences in body composition including body fat distribution, cardiovascular risk factors, psychosocial health, bone mineral content and density of femur, lumbar spine and total body and food intake. Discussion Our preliminary data suggest that the children were representative of Swiss children with respect to sex, socio-demographic status, and body mass index. Short-term results can be expected by the beginning of 2007. We hypothesized that our intervention will lead to an increase in PA, fitness and overall health. Based on our data, we aim to provide important information regarding the influence of such an intervention on these outcome measures in school-aged children and to provide nationwide guidelines to improve PA in children.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N -glycosylation and O - and C -mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1 , ALG2 , and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Background: Epithelial to mesenchymal transition (EMT) is a key process in carcinogenesis of head and neck squamous cell carcinoma (HNSCC), contributing to tumor invasiveness, distant metastasis, and recurrence. Exosomes are known mediators and regulators of EMT. Here, we analyze the impact of exosomes that were primed by conventional therapy on EMT modulation., Methods: Plasmas of n = 22 HNSCC patients were collected before and after standard of care surgery and adjuvant or primary (chemo)radiotherapy. Exosomes were isolated by size exclusion chromatography. Upon co-incubation of exosomes with HNSCC cells, the cellular EMT profile was analyzed by flow cytometry and RT-qPCR. Wound healing assays were performed to evaluate migratory potential of exosome-treated cells., Results: Reduction of total exosome protein after therapy and in vitro exosome induced EMT profiles were dependent on the type of treatment. Exosomal TFG-β and miRNA cargo were partly responsible for observed exosome induced EMT changes. Exosomes from recurrent patients induced higher tumor cell migration after therapy than exosomes from disease-free patients., Conclusions: HNSCC patients' exosomes from timepoints before and after therapy were able to confer therapy induced EMT modulation in vitro and have the potential to monitor the EMT process. Exosome induced changes in migratory potential emerged as discriminants of therapy outcome., 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 Hofmann, Waizenegger, Röth, Schmitteckert, Engelhardt, Schuler, Laban, Hoffmann, Brunner and Theodoraki.)

Irritable bowel syndrome (IBS) is a gut-brain disorder of multifactorial origin. Evidence of disturbed serotonergic function in IBS accumulated for the 5-HT 3 receptor family. 5-HT 3 Rs are encoded by HTR3 genes and control GI function, and peristalsis and secretion, in particular. Moreover, 5-HT 3 R antagonists are beneficial in the treatment of diarrhea predominant IBS (IBS-D). We previously reported on functionally relevant SNPs in HTR3A c.-42C > T (rs1062613), HTR3C p.N163K (rs6766410), and HTR3E c.*76G > A (rs56109847 = rs62625044) being associated with IBS-D, and the HTR3B variant p.Y129S (rs1176744) was also described within the context of IBS. We performed a multi-center study to validate previous results and provide further evidence for the relevance of HTR3 genes in IBS pathogenesis. Therefore, genotype data of 2682 IBS patients and 9650 controls from 14 cohorts (Chile, Germany (2), Greece, Ireland, Spain, Sweden (2), the UK (3), and the USA (3)) were taken into account. Subsequent meta-analysis confirmed HTR3E c.*76G > A (rs56109847 = rs62625044) to be associated with female IBS-D (OR = 1.58; 95% CI (1.18, 2.12)). Complementary expression studies of four GI regions (jejunum, ileum, colon, sigmoid colon) of 66 IBS patients and 42 controls revealed only HTR3E to be robustly expressed. On top, HTR3E transcript levels were significantly reduced in the sigma of IBS patients (p = 0.0187); more specifically, in those diagnosed with IBS-D (p = 0.0145). In conclusion, meta-analysis confirmed rs56109847 = rs62625044 as a risk factor for female IBS-D. Expression analysis revealed reduced HTR3E levels in the sigmoid colon of IBS-D patients, which underlines the relevance of HTR3E in the pathogenesis of IBS-D., (© 2022. The Author(s).)

Background: Head and neck squamous cell carcinomas (HNSCC) lack tumor-specific biomarkers. Exosomes from HNSCC patients carry immunomodulatory molecules, and correlate with clinical parameters. We compared miRNA profiles of plasma- and saliva-derived exosomes to reveal liquid biomarker candidates for HNSCC. Methods: Exosomes were isolated by differential ultracentrifugation from corresponding plasma and saliva samples from 11 HNSCC patients and five healthy donors (HD). Exosomal miRNA profiles, as determined by nCounter ® SPRINT technology, were analyzed regarding their diagnostic and prognostic potential, correlated to clinical data and integrated into network analysis. Results: 119 miRNAs overlapped between plasma- and saliva-derived exosomes of HNSCC patients, from which 29 tumor-exclusive miRNAs, associated with TP53 , TGFB1, PRDM1, FOX O 1 and CDH1 signaling, were selected. By intra-correlation of tumor-exclusive miRNAs from plasma and saliva, top 10 miRNA candidates with the strongest correlation emerged as diagnostic panels to discriminate cancer and healthy as well as potentially prognostic panels for disease-free survival (DFS). Further, exosomal miRNAs were differentially represented in human papillomavirus (HPV) positive and negative as well as low and high stage disease. Conclusion: A plasma- and a saliva-derived panel of tumor-exclusive exosomal miRNAs hold great potential as liquid biopsy for discrimination between cancer and healthy as well as HPV status and disease stage. Exosomal miRNAs from both biofluids represent a promising tool for future biomarker studies, emphasizing the possibility to substitute plasma by less-invasive saliva collection., 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 Hofmann, Abou Kors, Ezić, Niesler, Röth, Ludwig, Laban, Schuler, Hoffmann, Brunner, Medyany and Theodoraki.)

Background: Exosomes contribute to immunosuppression in head and neck squamous cell carcinoma (HNSCC), a tumor entity which lacks specific tumor biomarkers. Plasma-derived exosomes from HNSCC patients correlate with clinical parameters and have potential as liquid biopsy. Here, we investigate the cargo and functional profile of saliva-derived exosomes from HNSCC patients and their potential as non-invasive biomarkers for disease detection and immunomodulation., Methods: Exosomes were isolated from saliva of HNSCC patients ( n = 21) and healthy donors (HD, n = 12) by differential ultracentrifugation. Surface values of immune checkpoints and tumor associated antigens on saliva-derived exosomes were analyzed by bead-based flow cytometry using CD63 capture. Upon co-incubation with saliva-derived exosomes, activity and proliferation of T cells were assessed by flow cytometry (CD69 expression, CFSE assay). Adenosine levels were measured by mass spectrometry after incubation of saliva-derived exosomes with exogenous ATP. miRNA profiling of saliva-derived exosomes was performed using the nCounter ® SPRINT system., Results: Saliva-derived, CD63-captured exosomes from HNSCC patients carried high amounts of CD44v3, PDL1 and CD39. Compared to plasma, saliva was rich in tumor-derived, CD44v3 + exosomes and poor in hematopoietic cell-derived, CD45 + exosomes. CD8 + T cell activity was attenuated by saliva-derived exosomes from HNSCC patients, while proliferation of CD4 + T cells was not affected. Further, saliva-derived exosomes produced high levels of immunosuppressive adenosine. 62 HD- and 31 HNSCC-exclusive miRNAs were identified. Samples were grouped in "Healthy" and "Cancer" based on their saliva-derived exosomal miRNA profile, which was further found to be involved in RAS/MAPK, NF-κB complex, Smad2/3, and IFN-α signaling., Conclusions: Saliva-derived exosomes from HNSCC patients were enriched in tumor-derived exosomes whose cargo and functional profile reflected an immunosuppressive TME. Surface values of CD44v3, PDL1 and CD39 on CD63-captured exosomes, adenosine production and the miRNA cargo of saliva-derived exosomes emerged as discriminators of disease and emphasized their potential as liquid biomarkers specific for HNSCC., 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 Hofmann, Medyany, Ezić, Lotfi, Niesler, Röth, Engelhardt, Laban, Schuler, Hoffmann, Brunner, Jackson and Theodoraki.)

Clinical studies in glioblastoma and pancreatic carcinoma patients strongly support the further development of H-1 protoparvovirus (H-1PV)-based anticancer therapies. The identification of cellular factors involved in the H-1PV life cycle may provide the knowledge to improve H-1PV anticancer potential. Recently, we showed that sialylated laminins mediate H-1PV attachment at the cell membrane. In this study, we revealed that H-1PV also interacts at the cell surface with galectin-1 and uses this glycoprotein to enter cancer cells. Indeed, knockdown/out of LGALS1, the gene encoding galectin-1, strongly decreases the ability of H-1PV to infect and kill cancer cells. This ability is rescued by the re-introduction of LGALS1 into cancer cells. Pre-treatment with lactose, which is able to bind to galectins and modulate their cellular functions, decreased H-1PV infectivity in a dose dependent manner. In silico analysis reveals that LGALS1 is overexpressed in various tumours including glioblastoma and pancreatic carcinoma. We show by immunohistochemistry analysis of 122 glioblastoma biopsies that galectin-1 protein levels vary between tumours, with levels in recurrent glioblastoma higher than those in primary tumours or normal tissues. We also find a direct correlation between LGALS1 transcript levels and H-1PV oncolytic activity in 53 cancer cell lines from different tumour origins. Strikingly, the addition of purified galectin-1 sensitises poorly susceptible GBM cell lines to H-1PV killing activity by rescuing cell entry. Together, these findings demonstrate that galectin-1 is a crucial determinant of the H-1PV life cycle.

Irritable bowel syndrome (IBS) is a gut-brain disorder in which symptoms are shaped by serotonin acting centrally and peripherally. The serotonin transporter gene SLC6A4 has been implicated in IBS pathophysiology, but the underlying genetic mechanisms remain unclear. We sequenced the alternative P2 promoter driving intestinal SLC6A4 expression and identified single nucleotide polymorphisms (SNPs) that were associated with IBS in a discovery sample. Identified SNPs built different haplotypes, and the tagging SNP rs2020938 seems to associate with constipation-predominant IBS (IBS-C) in females. rs2020938 validation was performed in 1978 additional IBS patients and 6,038 controls from eight countries. Meta-analysis on data from 2,175 IBS patients and 6,128 controls confirmed the association with female IBS-C. Expression analyses revealed that the P2 promoter drives SLC6A4 expression primarily in the small intestine. Gene reporter assays showed a functional impact of SNPs in the P2 region. In silico analysis of the polymorphic promoter indicated differential expression regulation. Further follow-up revealed that the major allele of the tagging SNP rs2020938 correlates with differential SLC6A4 expression in the jejunum and with stool consistency, indicating functional relevance. Our data consolidate rs2020938 as a functional SNP associated with IBS-C risk in females, underlining the relevance of SLC6A4 in IBS pathogenesis., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: There is increasing evidence that Parkinson's disease (PD) might start in the gut, thus involving and compromising also the enteric nervous system (ENS). At the clinical onset of the disease the majority of dopaminergic neurons in the midbrain is already destroyed, so that the lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use a transgenic A30P-α-synuclein-overexpressing PD mouse model to identify appropriate candidate markers in the gut before hallmark symptoms begin to manifest., Methods: Based on a gait analysis and striatal dopamine levels, we defined 2-month-old A30P mice as pre-symptomatic (psA30P), since they are not showing any motoric impairments of the skeletal neuromuscular system and no reduced dopamine levels, but an intestinal α-synuclein pathology. Mice at this particular age were further used to analyze functional and molecular alterations in both, the gastrointestinal tract and the ENS, to identify early pathological changes. We examined the gastrointestinal motility, the molecular composition of the ENS, as well as the expression of regulating miRNAs. Moreover, we applied A30P-α-synuclein challenges in vitro to simulate PD in the ENS., Results: A retarded gut motility and early molecular dysregulations were found in the myenteric plexus of psA30P mice. We found that i.e. neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are significantly altered in the psA30P, thus representing potential biomarkers for early PD. Many of the dysregulated miRNAs found in the psA30P mice are reported to be changed in PD patients as well, either in blood, cerebrospinal fluid or brain tissue. Interestingly, the in vitro approaches delivered similar changes in the ENS cultures as seen in the transgenic animals, thus confirming the data from the mouse model., Conclusions: These findings provide an interesting and novel approach for the identification of appropriate biomarkers in men.

Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics Laboratories.

Intrinsic malignant brain tumors, such as glioblastomas are frequently resistant to immune checkpoint blockade (ICB) with few hypermutated glioblastomas showing response. Modeling patient-individual resistance is challenging due to the lack of predictive biomarkers and limited accessibility of tissue for serial biopsies. Here, we investigate resistance mechanisms to anti-PD-1 and anti-CTLA-4 therapy in syngeneic hypermutated experimental gliomas and show a clear dichotomy and acquired immune heterogeneity in ICB-responder and non-responder tumors. We made use of this dichotomy to establish a radiomic signature predicting tumor regression after pseudoprogression induced by ICB therapy based on serial magnetic resonance imaging. We provide evidence that macrophage-driven ICB resistance is established by CD4 T cell suppression and T reg expansion in the tumor microenvironment via the PD-L1/PD-1/CD80 axis. These findings uncover an unexpected heterogeneity of response to ICB in strictly syngeneic tumors and provide a rationale for targeting PD-L1-expressing tumor-associated macrophages to overcome resistance to ICB.

The interaction between the mammalian host and its resident gut microbiota is known to license adaptive immune responses. Nutritional constituents strongly influence composition and functional properties of the intestinal microbial communities. Here, we report that omission of a single essential amino acid - tryptophan - from the diet abrogates CNS autoimmunity in a mouse model of multiple sclerosis. Dietary tryptophan restriction results in impaired encephalitogenic T cell responses and is accompanied by a mild intestinal inflammatory response and a profound phenotypic shift of gut microbiota. Protective effects of dietary tryptophan restriction are abrogated in germ-free mice, but are independent of canonical host sensors of intracellular tryptophan metabolites. We conclude that dietary tryptophan restriction alters metabolic properties of gut microbiota, which in turn have an impact on encephalitogenic T cell responses. This link between gut microbiota, dietary tryptophan and adaptive immunity may help to develop therapeutic strategies for protection from autoimmune neuroinflammation.

Irritable bowel syndrome (IBS) is a gut-brain disorder involving alterations in intestinal sensitivity and motility. Serotonin 5-HT 4 receptors are promising candidates in IBS pathophysiology since they regulate gut motor function and stool consistency, and targeted 5-HT 4 R selective drug intervention has been proven beneficial in subgroups of patients. We identified a single nucleotide polymorphism (SNP) (rs201253747) c.*61 T > C within the 5-HT 4 receptor gene HTR4 to be predominantly present in diarrhoea-IBS patients (IBS-D). It affects a binding site for the miR-16 family and miR-103/miR-107 within the isoforms HTR4b/i and putatively impairs HTR4 expression. Subsequent miRNA-profiling revealed downregulation of miR-16 and miR-103 in the jejunum of IBS-D patients correlating with symptoms. In vitro assays confirmed expression regulation via three 3'UTR binding sites. The novel isoform HTR4b_2 lacking two of the three miRNA binding sites escapes miR-16/103/107 regulation in SNP carriers. We provide the first evidence that HTR4 expression is fine-tuned by miRNAs, and that this regulation is impaired either by the SNP c.*61 T > C or by diminished levels of miR-16 and miR-103 suggesting that HTR4 might be involved in the development of IBS-D.

Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development.

Human growth is influenced not only by environmental and internal factors but also by a large number of different genes. One of these genes, SHOX, is believed to play a major role in growth, since defects in this homeobox-containing gene on the sex chromosomes lead to syndromal short stature (Leri-Weill dyschondrosteosis, Langer mesomelic dysplasia, and Turner syndrome) as well as to idiopathic short stature. We have analyzed 118 unrelated patients with Leri-Weill dyschondrosteosis and >1,500 patients with idiopathic short stature for deletions encompassing SHOX. Deletions were detected in 34% of the patients with Leri-Weill dyschondrosteosis and in 2% of the patients with idiopathic short stature. For 27 patients with Leri-Weill dyschondrosteosis and for 6 with idiopathic short stature, detailed deletion mapping was performed. Analysis was performed by polymerase chain reaction with the use of pseudoautosomal polymorphic markers and by fluorescence in situ hybridization with the use of cosmid clones. Here, we show that, although the identified deletions vary in size, the vast majority (73%) of patients tested share a distinct proximal deletion breakpoint. We propose that the sequence present within this proximal deletion breakpoint "hotspot" region predisposes to recurrent breaks.