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BackgroundAlzheimer's disease (AD) is a progressive, multifactorial, neurodegenerative disorder affecting >6 million Americans. Chronic, low-grade neuroinflammation, and insulin resistance may drive AD pathogenesis. We explored the neurophysiological and neuropsychological effects of NE3107, an oral, anti-inflammatory, insulin-sensitizing molecule, in AD.MethodsIn this phase 2, open-label study, 23 patients with mild cognitive impairment or mild dementia received 20-mg oral NE3107 twice daily for 3 months. Primary endpoints assessed changes from baseline in neurophysiological health and oxidative stress (glutathione level) using advanced neuroimaging analyses. Secondary endpoints evaluated changes from baseline in neuropsychological health using cognitive assessments, including the 11-item Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog11), Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment, Clinical Dementia Rating, Quick Dementia Rating Scale, Alzheimer's Disease Composite Score, and Global Rating of Change (GRC). Exploratory endpoints assessed changes from baseline in neuroinflammation biomarkers (tumor necrosis factor alpha, TNF-α) and AD (amyloid beta and phosphorylated tau [P-tau]).ResultsNE3107 was associated with clinician-rated improvements in cerebral blood flow and functional connectivity within the brain. In patients with MMSE ≥ 20 (mild cognitive impairment to mild AD; n = 17), NE3107 was associated with directional, but statistically nonsignificant, changes in brain glutathione levels, along with statistically significant improvements in ADAS-Cog11 (P = .017), Clinical Dementia Rating (P = .042), Quick Dementia Rating Scale (P = .002), Alzheimer's Disease Composite Score (P = .0094), and clinician-rated GRC (P

Tissue engineering strategies show great potential for repairing osteochondral defects in osteoarthritic joints; however, these approaches often rely on passaging cells multiple times to obtain enough cells to produce functional tissue. Unfortunately, monolayer expansion culture causes chondrocyte dedifferentiation, which is accompanied by a phenotypical and morphological shift in chondrocyte properties that leads to a reduction in the quality of de novo cartilage produced. Thus, the objective of this study was to evaluate transcriptional variations during in vitro expansion culture and determine how differences in cell phenotype from monolayer expansion alter development of functional engineered cartilage. We used an unbiased approach to explore genome-wide transcriptional differences in chondrocyte phenotype at passage 1 (P1), P3, and P5, and then seeded cells into hydrogel scaffolds at P3 and P5 to assess cells' abilities to produce cartilaginous extracellular matrix in three dimensional (3D). We identified distinct phenotypic differences, specifically for genes related to extracellular organization and cartilage development. Both P3 and P5 chondrocytes were able to produce chondrogenic tissue in 3D, with P3 cells producing matrix with greater compressive properties and P5 cells secreting matrix with higher glycosaminoglycan/DNA and collagen/DNA ratios. Furthermore, we identified 24 genes that were differentially expressed with passaging and enriched in human osteoarthritis (OA) genome-wide association studies, thereby prioritizing them as functionally relevant targets to improve protocols that recapitulate functional healthy cartilage with cells from adult donors. Specifically, we identified novel genes, such as TMEM190 and RAB11FIP4, which were enriched with human hip OA and may play a role in chondrocyte dedifferentiation. This work lays the foundation for several pathways and genes that could be modulated to enhance the efficacy for chondrocyte culture for tissue regeneration, which could have transformative impacts for cell-based cartilage repair strategies.

Poor bone quality is a major factor in skeletal fragility in elderly individuals. The molecular mechanisms that establish and maintain bone quality, independent of bone mass, are unknown but are thought to be primarily determined by osteocytes. We hypothesize that the age-related decline in bone quality results from the suppression of osteocyte perilacunar/canalicular remodeling (PLR), which maintains bone material properties. We examined bones from young and aged mice with osteocyte-intrinsic repression of TGFβ signaling (TβRIIocy-/-) that suppresses PLR. The control aged bone displayed decreased TGFβ signaling and PLR, but aging did not worsen the existing PLR suppression in male TβRIIocy-/- bone. This relationship impacted the behavior of collagen material at the nanoscale and tissue scale in macromechanical tests. The effects of age on bone mass, density, and mineral material behavior were independent of osteocytic TGFβ. We determined that the decline in bone quality with age arises from the loss of osteocyte function and the loss of TGFβ-dependent maintenance of collagen integrity.

Sub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate intactness scores: the remaining proportion of an intact reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the regions major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems.

Speech disfluency refers to the errors, pauses, or repetitions in speech production. Co-speech gestures are known to help resolve disfluency, suggesting a metacognitive involvement.Here we ask whether (1) disfluencies and gestures act as metacognitive cues in speech, and (2) they have different functions in conversational vs. non-conversational settings. Fifty participants responded to trivia questions, and rated their confidence in their answers (i.e. metacognitive judgement), either with a visible or a non-visible listener. They audibly elaborated on their answers during which we measured the frequency and type of disfluencies and co-speech gestures. We predict confidence ratings to change as a function of the rate of disfluency and the gestures produced by the participants. We also expect the rate of disfluencies and gestures change depending on the conversational setting. Our findings will contribute to understanding the multimodal nature of language and the role of metacognition in speech and gesture production.

Tuberculosis lung lesions are complex and harbor heterogeneous microenvironments that influence antibiotic effectiveness. Major strides have been made recently in understanding drug pharmacokinetics in pulmonary lesions, but the bacterial phenotypes that arise under these conditions and their contribution to drug tolerance are poorly understood. A pharmacodynamic marker called the RS ratio® quantifies ongoing rRNA synthesis based on the abundance of newly synthesized precursor rRNA relative to mature structural rRNA. Application of the RS ratio in the C3HeB/FeJ mouse model demonstrated that Mycobacterium tuberculosis populations residing in different tissue microenvironments are phenotypically distinct and respond differently to drug treatment with rifampin, isoniazid, or bedaquiline. This work provides a foundational basis required to address how anatomic and pathologic microenvironmental niches may contribute to long treatment duration and drug tolerance during the treatment of human tuberculosis.

