Ecuador’s annual mortality rate from SIDS is 0.4 per 100 000 people, 4 times higher than neighboring countries Peru, Bolivia, and Brazil. Modifying the infant sleep environment toward safe practice has been demonstrated to be the most effective risk reduction strategy in reducing mortality from SIDS and little is known about sleep practices in Ecuador. The purpose of this study is to describe baseline infant sleep intentions of pregnant women in a peri-urban, low resource community in Ecuador. We also aim to identify demographic and psychosocial factors associated with suboptimal sleep practices in this context to develop long-term strategies to identify infants with high risk for SIDS/SUID. A cross-sectional study design was employed with 100 women in their third trimester of pregnancy. The majority of women were partnered (82%), both parents had approximately 8 years of education, and over half reported that their incomes met or exceeded their basic needs (55%). Significant predictors of safer sleep intention included years of paternal education (P = .019) and income meeting their basic needs (P = .0049). For each additional year of paternal education, families were 23% more likely to report safer intended infant sleep practices. Compared to those whose income did not allow for basic needs, those who had sufficient income to meet (or exceed) basic needs were 425% more likely to report safer intended sleep practices. Targeted interventions to high-risk populations may reduce the burden of SIDS/SUID in this community.Project ECHO (Extension of Community Healthcare Outcomes) is an innovative model of online education which has been proposed to enhance access to palliative care in resource-limited settings. There is limited literature describing how health care providers in low-and middle-income countries benefit from and learn from this type of training. This qualitative description study explores the learning experiences of participants in a Project ECHO program on pediatric palliative care in South Asia through focus group discussions. Discussions were transcribed, coded, independently verified, and arranged into overarching themes. We identified learning themes including the importance of creating a supportive learning community; the opportunity to share ideas and experiences; gaining knowledge and skills, and access to additional learning materials. Designing future programs to ensure a supportive and interactive learning community with attention cultural challenges may enhance learning from future Project ECHO programs.

On average Black patients have longer LOS than comparable White patients. Longer hospital length of stay (LOS) may be associated with higher readmission risk. However, evidence suggests that the Hospital Readmission Reduction Program (HRRP) reduced overall racial differences in 30-day adjusted readmission risk. Yet, it is unclear whether the HRRP narrowed these LOS racial differences.

We examined the relationship between Medicare-insured Black-White differences in average, adjusted LOS (ALOS) and the HRRP’s implementation and evaluation periods.

Using 2009-2017 data from State Inpatient Dataset from New York, New Jersey, and Florida, we employed an interrupted time series analysis with multivariate generalized regression models controlling for patient, disease, and hospital characteristics. Results are reported per 100 admissions.

We found that for those discharged home, Black-White ALOS differences significantly widened by 4.15 days per 100 admissions (95% CI 1.19 to 7.11,

< 0.001) for targeted conditions from before to after the HRRP implementation period, but narrowed in the HRRP evaluation period by 1.84 days per 100 admissions for every year-quarter (95% CI -2.86 to -0.82,

< 0.001); for those discharged to non-home destinations, there was no significant change between HRRP periods, but ALOS differences widened over the study period. Black-White ALOS differences for non-targeted conditions remained unchanged regardless of HRRP phase and discharge destination.

Increased LOS for Black patients may have played a role in reducing Black-White disparities in 30-day readmission risks for targeted conditions among patients discharged to home.

