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Veteran access to sleep medicine is of paramount importance to the Veterans Health Administration (VA). To increase access, VA has created community referral policies and programs, as well as telehealth programs. In 2017, the Office of Rural Health (ORH) funded a TeleSleep initiative focused on reaching rural Veterans with unmet sleep needs. ORH provided 3-6 years of funding to help 19 hubs support 98 spoke sites serving rural Veterans. As ORH funding concluded, each hub identified its path to sustainment. This case study follows one TeleSleep hub in VAs western geographic region as it transitioned from ORH funding sustainment as a regional Sleep Clinical Resource Hub. This case study describes the real-world process of adaptation in care delivery strategies. One key area of adaptation revolved around whether to deliver care via the patients home facility or the providers home facility. In early 2021, the TeleSleep team implemented an innovative provider transfer model, where temporary reinforcements from the TeleSleep hub increased the workforce capacity of spoke sites, similar to the concept of locum tenens. In this provider transfer model, TeleSleep clinicians scheduled, documented, and billed for each encounter at the Veterans home facility. Positioning TeleSleep clinicians as local providers facilitated communication and referrals and promoted continuity and quality of care for Veterans in their home facility. This provider transfer model reduced the administrative burden of providers and schedulers and supported patient-side-only documentation of care. While this mirrors current locum tenens practice, transferring providers did not fit VAs financial model as implemented by the western regions Sleep Clinical Resource Hub. Therefore, in December 2021, VA aligned TeleSleep with VAs preferred practice of patient rather than provider transfers. In the patient transfer model, providers schedule and document in both the provider and patient electronic health records, and bill in the providers facility. However, reflecting on this period of innovation, TeleSleep team members concluded that the provider transfer model could improve patient safety and care coordination while reducing the administrative burden of frontline clinicians. Further research and development are needed to align the provider transfer model with VAs financial model.

Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing. In this study, we aimed to develop an osteoanabolic therapy which activates the Wnt/β-catenin pathway, a molecular driver of endochondral ossification. We hypothesize that using an mRNA-based therapeutic encoding β-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes. To optimize a delivery platform built on recent advancements in liposomal technologies, two FDA-approved ionizable phospholipids, DLin-MC3-DMA (MC3) and SM-102, were used to fabricate unique ionizable lipid nanoparticle (LNP) formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model. Using firefly luciferase mRNA as a reporter gene to track and quantify transfection, SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection, minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs. The generated β-cateninGOF mRNA encapsulated in SM-102 LNPs (SM-102-β-cateninGOF mRNA) showed bioactivity in vitro through upregulation of downstream canonical Wnt genes, axin2 and runx2. When testing SM-102-β-cateninGOF mRNA therapeutic in a murine tibia fracture model, histomorphometric analysis showed increased bone and decreased cartilage composition with the 45 μg concentration at 2 weeks post-fracture. μCT testing confirmed that SM-102-β-cateninGOF mRNA promoted bone formation in vivo, revealing significantly more bone volume over total volume in the 45 μg group. Thus, we generated a novel mRNA-based therapeutic encoding a β-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery.

BACKGROUND: Remote monitoring (RM) of patients with cardiovascular implantable electronic devices (CIEDs) (pacemakers and implantable cardioverter-defibrillators) has a Class 1, Level of Evidence A Heart Rhythm Society recommendation. Yet RM adherence varies widely across settings, and factors associated with variation are not understood. OBJECTIVE: The purpose of this study was to identify strategies for supporting RM across Veterans Health Administration (VHA) facilities. METHODS: In a national evaluation, we surveyed and interviewed 27 nurses, medical instrument technicians, and advanced practice providers across 26 VHA facilities (following approximately 15,000 CIED patients). Participants were selected based on overall patient adherence by facility, which ranged from 46%-96%. Questions covered RM adherence strategies, manufacturer resources, organizational characteristics, and workflows for optimizing adherence. RESULTS: All clinicians reported that RM adherence was extremely important (53.8%), very important (34.6%), or important (11.5%) for improving patient outcomes. High performing facilities prioritized consistent patient education about RM and evaluated nonadherence using dashboards and manufacturer web sites. High performing facilities instituted clear standard operating procedures that defined staff responsibilities and facilitated efficient contact with nonadherent patients and then family members by phone and then mail. Clinicians based at high performing facilities spent twice as many hours per week (9.1) on average managing RM adherence compared to other facilities (4.5). Effective communication (internally and with non-VHA care partners) and use of CIED manufacturer resources were essential. Facilities that were not high performing rarely used these strategies. CONCLUSION: Clinicians can support high RM adherence by emphasizing patient education, regularly assessing and addressing nonadherence using staff protocols, and engaging CIED manufacturers.

TGFβ signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFβ signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFβ and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFβ/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFβ therapy efficacy. Our data suggest that TGFβ works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants and recognized developmental toxicants that are detectable in placental tissues. Higher levels of in utero PBDE exposure have been associated with an increased risk of adverse birth outcomes. During pregnancy, cytotrophoblasts (CTBs) from the placenta play critical roles in the formation of the maternal-fetal interface via uterine invasion and vascular remodeling. The differentiation of these cells towards an invasive phenotype is crucial for proper placental development. We previously have shown that BDE-47 can impact CTB viability and hinder the ability of these cells to migrate and invade. To expand on potential toxicological mechanisms, we utilized quantitative proteomic approaches to identify changes in the global proteome of mid-gestation primary human CTBs after exposure to BDE-47. Using sequential window acquisition of all theoretical fragment-ion spectra (SWATH), we identified 3024 proteins in our CTB model of differentiation/invasion. Over 200 proteins were impacted as a function of BDE-47 exposure (1 μM and 5 μM) across the treatment period (15, 24, and 39 h). The differentially expressed molecules displayed time- and concentration-dependent changes in expression and were enriched in pathways associated with aggregatory and adhesive processes. Network analysis identified CYFIP1, a molecule previously unexplored in a placental context, to be dysregulated at BDE-47 concentrations previously seen to impact CTB migration/invasion. Our SWATH-MS dataset thus demonstrates BDE-47 impacts the global proteome of differentiating CTBs and serves as a valuable resource for further understanding of the relationship between environmental chemical exposures and placental development and function. AVAILABILITY OF DATA AND MATERIAL: Raw chromatograms are deposited on the MassIVE proteomic database (https://massive.ucsd.edu) under accession number MSV000087870. Normalized relative abundances are also available as Table S1.

STUDY OBJECTIVES: The Veterans Health Administration cares for many veterans with sleep disorders who live in rural areas. The Veterans Health Administrations Office of Rural Health funded the TeleSleep Enterprise-Wide Initiative (EWI) to improve access to sleep care for rural veterans through creation of national telehealth networks. METHODS: The TeleSleep EWI consists of (1) virtual synchronous care, (2) home sleep apnea testing, and (3) REVAMP (Remote Veterans Apnea Management Platform), a patient- and provider-facing web application that enabled veterans to actively engage with their sleep care and sleep care team. The TeleSleep EWI was designed as a hub-and-spoke model, where larger sites with established sleep centers care for smaller, rural sites with a shortage of providers. Structured formative evaluation for the TeleSleep EWI is supported by the Veterans Health Administrations Quality Enhancement Research Initiative and was critical in assessing outcomes and effectiveness of the program. RESULTS: The TeleSleep EWI launched with 7 hubs and 34 spokes (2017) and rapidly expanded to 13 hubs and 63 spokes (2020). The TeleSleep EWI resulted in a significant increase in rural veterans accessing sleep care by utilizing home sleep apnea testing to establish a diagnosis of obstructive sleep apnea and virtual care for follow-up. Rates of virtual care utilization were greater in hubs and spokes participating in the TeleSleep EWI compared with non-EWI sleep programs. Additionally, veterans expressed satisfaction with their virtual care TeleSleep experiences. CONCLUSIONS: The TeleSleep EWI successfully increased sleep care access for rural veterans, promoted adoption of virtual care services, and resulted in high patient satisfaction. CITATION: Chun VS, Whooley MA, Williams K, et al. Veterans Health Administration TeleSleep Enterprise-Wide Initiative 2017-2020: bringing sleep care to our nations veterans. J Clin Sleep Med. 2023;19(5):913-923.

This case study describes, for the time frame of June 2021 through August 2022, the U.S. Veterans Health Administration (VHA) organizational response to a manufacturers recall of positive airway pressure devices used in the treatment of sleep disordered breathing. VHA estimated it could take over a year for Veterans to receive replacement devices. Veterans awaiting a replacement faced a dilemma. They could continue using the recalled devices and bear the product safety risks that led to the recall, or they could stop using them and bear the risks of untreated sleep disordered breathing. Using a program monitoring approach, we report on the processes VHA put in place to respond to the recall. Specifically, we report on the strategic, service, and operational plans associated with VHAs response to the recall for Veterans needing replacement devices. In program monitoring, the strategic plan reflects the internal process objectives for the program. The service plan articulates how the delivery of services will intersect the customer journey. The operational plan describes how the programs resources and actions must support the service delivery plan. VHAs strategic plan featured a clinician-led, as opposed to primarily legal or administrative response to the recall. The recall response team also engaged with VHAs medical ethics service to articulate an ethical framework guiding the allocation of replacement devices under conditions of scarcity. This framework proposed allocating scarce devices to Veterans according to their clinical need. The service plan invited Veterans to schedule visits with sleep providers who could assess their clinical need and counsel them accordingly. The operational plan distributed devices according to clinical need as they became available. Monitoring our program processes in real time helped VHA launch and adapt its response to a recall affecting more than 700,000 Veterans.

Approximately 12% of histone H2B molecules in mammalian brain contain a modification wherein Asp25 is present as the D-enantiomer, and is mostly linked to Gly26 via the side-chain carboxyl. Here we (1) demonstrate the high specificity of a polyclonal antibody to this modification, and (2) use this Ab to demonstrate that this modification is enriched in brain relative to liver, thymus, and HeLa cells.

Open pedagogy is growing in popularity as an instructional method to decentralize classroom power dynamics, engage students, and provide greater meaning to student work. To investigate the impact of open pedagogy on motivation, interviews were conducted with first-year college students at a four-year liberal arts college after completing a semester-long project based on this pedagogical approach. Student responses were assessed using self-determination theory as a theoretical framework, particularly in relation to the motivation regulatory styles displayed by research participants. Results indicate that students experienced various forms of extrinsic motivation during the project based on open pedagogy, with autonomous forms of regulation being more prevalent than controlled regulation. Interview data also suggest that agency plays a role in mediating the internalization of student motivation. Based on these findings, suggestions are provided to the design of assignments in general and open pedagogy specifically to enhance development of autonomous forms of motivation.

Introduction: Impaired fracture healing, specifically non-union, has been found to occur up to 14% in tibial shaft fractures. The current standard of care to treat non-union often requires additional surgeries which can result in long recovery times. Injectable-based therapies to accelerate fracture healing have the potential to mitigate the need for additional surgeries. Gene therapies have recently undergone significant advancements due to developments in nanotechnology, which improve mRNA stability while reducing immunogenicity. Methods: In this study, we tested the efficacy of mineral coated microparticles (MCM) and fluoride-doped MCM (FMCM) to effectively deliver firefly luciferase (FLuc) mRNA lipoplexes (LPX) to the fracture site. Here, adult mice underwent a tibia fracture and stabilization method and all treatments were locally injected into the fracture. Level of osteogenesis and amount of bone formation were assessed using gene expression and histomorphometry respectively. Localized and systemic inflammation were measured through gene expression, histopathology scoring and measuring C-reactive protein (CRP) in the serum. Lastly, daily IVIS images were taken to track and measure transfection over time. Results: MCM-LPX-FLuc and FMCM-LPX-FLuc were not found to cause any cytotoxic effects when tested in vitro. When measuring the osteogenic potential of each mineral composition, FMCM-LPX-FLuc trended higher in osteogenic markers through qRT-PCR than the other groups tested in a murine fracture and stabilization model. Despite FMCM-LPX-FLuc showing slightly elevated il-1β and il-4 levels in the fracture callus, inflammation scoring of the fracture callus did not result in any differences. Additionally, an acute systemic inflammatory response was not observed in any of the samples tested. The concentration of MCM-LPX-FLuc and FMCM-LPX-FLuc that was used in the murine fracture model did not stimulate bone when analyzed through stereological principles. Transfection efficacy and kinetics of delivery platforms revealed that FMCM-LPX-FLuc prolongs the luciferase signal both in vitro and in vivo. Discussion: These data together reveal that FMCM-LPX-FLuc could serve as a promising mRNA delivery platform for fracture healing applications.

BACKGROUND: In fiscal year 2021, the Veterans Health Administration (VHA) provided care for sleep disorders to 599,966 Veterans, including 189,932 rural Veterans. To further improve rural access, the VA Office of Rural Health developed the TeleSleep Enterprise-Wide Initiative (EWI). TeleSleeps telemedicine strategies include tests for sleep apnea at the Veterans home rather than in a sleep lab; Clinical Video Telehealth applications; and other forms of virtual care. In 2017 and 2020, VHA provided 3-year start-up funding to launch new TeleSleep programs at rural-serving VA medical facilities. METHODS: In early 2022, we surveyed leaders of 24 sites that received TeleSleep funding to identify successes, failures, facilitators, and barriers relevant to sustaining TeleSleep implementations upon expiration of startup funding. We tabulated frequencies on the multiple choice questions in the survey, and, using the surveys critical incident framework, summarized the responses to open-ended questions. TeleSleep program leaders discussed the responses and synthesized recommendations for improvement. RESULTS: 18 sites reported sustainment, while six were on track. Sustainment involved medical centers or regional entities incorporating TeleSleep into their budgets. Facilitators included: demonstrating value; aligning with local priorities; and collaborating with spoke sites serving rural Veterans. Barriers included: misalignment with local priorities; and hiring delays. COVID was a facilitator, as it stimulated adoption of telehealth practices; and also a barrier, as it consumed attention and resources. Recommendations included: longer startup funding; dedicated funding for human resources to accelerate hiring; funders communicating with local facility leaders regarding how TeleSleep aligns with organizational priorities; hiring into job classifications aligned with market pay; and obtaining, from finance departments, projections and outcomes for the return on investment in TeleSleep.

Students acting as co-creators of academic material is growing in popularity as a pedagogical approach in higher education. With student engagement and persistence consistently being emphasized for student and institution well-being, educational praxis must foster engaged, high-retention student cohorts. This exploratory research uses a mixed-methods approach to examine the experience of students participating in a first-year course utilizing OER-enabled Pedagogy. Students considered how projects that were open impacted their perception of course engagement, satisfaction, and overall experience. Participants also evaluated their level of concern in sharing attributed academic work. A plurality of students preferred the project using OER-enabled Pedagogy, indicating it increased engagement and skills acquisition. The majority of students were unconcerned about sharing work publicly, even if their names were included. Themes that emerged from interviews included the motivational value of creating work potentially valuable to others, being given agency, and receiving public credit for their efforts.

COVID-19, in addition to disrupting the global education system in general, is widening the economic and racial gaps institutions have spent years trying to address. The economic reality is that students who work to support themselves, their families, and purchase educational materials needed to succeed have been disproportionately harmed. This article discusses how the global COVID-19 pandemic is compounding structural inequities inherent in higher education. This requires faculty to reevaluate their role as agents of change in a world that is fundamentally different than it was a short time ago. The experience of one liberal arts institution in the U.S. who moved all courses to free materials in under six months will be recounted as an example of what is possible during extraordinary circumstances if students are truly prioritized during strategic planning.

BackgroundAdherence to childhood immunization schedules is a function of various factors. Given the increased use of technology as a strategy to increase immunization coverage, it is important to investigate how parents perceive different forms of communication, including traditional means and text-message reminders. ObjectiveTo examine current forms of communication about immunization information, parents’ satisfaction levels with these communication modes, perceived barriers and benefits to using text messaging, and the ideal content of text messages for immunization reminders. MethodsStructured interviews were developed and approved by two Institutional Review Boards. A convenience sample of 50 parents was recruited from two local pediatric clinics. The study included a demographics questionnaire, the shortened form of the Test of Functional Health Literacy for Adults (S-TOFHLA), questions regarding benefits and barriers of text communication from immunization providers, and preferred content for immunization reminders. Content analyses were performed on responses to barriers, benefits, and preferred content (all Cohen’s kappas > 0.70). ResultsRespondents were mostly female (45/50, 90%), white non-Hispanic (31/50, 62%), between 20–41 years (mean = 29, SD 5), with one or two children (range 1–9). Nearly all (48/50, 96%) had an S-TOFHLA score in the “adequate” range. All parents (50/50, 100%) engaged in face-to-face contact with their child’s physician at appointments, 74% (37/50) had contact via telephone, and none of the parents (0/50, 0%) used email or text messages. Most parents were satisfied with the face-to-face (48/50, 96%) and telephone (28/50, 75%) communication. Forty-nine of the 50 participants (98%) were interested in receiving immunization reminders by text message, and all parents (50/50, 100%) were willing to receive general appointment reminders by text message. Parents made 200 comments regarding text-message reminders. Benefits accounted for 63.5% of comments (127/200). The remaining 37.5% (73/200) regarded barriers; however, no barriers could be identified by 26% of participants (13/50). Parents made 172 comments regarding preferred content of text-message immunization reminders. The most frequently discussed topics were date due (50/172, 29%), general reminder (26/172, 26%), and child’s name (21/172, 12%). ConclusionsMost parents were satisfied with traditional communication; however, few had experienced any alternative forms of communication regarding immunizations. Benefits of receiving text messages for immunization reminders far outweighed the barriers identified by parents. Few barriers identified were text specific. Those that were, centered on cost if parents did not have unlimited texting plans.

OER-enabled pedagogy, one form of open pedagogy, is gaining popularity as a method for increasing student engagement and motivation. Realising the potential of this approach, however, depends on faculty implementation and reducing resistance to change. This study explores the experience of instructors during their first and second courses when facilitating OER-enabled pedagogy. Specifically, the research sought to determine how their view of the approach and teaching practices changed between classes, and if this prescribed experience increased their likelihood of using OER-enabled pedagogy in other classes. Interviews with faculty suggest that when teaching OER-enabled pedagogy for the first time, there is a lack of understanding of the value of this approach impacting their perceived mentorship of students. By the second iteration, however, instructors recognise an evolution in their view of teaching generally as well as OER-enabled pedagogy more specifically. This growth increases the quality of interaction with students and fosters broader use of open practices.

Wicked problems are large, complex problems involving multiple perspectives that present substantial future challenges. These challenges can be overwhelming for learners and pose difficulties in teaching for instructors. Herein a solutions-oriented teaching strategy that amalgamates proven active learning strategies is presented along with a step-by-step guide and materials list. Evidence of student learning is provided. This strategy provides students the opportunity to view complex, wicked problems from multiple perspectives and to visualize their role in future solutions.

To aid students during COVID-19, the University of Pikeville transitioned all undergraduate classes to no-cost alternatives for course textbooks. Additionally, the academic calendar was modified for the first time in the institution's 130-year history from a traditional 16- week semester to 8-week block scheduling. This case study explores strategies, approaches to corresponding with constituents, and lessons learned in leading an institution through two major curricular changes in under six months in response to a global pandemic.

SignificanceBisphenol A (BPA), found in many plastic products, has weak estrogenic effects that can be harmful to human health. Thus, structurally related replacements-bisphenol S (BPS) and bisphenol F (BPF)-are coming into wider use with very few data about their biological activities. Here, we compared the effects of BPA, BPS, and BPF on human mammary organoids established from normal breast tissue. BPS disrupted organoid architecture and induced supernumerary branching. At a proteomic level, the bisphenols altered the abundance of common targets and those that were unique to each compound. The latter included proteins linked to tumor-promoting processes. These data highlighted the importance of testing the human health effects of replacements that are structurally related to chemicals of concern.

Single-cell RNA-seq (scRNA-seq) has revolutionized modern genomics, but the large size of myotubes and myofibers has restricted use of scRNA-seq in skeletal muscle. For the study of muscle, single-nucleus RNA-seq (snRNA-seq) has emerged not only as an alternative to scRNA-seq, but as a novel method providing valuable insights into multinucleated cells such as myofibers. Nuclei within myofibers specialize at junctions with other cell types such as motor neurons. Nuclear heterogeneity plays important roles in certain diseases such as muscular dystrophies. We survey current methods of high-throughput single cell and subcellular resolution transcriptomics, including single-cell and single-nucleus RNA-seq and spatial transcriptomics, applied to satellite cells, myoblasts, myotubes and myofibers. We summarize the major myonuclei subtypes identified in homeostatic and regenerating tissue including those specific to fiber type or at junctions with other cell types. Disease-specific nucleus populations were found in two muscular dystrophies, FSHD and Duchenne muscular dystrophy, demonstrating the importance of performing transcriptome studies at the single nucleus level in muscle.

Labeling social groups can increase social essentialism (e.g., beliefs that group members are fundamentally the same), leading to greater discrimination and stigmatization. Labels can also increase stigma about mental illness (MI). Some mental health professionals claim that using "person-first" language can reduce stigma, but there is little empirical support for this, and no studies have investigated the relation between person-first language and social essentialism. Here, 513 adults read vignettes describing characters with MI, using person-first (e.g., "a person with autism"), identity-first (e.g., "an autistic person"), or generic noun language (e.g., "an autistic"). We assessed participants' stigmatizing and essentialist beliefs about characters and their MI. Reported stigma and essentialism were correlated. Person-first language reduced stigmatizing beliefs about individuals with some disorders, e.g., depression, but not others, e.g., autism. Relative to generic nouns, person-first language reduced essentialist beliefs about real mental illnesses, but not novel ones.

BackgroundObstructive sleep apnea is common among rural Veterans, however, access to diagnostic sleep testing, sleep specialists, and treatment devices is limited. To improve access to sleep care, the Veterans Health Administration (VA) implemented a national sleep telemedicine program. The TeleSleep program components included: 1) virtual clinical encounters; 2) home sleep apnea testing; and 3) web application for Veterans and providers to remotely monitor symptoms, sleep quality and use of positive airway pressure (PAP) therapy. This study aimed to identify factors impacting Veteran's participation, satisfaction and experience with the TeleSleep program as part of a quality improvement initiative.MethodsSemi-structured interview questions elicited patient perspectives and preferences regarding accessing and engaging with TeleSleep care. Rapid qualitative and matrix analysis methods for health services research were used to organize and describe the qualitative data.ResultsThirty Veterans with obstructive sleep apnea (OSA) recruited from 6 VA telehealth "hubs" participated in interviews. Veterans reported positive experiences with sleep telemedicine, including improvements in sleep quality, other health conditions, and quality of life. Access to care improved as a result of decreased travel burden and ability of both clinicians and Veterans to remotely monitor and track personal sleep data. Overall experiences with telehealth technology were positive. Veterans indicated a strong preference for VA over non-VA community-based sleep care. Patient recommendations for change included improving scheduling, continuity and timeliness of communication, and the equipment refill process.ConclusionsThe VA TeleSleep program improved patient experiences across multiple aspects of care including a reduction in travel burden, increased access to clinicians and remote monitoring, and patient-reported health and quality of life outcomes, though some communication and continuity challenges remain. Implementing telehealth services may also improve the experiences of patients served by other subspecialties or healthcare systems.

The rise in throughput and quality of long-read sequencing should allow unambiguous identification of full-length transcript isoforms. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here we develop and characterize long-read Split-seq (LR-Split-seq), which uses combinatorial barcoding to sequence single cells with long reads. Applied to the C2C12 myogenic system, LR-split-seq associates isoforms to cell types with relative economy and design flexibility. We find widespread evidence of changing isoform expression during differentiation including alternative transcription start sites (TSS) and/or alternative internal exon usage. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells.

Facioscapulohumeral dystrophy (FSHD) is associated with the upregulation of the DUX4 transcription factor and its target genes. However, low-frequency DUX4 upregulation in patient myocytes is difficult to detect and examining the relationship and dynamics of DUX4 and target gene expression has been challenging. Using RNAScope in situ hybridization with highly specific probes, we detect the endogenous DUX4 and target gene transcripts in situ in patient skeletal myotubes during 13-day differentiation in vitro. We found that the endogenous DUX4 transcripts primarily localize as foci in one or two nuclei as compared with the accumulation of the recombinant DUX4 transcripts in the cytoplasm. We also found the continuous increase of DUX4 and target gene-positive myotubes after Day 3, arguing against its expected immediate cytotoxicity. Interestingly, DUX4 and target gene expression become discordant later in differentiation with the increase of DUX4-positive/target gene-negative as well as DUX4-negative/target gene-positive myotubes. Depletion of DUX4-activated transcription factors, DUXA and LEUTX, specifically repressed a DUX4-target gene, KDM4E, later in differentiation, suggesting that after the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, the study provides important new insights into the dynamics of the DUX4 transcriptional network in FSHD patient myocytes.

Unlike English, German contains single words for “the day after tomorrow” (übermorgen) and “the day before yesterday” (vorgestern). How might these cross-linguistic differences influence children’s acquisition of time words? Prior work shows that English-speaking preschoolers learn the deictic status of time words (e.g., yesterday was in the past) long before learning their precise temporal locations (e.g., yesterday was exactly one day ago). Here we ask whether the set of time words influences children’s understanding of proximal (yesterday/tomorrow) and distal (day before yesterday/day after tomorrow) terms. English- and German-speaking 3- to 7-year-olds (N = 253) marked the temporal location of each term relative to today on a calendar template. While children in both language groups demonstrated equal knowledge of deictic status, German speakers were more likely to have precise meanings for proximal and distal items, suggesting that having more alternative time words available may help narrow the scope of children’s meanings.

Number line estimation (NLE) tasks are widely used to investigate numerical cognition, learning, and development, and as an instructional tool. Interpretation of these tasks generally involves an implicit expectation that responses are driven by the overall magnitudes of target numerals, in the sense that the particular digits conveying those magnitudes are unimportant. However, recent evidence shows that numbers with similar magnitudes but different leftmost digits are estimated very differently. For example, "798" is placed systematically much too far to the left of "801" in a 0-1000 NLE task by children aged 7-11 and adults. Here we ask whether this left digit effect generalizes to two-digit numerals in a 0-100 NLE task and whether it emerges in younger children. Children aged 5-8 (Study 1, N = 73), adults (Study 2, N = 44), and children aged 9-11 (Study 3, N = 27) completed a standard 0-100 NLE task on a touchscreen tablet. We observed left digit effects for two-digit numerals in children aged 8-11 and adults, with large effect sizes, demonstrating that these effects generalize to smaller numerical ranges. Left digit effects were not apparent in 5- to 7-year-olds, suggesting that these effects do not emerge at younger ages for smaller, more familiar numerical ranges. We discuss developmental emergence of left digit effects in number line estimation and implications within and beyond the field of cognitive development.

Reversing brain aging may be possible through systemic interventions such as exercise. We found that administration of circulating blood factors in plasma from exercised aged mice transferred the effects of exercise on adult neurogenesis and cognition to sedentary aged mice. Plasma concentrations of glycosylphosphatidylinositol (GPI)-specific phospholipase D1 (Gpld1), a GPI-degrading enzyme derived from liver, were found to increase after exercise and to correlate with improved cognitive function in aged mice, and concentrations of Gpld1 in blood were increased in active, healthy elderly humans. Increasing systemic concentrations of Gpld1 in aged mice ameliorated age-related regenerative and cognitive impairments by altering signaling cascades downstream of GPI-anchored substrate cleavage. We thus identify a liver-to-brain axis by which blood factors can transfer the benefits of exercise in old age.

FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes ("FSHD-Lo" and "FSHD Hi", respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression.

BackgroundYouth with autism spectrum disorder (ASD) represent a growing population with significant service needs. Prominent among these needs are high rates of co-occurring psychiatric conditions that contribute to increased functional impairments and often necessitate mental health services. Executive functioning deficits are associated with ASD as well as common co-occurring conditions (e.g., attention-deficit/hyperactivity disorder) and an evidence-based intervention has been developed and tested to address executive functioning within the school context. There is an urgent need to implement indicated evidence-based interventions for youth with ASD receiving care in community mental health settings. Interventions that optimally "fit" the mental health services context as well as the complex and co-occurring mental health needs of these youth have the potential to improve key clinical outcomes for this high priority population.MethodsThis mixed-methods developmental study will apply the Exploration, Preparation, Implementation, Sustainment implementation framework and a community-academic partnership approach to systematically adapt and test an evidence-based executive functioning intervention for youth with ASD for delivery in community mental health settings. Specific aims are to (1) conduct a need and context assessment to inform the systematic adaptation an executive functioning evidence-based intervention; (2) systematically adapt the clinical intervention and develop a corresponding implementation plan, together entitled "Executive Functioning for Enhancing Community-based Treatment for ASD," (EFFECT for ASD); and (3) conduct a feasibility pilot test of EFFECT for ASD in community mental health settings.DiscussionTailoring evidence-based interventions for delivery in community-based mental health services for youth with ASD has the potential to increase quality of care and improve child outcomes. Results from the current study will serve as the foundation for large-scale hybrid implementation and effectiveness trials and a generalizable approach for different service systems of care and clinical populations.Trial registrationClinicaltrials.gov, NCT04295512.