The present study investigates a potential method of optimizing effective strategies for the functional lateralization of the dorsolateral prefrontal cortex (dlPFC) while in a scanner. Effective hemisphere lateralization of the dlPFC is crucial for lowering the functional risks connected to specific interventions (such as neurosurgery and transcranial magnetic stimulation (TMS), as well as increasing the effectiveness of a given intervention by figuring out the optimal location. This task combines elements of creative problem solving, executive decision making based on an internal rule set, and working memory. A retrospective analysis was performed on a total of 58 unique participants (34 males, 24 females, Mage = 42.93 years, SDage = 16.38). Of these participants, 47 were classified as right-handed, 7 were classified as left-handed, and 4 were classified as ambidextrous, according to the Edinburgh Handedness Inventory. The imaging data were qualitatively judged by two trained, blinded investigators (neurologist and neuropsychologist) for dominant handedness (primary motor cortex) and dominant dorsolateral prefrontal cortex (dlPFC). The results demonstrated that 21.4% of right-handed individuals showed a dominant dlPFC localized to the right hemisphere rather than the assumed left, and 16.7% of left-handers were dominant in their left hemisphere. The task completed in the scanner might be an efficient method for localizing a potential dlPFC target for the purpose of brain stimulation (e.g., TMS), though further study replications are needed to extend and validate these findings.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are approved for multiple cancers but can result in ICI-associated myocarditis, an infrequent but life-threatening condition. Elevations in cardiac biomarkers, specifically troponin-I (cTnI), troponin-T (cTnT), and creatine kinase (CK), are used for diagnosis. However, the association between temporal elevations of these biomarkers with disease trajectory and outcomes has not been established. METHODS: We analyzed the diagnostic accuracy and prognostic performances of cTnI, cTnT, and CK in patients with ICI myocarditis (n=60) through 1-year follow-up in 2 cardio-oncology units (APHP Sorbonne, Paris, France and Heidelberg, Germany). A total of 1751 (1 cTnT assay type), 920 (4 cTnI assay types), and 1191 CK sampling time points were available. Major adverse cardiomyotoxic events (MACE) were defined as heart failure, ventricular arrhythmia, atrioventricular or sinus block requiring pacemaker, respiratory muscle failure requiring mechanical ventilation, and sudden cardiac death. Diagnostic performance of cTnI and cTnT was also assessed in an international ICI myocarditis registry. RESULTS: Within 72 hours of admission, cTnT, cTnI, and CK were increased compared with upper reference limits (URLs) in 56 of 57 (98%), 37 of 42 ([88%] P=0.03 versus cTnT), and 43 of 57 ([75%] P

Aging is associated with changes in circulating levels of various molecules, some of which remain undefined. We find that concentrations of circulating taurine decline with aging in mice, monkeys, and humans. A reversal of this decline through taurine supplementation increased the health span (the period of healthy living) and life span in mice and health span in monkeys. Mechanistically, taurine reduced cellular senescence, protected against telomerase deficiency, suppressed mitochondrial dysfunction, decreased DNA damage, and attenuated inflammaging. In humans, lower taurine concentrations correlated with several age-related diseases and taurine concentrations increased after acute endurance exercise. Thus, taurine deficiency may be a driver of aging because its reversal increases health span in worms, rodents, and primates and life span in worms and rodents. Clinical trials in humans seem warranted to test whether taurine deficiency might drive aging in humans.

BackgroundComplex systems models of breast cancer have previously focused on prediction of prognosis and clinical events for individual women. There is a need for understanding breast cancer at the population level for public health decision-making, for identifying gaps in epidemiologic knowledge and for the education of the public as to the complexity of this most common of cancers.Methods and findingsWe developed an agent-based model of breast cancer for the women of the state of California using data from the U.S. Census, the California Health Interview Survey, the California Cancer Registry, the National Health and Nutrition Examination Survey and the literature. The model was implemented in the Julia programming language and R computing environment. The Paradigm II model development followed a transdisciplinary process with expertise from multiple relevant disciplinary experts from genetics to epidemiology and sociology with the goal of exploring both upstream determinants at the population level and pathophysiologic etiologic factors at the biologic level. The resulting model reproduces in a reasonable manner the overall age-specific incidence curve for the years 2008-2012 and incidence and relative risks due to specific risk factors such as BRCA1, polygenic risk, alcohol consumption, hormone therapy, breastfeeding, oral contraceptive use and scenarios for environmental toxin exposures.ConclusionsThe Paradigm II model illustrates the role of multiple etiologic factors in breast cancer from domains of biology, behavior and the environment. The value of the model is in providing a virtual laboratory to evaluate a wide range of potential interventions into the social, environmental and behavioral determinants of breast cancer at the population level.

In cognitive science, research about mental imagery is often limited to visual, often overlooking olfactory imagery. In this study, we examined the relationship between beauty and olfactory imagery evoked by haiku. We used an odor priming commonly used in cognitive science to measure olfaction so that we could examine the effects of environmental aromas on the aesthetic experience. 44 participants were asked to evaluate 30 haikus. Half of them were exposed to a cypress aroma while the other half had no aroma exposure. The results showed that olfactory imagery during haiku appreciation positively influenced the beauty of haiku, and higher olfactory imagery ability led to a deeper immersion in the haiku. Odor priming did not affect evaluations, but it did affect gaze bias as measured by eye tracking. This is the first time to demonstrate the influence of olfactory imagery on aesthetic evaluation in the psychology of aesthetics.

Introduction: This study intended to evaluate the safety and possible therapeutic effect of transcranial infrared laser stimulation (TILS) based on photobiomodulation (PBM) among patients with traumatic brain injury (TBI). Methods: Eleven participants who were diagnosed with TBI after full neurological examination and MRI evaluation by a board-certified neurologist completed five to eight 20-minute TILS sessions using the Cytonsys CytonPro-5000 apparatus (pilot laser control, focused wavelength of 1064 nm, maximum output power of 10W, maximum optical power density of 500 mW/cm2, effective area 4.5 cm2 in diameter). Per TILS session, participants underwent a laser dose of 250 mW/cm2 continuous laser wave to each hemisphere using predetermined patient-specific coordinates. Structural imaging was used to neuronavigate individual treatment targets in the frontal cortex (Brodmann area 10). The primary safety measure for this study was the occurrence of adverse events (AEs) or serious adverse events (SAEs). The primary efficacy outcome measure was the participant-rated global rating of change (GRC) post-intervention. Secondary outcome measures included a battery of neuropsychological testing and mood questionnaires done both pre- and post-intervention. Results: All patients enrolled in this study protocol were able to tolerate the study procedures without any AEs or SAEs. Nine out of eleven participants had clinically significant improvements in GRC score (≥ +2). Neuropsychological testing and mood questionnaire outcomes also suggested a positive therapeutic effect. Conclusion: This study provides preliminary evidence supporting the safety and potential efficacy of TILS as a non-invasive clinical intervention for individuals with TBI.

BackgroundZebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create 'knockout' models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies.MethodsTo systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression.ResultsWe compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is a potentially fatal infectious disease requiring long treatment duration with multiple antibiotics and against which there is no reliable cure. Among the factors that have hampered the development of adequate drug regimens is the lack of an animal model that reproduces the NTM lung pathology required for studying antibiotic penetration and efficacy. Given the documented similarities between tuberculosis and NTM immunopathology in patients, we first determined that the rabbit model of active tuberculosis reproduces key features of human NTM-PD and provides an acceptable surrogate model to study lesion penetration. We focused on clarithromycin, a macrolide and pillar of NTM-PD treatment, and explored the underlying causes of the disconnect between its favorable potency and pharmacokinetics and inconsistent clinical outcome. To quantify pharmacokinetic-pharmacodynamic target attainment at the site of disease, we developed a translational model describing clarithromycin distribution from plasma to lung lesions, including the spatial quantitation of clarithromycin and azithromycin in mycobacterial lesions of two patients on long-term macrolide therapy. Through clinical simulations, we visualized the coverage of clarithromycin in plasma and four disease compartments, revealing heterogeneous bacteriostatic and bactericidal target attainment depending on the compartment and the corresponding potency against nontuberculous mycobacteria in clinically relevant assays. Overall, clarithromycin's favorable tissue penetration and lack of bactericidal activity indicated that its clinical activity is limited by pharmacodynamic, rather than pharmacokinetic, factors. Our results pave the way toward the simulation of lesion pharmacokinetic-pharmacodynamic coverage by multidrug combinations to enable the prioritization of promising regimens for clinical trials.

Disease gene discovery on chromosome (chr) X is challenging owing to its unique modes of inheritance. We undertook a systematic analysis of human chrX genes. We observe a higher proportion of disorder-associated genes and an enrichment of genes involved in cognition, language, and seizures on chrX compared to autosomes. We analyze gene constraints, exon and promoter conservation, expression, and paralogues, and report 127 genes sharing one or more attributes with known chrX disorder genes. Using machine learning classifiers trained to distinguish disease-associated from dispensable genes, we classify 247 genes, including 115 of the 127, as having high probability of being disease-associated. We provide evidence of an excess of variants in predicted genes in existing databases. Finally, we report damaging variants in CDK16 and TRPC5 in patients with intellectual disability or autism spectrum disorders. This study predicts large-scale gene-disease associations that could be used for prioritization of X-linked pathogenic variants.

Soccer-related ocular injuries, especially retinal injuries, have attracted increasing attention. The mechanics of a flying soccer ball have induced abnormally higher retinal stresses and strains, and their correlation with retinal injuries has been characterized using the finite element (FE) method. However, FE simulations demand solid mechanical expertise and extensive computational time, both of which are difficult to adopt in clinical settings. This study proposes a framework that combines FE analysis with a machine learning (ML) approach for the fast prediction of retina mechanics. Different impact scenarios were simulated using the FE method to obtain the von Mises stress map and the maximum principal strain map in the posterior retina. These stress and strain patterns, along with their input parameters, were used to train and test a partial least squares regression (PLSR) model to predict the soccer-induced retina stress and strain in terms of distributions and peak magnitudes. The peak von Mises stress and maximum principal strain prediction errors were 3.03% and 9.94% for the frontal impact and were 9.08% and 16.40% for the diagonal impact, respectively. The average prediction error of von Mises stress and the maximum principal strain were 15.62% and 21.15% for frontal impacts and were 10.77% and 21.78% for diagonal impacts, respectively. This work provides a surrogate model of FE analysis for the fast prediction of the dynamic mechanics of the retina in response to the soccer impact, which could be further utilized for developing a diagnostic tool for soccer-related ocular trauma.

Research shows that participants predict their memory performance to be lower when they experience disfluencies during encoding, even though encoding disfluency does not always affect actual memory performance. Bizarre statements are typically encoded slower than common statements, which constitutes an example of an encoding disfluency. The current study investigated how disfluencies during encoding for bizarre and common statements affect actual and predicted memory performance from a metacognitive perspective. Across two experiments under intentional learning instructions, participants made either memory predictions or vividness ratings for bizarre and common statements, followed by a free recall task. Participants predicted to remember common statements more than bizarre statements for both Experiment 1 (self-paced encoding) and Experiment 2 (experimenter-paced encoding), even though the actual memory performance was higher for bizarre than common statements. This demonstrates a metacognitive illusion for the bizarreness effect, similar to other manipulations of encoding disfluency.

Amikacin and kanamycin are second-line injectables used in the treatment of multidrug-resistant tuberculosis (MDR-TB) based on the clinical utility of streptomycin, another aminoglycoside and first-line anti-TB drug. While streptomycin was tested as a single agent in the first controlled TB clinical trial, introduction of amikacin and kanamycin into MDR-TB regimens was not preceded by randomized controlled trials. A recent large retrospective meta-analysis revealed that compared with regimens without any injectable drug, amikacin provided modest benefits, and kanamycin was associated with worse outcomes. Although their long-term use can cause irreversible ototoxicity, they remain part of MDR-TB regimens because they have a role in preventing emergence of resistance to other drugs. To quantify the contribution of amikacin and kanamycin to second-line regimens, we applied two-dimensional matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging in large lung lesions, quantified drug exposure in lung and in lesions of rabbits with active TB, and measured the concentrations required to kill or inhibit growth of the resident bacterial populations. Using these metrics, we applied site-of-action pharmacokinetic and pharmacodynamic (PK-PD) concepts and simulated drug coverage in patients' lung lesions. The results provide a pharmacological explanation for the limited clinical utility of both agents and reveal better PK-PD lesion coverage for amikacin than kanamycin, consistent with retrospective data of contribution to treatment success. Together with recent mechanistic studies dissecting antibacterial activity from aminoglycoside ototoxicity, the limited but rapid penetration of streptomycin, amikacin, and kanamycin to the sites of TB disease supports the development of analogs with improved efficacy and tolerability.

Amikacin and kanamycin are second-line injectables used in the treatment of multidrug-resistant tuberculosis (MDR-TB) based on the clinical utility of streptomycin, another aminoglycoside and first-line anti-TB drug. While streptomycin was tested as a single agent in the first controlled TB clinical trial, introduction of amikacin and kanamycin into MDR-TB regimens was not preceded by randomized controlled trials. A recent large retrospective meta-analysis revealed that compared with regimens without any injectable drug, amikacin provided modest benefits, and kanamycin was associated with worse outcomes. Although their long-term use can cause irreversible ototoxicity, they remain part of MDR-TB regimens because they have a role in preventing emergence of resistance to other drugs. To quantify the contribution of amikacin and kanamycin to second-line regimens, we applied two-dimensional matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging in large lung lesions, quantified drug exposure in lung and in lesions of rabbits with active TB, and measured the concentrations required to kill or inhibit growth of the resident bacterial populations. Using these metrics, we applied site-of-action pharmacokinetic and pharmacodynamic (PK-PD) concepts and simulated drug coverage in patients' lung lesions. The results provide a pharmacological explanation for the limited clinical utility of both agents and reveal better PK-PD lesion coverage for amikacin than kanamycin, consistent with retrospective data of contribution to treatment success. Together with recent mechanistic studies dissecting antibacterial activity from aminoglycoside ototoxicity, the limited but rapid penetration of streptomycin, amikacin, and kanamycin to the sites of TB disease supports the development of analogs with improved efficacy and tolerability.

Emerging evidence links genes within human-specific segmental duplications (HSDs) to traits and diseases unique to our species. Strikingly, despite being nearly identical by sequence (>98.5%), paralogous HSD genes are differentially expressed across human cell and tissue types, though the underlying mechanisms have not been examined. We compared cross-tissue mRNA levels of 75 HSD genes from 30 families between humans and chimpanzees and found expression patterns consistent with relaxed selection on or neofunctionalization of derived paralogs. In general, ancestral paralogs exhibited greatest expression conservation with chimpanzee orthologs, though exceptions suggest certain derived paralogs may retain or supplant ancestral functions. Concordantly, analysis of long-read isoform sequencing data sets from diverse human tissues and cell lines found that about half of derived paralogs exhibited globally lower expression. To understand mechanisms underlying these differences, we leveraged data from human lymphoblastoid cell lines (LCLs) and found no relationship between paralogous expression divergence and post-transcriptional regulation, sequence divergence, or copy-number variation. Considering cis-regulation, we reanalyzed ENCODE data and recovered hundreds of previously unidentified candidate CREs in HSDs. We also generated large-insert ChIP-sequencing data for active chromatin features in an LCL to better distinguish paralogous regions. Some duplicated CREs were sufficient to drive differential reporter activity, suggesting they may contribute to divergent cis-regulation of paralogous genes. This work provides evidence that cis-regulatory divergence contributes to novel expression patterns of recent gene duplicates in humans.

Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a putative iron-containing non-heme oxygenase of unknown specificity and biological significance. We report 25 families containing 34 individuals with neurological disease associated with biallelic HPDL variants. Phenotypes ranged from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spasticity and global developmental delays, sometimes complicated by episodes of neurological and respiratory decompensation. Variants included bona fide pathogenic truncating changes, although most were missense substitutions. Functionality of variants could not be determined directly as the enzymatic specificity of HPDL is unknown; however, when HPDL missense substitutions were introduced into 4-hydroxyphenylpyruvate dioxygenase (HPPD, an HPDL orthologue), they impaired the ability of HPPD to convert 4-hydroxyphenylpyruvate into homogentisate. Moreover, three additional sets of experiments provided evidence for a role of HPDL in the nervous system and further supported its link to neurological disease: (i) HPDL was expressed in the nervous system and expression increased during neural differentiation; (ii) knockdown of zebrafish hpdl led to abnormal motor behaviour, replicating aspects of the human disease; and (iii) HPDL localized to mitochondria, consistent with mitochondrial disease that is often associated with neurological manifestations. Our findings suggest that biallelic HPDL variants cause a syndrome varying from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spastic tetraplegia associated with global developmental delays.

Little is known about the mutational processes that shape the genetic landscape of gliomas. Numerous mutational processes leave marks on the genome in the form of mutations, copy number alterations, rearrangements or their combinations. To explore gliomagenesis, we hypothesized that gliomas with different underlying oncogenic mechanisms would have differences in the burden of various forms of these genomic alterations. This was an analysis on adult diffuse gliomas, but IDH-mutant gliomas as well as diffuse midline gliomas H3-K27M were excluded to search for the possible presence of new entities among the very heterogenous group of IDH-WT glioblastomas. The cohort was divided into two molecular subsets: (1) Molecularly-defined GBM (mGBM) as those that carried molecular features of glioblastomas (including TERT promoter mutations, 7/10 pattern, or EGFR-amplification), and (2) those who did not (others). Whole exome sequencing was performed for 37 primary tumors and matched blood samples as well as 8 recurrences. Single nucleotide variations (SNV), short insertion or deletions (indels) and copy number alterations (CNA) were quantified using 5 quantitative metrics (SNV burden, indel burden, copy number alteration frequency-wGII, chromosomal arm event ratio-CAER, copy number amplitude) as well as 4 parameters that explored underlying oncogenic mechanisms (chromothripsis, double minutes, microsatellite instability and mutational signatures). Findings were validated in the TCGA pan-glioma cohort. mGBM and Others differed significantly in their SNV (only in the TCGA cohort) and CNA metrics but not indel burden. SNV burden increased with increasing age at diagnosis and at recurrences and was driven by mismatch repair deficiency. On the contrary, indel and CNA metrics remained stable over increasing age at diagnosis and with recurrences. Copy number alteration frequency (wGII) correlated significantly with chromothripsis while CAER and CN amplitude correlated significantly with the presence of double minutes, suggesting separate underlying mechanisms for different forms of CNA.

ObjectivesTo compare regional differences in subchondral trabecular structure using high-resolution MRI in meniscus-covered/meniscus-uncovered tibia in cadaveric knees with intact/torn menisci.Materials and methods3D proton density CUBE MRI of 6 cadaveric knees without significant osteoarthritis (OA) was acquired, 0.25-mm resolution. Menisci were evaluated and classified intact or torn. MR data were transferred to ImageJ program to segment tibial 3D volume of interest (VOI). Data was subdivided into meniscus-covered/meniscus-uncovered regions. Segmented VOI was classified into binary data, trabeculae/bone marrow. The trabecular bone data was used to measure MR biomarkers (apparent subchondral plate-connected bone density (adapted from spine MR), apparent trabecular bone volume fraction, apparent mean trabecular thickness, apparent connectivity density, and structure model index (SMI)). Mean value of parameters was analyzed for the effects of meniscal tear/tibial coverage.ResultsNine torn menisci and 3 intact menisci were present. MR measures of bone varied significantly due to meniscal coverage/tear. Subchondral plate-connected bone density under covered meniscus regions increased from 10.9 to 23.5% with meniscal tear. Values increased in uncovered regions, 19.3% (intact) and 32.4% (torn). This reflects higher density when uncovered (p = 0.048) with meniscal tear (p = 0.007). Similar patterns were found for trabecular bone fraction (coverage p

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

This study was conducted in treatment-naive adults with drug-susceptible pulmonary tuberculosis in Port-au-Prince, Haiti, to assess the safety, bactericidal activity, and pharmacokinetics of nitazoxanide (NTZ). This was a prospective phase II clinical trial in 30 adults with pulmonary tuberculosis. Twenty participants received 1 g of NTZ orally twice daily for 14 days. A control group of 10 participants received standard therapy over 14 days. The primary outcome was the change in time to culture positivity (TTP) in an automated liquid culture system. The most common adverse events seen in the NTZ group were gastrointestinal complaints and headache. The mean change in TTP in sputum over 14 days in the NTZ group was 3.2 h ± 22.6 h and was not statistically significant (P = 0.56). The mean change in TTP in the standard therapy group was significantly increased, at 134 h ± 45.2 h (P

BackgroundDedicator of cytokinesis 8 (DOCK8) deficiency is the main cause of the autosomal recessive hyper-IgE syndrome (HIES). We previously reported the selective loss of group 3 innate lymphoid cell (ILC) number and function in a Dock8-deficient mouse model. In this study, we sought to test whether DOCK8 is required for the function and maintenance of ILC subsets in humans.MethodsPeripheral blood ILC1-3 subsets of 16 DOCK8-deficient patients recruited at the pretransplant stage, and seven patients with autosomal dominant (AD) HIES due to STAT3 mutations, were compared with those of healthy controls or post-transplant DOCK8-deficient patients (n = 12) by flow cytometry and real-time qPCR. Sorted total ILCs from DOCK8- or STAT3-mutant patients and healthy controls were assayed for survival, apoptosis, proliferation, and activation by IL-7, IL-23, and IL-12 by cell culture, flow cytometry, and phospho-flow assays.ResultsDOCK8-deficient but not STAT3-mutant patients exhibited a profound depletion of ILC3s, and to a lesser extent ILC2s, in their peripheral blood. DOCK8-deficient ILC1-3 subsets had defective proliferation, expressed lower levels of IL-7R, responded less to IL-7, IL-12, or IL-23 cytokines, and were more prone to apoptosis compared with those of healthy controls.ConclusionDOCK8 regulates human ILC3 expansion and survival, and more globally ILC cytokine signaling and proliferation. DOCK8 deficiency leads to loss of ILC3 from peripheral blood. ILC3 deficiency may contribute to the susceptibility of DOCK8-deficient patients to infections.

Recent efforts to comprehensively characterize great ape genetic diversity using short-read sequencing and single-nucleotide variants have led to important discoveries related to selection within species, demographic history, and lineage-specific traits. Structural variants (SVs), including deletions and inversions, comprise a larger proportion of genetic differences between and within species, making them an important yet understudied source of trait divergence. Here, we used a combination of long-read and -range sequencing approaches to characterize the structural variant landscape of two additional Pan troglodytes verus individuals, one of whom carries 13% admixture from Pan troglodytes troglodytes. We performed optical mapping of both individuals followed by nanopore sequencing of one individual. Filtering for larger variants (>10 kbp) and combined with genotyping of SVs using short-read data from the Great Ape Genome Project, we identified 425 deletions and 59 inversions, of which 88 and 36, respectively, were novel. Compared with gene expression in humans, we found a significant enrichment of chimpanzee genes with differential expression in lymphoblastoid cell lines and induced pluripotent stem cells, both within deletions and near inversion breakpoints. We examined chromatin-conformation maps from human and chimpanzee using these same cell types and observed alterations in genomic interactions at SV breakpoints. Finally, we focused on 56 genes impacted by SVs in >90% of chimpanzees and absent in humans and gorillas, which may contribute to chimpanzee-specific features. Sequencing a greater set of individuals from diverse subspecies will be critical to establish the complete landscape of genetic variation in chimpanzees.

Patients with relapsed/refractory multiple myeloma (RRMM) for whom the benefits of lenalidomide have been exhausted in early treatment lines need effective therapies. In cohort A of the phase 2 MM-014 trial, we examined the safety and efficacy of pomalidomide plus low-dose dexamethasone immediately after lenalidomide-based treatment failure in patients with RRMM and two prior lines of therapy. Pomalidomide 4 mg was given on days 1 to 21 of 28-day cycles. Dexamethasone 40 mg (20 mg for patients aged >75 years) was given on days 1, 8, 15 and 22 of 28-day cycles. The primary endpoint was overall response rate (ORR), and secondary endpoints included progression-free survival (PFS), overall survival (OS) and safety. The intention-to-treat population comprised 56 patients; all received prior lenalidomide (87·5% lenalidomide refractory) and 39 (69·6%) received prior bortezomib. ORR was 32·1% (28·2% in the prior-bortezomib subgroup). Median PFS was 12·2 months (7·9 months in the prior-bortezomib subgroup). Median OS was 41·7 months (38·6 months in the prior-bortezomib subgroup). The most common grade 3/4 treatment-emergent adverse events were anaemia (25·0%), pneumonia (14·3%) and fatigue (14·3%). These findings support earlier sequencing of pomalidomide-based therapy in lenalidomide-pretreated patients with RRMM, including those who have become refractory to lenalidomide. Trial registration: www.ClinicalTrials.gov identifier NCT01946477.

Patients with relapsed/refractory multiple myeloma (RRMM) for whom the benefits of lenalidomide have been exhausted in early treatment lines need effective therapies. In cohort A of the phase 2 MM-014 trial, we examined the safety and efficacy of pomalidomide plus low-dose dexamethasone immediately after lenalidomide-based treatment failure in patients with RRMM and two prior lines of therapy. Pomalidomide 4 mg was given on days 1 to 21 of 28-day cycles. Dexamethasone 40 mg (20 mg for patients aged >75 years) was given on days 1, 8, 15 and 22 of 28-day cycles. The primary endpoint was overall response rate (ORR), and secondary endpoints included progression-free survival (PFS), overall survival (OS) and safety. The intention-to-treat population comprised 56 patients; all received prior lenalidomide (87·5% lenalidomide refractory) and 39 (69·6%) received prior bortezomib. ORR was 32·1% (28·2% in the prior-bortezomib subgroup). Median PFS was 12·2 months (7·9 months in the prior-bortezomib subgroup). Median OS was 41·7 months (38·6 months in the prior-bortezomib subgroup). The most common grade 3/4 treatment-emergent adverse events were anaemia (25·0%), pneumonia (14·3%) and fatigue (14·3%). These findings support earlier sequencing of pomalidomide-based therapy in lenalidomide-pretreated patients with RRMM, including those who have become refractory to lenalidomide. Trial registration: www.ClinicalTrials.gov identifier NCT01946477.

In recent years, zebrafish have become commonly used as a model for studying human traits and disorders. Their small size, high fecundity, and rapid development allow for more high-throughput experiments compared to other vertebrate models. Given that zebrafish share >70% gene homologs with humans and their genomes can be readily edited using highly efficient CRISPR methods, we are now able to rapidly generate mutations impacting practically any gene of interest. Unfortunately, our ability to phenotype mutant larvae has not kept pace. To address this challenge, we have developed a protocol that obtains multiple phenotypic measurements from individual zebrafish larvae in an automated and parallel fashion, including morphological features (i.e., body length, eye area, and head size) and movement/behavior. By assaying wild-type zebrafish in a variety of conditions, we determined optimal parameters that avoid significant developmental defects or physical damage; these include morphological imaging of larvae at two time points [3 days post fertilization (dpf) and 5 dpf] coupled with motion tracking of behavior at 5 dpf. As a proof-of-principle, we tested our approach on two novel CRISPR-generated mutant zebrafish lines carrying predicted null-alleles of syngap1b and slc7a5, orthologs to two human genes implicated in autism-spectrum disorder, intellectual disability, and epilepsy. Using our optimized high-throughput phenotyping protocol, we recapitulated previously published results from mouse and zebrafish models of these candidate genes. In summary, we describe a rapid parallel pipeline to characterize morphological and behavioral features of individual larvae in a robust and consistent fashion, thereby improving our ability to better identify genes important in human traits and disorders.

Recent efforts to comprehensively characterize great ape genetic diversity using short-read sequencing and single-nucleotide variants have led to important discoveries related to selection within species, demographic history, and lineage-specific traits. Structural variants (SVs), including deletions and inversions, comprise a larger proportion of genetic differences between and within species, making them an important yet understudied source of trait divergence. Here, we used a combination of long-read and -range sequencing approaches to characterize the structural variant landscape of two additional Pan troglodytes verus individuals, one of whom carries 13% admixture from Pan troglodytes troglodytes. We performed optical mapping of both individuals followed by nanopore sequencing of one individual. Filtering for larger variants (>10 kbp) and combined with genotyping of SVs using short-read data from the Great Ape Genome Project, we identified 425 deletions and 59 inversions, of which 88 and 36, respectively, were novel. Compared with gene expression in humans, we found a significant enrichment of chimpanzee genes with differential expression in lymphoblastoid cell lines and induced pluripotent stem cells, both within deletions and near inversion breakpoints. We examined chromatin-conformation maps from human and chimpanzee using these same cell types and observed alterations in genomic interactions at SV breakpoints. Finally, we focused on 56 genes impacted by SVs in >90% of chimpanzees and absent in humans and gorillas, which may contribute to chimpanzee-specific features. Sequencing a greater set of individuals from diverse subspecies will be critical to establish the complete landscape of genetic variation in chimpanzees.

In recent years, zebrafish have become commonly used as a model for studying human traits and disorders. Their small size, high fecundity, and rapid development allow for more high-throughput experiments compared to other vertebrate models. Given that zebrafish share >70% gene homologs with humans and their genomes can be readily edited using highly efficient CRISPR methods, we are now able to rapidly generate mutations impacting practically any gene of interest. Unfortunately, our ability to phenotype mutant larvae has not kept pace. To address this challenge, we have developed a protocol that obtains multiple phenotypic measurements from individual zebrafish larvae in an automated and parallel fashion, including morphological features (i.e., body length, eye area, and head size) and movement/behavior. By assaying wild-type zebrafish in a variety of conditions, we determined optimal parameters that avoid significant developmental defects or physical damage; these include morphological imaging of larvae at two time points [3 days post fertilization (dpf) and 5 dpf] coupled with motion tracking of behavior at 5 dpf. As a proof-of-principle, we tested our approach on two novel CRISPR-generated mutant zebrafish lines carrying predicted null-alleles of syngap1b and slc7a5, orthologs to two human genes implicated in autism-spectrum disorder, intellectual disability, and epilepsy. Using our optimized high-throughput phenotyping protocol, we recapitulated previously published results from mouse and zebrafish models of these candidate genes. In summary, we describe a rapid parallel pipeline to characterize morphological and behavioral features of individual larvae in a robust and consistent fashion, thereby improving our ability to better identify genes important in human traits and disorders.

PurposeThe role of chemotherapy (CT) and radiotherapy (RT) for management of extraskeletal osteosarcoma (ESOS) remains controversial. We examined disease outcomes for ESOS patients and investigated the association between CT/RT with recurrence and survival.Patients and methodsRetrospective review at 25 international sarcoma centers identified patients ≥18 years old treated for ESOS from 1971 to 2016. Patient/tumour characteristics, treatment, local/systemic recurrence, and survival data were collected. Kaplan-Meier survival and Cox proportional-hazards regression and cumulative incidence competing risks analysis were performed.Results370 patients with localized ESOS treated definitively with surgery presented with mainly deep tumours (n = 294, 80%). 122 patients underwent surgical resection alone, 96 (26%) also received CT, 70 (19%) RT and 82 (22%) both adjuvants. Five-year survival for patients with localized ESOS was 56% (95% CI 51%-62%). Almost half of patients (n = 173, 47%) developed recurrence: local 9% (35/370), distant 28% (102/370) or both 10% (36/370). Considering death as a competing event, there was no significant difference in cumulative incidence of local or systemic recurrence between patients who received CT, RT, both or neither (local p = 0.50, systemic p = 0.69). Multiple regression Cox analysis showed a significant association between RT and decreased local recurrence (HR 0.46 [95% CI 0.26-0.80], p = 0.01).ConclusionAlthough the use of RT significantly decreased local recurrences, CT did not decrease the risk of systemic recurrence, and neither CT, nor RT nor both were associated with improved survival in patients with localized ESOS. Our results do not support the use of CT; however, adjuvant RT demonstrates benefit in patients with locally resectable ESOS.

Silver fir (Abies alba Mill.) is a keystone conifer of European montane forest ecosystems that has experienced large fluctuations in population size during during the Quaternary and, more recently, due to land-use change. To forecast the species' future distribution and survival, it is important to investigate the genetic basis of adaptation to environmental change, notably to extreme events. For this purpose, we here provide a first draft genome assembly and annotation of the silver fir genome, established through a community-based initiative. DNA obtained from haploid megagametophyte and diploid needle tissue was used to construct and sequence Illumina paired-end and mate-pair libraries, respectively, to high depth. The assembled A. alba genome sequence accounted for over 37 million scaffolds corresponding to 18.16 Gb, with a scaffold N50 of 14,051 bp. Despite the fragmented nature of the assembly, a total of 50,757 full-length genes were functionally annotated in the nuclear genome. The chloroplast genome was also assembled into a single scaffold (120,908 bp) that shows a high collinearity with both the A. koreana and A. sibirica complete chloroplast genomes. This first genome assembly of silver fir is an important genomic resource that is now publicly available in support of a new generation of research. By genome-enabling this important conifer, this resource will open the gate for new research and more precise genetic monitoring of European silver fir forests.

Silver fir (Abies alba Mill.) is a keystone conifer of European montane forest ecosystems that has experienced large fluctuations in population size during during the Quaternary and, more recently, due to land-use change. To forecast the species' future distribution and survival, it is important to investigate the genetic basis of adaptation to environmental change, notably to extreme events. For this purpose, we here provide a first draft genome assembly and annotation of the silver fir genome, established through a community-based initiative. DNA obtained from haploid megagametophyte and diploid needle tissue was used to construct and sequence Illumina paired-end and mate-pair libraries, respectively, to high depth. The assembled A. alba genome sequence accounted for over 37 million scaffolds corresponding to 18.16 Gb, with a scaffold N50 of 14,051 bp. Despite the fragmented nature of the assembly, a total of 50,757 full-length genes were functionally annotated in the nuclear genome. The chloroplast genome was also assembled into a single scaffold (120,908 bp) that shows a high collinearity with both the A. koreana and A. sibirica complete chloroplast genomes. This first genome assembly of silver fir is an important genomic resource that is now publicly available in support of a new generation of research. By genome-enabling this important conifer, this resource will open the gate for new research and more precise genetic monitoring of European silver fir forests.

Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of "universal" drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone. In the absence of clinical data comparing their side-by-side efficacies in controlled MDR-TB trials, a pharmacological rationale is needed to guide the selection of the most efficacious fluoroquinolone. The present studies were designed to test the hypothesis that fluoroquinolone concentrations (pharmacokinetics) and activity (pharmacodynamics) at the site of infection are better predictors of efficacy than the plasma concentrations and potency measured in standard growth inhibition assays and are better suited to determinations of whether one of the fluoroquinolones outperforms the others in rabbits with active TB. We first measured the penetration of these fluoroquinolones in lung lesion compartments, and their potency against bacterial populations that reside in each compartment, to compute lesion-centric pharmacokinetic-pharmacodynamic (PK/PD) parameters. PK modeling methods were used to quantify drug penetration from plasma to tissues at human-equivalent doses. On the basis of these metrics, moxifloxacin emerged with a clear advantage, whereas plasma-based PK/PD favored levofloxacin (the ranges of the plasma AUC/MIC ratio [i.e., the area under the concentration-time curve over 24 h in the steady state divided by the MIC] are 46 to 86 for moxifloxacin and 74 to 258 for levofloxacin). A comparative efficacy trial in the rabbit model of active TB demonstrated the superiority of moxifloxacin in reducing bacterial burden at the lesion level and in sterilizing cellular and necrotic lesions. Collectively, these results show that PK/PD data obtained at the site of infection represent an adequate predictor of drug efficacy against TB and constitute the baseline required to explore synergies, antagonism, and drug-drug interactions in fluoroquinolone-containing regimens.

Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of "universal" drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone. In the absence of clinical data comparing their side-by-side efficacies in controlled MDR-TB trials, a pharmacological rationale is needed to guide the selection of the most efficacious fluoroquinolone. The present studies were designed to test the hypothesis that fluoroquinolone concentrations (pharmacokinetics) and activity (pharmacodynamics) at the site of infection are better predictors of efficacy than the plasma concentrations and potency measured in standard growth inhibition assays and are better suited to determinations of whether one of the fluoroquinolones outperforms the others in rabbits with active TB. We first measured the penetration of these fluoroquinolones in lung lesion compartments, and their potency against bacterial populations that reside in each compartment, to compute lesion-centric pharmacokinetic-pharmacodynamic (PK/PD) parameters. PK modeling methods were used to quantify drug penetration from plasma to tissues at human-equivalent doses. On the basis of these metrics, moxifloxacin emerged with a clear advantage, whereas plasma-based PK/PD favored levofloxacin (the ranges of the plasma AUC/MIC ratio [i.e., the area under the concentration-time curve over 24 h in the steady state divided by the MIC] are 46 to 86 for moxifloxacin and 74 to 258 for levofloxacin). A comparative efficacy trial in the rabbit model of active TB demonstrated the superiority of moxifloxacin in reducing bacterial burden at the lesion level and in sterilizing cellular and necrotic lesions. Collectively, these results show that PK/PD data obtained at the site of infection represent an adequate predictor of drug efficacy against TB and constitute the baseline required to explore synergies, antagonism, and drug-drug interactions in fluoroquinolone-containing regimens.

Stroke is a major cause of death and disability in the United States and around the world with limited therapeutic option. Here, we discuss the critical role of mitochondria in stem cell-mediated rescue of stroke brain by highlighting the concept that deleting the mitochondria from stem cells abolishes the cells' regenerative potency. The application of innovative approaches entailing generation of mitochondria-voided stem cells as well as pharmacological inhibition of mitochondrial function may elucidate the mechanism underlying transfer of healthy mitochondria to ischemic cells, thereby providing key insights in the pathology and treatment of stroke and other brain disorders plagued with mitochondrial dysfunctions.

Biological materials such as chiton tooth, squid beak, and byssal threads of bivalves have inspired the development of new technologies. To this end, we have characterized the acellular components in the buccal mass of the terrestrial slug Ariolimax californicus (banana slug). These components are the radula, the jaw, and the odontophore. In the radula, calcium-rich denticles are tightly interlocked one to the other on top of a nanofibrous chitin membrane. The jaw has a nanostructured morphology made of chitin to achieve compression resistance and is directly linked to the foregut cuticle, which has a protective nanofibrous structure. Finally, in the odontophore, we observed a structurally elastic microstructure that interfaces soft tissues with a highly stressed radula membrane. Based on those observations, we discuss the interaction between these components and highlight how the materials in these task-specific components have evolved. This structure-properties-function study of the A. californicus' buccal mass may aid in the design and fabrication of novel bioinspired materials.

Biological materials such as chiton tooth, squid beak, and byssal threads of bivalves have inspired the development of new technologies. To this end, we have characterized the acellular components in the buccal mass of the terrestrial slug Ariolimax californicus (banana slug). These components are the radula, the jaw, and the odontophore. In the radula, calcium-rich denticles are tightly interlocked one to the other on top of a nanofibrous chitin membrane. The jaw has a nanostructured morphology made of chitin to achieve compression resistance and is directly linked to the foregut cuticle, which has a protective nanofibrous structure. Finally, in the odontophore, we observed a structurally elastic microstructure that interfaces soft tissues with a highly stressed radula membrane. Based on those observations, we discuss the interaction between these components and highlight how the materials in these task-specific components have evolved. This structure-properties-function study of the A. californicus' buccal mass may aid in the design and fabrication of novel bioinspired materials.

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in the medial compartment of OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. As a step toward evaluating the causality of PLR suppression in OA, we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact, using Cre recombinase driven by the 9.6-kb DMP1 promoter. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content, change chondrocyte production of collagen II, aggrecan, and MMP13, and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, defects in PLR coincide with cartilage defects. Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis, likely via its effects on PLR. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis.

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in the medial compartment of OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. As a step toward evaluating the causality of PLR suppression in OA, we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact, using Cre recombinase driven by the 9.6-kb DMP1 promoter. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content, change chondrocyte production of collagen II, aggrecan, and MMP13, and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, defects in PLR coincide with cartilage defects. Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis, likely via its effects on PLR. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis.

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

Neuronal migration defects, including pachygyria, are among the most severe developmental brain defects in humans. Here, we identify biallelic truncating mutations in CTNNA2, encoding αN-catenin, in patients with a distinct recessive form of pachygyria. CTNNA2 was expressed in human cerebral cortex, and its loss in neurons led to defects in neurite stability and migration. The αN-catenin paralog, αE-catenin, acts as a switch regulating the balance between β-catenin and Arp2/3 actin filament activities1. Loss of αN-catenin did not affect β-catenin signaling, but recombinant αN-catenin interacted with purified actin and repressed ARP2/3 actin-branching activity. The actin-binding domain of αN-catenin or ARP2/3 inhibitors rescued the neuronal phenotype associated with CTNNA2 loss, suggesting ARP2/3 de-repression as a potential disease mechanism. Our findings identify CTNNA2 as the first catenin family member with biallelic mutations in humans, causing a new pachygyria syndrome linked to actin regulation, and uncover a key factor involved in ARP2/3 repression in neurons.

Neuronal migration defects, including pachygyria, are among the most severe developmental brain defects in humans. Here, we identify biallelic truncating mutations in CTNNA2, encoding αN-catenin, in patients with a distinct recessive form of pachygyria. CTNNA2 was expressed in human cerebral cortex, and its loss in neurons led to defects in neurite stability and migration. The αN-catenin paralog, αE-catenin, acts as a switch regulating the balance between β-catenin and Arp2/3 actin filament activities1. Loss of αN-catenin did not affect β-catenin signaling, but recombinant αN-catenin interacted with purified actin and repressed ARP2/3 actin-branching activity. The actin-binding domain of αN-catenin or ARP2/3 inhibitors rescued the neuronal phenotype associated with CTNNA2 loss, suggesting ARP2/3 de-repression as a potential disease mechanism. Our findings identify CTNNA2 as the first catenin family member with biallelic mutations in humans, causing a new pachygyria syndrome linked to actin regulation, and uncover a key factor involved in ARP2/3 repression in neurons.

Metazoan development depends on tightly regulated gene expression programs that instruct progenitor cells to adopt specialized fates. Recent work found that posttranslational modifications, such as monoubiquitylation, can determine cell fate also independently of effects on transcription, yet how monoubiquitylation is implemented during development is poorly understood. Here, we have identified a regulatory circuit that controls monoubiquitylation-dependent neural crest specification by the E3 ligase CUL3 and its substrate adaptor KBTBD8. We found that CUL3KBTBD8 monoubiquitylates its essential targets only after these have been phosphorylated in multiple motifs by CK2, a kinase whose levels gradually increase during embryogenesis. Its dependency on multisite phosphorylation allows CUL3KBTBD8 to convert the slow rise in embryonic CK2 into decisive recognition of ubiquitylation substrates, which in turn is essential for neural crest specification. We conclude that multisite dependency of an E3 ligase provides a powerful mechanism for switch-like cell fate transitions controlled by monoubiquitylation.

β-Ga2O3 thin films were grown on n-type GaN substrates using the sol–gel method. The forward-biased temperature dependent current–voltage (I–V–T) characteristics of Ni/β-Ga2O3/GaN structure have been investigated in the temperature range of 298–473 K. The apparent barrier height (ϕap) increased while the ideality factor (n) decreased with the increase in temperature. Such a temperature dependent behavior of ϕap and n was explained by the inhomogeneity of ϕap, which obeyed Gaussian distribution with zero-bias mean barrier height (ϕ¯ B0) of 1.02 ± 0.02 eV and standard deviation (σs0) of 153 ± 0.04 mV. Subsequently, ϕ¯ B0 and Richardson constant A* were obtained from the slope and intercept of the modified Richardson plot as 0.99 ± 0.01 e V and 67.2 A cm−2 K−2, respectively. The ϕ¯ B0 obtained from the modified Richardson plot was in good agreement with the theoretical value calculated from the work function of Ni and electron affinity of β-Ga2O3. The I–V–T characteristics of Ni/β-Ga2O3/GaN MOS structures can be successfully explained by the thermionic emission theory with a single Gaussian distribution of the barrier height.

β-Ga2O3 thin films were grown on n-type GaN substrates using the sol–gel method. The forward-biased temperature dependent current–voltage (I–V–T) characteristics of Ni/β-Ga2O3/GaN structure have been investigated in the temperature range of 298–473 K. The apparent barrier height (ϕap) increased while the ideality factor (n) decreased with the increase in temperature. Such a temperature dependent behavior of ϕap and n was explained by the inhomogeneity of ϕap, which obeyed Gaussian distribution with zero-bias mean barrier height (ϕ¯ B0) of 1.02 ± 0.02 eV and standard deviation (σs0) of 153 ± 0.04 mV. Subsequently, ϕ¯ B0 and Richardson constant A* were obtained from the slope and intercept of the modified Richardson plot as 0.99 ± 0.01 e V and 67.2 A cm−2 K−2, respectively. The ϕ¯ B0 obtained from the modified Richardson plot was in good agreement with the theoretical value calculated from the work function of Ni and electron affinity of β-Ga2O3. The I–V–T characteristics of Ni/β-Ga2O3/GaN MOS structures can be successfully explained by the thermionic emission theory with a single Gaussian distribution of the barrier height.

Wingless-2 (wg-2) is an autosomal recessive mutation in chicken that results in an embryonic lethal condition. Affected individuals exhibit a multisystem syndrome characterized by absent wings, truncated legs, and craniofacial, kidney, and feather malformations. Previously, work focused on phenotype description, establishing the autosomal recessive pattern of Mendelian inheritance and placing the mutation on an inbred genetic background to create the congenic line UCD Wingless-2.331. The research described in this paper employed the complementary tools of breeding, genetics, and genomics to map the chromosomal location of the mutation and successively narrow the size of the region for analysis of the causative element. Specifically, the wg-2 mutation was initially mapped to a 7 Mb region of chromosome 12 using an Illumina 3 K SNP array. Subsequent SNP genotyping and exon sequencing combined with analysis from improved genome assemblies narrowed the region of interest to a maximum size of 227 kb. Within this region, 3 validated and 3 predicted candidate genes are found, and these are described. The wg-2 mutation is a valuable resource to contribute to an improved understanding of the developmental pathways involved in chicken and avian limb development as well as serving as a model for human development, as the resulting syndrome shares features with human congenital disorders.