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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.

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.

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.

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.

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds.

We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of β2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process.

Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation.

BackgroundThe carbohydrate portions of salivary glycoproteins play important roles, including mediating bacterial and leukocyte adhesion. Salivary glycosylation is complex. Many of its glycoproteins present ABO and Lewis blood group determinants. An individual's genetic complement and secretor status govern the expression of blood group antigens. We queried the extent to which salivary glycosylation varies according to blood group and secretor status. First, we screened submandibular/sublingual and parotid salivas collected as ductal secretions for reactivity with a panel of 16 lectins. We selected three lectins that reacted with the largest number of glycoproteins and one that recognized uncommon lactosamine-containing structures. Ductal salivas representing a secretor with complex blood group expression and a nonsecretor with a simple pattern were separated by SDS-PAGE. Gel slices were trypsin digested and the glycopeptides were individually separated on each of the four lectins. The bound fractions were de-N-glycosylated. LC-MS/MS identified the original glycosylation sites, the peptide sequences, and the parent proteins.ResultsThe results revealed novel salivary N-glycosites and glycoproteins not previously reported. As compared to the secretor, nonsecretor saliva had higher levels of N-glycosylation albeit with simpler structures.ConclusionsTogether, the results suggested a molecular basis for inter-individual variations in salivary protein glycosylation with functional implications for oral health.

Community-based mental health (CMH) services play an important, but relatively understudied role in the identification and treatment of youth with autism spectrum disorder (ASD) who may be receiving care for other psychiatric conditions. Little is known about the role of standardized ASD assessment measures administered by providers working in generalist community-based mental health (CMH) settings. This pilot study extracted data from three CMH clinics to examine the use of the Autism Diagnostic Observation Schedule (ADOS) by 17 CMH providers who received ASD assessment training with 62 youth (Mean = 10.69 years) referred for an ASD diagnostic evaluation. Results indicated that 57% of youths assessed ultimately received an ASD diagnosis. All cases given a final ASD diagnosis were classified as "Autism" or "ASD" on the ADOS. Seventy percent of youth who did not receive a final ASD diagnosis were classified as "Non-Spectrum" on the ADOS. In these false positive cases, report narratives indicated that social communication difficulties identified on the ADOS were explained by symptoms of other mental health conditions (e.g., ADHD, anxiety). Future research is needed to examine the utility of the ADOS when used by CMH providers to facilitate CMH capacity to identify ASD.

HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specificmemory B cells encoding Abs that mediate ADCC froma subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activitywhen compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise fromunique B-cell lineages in the same individual. Further, thesemAbsmediate potent plasma IgG-specificADCC breadth and potency and contribute to ADCC activity in other HIV-infected individuals.

Endometriosis, ectopic growth of the uterine lining (endometrium), which affects 6-11% of reproductive age women, is associated with pelvic pain and infertility. We investigated the peritoneal fluid (PF), urine and omental fat (OF) proteomes of women with endometriosis vs. individuals with no surgically visualized endometriosis. All participants were enrolled in the NICHD-funded ENDO Study. A two-step proteomic study was performed. The first, a broad survey, employed a semi-quantitative gel LC-mass spectrometry (MS) workflow: SDS PAGE fractionation, trypsin digestion and LC-MS/MS. The results showed sample integrity but failed to detect any differences between women with and without endometriosis. The second step was a quantitative analysis of OF samples. We employed another sample set (n=30) from women ± disease and isobaric mass-tag (iTRAQ) chemistry to label peptides and 2D LC-MS/MS for protein identification and quantification. Three proteins-matrix metalloproteinase-9, neutrophil elastase, and FAM49B-were significantly lower in abundance in samples from women with endometriosis. Interestingly, neutrophil elastase and FAM49B levels were associated with higher levels of a subset of endocrine disrupting chemicals (EDCs) that were previously measured in the same samples. The results of these experiments showed the feasibility of associating endometriosis with changes in the OF protein repertoire and EDC levels.Biological significanceEndometriosis, pathological growth of the uterine lining, is associated with significant morbidities, including pain and infertility. However, the causes of this common condition are poorly understood. This study determined whether endometriosis was associated with changes in the protein composition of peritoneal fluid, urine and/or omental fat. A protein of unknown function (FAM49B) and two proteinases (metalloproteinase-9, neutrophil elastase) were down regulated in OF samples from women with versus without endometriosis. These findings suggested proteinase imbalances at sites that were distant from the endometriotic lesions. Additionally, FAM49B and neutrophil elastase levels were associated with higher levels of a subset of environmental chemicals that were quantified in the same samples, suggesting other possible associations. Thus, this work generated hypotheses that will be tested in further studies.

BackgroundHepatoblastoma is a rare pediatric liver tumor that has significantly increased in incidence over the last several decades. The International Agency for Research on Cancer (IARC) recently classified hepatoblastoma as a tobacco-related cancer. Parental alcohol use has shown no association. We examined associations between parental tobacco and alcohol use around the time of pregnancy and hepatoblastoma in a large case-control study.MethodsMaternal interviews were completed for 383 cases diagnosed in the United States during 2000-2008. Controls (n = 387) were identified through U.S. birth registries and frequency-matched to cases on birth weight, birth year, and region of residence. We used unconditional logistic regression to calculate ORs and 95% confidence intervals (CI) for associations between parental smoking and maternal drinking and offspring hepatoblastoma.ResultsWe found no association between hepatoblastoma and maternal smoking at any time (OR, 1.0; 95% CI, 0.7-1.4), within the year before pregnancy (OR, 1.1; 95% CI, 0.8-1.6), early in pregnancy (OR, 1.0; 95% CI, 0.7-1.6), or throughout pregnancy (OR, 0.9; 95% CI, 0.5-1.6). We observed marginally positive associations between hepatoblastoma and paternal smoking in the year before pregnancy (OR, 1.4; 95% CI, 1.0-2.0) and during pregnancy (OR, 1.4; 95% CI, 0.9-2.0). Maternal alcohol use was not associated with hepatoblastoma.ConclusionOur results do not provide evidence for an etiologic relationship between maternal smoking or drinking and hepatoblastoma, and only weak evidence for an association for paternal smoking in the year before pregnancy.ImpactOur study provides limited support for hepatoblastoma as a tobacco-related cancer; however, it remains wise to counsel prospective parents on the merits of smoking cessation.

Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue.