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Adeno-associated virus (AAV)-mediated gene transfer has shown promise in rescuing mouse models of genetic hearing loss, but how viral capsid and promoter selection affects efficacy is poorly characterized. Here, we tested combinations of AAVs and promoters to deliver Tmprss3, mutations in which are associated with hearing loss in humans. Tmprss3tm1/tm1 mice display severe cochlear hair cell degeneration, loss of auditory brainstem responses, and delayed loss of spiral ganglion neurons. Under the ubiquitous CAG promoter and AAV-KP1 capsid, Tmprss3 overexpression caused striking cytotoxicity in vitro and in vivo and failed to rescue degeneration or dysfunction of the Tmprss3tm1/tm1 cochlea. Reducing the dosage or using AAV-DJ-CAG-Tmprss3 diminished cytotoxicity without rescue of the Tmprss3tm1/tm1 cochlea. Finally, the combination of AAV-KP1 capsid and the EF1α promoter prevented cytotoxicity and reduced hair cell degeneration, loss of spiral ganglion neurons, and improved hearing thresholds in Tmprss3tm1/tm1 mice. Together, our study illustrates toxicity of exogenous genes and factors governing rescue efficiency, and suggests that cochlear gene therapy likely requires precisely targeted transgene expression.

Cochlear implants (CI) have revolutionized the treatment of patients with severe to profound sensory hearing loss by providing a method of bypassing normal hearing to directly stimulate the auditory nerve. A further advance in the field has been the introduction of “hearing preservation” surgery, whereby the CI electrode array (EA) is carefully inserted to spare damage to the delicate anatomy and function of the cochlea. Preserving residual function of the inner ear allows patients to receive maximal benefit from the CI and to combine CI electric stimulation with acoustic hearing, offering improved postoperative speech, hearing, and quality of life outcomes. However, under the current paradigm of implant surgery, where EAs are inserted by hand, the cochlea cannot be reliably spared from damage. Robotics-assisted EA insertion is an emerging technology that may overcome fundamental human kinetic limitations that prevent consistency in achieving steady and slow EA insertion. This review begins by describing the relationship between EA insertion speed and generation of intracochlear forces and pressures. The various mechanisms by which these intracochlear forces can damage the cochlea and lead to worsened postoperative outcomes are discussed. The constraints of manual insertion technique are compared to robotics-assisted methods, followed by an overview of the current and future state of robotics-assisted EA insertion.

Abstract Somatic and germline gain‐of‐function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor‐prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss‐of‐function of the proto‐oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure‐based prediction and functional tests using human knock‐in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress‐induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis.

Fibrillin-1 and fibrillin-2, encoded by FBN1 and FBN2, respectively, play significant roles in elastic fiber assembly, with pathogenic variants causing a diverse group of connective tissue disorders such as Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCD). Different genomic variations may lead to heterogeneous phenotypic features and functional consequences. Recent high-throughput sequencing modalities have allowed detection of novel variants that may guide the care for patients and inform the genetic counseling for their families. We performed clinical phenotyping for two newborn infants with complex congenital heart defects. For genetic investigations, we employed next-generation sequencing strategies including whole-genome Single-Nucleotide Polymorphism (SNP) microarray for infant A with valvular insufficiency, aortic sinus dilatation, hydronephrosis, and dysmorphic features, and Trio whole-exome sequencing (WES) for infant B with dextro-transposition of the great arteries (D-TGA) and both parents. Infant A is a term male with neonatal marfanoid features, left-sided hydronephrosis, and complex congenital heart defects including tricuspid regurgitation, aortic sinus dilatation, patent foramen ovale, patent ductus arteriosus, mitral regurgitation, tricuspid regurgitation, aortic regurgitation, and pulmonary sinus dilatation. He developed severe persistent pulmonary hypertension and worsening acute hypercapnic hypoxemic respiratory failure, and subsequently expired on day of life (DOL) 10 after compassionate extubation. Cytogenomic whole-genome SNP microarray analysis revealed a deletion within the FBN1 gene spanning exons 7–30, which overlapped with the exon deletion hotspot region associated with neonatal Marfan syndrome. Infant B is a term male prenatally diagnosed with isolated D-TGA. He required balloon atrial septostomy on DOL 0 and subsequent atrial switch operation, atrial septal defect repair, and patent ductus arteriosus ligation on DOL 5. Trio-WES revealed compound heterozygous c.518C>T and c.8230T>G variants in the FBN2 gene. Zygosity analysis confirmed each of the variants was inherited from one of the parents who were healthy heterozygous carriers. Since his cardiac repair at birth, he has been growing and developing well without any further hospitalization. Our study highlights novel FBN1/FBN2 variants and signifies the phenotype–genotype association in two infants affected with complex congenital heart defects with and without dysmorphic features. These findings speak to the importance of next-generation high-throughput genomics for novel variant detection and the phenotypic variability associated with FBN1/FBN2 variants, particularly in the neonatal period, which may significantly impact clinical care and family counseling.

Hydrocarbon industry flaring practices emit large amounts of unburned hydrocarbons and climate forcers, creating environmental and social impacts. The objective of this study is to thoroughly compare different flaring technologies in situations where the industry is unable to avoid flaring. An integrated methodology for assessing two flare technologies in terms of Health-Environment-Safety (HES) impacts, economic performance, and safety benefits is proposed. The methodology includes stoichiometric calculations of combustion reactions, pollutants emission estimation, flare dimensioning for an equivalent inflow stream to be flared, and economic scenarios for assessing Enclosed Ground Flares (EGFs) versus Open Flame Flares (OFFs). The methodology was applied to a Mexican hydrocarbon installation that currently operates OFFs with a historical record of low combustion efficiency. If an EGF were to replace the existing OFF, there would be a significant emissions abatement that depends on their realistic operating efficiencies. Furthermore, the EGF has the potential to utilize 1.55thermal GW for energy applications, which is currently being wasted as the OFF releases it into the environment by flaring 3.75 Mm3/d. The safety radius required for the OFF is 9.3 times that required for the EGF of equivalent capacity. Over a 30-year period for the case study, the cumulative investment and O&M costs for EGF are about 23% less than for OFF. The consequence analysis with the PHAST software found that various failure conditions can be contained within the EGF combustion chamber without causing significant damage to the facility. However, the impacts of disturbances in the OFF such as gas clouds, flash fires, and explosions are around the torch. All these findings indicate that replacing OFF with EGF would be a safer and more economical solution in the future, as it also helps reduce pollutant emissions.

The moving target defense (MTD) is a proactive cybersecurity defense technique that constantly changes potentially vulnerable points to be attacked, to confuse the attackers, making it difficult for attackers to infer the system configuration and nullify reconnaissance activities to a victim system. We consider an MTD strategy for software-defined networking (SDN) environment where every SDN switch is controlled by a central SDN controller. As the MTD may incur excessive usage of the network/system resources for cybersecurity purposes, we propose to perform the MTD operations adaptively according to the security risk assessment based on a Bayesian attack graph (BAG) analysis. For accurate BAG analysis, we model random and weakest-first attack behaviors and incorporate the derived analytical models into the BAG analysis. Using the BAG analysis result, we formulate a knapsack problem to determine the optimal set of vulnerabilities to be reconfigured under a constraint of SDN reconfiguration overhead. The experiment results prove that the proposed MTD strategy outperforms the full MTD and random MTD counterparts in terms of the maximum/average of attack success probabilities and the number of SDN reconfiguration updates.

The integrator complex is required for the synthesis of protein coding and non-coding RNA and contains the protein INTS13. Here, the authors find germline mutations in INTS13 in two families with oral facial digital syndrome and show that the mutation affects the c-terminal domain of the protein and disrupts cilliogenesis.

A method to isolate and sequence individual nuclei from human and mouse muscle biopsies provides further insight into the mechanisms of dystrophin loss and repair, in the context of Duchenne muscular dystrophy.

This study applies stable carbon isotope labelling to study polymer biodegradation in soils. This labelling enables accurate and precise tracking of polymer carbon during biodegradation and, thereby, provides a holistic picture of this process.

Duchenne Muscular Dystrophy (DMD)’s complex multi-system pathophysiology, coupled with the cost-prohibitive logistics of multi-year drug screening and follow-up, has hampered the pursuit of new therapeutic approaches. Here we conducted a systematic historical and text mining-based pilot feasibility study to explore the potential of established or previously tested drugs as prospective DMD therapeutic agents. Our approach utilized a Swanson linking-inspired method to uncover meaningful yet largely hidden deep semantic connections between pharmacologically significant DMD targets and drugs developed for unrelated diseases. Specifically, we focused on molecular target-based MeSH terms and categories as high-yield bioinformatic proxies, effectively tagging relevant literature with categorical metadata. To identify promising leads, we comprehensively assembled published reports from 2011 and sampling from subsequent years. We then determined the earliest year when distinct MeSH terms or category labels of the relevant cellular target were referenced in conjunction with the drug, as well as when the pertinent target itself was first conclusively identified as holding therapeutic value for DMD. By comparing the earliest year when the drug was identifiable as a DMD treatment candidate with that of the first actual report confirming this, we computed an Index of Delayed Discovery (IDD), which serves as a metric of Swanson-linked latent knowledge. Using these findings, we identified data from previously unlinked articles subsetted via MeSH-derived Swanson linking or from target classes within the DrugBank repository. This enabled us to identify new but untested high-prospect small-molecule candidates that are of particular interest in repurposing for DMD and warrant further investigations.

Muscle damage and fibro-fatty replacement of skeletal muscles is a main pathologic feature of Duchenne muscular dystrophy (DMD) with more proximal muscles affected earlier and more distal affected later in the disease course, suggesting that different skeletal muscle groups possess distinctive characteristics that influence their susceptibility to disease. To explore transcriptomic factors driving differential gene expression and modulating DMD skeletal muscle severity, we characterized the transcriptome of vastus lateralis (VL), a more proximal and susceptible muscle, relative to tibialis anterior (TA), a more distal and protected muscle, in 15 healthy individuals using bulk RNA sequencing to identify gene expression differences that may mediate their relative susceptibility to damage with loss of dystrophin. Matching single nuclei RNA sequencing data was generated for 3 of the healthy individuals, to infer cell composition in the bulk RNA sequencing dataset and to improve mapping of differentially expressed genes to their cell source of expression. A total of 3,410 differentially expressed genes were identified and mapped to cell type using single nuclei RNA sequencing of muscle, including long non-coding RNAs and protein coding genes. There was an enrichment of genes involved in calcium release from the sarcoplasmic reticulum, particularly in the myofibers and these myofiber genes were higher in the VL. There was an enrichment of genes in “Collagen-Containing Extracellular Matrix” expressed by fibroblasts, endothelial, smooth muscle and pericytes, with most genes higher in the TA, as well as genes in “Regulation Of Apoptotic Process” expressed across all cell types. Previously reported genetic modifiers were also enriched within the differentially expressed genes. We also identify 6 genes with differential isoform usage between the VL and TA. Lastly, we integrate our findings with DMD RNA sequencing data from the TA, and identify “Collagen-Containing Extracellular Matrix” and “Negative Regulation Of Apoptotic Process” as differentially expressed between DMD compared to healthy. Collectively, these findings propose novel candidate mechanisms that may mediate differential muscle susceptibility in muscular dystrophies and provide new insight into potential therapeutic targets.