Increased LOS for Black patients may have played a role in reducing Black-White disparities in 30-day readmission risks for targeted conditions among patients discharged to home.Successful implementation of cooling strategies obviously depends on identifying effective interventions, but in industrial settings, it is equally important to consider feasibility and economic viability. Many cooling interventions are available, but the decision processes affecting adoption by end-users are not well elucidated. We therefore arranged two series of meetings with stakeholders to identify knowledge gaps, receive feedback on proposed cooling interventions, and discuss factors affecting implementation of heat-health interventions. This included four meetings attended by employers, employees, and health and safety officers (n = 41), and three meetings attended primarily by policy makers (n = 74), with feedback obtained via qualitative and quantitative questionnaires and focus group discussions. On a 10-point scale, both employers and employees valued worker safety (9.1 ± 1.8; mean±SD) and health (8.5 ± 1.9) as more important than protecting company profits (6.3 ± 2.3). Of the respondents, 41% were unaware of any cooling strategies at their company and of those who were aware, only 30% thought the interventions were effective. Following presentation of proposed interventions, the respondents rated „facilitated hydration”, „optimization of clothing/protective equipment”, and „rescheduling of work tasks” as the top-three preferred solutions. The main barriers for adopting cooling interventions were cost, feasibility, employer perceptions, and legislation. In conclusion, preventing negative health and safety effects was deemed to be more important than preventing productivity loss. Regardless of work sector or occupation, both health and wealth were emphasized as important parameters and considered as somewhat interrelated. However, a large fraction of the European worker force lacks information on effective measures to mitigate occupational heat stress. List of abbreviations OH-Stress Occupational heat stress; WBGT Wet Bulb Globe Temperature.Heat strain impairs performance across a broad spectrum of sport disciplines. The impeding effects of hyperthermia and dehydration are often ascribed to compromised cardiovascular and muscular functioning, but expert performance also depends on appropriately tuned sensory, motor and cognitive processes. Considering that hyperthermia has implications for central nervous system (CNS) function and fatigue, it is highly relevant to analyze how heat stress forecasted for the upcoming Olympics may influence athletes. This paper proposes and demonstrates the use of a framework combining expected weather conditions with a heat strain and motor-cognitive model to analyze the impact of heat and associated factors on discipline- and scenario-specific performances during the Tokyo 2021 games. We pinpoint that hyperthermia-induced central fatigue may affect prolonged performances and analyze how hyperthermia may impair complex motor-cognitive performance, especially when accompanied by either moderate dehydration or exposure to severe solar radiation. Interestingly, several short explosive performances may benefit from faster cross-bridge contraction velocities at higher muscle temperatures in sport disciplines with little or no negative heat-effect on CNS fatigue or motor-cognitive performance. In the analyses of scenarios and Olympic sport disciplines, we consider thermal impacts on „motor-cognitive factors” such as decision-making, maximal and fine motor-activation as well as the influence on central fatigue and pacing. From this platform, we also provide perspectives on how athletes and coaches can identify risks for their event and potentially mitigate negative motor-cognitive effects for and optimize performance in the environmental settings projected.Due to time and logistical constraints sweat samples cannot always be analyzed immediately. The purpose of this study was to investigate the effect of storage temperature and duration on sweat electrolyte and metabolite concentrations. Twelve participants cycled for 60 min at 40 W.m-2 in 33°C and 65% RH. Using the absorbent patch technique, six sweat samples were collected from the posterior torso. Sweat from the six samples was mixed, divided again over six samples and placed in sealed vials. Sweat sodium, chloride, potassium, ammonia, lactate and urea concentrations in one sample were determined immediately. Two samples were stored at room temperature (~25°C, 42% RH) for 7 and 28 days respectively. The remaining samples were frozen at -20°C for 1 h, 7 or 28 days respectively before analysis. Sweat sodium, chloride, potassium and urea concentrations were not affected by storage temperature and duration. Sweat lactate decreased (-1.8 ± 1.8 mmol.L-1, P = 0.007) and ammonia concentrations increased (5.1 ± 3.9 mmol.L-1, P = 0.017) after storage for 28 days at 25°C only. The storage temperature and duration did not affect sodium, chloride, potassium and urea concentrations. However, sweat samples should not be stored for longer than 7 days at 25°C to obtain reliable sweat lactate and ammonia concentrations. When samples are frozen at -20°C, the storage duration could be extended to 28 days for these components.The environmental conditions during the Tokyo Olympic and Paralympic Games are expected to be challenging, which increases the risk for participating athletes to develop heat-related illnesses and experience performance loss. To allow safe and optimal exercise performance of Dutch elite athletes, the Thermo Tokyo study aimed to determine thermoregulatory responses and performance loss among elite athletes during exercise in the heat, and to identify personal, sports-related, and environmental factors that