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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nucleases and human induced pluripotent stem cell (iPSC) technology can reveal deep insight into the genetic and molecular bases of human biology and disease. Undesired editing outcomes, both on-target (at the edited locus) and off-target (at other genomic loci) hinder the application of CRISPR-Cas nucleases. We developed Off-flow, a Nextflow-coded bioinformatic workflow that takes a specific guide sequence and Cas protein input to call four separate off-target prediction programs (CHOPCHOP, Cas-Offinder, CRISPRitz, CRISPR-Offinder) to output a comprehensive list of predicted off-target sites. We applied it to whole genome sequencing (WGS) data to investigate the occurrence of unintended effects in human iPSCs that underwent repair or insertion of disease-related variants by homology-directed repair. Off-flow identified a 3-base-pair-substitution and a mono-allelic genomic deletion at the target loci, KCNQ2, in 2 clones. Unbiased WGS analysis further identified off-target missense variants and a mono-allelic genomic deletion at the targeted locus, GNAQ, in 10 clones. On-target substitution and deletions had escaped standard PCR and Sanger sequencing analysis, while missense variants at other genomic loci were not detected by Off-flow. We used these results to filter out iPSC clones for subsequent functional experiments. Off-flow, which we make publicly available, works for human and mouse genomes currently and can be adapted for other genomes. Off-flow and WGS analysis can improve the integrity of studies using CRISPR/Cas-edited cells and animal models.

A climate change assessment for streamflow availability of the selected rivers in Turkey is presented. Using an Index Basin Mapping (IBM) approach, climate change information is transferred across hydrologically similar rivers. This approach maps rivers of interest without downscaled climate information to index rivers where climate projections are available. Then, monthly perturbation ratios of index rivers, conveying projected climatic changes, are applied to the mapped rivers. Climate change effects on monthly streamflow availability and timing in eight selected rivers are evaluated under 20 scenarios produced from 10 General Circulation Models (GCMs) under medium and high emission cases. Results show that winter streamflow availability will increase due to more precipitation falling as rainfall rather than snowfall. Spring and summer streamflow availability will decrease due to reduced snowmelt runoff. Monthly streamflow variability will increase in all evaluated rivers. Out of eight selected rivers, the Çoruh, Yeşilırmak and Zamantı Rivers will be the most affected by climatic changes, with 14.6, 4.1, and 5.6% reductions in overall water availability under the high emission ensemble scenario. Overall water availability is projected increase in the Ceyhan and Göksu Rivers with climate change. Increased monthly streamflow variability can complicate water management in the region. HIGHLIGHTS Applicability of river mapping between hydroclimatologically similar basins.; Monthly perturbation ratios that reflect seasonal shift and water quantity.; Climate change assessment on timing and magnitude of streamflow availability.; Water availability reduced in spring but increased in winter with climatic changes in the Mediterranean rivers.;

Abstract Background Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene and dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood–brain barrier (BBB). Methods We generated TSC disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. Results Using microphysiological systems, we demonstrate that a BBB generated from TSC2 heterozygous mutant cells shows increased permeability. This can be rescued by wild type astrocytes or by treatment with rapamycin, an mTOR kinase inhibitor. Conclusion Our results demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of cell lineages contributing to TSC pathogenesis and informs future therapeutics.

Abstract Objective PTEN, a known tumor suppressor gene, is a mediator of neurodevelopment. Individuals with germline pathogenic variants in the PTEN gene, molecularly defined as PTEN hamartoma tumor syndrome (PHTS), experience a variety of neurological and neuropsychiatric challenges during childhood, including autism spectrum disorder (ASD). However, the frequency and nature of seizures and the utilization of allied health services have not been described. Methods Young patients with PHTS and sibling controls were recruited across five centers in the United States and followed every 6–12 months for a mean of 2.1 years. In addition to the history obtained from caregivers, neurodevelopmental evaluations and structured dysmorphology examinations were conducted, and brain MRI findings, received therapies, and epilepsy characteristics were reported. Results One hundred and seven patients with PHTS (median age 8.7 years; range 3–21 years) and 38 controls were enrolled. ASD and epilepsy were frequent among patients with PHTS (51% and 15%, respectively), with generalized epilepsy strongly associated with ASD. Patients with epilepsy often required two antiseizure medications. Neuroimaging revealed prominent perivascular spaces and decreased peritrigonal myelination in individuals with PHTS‐ASD. Allied therapy use was frequent and involved physical, occupational, speech, and social skills therapies, with 89% of all patients with PHTS, regardless of ASD diagnosis, utilizing at least one service. Interpretation This prospective, longitudinal study highlights the wide neurological spectrum seen in young individuals with PHTS. ASD is common in PHTS, comorbid with epilepsy, and allied health services are used universally. Our findings inform care discussions with families about neurological outcomes in PHTS.

Abstract Background Phelan-McDermid syndrome (PMS) is a genetic neurodevelopmental disorder caused by SHANK3 haploinsufficiency and is associated with an increased risk for seizures. Previous literature indicates that around one third of individuals with PMS also have epilepsy or seizures, with a wide range of types and ages of onset. Investigating the impact of seizures on intellectual and adaptive functioning for PMS is a primary concern for caregivers and is important to understanding the natural history of this syndrome. Methods We report on results from 98 individuals enrolled in a prospective, longitudinal study. We detailed seizure frequency, type, and age of onset, and we analyzed seizure occurrence with best estimate IQ, adaptive functioning, clinical features, and genotype. We conducted multiple linear regression analyses to assess the relationship between the presence of seizures and the Vineland Adaptive Behavior Scale, Second Edition (VABS-II) Adaptive Behavior Composite score and the best estimate full-scale IQ. We also performed Chi-square tests to explore associations between seizure prevalence and genetic groupings. Finally, we performed Chi-square tests and t-tests to explore the relationship between seizures and demographic features, features that manifest in infancy, and medical features. Results Seizures were present in 41% of the cohort, and age of onset was widely variable. The presence of seizures was associated with significantly lower adaptive and intellectual functioning. Genotype–phenotype analyses were discrepant, with no differences in seizure prevalence across genetic classes, but with more genes included in deletions of participants with 22q13 deletions and seizures compared to those with 22q13 deletions and no seizures. No clinical associations were found between the presence of seizures and sex, history of pre- or neonatal complications, early infancy, or medical features. In this cohort, generalized seizures were associated with developmental regression, which is a top concern for PMS caregivers. Conclusions These results begin to eludicate correlates of seizures in individuals with PMS and highlight the importance of early seizure management. Importantly, presence of seizures was associated with adaptive and cognitive functioning. A larger cohort might be able to identify additional associations with medical features. Genetic findings suggest an increased capability to realize genotype–phenotype relationships when deletion size is taken into account.

Abstract Background Succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a model neurometabolic disease at the fulcrum of translational research within the Boston Children’s Hospital Intellectual and Developmental Disabilities Research Centers (IDDRC), including the NIH-sponsored natural history study of clinical, neurophysiological, neuroimaging, and molecular markers, patient-derived induced pluripotent stem cells (iPSC) characterization, and development of a murine model for tightly regulated, cell-specific gene therapy. Methods SSADHD subjects underwent clinical evaluations, neuropsychological assessments, biochemical quantification of γ-aminobutyrate (GABA) and related metabolites, electroencephalography (standard and high density), magnetoencephalography, transcranial magnetic stimulation, magnetic resonance imaging and spectroscopy, and genetic tests. This was parallel to laboratory molecular investigations of in vitro GABAergic neurons derived from induced human pluripotent stem cells (hiPSCs) of SSADHD subjects and biochemical analyses performed on a versatile murine model that uses an inducible and reversible rescue strategy allowing on-demand and cell-specific gene therapy. Results The 62 SSADHD subjects [53% females, median (IQR) age of 9.6 (5.4–14.5) years] included in the study had a reported symptom onset at ∼ 6 months and were diagnosed at a median age of 4 years. Language developmental delays were more prominent than motor. Autism, epilepsy, movement disorders, sleep disturbances, and various psychiatric behaviors constituted the core of the disorder’s clinical phenotype. Lower clinical severity scores, indicating worst severity, coincided with older age (R= -0.302, p = 0.03), as well as age-adjusted lower values of plasma γ-aminobutyrate (GABA) (R = 0.337, p = 0.02) and γ-hydroxybutyrate (GHB) (R = 0.360, p = 0.05). While epilepsy and psychiatric behaviors increase in severity with age, communication abilities and motor function tend to improve. iPSCs, which were differentiated into GABAergic neurons, represent the first in vitro neuronal model of SSADHD and express the neuronal marker microtubule-associated protein 2 (MAP2), as well as GABA. GABA-metabolism in induced GABAergic neurons could be reversed using CRISPR correction of the pathogenic variants or mRNA transfection and SSADHD iPSCs were associated with excessive glutamatergic activity and related synaptic excitation. Conclusions Findings from the SSADHD Natural History Study converge with iPSC and animal model work focused on a common disorder within our IDDRC, deepening our knowledge of the pathophysiology and longitudinal clinical course of a complex neurodevelopmental disorder. This further enables the identification of biomarkers and changes throughout development that will be essential for upcoming targeted trials of enzyme replacement and gene therapy.

Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is an ultra-rare autosomal recessive neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. Here, we report the generation and characterization of human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of three unrelated SSADHD patients – one female and two males with the CRISPR-corrected isogenic controls. These individuals are clinically diagnosed and are being followed in a longitudinal clinical study.

Climate change and global warming are expected to affect water resources management and planning, requiring adaptations to changing conditions. Therefore, it is very important, especially for decision-makers, to identify demand deficits due to less water availability with climate change that may occur in the existing water supply system in advance. FEHEM, a hydroeconomic optimization model of the integrated reservoir system of the Upper Euphrates Basin, which is the largest and main basin providing water flow to the Euphrates River, is developed. Using a 45-year historical hydrological dataset, water management and hydroelectric operations are evaluated with a linear programming model at monthly time steps. The effects of climate change on the Upper Euphrates Basin are evaluated under low and high carbon emission scenarios. According to the average of the different climate scenarios studied in the model, the average decrease in flows is 37.5%. With climate change, peak flows will occur about 1–2 months earlier on average. As a result of these hydrological changes, the total amount of energy production in the basin will decrease by about 54% and energy revenue by the same percentage. HIGHLIGHTS Two climate scenarios, one with high and the other with low emissions, were studied in the Upper Euphrates Basin. In both scenarios, there was a 37.5% reduction in flows on average.; Moreover, the production of 10 large storage hydroelectric power plants in the Upper Euphrates Basin will decrease by approximately 50% according to different climate scenarios.; The break in energy income in the basin caused by climate change was calculated.;

Abstract Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein trafficking in adapter protein complex 4 (AP-4) deficiency, a rare but prototypical form of childhood-onset hereditary spastic paraplegia characterized by mislocalization of the autophagy protein ATG9A. Using high-content microscopy and an automated image analysis pipeline, we screened a diversity library of 28,864 small molecules and identified a lead compound, BCH-HSP-C01, that restored ATG9A pathology in multiple disease models, including patient-derived fibroblasts and induced pluripotent stem cell-derived neurons. We used multiparametric orthogonal strategies and integrated transcriptomic and proteomic approaches to delineate potential mechanisms of action of BCH-HSP-C01. Our results define molecular regulators of intracellular ATG9A trafficking and characterize a lead compound for the treatment of AP-4 deficiency, providing important proof-of-concept data for future studies.

Abstract Background The aim of this study was to evaluate the effectiveness of current ovarian reserve tests in unexplained infertile cases. Material and methods This case–control study was conducted on 70 unexplained infertile women who were included in a tertiary university hospital. Both groups of basal FSH, estradiol, antimullerian hormone (AMH), inhibin B, ovarian volume, total antral follicle count (AFC), ovarian volume, and ovarian stromal blood flow (peak systolic velocity (PSV), S/D (systole and diastole ratio), resistance index (RI), and pulsatility index (PI)) values were compared. Results The mean AMH, inhibin B, PSV, and stromal blood flow values of the control group patients were higher than those in the unexplained infertility group. However, the values of the means of RI and PI of the cases in the control group were lower than those in the infertility group. When PI’s value was ≥ 2.00, its sensitivity was 65.7%, and its specificity was 64.3%. In the case of the RI, its value was ≥ 0.745, its sensitivity was 65.7%, and its specificity was 62.9%. In order to show ovarian reserve in unexplained infertile cases, the sensitivity values can be sorted from high to low as follows: PI > RI > estradiol > FSH > ovarian volume > AFC > inhibin B > stromal blood flow > PSV > S/D. Conclusion In the unexplained infertile patient group with normal ovarian reserve test results, basal estradiol, decreased PI, and RI values may be used as good ovarian reserve predictors.

Abstract Objective Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder caused by autosomal‐dominant pathogenic variants in either the TSC1 or TSC2 gene, and it is characterized by hamartomas in multiple organs, such as skin, kidney, lung, and brain. These changes can result in epilepsy, learning disabilities, and behavioral complications, among others. The mechanistic link between TSC and the mechanistic target of the rapamycin (mTOR) pathway is well established, thus mTOR inhibitors can potentially be used to treat the clinical manifestations of the disorder, including epilepsy. Methods In this study, we tested the efficacy of a novel mTOR catalytic inhibitor (here named Tool Compound 1 or TC1) previously reported to be more brain‐penetrant compared with other mTOR inhibitors. Using a well‐characterized hypomorphic Tsc2 mouse model, which displays a translationally relevant seizure phenotype, we tested the efficacy of TC1. Results Our results show that chronic treatment with this novel mTOR catalytic inhibitor (TC1), which affects both the mTORC1 and mTORC2 signaling complexes, reduces seizure burden, and extends the survival of Tsc2 hypomorphic mice, restoring species typical weight gain over development. Interpretation Novel mTOR catalytic inhibitor TC1 exhibits a promising therapeutic option in the treatment of TSC.

Introduction: Intra-articular Hyaluronic acid (IAHA) injection provides intra-articular (IA) anti-inflammatory effect and improves synovial fluid flow dynamics and viscoelasticity. The aim of this prospective observational, clinical study was to evaluate the safety and efficacy of Hyaluronic Acid (HA) (40mg/2ml) 3 times with one week interval for the treatment of osteoarthritis (OA). Methods and materials: A total of 105 adults with OA were enrolled in the study. Each patient received 3 injection cycles of HA with one week interval. WOMAC OA Index was used to assess the effectiveness of injection and SF-36 to observe the change in quality of life. Physical examination was performed before injection and at the 3rd and 6th months following injection. Results: WOMAC score showed significant reduction from baseline (61.6) to 3rd treatment procedure visit (32.1), 3 months (16.2) and 6 months follow up visit (4.8) respectively (p

Hydro-economic optimization models are common in hydropower reservoir modeling to aid system operators and planners. In these models, operations are driven by the economic value and constrained by the availability of water. The objective is to either minimize total costs or maximize total benefits. In this study, a hydro-economic optimization model for the integrated reservoir system of the Upper Euphrates Basin, with major tributaries providing water flow to the Euphrates River, is introduced. These model the 10 large-scale reservoirs of the basin with a total installed capacity of 3,255 MW. Water management and hydropower decision-making operations are evaluated with a piecewise linear programming algorithm in monthly time steps using a 45-year historical hydrology between 1971 and 2016. The model aims to maximize hydropower revenue over a long-term time horizon with energy prices varying by month. Reservoir storage and turbine release decisions are optimized for multiple hydropower plants connected in serial or parallel. Hydropower generation, revenue, reservoir storage, capacity ratios and generation reliability results are analyzed. Results show that these hydropower plants generate about 9,481 Gigawatt hour (GWh) of energy with an average turbine capacity use of 36% and obtain a revenue of 620 million $ per year. HIGHLIGHTS With the Upper Euphrates Basin Hydro-economic Model (FEHEM), a hydro-economic optimization model covering a 45-year period of hydrological datasets was developed.; With the FEHEM, cost estimates can be developed for adaptation projects to be carried out in the Euphrates Basin water system under future climatic conditions and adaptation strategies that minimize the costs of increased hydrological variability can be developed.;

Abstract Background Tuberous sclerosis complex (TSC) is associated with a wide range of physical manifestations for which international clinical recommendations for diagnosis and management have been established. TSC is, however, also associated with a wide range of TSC-Associated Neuropsychiatric Disorders (TAND) that are typically under-identified and under-treated yet associated with a profound burden of disease. The contemporary evidence base for the identification and treatment of TAND is much more limited and, to date, consensus recommendations for the diagnosis and management of TAND have also been limited and non-specific. Methods The TANDem project was launched with an international, interdisciplinary, and participatory consortium of 24 individuals, including TSC family representatives, from all World Health Organization (WHO) regions but one. One of the aims of the TANDem project was to generate consensus recommendations for the identification and treatment of TAND. At the time of this project, no internationally adopted standard methodology and methodological checklists existed for the generation of clinical practice recommendations. We therefore developed our own systematic procedure for evidence review and consensus-building to generate evidence-informed consensus recommendations of relevance to the global TSC community. Results At the heart of the consensus recommendations are ten core principles surrounded by cluster-specific recommendations for each of the seven natural TAND clusters identified in the literature (autism-like, dysregulated behavior, eat/sleep, mood/anxiety, neuropsychological, overactive/impulsive, and scholastic) and a set of wraparound psychosocial cluster recommendations. The overarching recommendation is to “screen” for TAND at least annually, to “act” using appropriate next steps for evaluation and treatment, and to “repeat” the process to ensure early identification and early intervention with the most appropriate biological, psychological, and social evidence-informed approaches to support individuals with TSC and their families. Conclusions The consensus recommendations should provide a systematic framework to approach the identification and treatment of TAND for health, educational, social care teams and families who live with TSC. To ensure global dissemination and implementation of these recommendations, partnerships with the international TSC community will be important. One of these steps will include the generation of a “TAND toolkit” of “what to seek” and “what to do” when difficulties are identified in TAND clusters.

This study tested the effect of Companilactobacillus paralimentarius E-106, Lactiplantibacillus paraplantarum N-15 and Lactiplantibacillus plantarum SC-9 on the amount of Maillard reaction and aroma profile in bread making with main bread quality parameters. The specific volumes of sourdough and control breads were in the range of 2.97–3.04 cm3/g, and the control II bread had the highest hardness values on all days. The FAST index value was determined to be between 40.48% and 81.22% in all breads. The FAST index value was found to be higher in the control breads than in the sourdough breads. In the volatile compounds analysis, 72 volatile compounds were detected. The variety of volatile compounds in the breads with sourdough addition was higher than the control breads. Among the tested strains, Companilactobacillus paralimentarius E-106 demonstrated superior properties for bread characteristics in comparison to other strains as a type II sourdough starter. In summary, improved aroma profile and decreased Maillard reaction products can be provided by sourdough addition without changing the bread quality, along with meeting consumer demand for less additive use.

Predicting missing historical or forecasting streamflows for future periods is a challenging task. This paper presents open-source data-driven machine learning models for streamflow prediction. The Random Forests algorithm is employed and the results are compared with other machine learning algorithms. The developed models are applied to the Kızılırmak River, Turkey. First model is built with streamflow of a single station (SS), and the second model is built with streamflows of multiple stations (MS). The SS model uses input parameters derived from one streamflow station. The MS model uses streamflow observations of nearby stations. Both models are tested to estimate missing historical and predict future streamflows. Model prediction performances are measured by root mean squared error (RMSE), Nash–Sutcliffe efficiency (NSE), coefficient of determination (R2), and percent bias (PBIAS). The SS model has an RMSE of 8.54, NSE and R2 of 0.98, and PBIAS of 0.7% for the historical period. The MS model has an RMSE of 17.65, NSE of 0.91, R2 of 0.93, and PBIAS of −13.64% for the future period. The SS model is useful to estimate missing historical streamflows, while the MS model provides better predictions for future periods, with its ability to better catch flow trends. HIGHLIGHTS An open-source machine learning model to estimate streamflow.; Use of single- and multi-streamflow station datasets as inputs are compared.; A single streamflow station can be used to estimate its missing flows in a historical period.; A multi-station model can better capture streamflow trends.;

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. SSADHD leads to impaired GABA metabolism and results in accumulation of GABA and γ-hydroxybutyrate (GHB), which alter neurotransmission and are thought to lead to neurobehavioral symptoms. However, why increased inhibitory neurotransmitters lead to seizures remains unclear. We used induced pluripotent stem cells from SSADHD patients (one female and two male) and differentiated them into GABAergic and glutamatergic neurons. SSADHD iGABA neurons show altered GABA metabolism and concomitant changes in expression of genes associated with inhibitory neurotransmission. In contrast, glutamatergic neurons display increased spontaneous activity and upregulation of mitochondrial genes. CRISPR correction of the pathogenic variants or SSADHD mRNA expression rescue various metabolic and functional abnormalities in human neurons. Our findings uncover a previously unknown role for SSADHD in excitatory human neurons and provide unique insights into the cellular and molecular basis of SSADHD and potential therapeutic interventions.

Abstract Background Individuals with PTEN hamartoma tumor syndrome (PHTS) demonstrate a distinct neurobehavioral profile suggesting primary disruption of frontal lobe symptoms, with more severe cognitive deficits in those with associated autism spectrum disorder (ASD) that extend to other areas of neurobehavioral function as well (e.g., adaptive behavior, sensory deficits). The current study sought to characterize longitudinal neurobehavioral profiles in individuals with PHTS who completed serial assessments (2–3 evaluations) over a 2-year time period. Methods Comprehensive neurobehavioral evaluations were conducted on 92 participants (age range 6–21) with PHTS and/or ASD. Spaghetti plots and linear mixed effects models were used to visualize the individual patient profiles and group trends and examine the group differences in cognitive/behavioral test scores over time. Practice-adjusted reliable change indices (RCIs) and standardized regression-based change scores (SRBs) were calculated for those measures in the battery with adequate sample sizes and test–retest reliabilities for future use in assessing neurobehavioral change in children and young adults with PHTS. Results Wide individual differences were observed at baseline across all measures. Encouragingly, baseline differences between patient groups persisted at the same magnitude over a 2-year time period with no differences in longitudinal neurobehavioral profiles within any one group. Test–retest reliabilities were generally high, ranging from 0.62 to 0.97, and group mean change from baseline to 12 months was small (range − 3.8 to 3.7). A Microsoft Excel calculator was created that clinicians and researchers can use to automatically calculate RCI and SRB thresholds at both 80% and 90% confidence intervals using test scores from a given child or young adult with PHTS. Conclusions Our results suggest that the neurobehavioral phenotypes observed in individuals with PHTS remain relatively stable over time, even in those with ASD. The RCIs and SRBs provided can be used in future research to examine patient outcomes at the individual level as well as to detect negative deviations from the expected trajectory that can be used to inform intervention strategies.

Interest in gene-based therapies for neurodevelopmental disorders is increasing exponentially, driven by the rise in recognition of underlying genetic etiology, progress in genomic technology, and recent proof of concept in several disorders. The current prioritization of one genetic disorder over another for development of therapies is driven by competing interests of pharmaceutical companies, advocacy groups, and academic scientists. Although these are all valid perspectives, a consolidated framework will facilitate more efficient and rational gene therapy development. Here we outline features of Mendelian neurodevelopmental disorders that warrant consideration when determining suitability for gene therapy. These features fit into four broad domains: genetics, preclinical validation, clinical considerations, and ethics. We propose a simple mnemonic, GENE TARGET, to remember these features and illustrate how they could be scored using a preliminary scoring rubric. In this suggested rubric, for a given disorder, scores for each feature may be added up to a composite GENE TARGET suitability (GTS) score. In addition to proposing a systematic method to evaluate and compare disorders, our framework helps identify gaps in the translational pipeline for a given disorder, which can inform prioritization of future research efforts.

Summary: Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder, but new therapies have been impeded by a lack of understanding of the pathological mechanisms. Tuberous sclerosis complex (TSC) and fragile X syndrome are associated with alterations in the mechanistic target of rapamycin (mTOR) and fragile X messenger ribonucleoprotein 1 (FMRP), which have been implicated in the development of ASD. Previously, we observed that transcripts associated with FMRP were down-regulated in TSC2-deficient neurons. In this study, we find that FMRP turnover is dysregulated in TSC2-deficient rodent primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons and is dependent on the E3 ubiquitin ligase anaphase-promoting complex. We also demonstrate that overexpression of FMRP can partially rescue hyperexcitability in TSC2-deficient iPSC-derived neurons. These data indicate that FMRP dysregulation represents an important pathological mechanism in the development of abnormal neuronal activity in TSC and illustrate a molecular convergence between these two neurogenetic disorders.

Today, as the demand for energy increases based on the increasing population and improving technology, resorting to new energy sources has become a necessity. Due to the rapid consumption of fossil fuels and the responsibility of humanity for the environment, Renewable Energy Sources (RES) are of a quality that may respond to this necessity. But the RES such as sun and wind vary depending on the weather conditions. Considering such variance, Hybrid Power Systems (HPS) are suggested in order to assure reliability and continuity in energy generation. For the sake of strengthening the HPS that are dependent on weather conditions, it is considered to increase the system's reliability and continuity through the inclusion in the HPS of cattle biomass reserves in the area. In this paper, it was studied on modeling of HPS based on sun, wind and biogas that will meet the electric power demand of the cattle farm located at Afyonkarahisar, Turkey. The two decades' animal population and load value changes were estimated through Genetic Algorithm (GA), and the HPS model was investigated under different scenarios considering sustainable energy and environment objectives, and the analyses were performed considering the changes in economic parameters.

Spastic paraplegia 50 (SPG50) is an ultrarare childhood-onset neurological disorder caused by biallelic loss-of-function variants in the AP4M1 gene. SPG50 is characterized by progressive spastic paraplegia, global developmental delay, and subsequent intellectual disability, secondary microcephaly, and epilepsy. We preformed preclinical studies evaluating an adeno-associated virus (AAV)/AP4M1 gene therapy for SPG50 and describe in vitro studies that demonstrate transduction of patient-derived fibroblasts with AAV2/AP4M1, resulting in phenotypic rescue. To evaluate efficacy in vivo, Ap4m1-KO mice were intrathecally (i.t.) injected with 5 × 1011, 2.5 × 1011, or 1.25 × 1011 vector genome (vg) doses of AAV9/AP4M1 at P7–P10 or P90. Age- and dose-dependent effects were observed, with early intervention and higher doses achieving the best therapeutic benefits. In parallel, three toxicology studies in WT mice, rats, and nonhuman primates (NHPs) demonstrated that AAV9/AP4M1 had an acceptable safety profile up to a target human dose of 1 × 1015 vg. Of note, similar degrees of minimal-to-mild dorsal root ganglia (DRG) toxicity were observed in both rats and NHPs, supporting the use of rats to monitor DRG toxicity in future i.t. AAV studies. These preclinical results identify an acceptably safe and efficacious dose of i.t.-administered AAV9/AP4M1, supporting an investigational gene transfer clinical trial to treat SPG50.

BackgroundRecruitment into clinical trials is a challenging process, with as many as 40% of studies failing to meet their target sample sizes. The principles of direct-to-consumer (DTC) advertising rely upon novel marketing strategies. The ability to reach expansive audiences in the web-based realm presents a unique opportunity for researchers to overcome various barriers to enrollment in clinical trials. Research has investigated the use of individual web-based platforms to aid in recruitment and accrual into trials; however, a gap in the literature exists, whereby multiple mass communication platforms have yet to be investigated across a range of clinical trials. ObjectiveThere is a need to better understand how individual factors combine to collectively influence trial recruitment. We aimed to test whether DTC recruitment of potentially eligible study participants via social media platforms (eg, Facebook [Meta Platforms Inc] and Twitter [Twitter Inc]) was an effective strategy or whether this acted as an enhancement to traditional (eg, email via contact registries) recruitment strategies through established clinical research sites. MethodsThis study tested multiple DTC web-based recruitment efforts (Facebook, Twitter, email, and patient advocacy group [PAG] involvement) across 6 national and international research studies from 5 rare disease consortia. Targeted social media messaging, social media management software, and individual study websites with prescreening questions were used in the Protocol for Increasing Accrual Using Social Media (PRISM). ResultsIn total, 1465 PRISM website referrals occurred across all 6 studies. Organic (unpaid) Facebook posts (676/1465, 46.14%) and Rare Diseases Clinical Research Network patient contact registry emails (461/1465, 31.47%) represented the most successful forms of engagement. PRISM was successful in accumulating a 40.1% (136/339) lead generation (those who screened positive and consented to share their contact information to be contacted by a clinical site coordinator). Despite the large number of leads generated from PRISM recruitment efforts, the number of patients who were subsequently enrolled in studies was low. Across 6 studies, 3 participants were ultimately enrolled, meaning that 97.8% (133/136) of leads dropped off. ConclusionsThe results indicate that although accrual results were low, this is consistent with previously documented challenges of studying populations with rare diseases. Targeted messaging integrated throughout the recruitment process (eg, referral, lead, and accrual) remains an area for further research. Key elements to consider include structuring the communicative workflow in such a way that PAG involvement is central to the process, with clinical site coordinators actively involved after an individual consents to share their contact information. Customized approaches are needed for each population and research study, with observational studies best suited for social media recruitment. As evidenced by lead generation, results suggest that web-based recruitment efforts, coupled with targeted messaging and PAG partnerships, have the potential to supplement clinical trial accrual.

Abstract CAPN1‐associated hereditary spastic paraplegia (SPG76) is a rare and clinically heterogenous syndrome due to loss of calpain‐1 function. Here we illustrate a translational approach to the case of an 18‐year‐old patient who first presented with psychiatric symptoms followed by spastic gait, intention tremor, and neurogenic bladder dysfunction, consistent with a complex form of HSP. Exome sequencing showed compound‐heterozygous missense variants in CAPN1 (NM_001198868.2: c.1712A>G (p.Asn571Ser)/c.1991C>T (p.Ser664Leu)) and a previously reported heterozygous stop‐gain variant in RCL1. In silico analyses of the CAPN1 variants predicted a deleterious effect and in vitro functional studies confirmed reduced calpain‐1 activity and dysregulated downstream signaling. These findings support a diagnosis of SPG76 and highlight that the psychiatric symptoms can precede the motor symptoms in HSP. Our results also suggest that multiple genes can potentially contribute to complex neuropsychiatric diseases.

Abstract Rett syndrome is associated with severe functional impairments and many comorbidities, each in urgent need of treatments. Mutations in the MECP2 gene were identified as causing Rett syndrome in 1999. Over the past 20 years there has been an abundance of preclinical research with some studies leading to human clinical trials. Despite this, few viable therapeutic options have emerged from this investment of effort. Reasons for this lack of success as they relate both to preclinical research and the clinical trial landscape are discussed. Considering what needs to be done to promote further success in the field, we take a positive and constructive approach and introduce the concept of clinical trial readiness and its necessary ingredients for Rett syndrome. These include: listening to the needs of families; support from advocacy groups; optimising use of existing clinic infrastructures and available natural history data; and, finally, the validation of existing outcome measures and/or the development and validation of new measures. We conclude by reiterating the need for a collaborative and coordinated approach amongst the many different stakeholder groups and the need to engage in new types of trial design which could be much more efficient, less costly and much less burdensome on families.

Abstract Background Computational phenotypes are most often combinations of patient billing codes that are highly predictive of disease using electronic health records (EHR). In the case of rare diseases that can only be diagnosed by genetic testing, computational phenotypes identify patient cohorts for genetic testing and possible diagnosis. This article details the validation of a computational phenotype for PTEN hamartoma tumor syndrome (PHTS) against the EHR of patients at three collaborating clinical research centers: Boston Children's Hospital, Children's National Hospital, and the University of Washington. Methods A combination of billing codes from the International Classification of Diseases versions 9 and 10 (ICD-9 and ICD-10) for diagnostic criteria postulated by a research team at Cleveland Clinic was used to identify patient cohorts for genetic testing from the clinical data warehouses at the three research centers. Subsequently, the EHR—including billing codes, clinical notes, and genetic reports—of these patients were reviewed by clinical experts to identify patients with PHTS. Results The PTEN genetic testing yield of the computational phenotype, the number of patients who needed to be genetically tested for incidence of pathogenic PTEN gene variants, ranged from 82 to 94% at the three centers. Conclusions Computational phenotypes have the potential to enable the timely and accurate diagnosis of rare genetic diseases such as PHTS by identifying patient cohorts for genetic sequencing and testing.

Abstract Background Tuberous sclerosis complex (TSC)–associated neuropsychiatric disorders (TAND) is an umbrella term for the behavioural, psychiatric, intellectual, academic, neuropsychological and psychosocial manifestations of TSC. Although TAND affects 90% of individuals with TSC during their lifetime, these manifestations are relatively under-assessed, under-treated and under-researched. We performed a comprehensive scoping review of all TAND research to date (a) to describe the existing TAND research landscape and (b) to identify knowledge gaps to guide future TAND research. Methods The study was conducted in accordance with stages outlined within the Arksey and O’Malley scoping review framework. Ten research questions relating to study characteristics, research design and research content of TAND levels and clusters were examined. Results Of the 2841 returned searches, 230 articles published between 1987 and 2020 were included (animal studies = 30, case studies = 47, cohort studies = 153), with more than half published since the term TAND was coined in 2012 (118/230; 51%). Cohort studies largely involved children and/or adolescents (63%) as opposed to older adults (16%). Studies were represented across 341 individual research sites from 45 countries, the majority from the USA (89/341; 26%) and the UK (50/341; 15%). Only 48 research sites (14%) were within low–middle income countries (LMICs). Animal studies and case studies were of relatively high/high quality, but cohort studies showed significant variability. Of the 153 cohort studies, only 16 (10%) included interventions. None of these were non-pharmacological, and only 13 employed remote methodologies (e.g. telephone interviews, online surveys). Of all TAND clusters, the autism spectrum disorder–like cluster was the most widely researched (138/230; 60%) and the scholastic cluster the least (53/200; 27%). Conclusions Despite the recent increase in TAND research, studies that represent participants across the lifespan, LMIC research sites and non-pharmacological interventions were identified as future priorities. The quality of cohort studies requires improvement, to which the use of standardised direct behavioural assessments may contribute. In human studies, the academic level in particular warrants further investigation. Remote technologies could help to address many of the TAND knowledge gaps identified.

Abstract Objectives Cerebral palsy (CP) is the most common childhood motor disability, yet its link to single‐gene disorders is under‐characterized. To explore the genetic landscape of CP, we conducted whole exome sequencing (WES) in a cohort of patients with CP. Methods We performed comprehensive phenotyping and WES on a prospective cohort of individuals with cryptogenic CP (who meet criteria for CP; have no risk factors), non‐cryptogenic CP (who meet criteria for CP; have at least one risk factor), and CP masqueraders (who could be diagnosed with CP, but have regression/progressive symptoms). We characterized motor phenotypes, ascertained medical comorbidities, and classified brain MRIs. We analyzed WES data using an institutional pipeline. Results We included 50 probands in this analysis (20 females, 30 males). Twenty‐four had cryptogenic CP, 20 had non‐cryptogenic CP, five had CP masquerader classification, and one had unknown classification. Hypotonic‐ataxic subtype showed a difference in prevalence across the classification groups (p = 0.01). Twenty‐six percent of participants (13/50) had a pathogenic/likely pathogenic variant in 13 unique genes (ECHS1, SATB2, ZMYM2, ADAT3, COL4A1, THOC2, SLC16A2, SPAST, POLR2A, GNAO1, PDHX, ACADM, ATL1), including one patient with two genetic disorders (ACADM, PDHX) and two patients with a SPAST‐related disorder. The CP masquerader category had the highest diagnostic yield (n = 3/5, 60%), followed by the cryptogenic CP category (n = 7/24, 29%). Fifteen percent of patients with non‐cryptogenic CP (n = 3/20) had a Mendelian disorder on WES. Interpretation WES demonstrated a significant prevalence of Mendelian disorders in individuals clinically diagnosed with CP, including in individuals with known CP risk factors.

Davies et al. identify a putative mechanism underlying the childhood neurological disorder AP-4 deficiency syndrome. In the absence of AP-4, an enzyme that makes 2-AG is not transported to the axon, leading to axonal growth defects, which can be rescued by inhibition of 2-AG breakdown.

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates the mechanistic Target of Rapamycin Complex 1 (mTORC1). TSC research has focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, non-canonical functions of TSC2 are not well studied, and the potential disease-relevant biological mechanisms of mutations affecting these functions are not well understood. We observed aberrant multipolar mitotic division, a novel phenotype, in TSC2 mutant iPSCs. The multipolar phenotype is not meaningfully affected by treatment with the inhibitor rapamycin. We further observed dominant negative activity of the mutant form of TSC2 in producing the multipolar division phenotype. These data expand the knowledge of TSC2 function and pathophysiology which will be highly relevant to future treatments for patients with TSC.

Summary: Phelan-McDermid syndrome (PMS) is a rare neurodevelopmental disorder caused at least in part by haploinsufficiency of the SHANK3 gene, due to sequence variants in SHANK3 or subtelomeric 22q13.3 deletions. Phenotypic differences have been reported between PMS participants carrying small “class I” mutations and large “class II” mutations; however, the molecular perturbations underlying these divergent phenotypes remain obscure. Using peripheral blood transcriptome and serum metabolome profiling, we examined the molecular perturbations in the peripheral circulation associated with a full spectrum of PMS genotypes spanning class I (n = 37) and class II mutations (n = 39). Transcriptomic data revealed 52 genes with blood expression profiles that tightly scale with 22q.13.3 deletion size. Furthermore, we uncover 208 underexpressed genes in PMS participants with class II mutations, which were unchanged in class I mutations. These genes were not linked to 22q13.3 and were strongly enriched for glycosphingolipid metabolism, NCAM1 interactions, and cytotoxic natural killer (NK) immune cell signatures. In silico predictions estimated a reduction in CD56+ CD16– NK cell proportions in class II mutations, which was validated by mass cytometry time of flight. Global metabolomics profiling identified 24 metabolites that were significantly altered in PMS participants with class II mutations and confirmed a general reduction in sphingolipid metabolism. Collectively, these results provide new evidence linking PMS participants carrying class II mutations with decreased expression of cytotoxic cell signatures, reduced relative proportions of NK cells, and lower sphingolipid metabolism. These findings highlight alternative avenues for therapeutic development and offer new mechanistic insights supporting genotype-to-phenotype associations in PMS.

Abstract Background Phelan McDermid syndrome (PMS) is a neurogenetic condition associated with a high prevalence of intellectual disability (ID) and autism spectrum disorder (ASD). This study provides a more comprehensive and quantitative profile of repetitive behaviors within the context of ID seen with the condition. Methods Individuals age 3–21 years with a confirmed PMS diagnosis participated in a multicenter observational study evaluating the phenotype and natural history of the disorder. We evaluated data collected from this study pertaining to repetitive behaviors from the Repetitive Behavior Scales-Revised (RBS-R). Results There were n = 90 participants who were part of this analysis. Forty-seven percent (n = 42/90) were female, and the average age at baseline evaluation was 8.88 ± 4.72 years. The mean best estimate IQ of the cohort was 26.08 ± 17.67 (range = 3.4–88), with n = 8 with mild ID (or no ID), n = 20 with moderate ID, and n = 62 with severe-profound ID. The RBS-R total overall score was 16.46 ± 13.9 (compared to 33.14 ± 20.60 reported in previous studies of ASD) (Lam and Aman, 2007), and the total number of items endorsed was 10.40 ± 6.81 (range = 0–29). After statistical correction for multiple comparisons, IQ correlated with the RBS-R stereotypic behavior subscale score (r s = − 0.33, unadjusted p = 0.0014, adjusted p = 0.01) and RBS-R stereotypic behavior total number of endorsed items (r s = − 0.32, unadjusted p = 0.0019, adjusted p = 0.01). IQ did not correlate with any other RBS-R subscale scores. Conclusions The RBS-R total overall score in a PMS cohort appears milder compared to individuals with ASD characterized in previous studies. Stereotypic behavior in PMS may reflect cognitive functioning.

Abstract Autism Spectrum Disorder (ASD) is genetically complex with ~100 copy number variants and genes involved. To try to establish more definitive genotype and phenotype correlations in ASD, we searched genome sequence data, and the literature, for recurrent predicted damaging sequence-level variants affecting single genes. We identified 18 individuals from 16 unrelated families carrying a heterozygous guanine duplication (c.3679dup; p.Ala1227Glyfs*69) occurring within a string of 8 guanines (genomic location [hg38]g.50,721,512dup) affecting SHANK3, a prototypical ASD gene (0.08% of ASD-affected individuals carried the predicted p.Ala1227Glyfs*69 frameshift variant). Most probands carried de novo mutations, but five individuals in three families inherited it through somatic mosaicism. We scrutinized the phenotype of p.Ala1227Glyfs*69 carriers, and while everyone (17/17) formally tested for ASD carried a diagnosis, there was the variable expression of core ASD features both within and between families. Defining such recurrent mutational mechanisms underlying an ASD outcome is important for genetic counseling and early intervention.

Abstract Background Tuberous Sclerosis Complex (TSC), a multi-system genetic disorder, is associated with a wide range of TSC-Associated Neuropsychiatric Disorders (TAND). Individuals have apparently unique TAND profiles, challenging diagnosis, psycho-education, and intervention planning. We proposed that identification of natural TAND clusters could lead to personalized identification and treatment of TAND. Two small-scale studies showed cluster and factor analysis could identify clinically meaningful natural TAND clusters. Here we set out to identify definitive natural TAND clusters in a large, international dataset. Method Cross-sectional, anonymized TAND Checklist data of 453 individuals with TSC were collected from six international sites. Data-driven methods were used to identify natural TAND clusters. Mean squared contingency coefficients were calculated to produce a correlation matrix, and various cluster analyses and exploratory factor analysis were examined. Statistical robustness of clusters was evaluated with 1000-fold bootstrapping, and internal consistency calculated with Cronbach’s alpha. Results Ward’s method rendered seven natural TAND clusters with good robustness on bootstrapping. Cluster analysis showed significant convergence with an exploratory factor analysis solution, and, with the exception of one cluster, internal consistency of the emerging clusters was good to excellent. Clusters showed good clinical face validity. Conclusions Our findings identified a data-driven set of natural TAND clusters from within highly variable TAND Checklist data. The seven natural TAND clusters could be used to train families and professionals and to develop tailored approaches to identification and treatment of TAND. Natural TAND clusters may also have differential aetiological underpinnings and responses to molecular and other treatments.

16p13.11 copy number variants (CNVs) have been associated with autism, schizophrenia, psychosis, intellectual disability, and epilepsy. The majority of 16p13.11 deletions or duplications occur within three well-defined intervals, and despite growing knowledge of the functions of individual genes within these intervals, the molecular mechanisms that underlie commonly observed clinical phenotypes remain largely unknown. Patient-derived, induced pluripotent stem cells (iPSCs) provide a platform for investigating the morphological, electrophysiological, and gene-expression changes that result from 16p13.11 CNVs in human-derived neurons. Patient derived iPSCs with varying sizes of 16p13.11 deletions and familial controls were differentiated into cortical neurons for phenotypic analysis. High-content imaging and morphological analysis of patient-derived neurons demonstrated an increase in neurite branching in patients compared with controls. Whole-transcriptome sequencing revealed expression level changes in neuron development and synaptic-related gene families, suggesting a defect in synapse formation. Subsequent quantification of synapse number demonstrated increased numbers of synapses on neurons derived from early-onset patients compared to controls. The identification of common phenotypes among neurons derived from patients with overlapping 16p13.11 deletions will further assist in ascertaining common pathways and targets that could be utilized for screening drug candidates. These studies can help to improve future treatment options and clinical outcomes for 16p13.11 deletion patients.

Summary: MECP2 loss-of-function mutations cause Rett syndrome, a neurodevelopmental disorder resulting from a disrupted brain transcriptome. How these transcriptional defects are decoded into a disease proteome remains unknown. We studied the proteome of Rett cerebrospinal fluid (CSF) to identify consensus Rett proteome and ontologies shared across three species. Rett CSF proteomes enriched proteins annotated to HDL lipoproteins, complement, mitochondria, citrate/pyruvate metabolism, synapse compartments, and the neurosecretory protein VGF. We used shared Rett ontologies to select analytes for orthogonal quantification and functional validation. VGF and ontologically selected CSF proteins had genotypic discriminatory capacity as determined by receiver operating characteristic analysis in Mecp2-/y and Mecp2−/+. Differentially expressed CSF proteins distinguished Rett from a related neurodevelopmental disorder, CDKL5 deficiency disorder. We propose that Mecp2 mutant CSF proteomes and ontologies inform putative mechanisms and biomarkers of disease. We suggest that Rett syndrome results from synapse and metabolism dysfunction.

Abstract Objective To confirm the critical factors affecting seizure susceptibility in acute pentylenetetrazole (PTZ) mouse epilepsy models and evaluate the prior literature for these factors. Methods Serial cohorts of wild‐type mice administered intraperitoneal (IP)‐PTZ were aggregated and analyzed by multivariate logistic regression for the effect of sex, age, background strain, dose, and physiologic stress (i.e., EEG implantation and/or single‐housing) on seizure response. We assessed the reporting of these factors in a comprehensive literature review over the last 10 years (2010–2020). Results We conducted aggregated analysis of pooled data of 307 mice (220 C57BL/6J mice and 87 mixed background mice; 202 males, 105 females) with median age of 10 weeks (range: 6–49 weeks) with acute PTZ injection (dose range 40–65 mg/kg). Significance in multivariate analysis was found between seizures and increased PTZ dose (odds ratio (OR) 1.149, 95% confidence interval (CI) 1.102–1.205), older age (OR 1.1, 95% CI 1.041–1.170), physiologic stress (OR 17.36, 95% CI 7.349–44.48), and mixed background strain (OR 0.4725, 95% CI 0.2315–0.9345). Literature review identified 97 papers using acute PTZ‐seizure models. Age, housing, sex, and background were omitted by 61% (59/97), 51% (49/97), 18% (17/97), and 8% (8/97) papers, respectively. Only 17% of publications specified all four factors (16/97). Interpretation Our analysis and literature review demonstrate a critical gap in standardization of acute PTZ‐induced seizure paradigm in mice. We recommend that future studies specify and control for age, background strain, sex, and housing conditions of experimental animals.

16p11.2 CNVs are associated with neurodevelopmental disorders. Here, the authors show that 16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro.

Abstract Background Phelan-McDermid Syndrome (PMS) is a rare condition caused by deletion or mutation of the SHANK3 gene. Individuals with PMS frequently present with intellectual disability, autism spectrum disorder, and other neurodevelopmental challenges. Electroencephalography (EEG) can provide a window into network-level function in PMS. Methods Here, we analyze EEG data collected across multiple sites in individuals with PMS (n = 26) and typically developing individuals (n = 15). We quantify oscillatory power, alpha-gamma phase-amplitude coupling strength, and phase bias, a measure of the phase of cross frequency coupling thought to reflect the balance of feedforward (bottom-up) and feedback (top-down) activity. Results We find individuals with PMS display increased alpha-gamma phase bias (U = 3.841, p

Abstract Background PTEN is a well-established risk gene for autism spectrum disorder (ASD). Yet, little is known about how PTEN mutations and associated molecular processes influence neurobehavioral function in mutation carriers with (PTEN-ASD) and without ASD (PTEN no-ASD). The primary aim of the present study was to examine group differences in peripheral blood-derived PTEN pathway protein levels between PTEN-ASD, PTEN no-ASD, and idiopathic macrocephalic ASD patients (macro-ASD). Secondarily, associations between protein levels and neurobehavioral functions were examined in the full cohort. Methods Patients were recruited at four tertiary medical centers. Peripheral blood-derived protein levels from canonical PTEN pathways (PI3K/AKT and MAPK/ERK) were analyzed using Western blot analyses blinded to genotype and ASD status. Neurobehavioral measures included standardized assessments of global cognitive ability and multiple neurobehavioral domains. Analysis of variance models examined group differences in demographic, neurobehavioral, and protein measures. Bivariate correlations, structural models, and statistical learning procedures estimated associations between molecular and neurobehavioral variables. To complement patient data, Western blots for downstream proteins were generated to evaluate canonical PTEN pathways in the PTEN-m3m4 mouse model. Results Participants included 61 patients (25 PTEN-ASD, 16 PTEN no-ASD, and 20 macro-ASD). Decreased PTEN and S6 were observed in both PTEN mutation groups. Reductions in MnSOD and increases in P-S6 were observed in ASD groups. Elevated neural P-AKT/AKT and P-S6/S6 from PTEN murine models parallel our patient observations. Patient PTEN and AKT levels were independently associated with global cognitive ability, and p27 expression was associated with frontal sub-cortical functions. As a group, molecular measures added significant predictive value to several neurobehavioral domains over and above PTEN mutation status. Limitations Sample sizes were small, precluding within-group analyses. Protein and neurobehavioral data were limited to a single evaluation. A small number of patients were excluded with invalid protein data, and cognitively impaired patients had missing data on some assessments. Conclusions Several canonical PTEN pathway molecules appear to influence the presence of ASD and modify neurobehavioral function in PTEN mutation patients. Protein assays of the PTEN pathway may be useful for predicting neurobehavioral outcomes in PTEN patients. Future longitudinal analyses are needed to replicate these findings and evaluate within-group relationships between protein and neurobehavioral measures. Trial registration ClinicalTrials.gov Identifier NCT02461446

Purpose: The study aimed to investigate the changes in choroidal thickness (CT), retinal nerve fiber layer thickness (RNFL), and visual field parameters in morbidly obese patients following bariatric surgery. Methods: The study included 40 morbidly obese patients with body mass indexes (BMI) ≥40 who had undergone bariatric surgery (Group 1) and 40 age-and sex-matched healthy subjects with normal BMI values (Group 2). RNFL and CT measurements by optical coherence tomography (OCT) and visual field test were performed preoperatively and the 1st, 6th, and 12th months postoperatively. CT measurements were obtained from the subfoveal, nasal (N), and temporal (T) regions at distances of 500 μm and 1,000 μm from the fovea. Results: No significant pathology was detected during ophthalmological examinations following bariatric surgery. The BMIs were found to be significantly lower in all of the periods after bariatric surgery (P < 0.0001). The CT measurements decreased significantly in all periods after bariatric surgery (P < 0.0001). No differences were found in terms of the mean RNFL thicknesses in all postoperative periods (P = 0.125). Visual field tests showed no significant changes during scheduled visits. (P = 0.877). No visual field defect was detected in any patient during the follow-up periods after bariatric surgery. Conclusion: These results have suggested that CT is positively correlated with BMI and decreased with a reduction in BMI progressively. Nutritional disorders resulting from malabsorption have not caused any nutritional optic neuropathy and visual field defect for at least the first postoperative year after bariatric surgery.

Mammalian tissues are highly heterogenous and complex, posing a challenge in understanding the molecular mechanisms regulating protein expression within various tissues. Recent studies have shown that translation at the level of the ribosome is highly regulated, and can vary independently of gene expression observed at a transcriptome level, as well as between cell populations, contributing to the diversity of mammalian tissues. Earlier methods that analyzed gene expression at the level of translation, such as polysomal- or ribosomal-profiling, required large amounts of starting material to isolate enough RNA for analysis by microarray or RNA-sequencing. Thus, rare or less abundant cell types within tissues were not able to be properly studied with these methods. Translating ribosome affinity purification (TRAP) utilizes the incorporation of an eGFP-affinity tag on the large ribosome subunit, driven by expression of cell-type specific Cre-lox promoters, to allow for identification and capture of transcripts from actively translating ribosomes in a cell-specific manner. As a result, TRAP offers a unique opportunity to evaluate the entire mRNA translation profile within a specific cell type, and increase our understanding regarding the cellular complexity of mammalian tissues.Graphical abstract: Schematic demonstrating TRAP protocol for identifying ribosome-bound transcripts specifically within cerebellar Purkinje cells.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder without effective treatments. Attempts at developing targetted therapies have been relatively unsuccessful, at least in part, because the genotypical and phenotypical variability of the disorder. Therefore, identification of biomarkers of response and patients’ stratification are high priorities. Administration of Insulin-like Growth Factor 1 (IGF-1) and related compounds leads to significant reversal of RTT-like symptoms in preclinical mouse models. However, improvements in corresponding clinical trials have not been consistent. A 20-weeks phase I open label trial of mecasermin (recombinant human IGF-1) in children with RTT demonstrated significant improvements in breathing phenotypes. However, a subsequent randomised controlled phase II trial did not show significant improvements in primary outcomes although two secondary clinical endpoints showed positive changes. To identify molecular biomarkers of response and surrogate endpoints, we used RNA sequencing to measure differential gene expression in whole blood samples of participants in the abovementioned phase I mecasermin trial. When all participants (n = 9) were analysed, gene expression was unchanged during the study (baseline vs. end of treatment, T0–T3). However, when participants were subclassified in terms of breathing phenotype improvement, specifically by their plethysmography-based apnoea index, individuals with moderate-severe apnoea and breathing improvement (Responder group) displayed significantly different transcript profiles compared to the other participants in the study (Mecasermin Study Reference group, MSR). Many of the differentially expressed genes are involved in the regulation of cell cycle processes and immune responses, as well as in IGF-1 signalling and breathing regulation. While the Responder group showed limited gene expression changes in response to mecasermin, the MSR group displayed marked differences in the expression of genes associated with inflammatory processes (e.g., neutrophil activation, complement activation) throughout the trial. Our analyses revealed gene expression profiles associated with severe breathing phenotype and its improvement after mecasermin administration in RTT, and suggest that inflammatory/immune pathways and IGF-1 signalling contribute to treatment response. Overall, these data support the notion that transcript profiles have potential as biomarkers of response to IGF-1 and related compounds.

Summary: The tuberous sclerosis complex (TSC) 1 and 2 proteins associate with TBC1D7 to form the TSC complex, which is an essential suppressor of mTOR complex 1 (mTORC1), a ubiquitous driver of cell and tissue growth. Loss-of-function mutations in TSC1 or TSC2, but not TBC1D7, give rise to TSC, a pleiotropic disorder with aberrant activation of mTORC1 in various tissues. Here, we characterize mice with genetic deletion of Tbc1d7, which are viable with normal growth and development. Consistent with partial loss of function of the TSC complex, Tbc1d7 knockout (KO) mice display variable increases in tissue mTORC1 signaling with increased muscle fiber size but with strength and motor defects. Their most pronounced phenotype is brain overgrowth due to thickening of the cerebral cortex, with enhanced neuron-intrinsic mTORC1 signaling and growth. Thus, TBC1D7 is required for full TSC complex function in tissues, and the brain is particularly sensitive to its growth-suppressing activities.

This study investigates the modelling of the individual growth curves of Romanov lambs using different equations and the data on the increase in live weight and selects the best model. For this purpose, the live weights of Romanov lambs that were brought to Gürçeşme Village, Niksar, Turkey, from Nikopol, Russia, were recorded from birth to day 180. In the study, the Cubic Spline, Logistic, Gompertz and Richard models were used. For the study, individual growth curves of a total of 278 (178 females, 100 males) lambs were modeled. For the selection of the best model, adjusted determination coefficient (R2 adj.), mean square error (MSE), Akaike information criteria (AIC) and Durbin-Watson (DW) values were used. In addition, attention was paid to the parameters and standard errors of the models. The results showed that the mean square error for the male lambs varied from 0.295 to 0.995, while it varied from 0.995 to 2.659 for the female lambs; the R2 adj. values were between 0.971 and 0.997 for the male lambs and 0.969 and 0.993 for the female lambs. The AIC values were between -37.12 and 0.094 for the male lambs and -0.196 and 122.12 for the female lambs. The DW values ranged from 1.86 to 2.44 for the female lambs and from 1.02 to 2.79 for the male lambs. Considering the MSE, R2 adj., AIC and DW values of the female lambs (0.295±1.195, 0.997±0.002, -37.12±0.001, 2.23±0.49, respectively) and male lambs (0.995±1.021, 0.993±0.001, -122.12±0.05, 2.31±0.19, respectively), the Cubic Spline model was determined to be the best model, while the Richard model was determined to be the worst fitting model both for the female (0.95±5.143, 0.971±0.002, 0.094±0.31, 2.41±0.01) and male (1.85±2.569, 0.969±0.011, -0.196±0.04, 2.79±0.05) lambs.

Abstract Autosomal recessive spastic paraplegia 52 is caused by biallelic mutations in AP4S1 which encodes a subunit of the adaptor protein complex 4 (AP‐4). Using next‐generation sequencing, we identified three novel unrelated SPG52 patients from a cohort of patients with cerebral palsy. The discovered variants in AP4S1 lead to reduced AP‐4 complex formation in patient‐derived fibroblasts. To further understand the role of AP4S1 in neuronal development and homeostasis, we engineered the first zebrafish model of AP‐4 deficiency using morpholino‐mediated knockdown of ap4s1. In this model, we discovered several phenotypes mimicking SPG52, including altered CNS development, locomotor deficits, and abnormal neuronal excitability.

Abstract Background Participant recruitment for clinical research studies remains a significant challenge for researchers. Novel approaches to recruitment are necessary to ensure that populations are easier to reach. In the context of rare diseases, social media provides a unique opportunity for connecting with patient groups that have representatively lower diagnosis rates when compared with more common diseases or illness. We describe the implementation of designing a patient-centered approach to message design for the purposes of recruiting patients for clinical research studies for rare disease populations. Methods Using an iterative research approach, we analyzed our previous experience of using web-based direct-to-patient recruitment methods to compare these online strategies with traditional center of excellence recruitment strategies. After choosing six research studies for inclusion in the previous study, in-depth, online interviews (n = 37) were conducted with patients represented in each disease category to develop and test recruitment message strategies for social media and a Web-based platform for patients to access study information and pre-screen. Finally, relationships were established with Patient Advocacy Groups representing each rare disease category to ensure further dissemination of recruitment materials via their own social media networks. Results Guided by social marketing theory, we created and tested various recruitment message designs. Three key message concepts preferred by patients emerged: (1) infographic; (2) positive emotional messages; and (3) educational information for sharing. A base study website was designed and created based on data from patient interviews. This website includes the option for potential participants to pre-screen and determine their eligibility for the study. Conclusions Study participants report wanting to be involved in the design and implementation of recruitment approaches for clinical research studies. The application of the aforementioned methods could aide in the evolution of clinical research practices for the recruitment of both rare and common diseases, where patient-centric approaches can help to create targeted messages designs that participants pre-test and support.

Abstract Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by epilepsy, intellectual disability, and benign tumors of the brain, heart, skin, and kidney. Animal models have contributed to our understanding of normal and abnormal human brain development, but the construction of models that accurately recapitulate a human pathology remains challenging. Recent advances in stem cell biology with the derivation of human-induced pluripotent stem cells (hiPSCs) from somatic cells from patients have opened new avenues to the study of TSC. This approach combined with gene-editing tools such as CRISPR/Cas9 offers the advantage of preserving patient-specific genetic background and the ability to generate isogenic controls by correcting a specific mutation. The patient cell line and the isogenic control can be differentiated into the cell type of interest to model various aspects of TSC. In this review, we discuss the remarkable capacity of these cells to be used as a model for TSC in two- and three-dimensional cultures, the potential variability in iPSC models, and highlight differences between findings reported to date.