Patterns of habitat selection for sympatric urial Ovis vignei and Siberian ibex Capra sibirica are poorly known, in part because there are few places where such overlap exists. Using envelope modeling methodology, we analyzed location data of these species in the Hindu Kush range along the Wakhan Valley of the Wakhan National Park (10,950 km2) in northeastern Afghanistan, recorded during field surveys in April-May of 2011, 2015, and 2018. Distribution models showed significant ecological niche differences (P 0) and showed the same avoidance of lower quality areas. Understanding selection criteria of habitat use by urial and ibex in Wakhan Valley, inhabited by over 14,000 people and their livestock (ca. 78,000), will enable adjustments to the protection schemes regarding the requirements of two key mountain ungulate species critical to the sustainability and conservation of this unique ecosystem. This type of information is very scarce in the literature for the sympatric mountain ungulates in Asia.

Abstract Objective Military veterans have high rates of noise‐induced hearing loss (NIHL) which is associated with more significant spiral ganglion neuronal loss. This study explores the relationship between NIHL and cochlear implant (CI) outcomes in veterans. Study Design Retrospective case series of veterans who underwent CI between 2019 and 2021. Setting Veterans Health Administration hospital. Methods AzBio Sentence Test, Consonant‐Nucleus‐Consonant (CNC) scores, and Speech, Spatial, and Qualities of Hearing Scale (SSQ) were measured pre‐ and postoperatively. Linear regression assessed relationships between outcomes and noise exposure history, etiology of hearing loss, duration of hearing loss, and Self‐Administered Gerocognitive Exam (SAGE) scores. Results Fifty‐two male veterans were implanted at an average (standard deviation) age of 75.0 (9.2) years without major complications. The average duration of hearing loss was 36.0 (18.4) years. The average time of hearing aid use was 21.2 (15.4) years. Noise exposure was reported in 51.3% of patients. Objectively, AzBio and CNC scores 6 months postoperatively showed significant improvement of 48% and 39%, respectively. Subjectively, average 6‐month SSQ scores showed significant improvement by 34 points (p

Technological advancement of vehicle platforms exposes opportunities for new attack paths and vulnerabilities. Static cyber defenses can help mitigate certain attacks, but those attacks must generally be known ahead of time, and the cyber defenses must be hand-crafted by experts. This research explores reinforcement learning (RL) as a path to achieve autonomous, intelligent cyber defense of vehicle control networks—namely, the controller area network (CAN) bus. We train an RL agent for the CAN bus using Toyota’s Portable Automotive Security Testbed with Adaptability (PASTA). We then apply the U.S. Army Combat Capabilities Development Command (DEVCOM) Army Research Laboratory’s methodology for quantitative measurement of cyber resilience to assess the agent’s effect on the vehicle testbed in a contested cyberspace environment. Despite all defenses having similar traditional performance measures, our RL agent averaged a 90% cyber resilience measurement during drive cycles executed on hardware versus 41% for a naïve static timing defense and 98% for the bespoke timing-based defense. Our results also show that an RL-based agent can detect and block injection attacks on a vehicle CAN bus in a laboratory environment with greater cyber resilience than prior learning approaches (1% for convolutional networks and 0% for recurrent networks). With further research, we believe there is potential for using RL in the autonomous intelligent cyber defense agent concept.

Abstract Background We aimed to define the clinical and variant spectrum and to provide novel molecular insights into the DHX30-associated neurodevelopmental disorder. Methods Clinical and genetic data from affected individuals were collected through Facebook-based family support group, GeneMatcher, and our network of collaborators. We investigated the impact of novel missense variants with respect to ATPase and helicase activity, stress granule (SG) formation, global translation, and their effect on embryonic development in zebrafish. SG formation was additionally analyzed in CRISPR/Cas9-mediated DHX30-deficient HEK293T and zebrafish models, along with in vivo behavioral assays. Results We identified 25 previously unreported individuals, ten of whom carry novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment, and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30, trigger SG formation, interfere with global translation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented with a milder clinical course, similar to an individual harboring a de novo mosaic HCM missense variant. Functionally, we established DHX30 as an ATP-dependent RNA helicase and as an evolutionary conserved factor in SG assembly. Based on the clinical course, the variant location, and type we establish two distinct clinical subtypes. DHX30 loss-of-function variants cause a milder phenotype whereas a severe phenotype is caused by HCM missense variants that, in addition to the loss of ATPase and helicase activity, lead to a detrimental gain-of-function with respect to SG formation. Behavioral characterization of dhx30-deficient zebrafish revealed altered sleep-wake activity and social interaction, partially resembling the human phenotype. Conclusions Our study highlights the usefulness of social media to define novel Mendelian disorders and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected individuals, clinicians, molecular genetics diagnostic laboratories, and research laboratories.

Abstract Background An identical homozygous missense variant in EIF3F, identified through a large-scale genome-wide sequencing approach, was reported as causative in nine individuals with a neurodevelopmental disorder, characterized by variable intellectual disability, epilepsy, behavioral problems and sensorineural hearing-loss. To refine the phenotypic and molecular spectrum of EIF3F-related neurodevelopmental disorder, we examined independent patients. Results 21 patients were homozygous and one compound heterozygous for c.694T>G/p.(Phe232Val) in EIF3F. Haplotype analyses in 15 families suggested that c.694T>G/p.(Phe232Val) was a founder variant. All affected individuals had developmental delays including delayed speech development. About half of the affected individuals had behavioral problems, altered muscular tone, hearing loss, and short stature. Moreover, this study suggests that microcephaly, reduced sensitivity to pain, cleft lip/palate, gastrointestinal symptoms and ophthalmological symptoms are part of the phenotypic spectrum. Minor dysmorphic features were observed, although neither the individuals’ facial nor general appearance were obviously distinctive. Symptoms in the compound heterozygous individual with an additional truncating variant were at the severe end of the spectrum in regard to motor milestones, speech delay, organic problems and pre- and postnatal growth of body and head, suggesting some genotype–phenotype correlation. Conclusions Our study refines the phenotypic and expands the molecular spectrum of EIF3F-related syndromic neurodevelopmental disorder.

Hearing loss and peripheral neuropathy are two clinical entities that are genetically and phenotypically heterogeneous and sometimes co-occurring. Using exome sequencing and targeted segregation analysis, we investigated the genetic etiology of peripheral neuropathy and hearing loss in a large Ashkenazi Jewish family. Moreover, we assessed the production of the candidate protein via western blotting of lysates from fibroblasts from an affected individual and an unaffected control. Pathogenic variants in known disease genes associated with hearing loss and peripheral neuropathy were excluded. A homozygous frameshift variant in the BICD1 gene, c.1683dup (p.(Arg562Thrfs*18)), was identified in the proband and segregated with hearing loss and peripheral neuropathy in the family. The BIDC1 RNA analysis from patient fibroblasts showed a modest reduction in gene transcripts compared to the controls. In contrast, protein could not be detected in fibroblasts from a homozygous c.1683dup individual, whereas BICD1 was detected in an unaffected individual. Our findings indicate that bi-allelic loss-of-function variants in BICD1 are associated with hearing loss and peripheral neuropathy. Definitive evidence that bi-allelic loss-of-function variants in BICD1 cause peripheral neuropathy and hearing loss will require the identification of other families and individuals with similar variants with the same phenotype.

Abstract Objective The risk of SARS‐CoV‐2 transmission to healthcare workers through airborne aerosolization during otologic surgery has not been characterized. The objective of this study was to describe and quantify the aerosol generation during common otologic procedures in both cadaveric surgical simulation and live patient surgery. Methods The number concentrations of generated aerosols in the particle size range of 0.30 to 10.0 μm were quantified using an optical particle sizer during both a cadaveric simulation of routine otologic procedures as well as cochlear implant surgery on live patients in the operating room. Results In the cadaveric simulation, temporalis fascia graft harvest using cold techniques (without electrocautery) (n = 4) did not generate aerosols above baseline concentrations. Tympanoplasty (n = 3) and mastoidectomy (n = 3) both produced statistically significant increases in concentrations of aerosols (P

Abstract Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches have poor precision, require large amounts of tissue, and are difficult to standardize. This paper presents an immuno-mass spectrometry imaging method using gadolinium (Gd)-labeled anti-dystrophin antibodies and laser ablation-inductively coupled plasma-mass spectrometry to simultaneously quantify and localize dystrophin in muscle sections. Gd is quantified as a proxy for the relative expression of dystrophin and was validated in murine and human skeletal muscle sections following k-means clustering segmentation, before application to DMD patients with different gene mutations where dystrophin expression was measured up to 100 µg kg−1 Gd. These results demonstrate that immuno-mass spectrometry imaging is a viable approach for pre-clinical to clinical research in DMD. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation.

In vitro models of patient-derived muscle allow for more efficient development of genetic medicines for the muscular dystrophies, which often present mutation-specific pathologies. One popular strategy to generate patient-specific myotubes involves reprogramming dermal fibroblasts to a muscle lineage through MyoD induction. However, creating physiologically relevant, reproducible tissues exhibiting multinucleated, aligned myotubes with organized striations is dependent on the introduction of physicochemical cues that mimic the native muscle microenvironment. Here, we engineered patient-specific control and dystrophic muscle tissues in vitro by culturing and differentiating MyoD–directly reprogrammed fibroblasts isolated from one healthy control subject, three patients with Duchenne muscular dystrophy (DMD), and two Limb Girdle 2A/R1 (LGMD2A/R1) patients on micromolded gelatin hydrogels. Engineered DMD and LGMD2A/R1 tissues demonstrated varying levels of defects in α-actinin expression and organization relative to control, depending on the mutation. In genetically relevant DMD tissues amenable to mRNA reframing by targeting exon 44 or 45 exclusion, exposure to exon skipping antisense oligonucleotides modestly increased myotube coverage and alignment and rescued dystrophin protein expression. These findings highlight the value of engineered culture substrates in guiding the organization of reprogrammed patient fibroblasts into aligned muscle tissues, thereby extending their value as tools for exploration and dissection of the cellular and molecular basis of genetic muscle defects, rescue, and repair.

Abstract Exome or genome sequencing was performed to identify the genetic etiology for the clinical presentation of global developmental delay, intellectual disability, and sensorimotor neuropathy with associated distal weakness in two unrelated families. A homozygous frameshift variant c.186delA (p.A63Qfs*3) in the NUDT2 gene was identified in cases 1 and 2 from one family and a third case from another family. Variants in NUDT2 were previously shown to cause intellectual disability, but here we expand the phenotype by demonstrating its association with distal upper and lower extremity weakness due to a sensorimotor polyneuropathy with demyelinating and/or axonal features.

Objective: To determine whether earlier hospital discharge is feasible and safe in selected patients with subarachnoid hemorrhage (SAH) using an outpatient “fast-track” protocol. Patients and Methods: We conducted a prospective quality improvement cohort study with the primary feasibility end point of patients with SAH deemed safe for discharge by treating team consensus. All patients received detailed education and outpatient transcranial Doppler monitoring; caregivers could contact the on-call team 24-7. Primary safety end points were adverse events after discharge and hospital readmission. Results: From January 1, 2010, to January 1, 2015, our center had 377 SAH diagnoses, of which 200 were included in the final cohort, 36 qualifying for fast-track early discharge. The 30-day readmission rate for fast-track patients was 11.0% (4 of 36) compared with 11.4% (18 of 164) for non–fast-track patients. The rate of delayed cerebral ischemia and stroke was 3% (1 of 36) in the fast-track group vs 25.0% (41 of 164) for the non–fast-track group. Adverse events occurred in 11.0% (4 of 36) of the fast-track group compared with 26.0% (43 of 164) in the non–fast-track group. The mean length of stay was reduced 60% from 15 days to 6.6 days in the fast-track group. Conclusion: Although our fast-track group was relatively small, data suggested early feasibility and safety in a carefully selected group of patients with SAH. Direct and indirect financial benefits of early discharge over a 5-year period were an estimated savings at least $864,000 in overall costs. A comparative effectiveness study is planned to replicate and validate these results using a larger multicenter design.

We report a case of hypertrophic cardiomyopathy and lactic acidosis in a 3-year-old female. Cardiac and skeletal muscles biopsies exhibited mitochondrial hyperplasia with decreased complex IV activity. Whole exome sequencing identified compound heterozygous variants, p.Arg333Trp and p.Val119Leu, in TSFM, a nuclear gene that encodes a mitochondrial translation elongation factor, resulting in impaired oxidative phosphorylation and juvenile hypertrophic cardiomyopathy.

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines. Keywords: Duchenne muscular dystrophy, combination therapy, dantrolene, dystrophin, exon skipping, armgo, ARM210, muscle, therapy

Speeding remains a major and consistent contributor to motor vehicle fatalities in the U.S. Using a parallel convergent mixed methods approach, we compared minor, moderate, and extreme U.S. speeder types (N = 293) to gain a better understanding of why U.S. drivers speed to the degree that they do. An online survey collected quantitative data on self-reported characteristics, speeding behaviors, and roadway behaviors after key events (collisions, speeding tickets) and given passenger presence. Analysis of qualitative data from participants explaining their roadway behaviors and beliefs identified several speeding-related themes. Themes shared to the same extent by all speeder types include not typically citing speeding a cause of a collision and good drivers not needing to change speeding behaviors (i.e., perceived mastery). However, the permanence and extent of speed reductions after key events and with passengers did vary by speeder type, with minor speeders making more extensive and permanent changes. Qualitative and quantitative data both underscore the perception that speeding is not a high priority threat to roadway safety. Findings suggest continued research using conservative and nuanced definitions of speeding is warranted to understand how similar perceptions result in a spectrum of speeding behaviors.

Abstract While intraosseous cranial myxoma is a rare pathology, it is important for providers to be aware of it, as early diagnosis and treatment is imperative for prognosis. Long‐term follow‐up is needed as high rates of recurrence have been documented.

That faculty matter to student learning is a widely accepted article of faith with substantial empirical support. Indeed, based on their review of several thousand studies of college student development, Pascarella and Terenzini (1991, 611) concluded that "there can be little doubt about the need for faculty members? acceptance of their roles and responsibilities for student learning and for their active involvement in students' lives." But as times change, so do student characteristics and aspirations as well as the demands on institutions and faculty, all of which presumably influence the nature and frequency of student-faculty contact, inside and outside the classroom. A particularly worrisome national trend is hiring part-time instructors--many of whom teach at two or more universities in the same academic term--in lieu of full-time faculty members with continuing contracts (Benjamin 2001). As a result, it's conceivable that face-to-face exchanges between students and faculty outside the classroom will decline because part-time faculty spend less time on campus and often do not have a designated space to meet with students after class. Relatively little is known about the effects of these and other trends and policies on student learning.

Summary: Mutations in the gene encoding the type II transmembrane protease 3 (TMPRSS3) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3-related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3-KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders. : The role of TMPRSS3 in auditory hair cell (HC) remains unclear. Nelson and colleagues generated stem cell-derived inner ear organoids with Tmprss3 mutations, which demonstrate that (1) inner ear organoids exhibit comparative effects of the genetic abnormality to its in vivo counterparts, (2) Tmprss3 mutations lead to HC apoptosis and reduced BK channels, and (3) TMPRSS3 localizes to the cell membrane. Keywords: deafness, degeneration, disease modeling, embryonic stem cell, hair cell, inner ear, organoid, TMPRSS3, vestibular system

Urban trees are often more sun- and wind-exposed than their forest-grown counterparts. These environmental differences can impact how many species grow–impacting trunk taper, crown spread, branch architecture, and other aspects of tree form. Given these differences, windthrow models derived from traditional forest production data sources may not be appropriate for urban forest management. Additionally, visual abnormalities historically labeled as “defects” in timber production, may not have a significant impact on tree failure potential. In this study, we look at urban tree failures associated with Hurricane Irma in Tampa, Florida, USA. We used spatial analysis to determine if patterns of failure existed among our inventoried trees. We also looked at risk assessment data to determine which visual defects were the most common and the most likely to be associated with branch or whole-tree failure. Results indicate that there was no spatial pattern associated with the observed tree failures–trees failed or withstood the storm as individuals. While some defects like decay and dead wood were associated with increased tree failure, other defects such as weak branch unions and poor branch architecture were less problematic.

Objectives To gather the perspectives of collegiate instructors regarding how concussion is managed within the college classroom. To introduce the themes surrounding collegiate return-to-learn (RTL) and the classroom management of students with concussion.Design Qualitative grounded theory.Setting Large, public university in the Midwest.Participants Twenty-three college instructors participated in a private, semistructured, audio-recorded, one-on-one interview. Participants included 12 males and 11 females. Interview recordings were transcribed verbatim, followed by an iterative process of open-coding and axial-coding, performed by two researchers.Results Three themes emerged from the coded data: (1) awareness—external knowledge of concussion and previous experiences, (2) legitimacy—medical note provided and no note provided and (3) accommodating the student—instructor’s role and feasibility of the accommodation. Psychosocial factors such as small class sizes, graduate-level students and an instructor’s empathy appeared to influence an instructor’s decision making when accommodating a student recovering from concussion.Conclusion These novel data provide foundational evidence regarding how college instructors perceive and subsequently manage concussion within the classroom, while also offering accuracy to aims of subsequent collegiate RTL investigationsArticle summary RTL is an emerging field within concussion management, yet is grossly underexplored within the college setting. By utilising a grounded theory approach, this article introduces the themes that dictate the landscape of RTL for a college student.

IntroductionMacrophage expressed gene 1 (MPEG1) is highly expressed in macrophages and other phagocytes. The gene encodes a bactericidal pore-forming protein, dubbed Perforin-2. Structural-, animal-, and cell-based studies have established that perforin-2 facilitates the destruction of phagocytosed microbes upon its activation within acidic phagosomes. Relative to wild-type controls, Mpeg1 knockout mice suffer significantly higher mortality rates when challenged with gram-negative or -positive pathogens. Only four variants of MPEG1 have been functionally characterized, each in association with pulmonary infections. Here we report a new MPEG1 non-sense variant in a patient with the a newly described association with persistent polymicrobial infections of the skin and soft tissue.Case DescriptionA young adult female patient was evaluated for recurrent abscesses and cellulitis of the breast and demonstrated a heterozygous, rare variant in MPEG1 p.Tyr430*. Multiple courses of broad-spectrum antimicrobials and surgical incision and drainage failed to resolve the infection. Functional studies revealed that the truncation variant resulted in significantly reduced capacity of the patient’s phagocytes to kill intracellular bacteria. Patient-derived macrophages responded to interferon gamma (IFN-γ) by significantly increasing the expression of MPEG1. IFN-γ treatment supported perforin-2 dependent bactericidal activity and wound healing.ConclusionsThis case expands the phenotype of MPEG1 deficiency to include severe skin and soft tissue infection. We showed that haploinsufficiency of perforin-2 reduced the bactericidal capacity of human phagocytes. Interferon-gamma therapy increases expression of perforin-2, which may compensate for such variants. Thus, treatment with IFN-γ could help prevent infections.

We investigate mutations in trβ2, a splice variant of thrb, identifying changes in function, structure, and behavior in larval and adult zebrafish retinas. Two N-terminus CRISPR mutants were identified. The first is a 6BP+1 insertion deletion frameshift resulting in a truncated protein. The second is a 3BP in frame deletion with intact binding domains. ERG recordings of isolated cone signals showed that the 6BP+1 mutants did not respond to red wavelengths of light while the 3BP mutants did respond. 6BP+1 mutants lacked optomotor and optokinetic responses to red/black and green/black contrasts. Both larval and adult 6BP+1 mutants exhibit a loss of red-cone contribution to the ERG and an increase in UV-cone contribution. Transgenic reporters show loss of cone trβ2 activation in the 6BP+1 mutant but increase in the density of cones with active blue, green, and UV opsin genes. Antibody reactivity for red-cone LWS1 and LWS2 opsin was absent in the 6BP+1 mutant, as was reactivity for arrestin3a. Our results confirm a critical role for trβ2 in long-wavelength cone development.

We report two brothers with severe global cognitive and motor delay, cortical visual impairment and sick sinus syndrome who were born to consanguineous parents. Standard genetic evaluations did not reveal the cause of their mental retardation. As expected, chromosomal microarray (CMA) revealed extensive regions of homozygosity. Exome sequencing revealed that both affected boys were homozygous for a nonsense mutation in the G-protein β5 (GNB5) gene (NM_016194.3:c.1032C > G; Tyr344Ter), and that the parents were carriers of this mutation. No other DNA variants that were explanatory for the sick sinus or the developmental delay/intellectual disability were identified, and no other clinical parameters are likely to have contributed to this unusual combination of phenotypes. The neurologic features of our patients are more severe than those of most of the other patients previously reported with GNB5 variants, probably because of the homozygous, complete loss-of-function (nonsense/stop-gain) nature of their variant, and their clinical course has been monitored for longer duration.

A new annually resolved sedimentary record of Southern Hemisphere mid-latitude hydroclimate was recovered from Lake Ohau, South Island, New Zealand, in March 2016. The Lake Ohau Climate History (LOCH) project acquired cores from two sites (LOCH-1 and -2) that preserve sequences of laminated mud that accumulated since the lake formed ∼  17 000 years ago. Cores were recovered using a purpose-built barge and drilling system designed to recover soft sediment from thick sedimentary sequences in lake systems up to 150 m deep. This system can be transported in two to three 40 ft long shipping containers and is suitable for use in a range of geographic locations. A comprehensive suite of data has been collected from the sedimentary sequence using state-of-the-art analytical equipment and techniques. These new observations of past environmental variability augment the historical instrumental record and are currently being integrated with regional climate and hydrological modelling studies to explore causes of variability in extreme/flood events over the past several millennia.

Abstract Background With the expanding use of next-gen sequencing (NGS) to diagnose the thousands of rare Mendelian genetic diseases, it is critical to be able to interpret individual DNA variation. To calculate the significance of finding a rare protein-altering variant in a given gene, one must know the frequency of seeing a variant in the general population that is at least as damaging as the variant in question. Methods We developed a general method to better interpret the likelihood that a rare variant is disease causing if observed in a given gene or genic region mapping to a described protein domain, using genome-wide information from a large control sample. Based on data from 2504 individuals in the 1000 Genomes Project dataset, we calculated the number of individuals who have a rare variant in a given gene for numerous filtering threshold scenarios, which may be used for calculating the significance of an observed rare variant being causal for disease. Additionally, we calculated mutational burden data on the number of individuals with rare variants in genic regions mapping to protein domains. Results We describe methods to use the mutational burden data for calculating the significance of observing rare variants in a given proportion of sequenced individuals. We present SORVA, an implementation of these methods as a web tool, and we demonstrate application to 20 relevant but diverse next-gen sequencing studies. Specifically, we calculate the statistical significance of findings involving multi-family studies with rare Mendelian disease and a large-scale study of a complex disorder, autism spectrum disorder. If we use the frequency counts to rank genes based on intolerance for variation, the ranking correlates well with pLI scores derived from the Exome Aggregation Consortium (ExAC) dataset (ρ = 0.515), with the benefit that the scores are directly interpretable. Conclusions We have presented a strategy that is useful for vetting candidate genes from NGS studies and allows researchers to calculate the significance of seeing a variant in a given gene or protein domain. This approach is an important step towards developing a quantitative, statistics-based approach for presenting clinical findings.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD “exon skipping” uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the results of statistical tests from both the quadriceps and diaphragm assessing contributions of dantrolene beyond AON, across all AON treatment groups, provide strong evidence that dantrolene modestly boosts exon skipping and dystrophin rescue while reducing muscle pathology in mdx mice (p < 0.0087). These findings support a trial of combination dantrolene/AON to increase exon-skipping efficacy and highlight the value of combinatorial approaches and Food and Drug Administration (FDA) drug re-purposing for discovery of unsuspected therapeutic application and rapid translation.

One of the key challenges in atmospheric chemistry is to reduce the uncertainty of biogenic volatile organic compound (BVOC) emission estimates from vegetation to the atmosphere. In Australia, eucalypt trees are a primary source of biogenic emissions, but their contribution to Australian air sheds is poorly quantified. The Model of Emissions of Gases and Aerosols from Nature (MEGAN) has performed poorly against Australian isoprene and monoterpene observations. Finding reasons for the MEGAN discrepancies and strengthening our understanding of biogenic emissions in this region is our focus. We compare MEGAN to the locally produced Australian Biogenic Canopy and Grass Emissions Model (ABCGEM), to identify the uncertainties associated with the emission estimates and the data requirements necessary to improve isoprene and monoterpene emissions estimates for the application of MEGAN in Australia. Previously unpublished, ABCGEM is applied as an online biogenic emissions inventory to model BVOCs in the air shed overlaying Sydney, Australia. The two models use the same meteorological inputs and chemical mechanism, but independent inputs of leaf area index (LAI), plant functional type (PFT) and emission factors. We find that LAI, a proxy for leaf biomass, has a small role in spatial, temporal and inter-model biogenic emission variability, particularly in urban areas for ABCGEM. After removing LAI as the source of the differences, we found large differences in the emission activity function for monoterpenes. In MEGAN monoterpenes are partially light dependent, reducing their dependence on temperature. In ABCGEM monoterpenes are not light dependent, meaning they continue to be emitted at high rates during hot summer days, and at night. When the light dependence of monoterpenes is switched off in MEGAN, night-time emissions increase by 90–100 % improving the comparison with observations, suggesting the possibility that monoterpenes emitted from Australian vegetation may not be as light dependent as vegetation globally. Targeted measurements of emissions from in situ Australian vegetation, particularly of the light dependence issue are critical to improving MEGAN for one of the world's major biogenic emitting regions.

Abstract Interleukin 33 (IL-33) is among the earliest-released cytokines in response to allergens that orchestrate type 2 immunity. The prolyl cis-trans isomerase PIN1 is known to induce cytokines for eosinophil survival and activation by stabilizing cytokines mRNAs, but the function of PIN1 in upstream signaling pathways in asthma is unknown. Here we show that interleukin receptor associated kinase M (IRAK-M) is a PIN1 target critical for IL-33 signaling in allergic asthma. NMR analysis and docking simulations suggest that PIN1 might regulate IRAK-M conformation and function in IL-33 signaling. Upon IL-33-induced airway inflammation, PIN1 is activated for binding with and isomerization of IRAK-M, resulting in IRAK-M nuclear translocation and induction of selected proinflammatory genes in dendritic cells. Thus, the IL-33-PIN1-IRAK-M is an axis critical for dendritic cell activation, type 2 immunity and IL-33 induced airway inflammation.

Passive air samplers (PASs) for gaseous mercury (Hg) were deployed for time periods between 1 month and 1 year at 20 sites across the globe with continuous atmospheric Hg monitoring using active Tekran instruments. The purpose was to evaluate the accuracy of the PAS vis-à-vis the industry standard active instruments and to determine a sampling rate (SR; the volume of air stripped of gaseous Hg per unit of time) that is applicable across a wide range of conditions. The sites spanned a wide range of latitudes, altitudes, meteorological conditions, and gaseous Hg concentrations. Precision, based on 378 replicated deployments performed by numerous personnel at multiple sites, is 3.6 ± 3.0 %1, confirming the PAS's excellent reproducibility and ease of use. Using a SR previously determined at a single site, gaseous Hg concentrations derived from the globally distributed PASs deviate from Tekran-based concentrations by 14.2 ± 10 %. A recalibration using the entire new data set yields a slightly higher SR of 0.1354 ± 0.016 m3 day−1. When concentrations are derived from the PAS using this revised SR the difference between concentrations from active and passive sampling is reduced to 8.8 ± 7.5 %. At the mean gaseous Hg concentration across the study sites of 1.54 ng m−3, this represents an ability to resolve concentrations to within 0.13 ng m−3. Adjusting the sampling rate to deployment specific temperatures and wind speeds does not decrease the difference in active–passive concentration further (8.7 ± 5.7 %), but reduces its variability by leading to better agreement in Hg concentrations measured at sites with very high and very low temperatures and very high wind speeds. This value (8.7 ± 5.7 %) represents a conservative assessment of the overall uncertainty of the PAS due to inherent uncertainties of the Tekran instruments. Going forward, the recalibrated SR adjusted for temperature and wind speed should be used, especially if conditions are highly variable or deviate considerably from the average of the deployments in this study (9.89 °C, 3.41 m s−1). Overall, the study demonstrates that the sampler is capable of recording background gaseous Hg concentrations across a wide range of environmental conditions with accuracy similar to that of industry standard active sampling instruments. Results at sites with active speciation units were inconclusive on whether the PASs take up total gaseous Hg or solely gaseous elemental Hg primarily because gaseous oxidized Hg concentrations were in a similar range as the uncertainty of the PAS. 1Subscripted numbers are not significant, but are reported to reduce rounding errors in subsequent studies (see Sect. 2.3 for details).

Abstract Background Disorders of sex development (DSD) have an estimated frequency of 0.5% of live births encompassing a variety of urogenital anomalies ranging from mild hypospadias to a discrepancy between sex chromosomes and external genitalia. In order to identify the underlying genetic etiology, we had performed exome sequencing in a subset of DSD cases with 46,XY karyotype and were able to identify the causative genetic variant in 35% of cases. While the genetic etiology was not ascertained in more than half of the cases, a large number of variants of unknown clinical significance (VUS) were identified in those exomes. Methods To investigate the relevance of these VUS in regards to the patient’s phenotype, we utilized a mouse model in which the presence of a Y chromosome from the poschiavinus strain (Y POS ) on a C57BL/6J (B6) background results in XY undervirilization and sex reversal, a phenotype characteristic to a large subset of human 46,XY DSD cases. We assessed gene expression differences between B6-Y B6 and undervirilized B6-Y POS gonads at E11.5 and identified 515 differentially expressed genes (308 underexpressed and 207 overexpressed in B6-Y POS males). Results We identified 15 novel candidate genes potentially involved in 46,XY DSD pathogenesis by filtering the list of human VUS-carrying genes provided by exome sequencing with the list of differentially expressed genes from B6-Y POS mouse model. Additionally, we identified that 7 of the 15 candidate genes were significantly underexpressed in the XY gonads of mice with suppressed Sox9 expression in Sertoli cells suggesting that some of the candidate genes may be downstream of a well-known sex determining gene, Sox9. Conclusion The use of a DSD-specific animal model improves variant interpretation by correlating human sequence variants with transcriptome variation.

This study explores how often faculty members encourage students to engage with campus, local, state, national, and global issues. Using data from the 2013 administration of the Faculty Survey of Student Engagement (FSSE), the results show that faculty members are more likely to encourage students to engage in state, national, or global issues than campus or local issues. Differences in faculty encouragement of civic engagement are also presented across gender, racial/ethnic identification, rank and employment status, and institutional affiliation, among other characteristics. Implications for practice are provided.

The SAFIRED (Savannah Fires in the Early Dry Season) campaign took place from 29 May until 30 June 2014 at the Australian Tropical Atmospheric Research Station (ATARS) in the Northern Territory, Australia. The purpose of this campaign was to investigate emissions from fires in the early dry season in northern Australia. Measurements were made of biomass burning aerosols, volatile organic compounds, polycyclic aromatic carbons, greenhouse gases, radon, speciated atmospheric mercury and trace metals. Aspects of the biomass burning aerosol emissions investigated included; emission factors of various species, physical and chemical aerosol properties, aerosol aging, micronutrient supply to the ocean, nucleation, and aerosol water uptake. Over the course of the month-long campaign, biomass burning signals were prevalent and emissions from several large single burning events were observed at ATARS.Biomass burning emissions dominated the gas and aerosol concentrations in this region. Dry season fires are extremely frequent and widespread across the northern region of Australia, which suggests that the measured aerosol and gaseous emissions at ATARS are likely representative of signals across the entire region of north Australia. Air mass forward trajectories show that these biomass burning emissions are carried north-west over the Timor Sea and could influence the atmosphere over Indonesia and the tropical atmosphere over the Indian Ocean. Here we present characteristics of the biomass burning observed at the sampling site and provide an overview of the more specific outcomes of the SAFIRED campaign.

Abstract Background Massively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient’s phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100–300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as insertions, deletions, inversions, and translocations. Methods To overcome these limitations, we used next-generation mapping (NGM) to image high molecular weight double-stranded DNA molecules (megabase size) with fluorescent tags in nanochannel arrays for de novo genome assembly. We investigated the capacity of this NGM platform to identify pathogenic SV in a series of patients diagnosed with Duchenne muscular dystrophy (DMD), due to large deletions, insertion, and inversion involving the DMD gene. Results We identified deletion, duplication, and inversion breakpoints within DMD. The sizes of deletions were in the range of 45–250 Kbp, whereas the one identified insertion was approximately 13 Kbp in size. This method refined the location of the break points within introns for cases with deletions compared to current polymerase chain reaction (PCR)-based clinical techniques. Heterozygous SV were detected in the known carrier mothers of the DMD patients, demonstrating the ability of the method to ascertain carrier status for large SV. The method was also able to identify a 5.1-Mbp inversion involving the DMD gene, previously identified by RNA sequencing. Conclusions We showed the ability of NGM technology to detect pathogenic structural variants otherwise missed by PCR-based techniques or chromosomal microarrays. NGM is poised to become a new tool in the clinical genetic diagnostic strategy and research due to its ability to sensitively identify large genomic variations.