contribute to the magnitude of these outcomes. For this purpose, Dutch Olympic and Paralympic athletes performed two personalized incremental exercise tests in simulated control (15°C, relative humidity (RH) 50%) and Tokyo (32°C, RH 75%) conditions, during which exercise performance and (thermo)physiological parameters were obtained. Thereafter, athletes were invited for an additional visit to conduct anthropometric, dual-energy X-ray absorptiometry (DXA), and 3D scan measurements. Collected data also served as input for a thermophysiological computer simulation model to estimate the impact of a wider range of environmental conditions on thermoregulatory responses. Findings of this study can be used to inform elite athletes and their coaches on how heat impacts their individual (thermo)physiological responses and, based on these data, advise which personalized countermeasures (i.e. heat acclimation, cooling interventions, rehydration plan) can be taken to allow safe and maximal performance in the challenging environmental conditions of the Tokyo 2020 Olympic and Paralympic Games.Contingency management (CM) has robust evidence of effectiveness as an adjunct to medication for opioid use disorders. However, CM implementation in opioid treatment programs has been limited by a myriad of well-documented barriers. One relatively unexplored barrier that may hinder CM implementation is health professional stigma toward patients with opioid use disorders. Qualitative interviews were conducted with 43 health professionals (21 leaders, 22 front-line counselors) from 11 different opioid treatment programs across Rhode Island to explore their familiarity with CM and to elucidate barriers and facilitators to CM implementation. Interviews were transcribed and coded by 3 independent raters using a reflexive team approach. Transcripts were analyzed for both a priori and emergent themes. Health professional stigma was identified as an emergent major theme with 4 distinct subthemes (a) distrust of patients (44%, N = 19); (b) infantilizing views about patients (19%, N = 8); (c) belief that patients do not deserve prizes (19%, N = 8); and (d) recognition of patient self-stigma and community-based stigma (23%, N = 10). In addition, we identified multiple instances of health professional use of potentially stigmatizing language toward patients with opioid use disorders via terms such as drug abuser, addict, and clean or dirty urine screens (70%, N = 30). Stigma themes were identified in 86% of the transcripts, highlighting potential targets for multilevel implementation strategies. Findings of this study suggest that multiple types of health professional stigma should be considered and proactively addressed in efforts by psychologists to implement CM and other evidence-based interventions in opioid treatment programs.Early coverage of myelomeningocele (MMC) defects within the post-delivery period is crucial for decreasing mortality rates. Herein, we report the case of a premature 5-day-old male neonate with large MMC defect successfully managed using a quadruple V-Y rotation advancement flap (butterfly technique), an effective surgical technique for large MCC defects.The observation of biological structures in live cells beyond the diffraction limit with super-resolution fluorescence microscopy is limited by the ability of fluorescence probes to permeate live cells and the effect of these probes, which are often toxic, on cellular behavior. Here we present a coherent confocal light scattering and absorption spectroscopic microscopy that for the first time enables the use of large numerical aperture optics to characterize structures in live cells down to 10 nm spatial scales, well beyond the diffraction limit. Not only does this new capability allow high resolution microscopy with light scattering contrast, but it can also be used with almost any light scattering spectroscopic application which employs lenses. We demonstrate that the coherent light scattering contrast based technique allows continuous temporal tracking of the transition from non-cancerous to an early cancerous state in live cells, without exogenous markers. We also use the technique to sense differences in the aggressiveness of cancer in live cells and for label free identification of different grades of cancer in resected tumor tissues.Imaging-based single-cell analysis is essential to study the expression level and functions of biomolecules at subcellular resolution. However, its low throughput has prevented the measurement of numerous cellular features from multiples cells in a rapid and efficient manner. Here we report 2.5D microscopy that significantly improves the throughput of fluorescence imaging systems while maintaining high-resolution and single-molecule sensitivity. Instead of sequential z-scanning, volumetric information is projected onto a 2D image plane in a single shot by engineering the emitted fluorescence light. Our approach provides an improved imaging speed and uniform focal response within a specific imaging depth, which enabled us to perform quantitative single-molecule RNA measurements over a 2×2 mm2 region within an imaging depth of ~5 μm for mammalian cells in 30 Hz volumetric frame rate with reduced photobleaching. Our microscope also offers the ability of multi-color imaging, depth control and super-resolution imaging.Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. In vitro, CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, in vivo infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable in vivo antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion in vivo. To investigate this in vitro versus in vivo discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of in vivo expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope.The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.The human small intestine is the key organ for absorption, metabolism, and excretion of orally administered drugs. To preclinically predict these reactions in drug discovery research, a cell model that can precisely recapitulate the in vivo human intestinal monolayer is desired. In this study, we developed a monolayer platform using human biopsy-derived duodenal organoids for application to pharmacokinetic studies. The human duodenal organoid-derived monolayer was prepared by a simple method in 3-8 days. It consisted of polarized absorptive cells and had tight junctions. It showed much higher cytochrome P450 (CYP)3A4 and carboxylesterase (CES)2 activities than did the existing models (Caco-2 cells). It also showed efflux activity of P-glycoprotein (P-gp) and inducibility of CYP3A4. Finally, its gene expression profile was closer to the adult human duodenum, compared to the profile of Caco-2 cells. Based on these findings, this monolayer assay system using biopsy-derived human intestinal organoids is likely to be widely adopted.Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency ( less then 0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (P KLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients.Muscle function and mass begin declining in adults long before evidence of sarcopenia and include reduced mitochondrial function, although much remains to be characterized. We found that mRNA decay factor AU-rich mRNA binding factor 1 (AUF1), which stimulates myogenesis, is strongly reduced in skeletal muscle of adult and older mice in the absence of evidence of sarcopenia. Muscle-specific adeno-associated virus (AAV)8-AUF1 gene therapy increased expression of AUF1, muscle function, and mass. AAV8 AUF1 muscle gene transfer in 12-month-old mice increased the levels of activated muscle stem (satellite) cells, increased muscle mass, reduced markers of muscle atrophy, increased markers of mitochondrial content and muscle fiber oxidative capacity, and enhanced exercise performance to levels of 3-month-old mice. With wild-type and AUF1 knockout mice and cultured myoblasts, AUF1 supplementation of muscle fibers was found to increase expression of Peroxisome Proliferator-activated Receptor Gamma Co-activator 1-alpha (PGC1α), a major effector of skeletal muscle mitochondrial oxidative metabolism. AUF1 stabilized and increased translation of the pgc1α mRNA, which is strongly reduced in adult muscle in the absence of AUF1 supplementation. Skeletal muscle-specific gene transfer of AUF1 therefore restores muscle mass, increases exercise endurance, and may provide a therapeutic strategy for age-related muscle loss.Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease caused by mutations in the CYP27A1 gene, encoding the sterol 27-hydroxylase. Disruption of the bile acid biosynthesis pathway and accumulation of toxic precursors such as cholestanol cause chronic diarrhea, bilateral juvenile cataracts, tissue deposition of cholestanol and cholesterol (xanthomas), and progressive motor/neuropsychiatric alterations. We have evaluated the therapeutic potential of adeno-associated virus (AAV) vectors expressing CYP27A1 in a CTX mouse model. We found that a vector equipped with a strong liver-specific promoter (albumin enhancer fused with the α1 anti-trypsin promoter) is well tolerated and shows therapeutic effect at relatively low doses (1.5 × 1012 viral genomes [vg]/kg), when less than 20% of hepatocytes overexpress the transgene. This vector restored bile acid metabolism and normalized the concentration of most bile acids in plasma. By contrast, standard treatment (oral chenodeoxycholic acid [CDCA]), while reducing cholestanol, did not normalize bile acid composition in plasma and resulted in supra-physiological levels of CDCA and its derivatives. At the transcriptional level, only the vector was able to avoid the induction of xenobiotic-induced pathways in mouse liver. In conclusion, the overexpression of CYP27A1 in a fraction of hepatocytes using AAV vectors is well tolerated and provides full metabolic restoration in Cyp27a1 -/- mice. These features make gene therapy a feasible option for the etiological treatment of CTX patients.Fragile X syndrome (FXS), a neurodevelopmental disorder with no known cure, is caused by a lack of expression of the fragile X mental retardation protein (FMRP). As a single-gene disorder, FXS is an excellent candidate for viral-vector-based gene therapy, although that is complicated by the existence of multiple isoforms of FMRP, whose individual cellular functions are unknown. We studied the effects of rat and mouse orthologs of human isoform 17, a major expressed isoform of FMRP. Injection of neonatal Fmr1 knockout rats and mice with adeno-associated viral vectors (AAV9 serotype) under the control of an MeCP2 mini-promoter resulted in widespread distribution of the FMRP transgenes throughout the telencephalon and diencephalon. Transgene expression occurred mainly in non-GABAergic neurons, with little expression in glia. Early postnatal treatment resulted in partial rescue of the Fmr1 KO rat phenotype, including improved social dominance in treated Fmr1 KO females and partial rescue of locomotor activity in males. Electro-encephalogram (EEG) recordings showed correction of abnormal slow-wave activity during the sleep-like state in male Fmr1 KO rats. These findings support the use of AAV-based gene therapy as a treatment for FXS and specifically demonstrate the potential therapeutic benefit of human FMRP isoform 17 orthologs.Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials.Monoclonal antibodies that target the inhibitory immune checkpoint axis consisting of programmed cell death protein 1 (PD-1) and its ligand, PD-L1, have changed the immune-oncology field. We identified K2, an anti-human PD-L1 single-domain antibody fragment, that can enhance T cell activation and tumor cell killing. In this study, the potential of different K2 formats as immune checkpoint blocking medicines was evaluated using a gene-based delivery approach. We showed that 2K2 and 3K2, a bivalent and trivalent K2 format generated using a 12 GS (glycine-serine) linker, were 313- and 135-fold more potent in enhancing T cell receptor (TCR) signaling in PD-1POS cells than was monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169- and 35-fold less potent in enhancing TCR signaling than was 2K2. 2K2 enhanced tumor cell killing in a 3D melanoma model, albeit to a lesser extent than avelumab. Therefore, an immunoglobulin (Ig)G1 antibody-like fusion protein was generated, referred to as K2-Fc. K2-Fc was significantly better than avelumab in enhancing tumor cell killing in the 3D melanoma model. Overall, this study describes K2-based immune checkpoint medicines, and it highlights the benefit of an IgG1 Fc fusion to K2 that gains bivalency, effector functions, and efficacy.Most gene therapy clinical trials that systemically administered adeno-associated virus (AAV) vector enrolled only patients without anti-AAV-neutralizing antibodies. However, laboratory tests to measure neutralizing antibodies varied among clinical trials and have not been standardized. In this study, we attempted to improve the sensitivity and reproducibility of a cell-based assay to detect neutralizing antibodies and to determine the detection threshold to predict treatment efficacy. Application of the secreted type of NanoLuc and AAV receptor-expressing cells reduced the multiplicity of infection (MOI) for AAV transduction and improved the sensitivity to detect neutralizing antibodies with a low coefficient of variation, whereas the detection threshold could not be improved by the reduction of MOI to less then 100. After human immunoglobulin administration into mice at various doses, treatment with high-dose AAV8 vector enabled evasion of the inhibitory effect of neutralizing antibodies. Conversely, gene transduction was slightly influenced in the mice treated with low-dose AAV8 vector, even when neutralizing antibodies were determined to be negative in the assay. In conclusion, we developed a reliable and sensitive cell-based assay to measure neutralizing antibodies against AAV and found that the appropriate MOI to detect marginal neutralizing antibodies was 100. Other factors, including noninhibitory antibodies, marginally influence in vivo transduction at low vector doses.Cardiac endothelial cells (ECs) are important targets for cardiovascular gene therapy. However, the approach of stably transducing ECs in vivo using different vectors, including adeno-associated virus (AAV), remains unexamined. Regarding this unmet need, two AAV libraries from DNA shuffling and random peptide display were simultaneously screened in a transgenic mouse model. Cardiac ECs were isolated by cell sorting for salvage of EC-targeting AAV. Two AAV variants, i.e., EC71 and EC73, enriched in cardiac EC, were further characterized for their tissue tropism. Both of them demonstrated remarkably enhanced transduction of cardiac ECs and reduced infection of liver ECs in comparison to natural AAVs after intravenous injection. Significantly, persistent transgene expression was maintained in mouse cardiac ECs in vivo for at least 4 months. The EC71 vector was selected for delivery of the endothelial nitric oxide synthase (eNOS) gene into cardiac ECs in a mouse model of myocardial infarction. Enhanced eNOS activity was observed in the mouse heart and lung, which was correlated with partially improved cardiac function. Taken together, two AAV capsids were evolved with more efficient transduction in cardiovascular endothelium in vivo, but their endothelial tropism might need to be further optimized for practical application to cardiac gene therapy.The transmission of information between tumor cells and other cell types in the tumor microenvironment plays an important role in tumor metastasis and is critically modulated by exosomes and other mediators. Tumor-derived exosomes can promote epithelial-mesenchymal transition, angiogenesis, immune escape, formation of the pre-metastatic microenvironment, and transmission of drug-resistant molecules, thereby promoting tumor growth, invasion, and metastasis. Integrins are important regulatory molecules on exosomes that can locate metastatic cells at the initial stage of metastasis and show good organotropism. This fact suggests that a clear understanding of the roles of exosomal integrins will be beneficial for future clinical applications. Follow-up studies on exosomes using continuously updated purification techniques and identification methods are extremely important. In addition to their potential as cancer biomarkers, exosomes also provide new research directions for precision medicine. Currently, exosomes have potential value in disease treatment and provide clinicians with more meaningful judgment standards.Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a lethal neuromuscular disease. Correction of DMD mutations in animal models has been achieved by CRISPR/Cas9 genome editing using Streptococcus pyogenes Cas9 (SpCas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the SpCas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Staphylococcus aureus Cas9 (SaCas9) to restore the open reading frame of exon 51, the most commonly affected out-of-frame exon in DMD. Editing of exon 51 in cardiomyocytes derived from human induced pluripotent stem cells revealed a strong preference for exon reframing via a two-nucleotide deletion. We adapted this system to express SaCas9 and sgRNA from a single AAV9 vector. Systemic delivery of this All-In-One AAV9 system restored dystrophin expression and improved muscle contractility in a mouse model of DMD with exon 50 deletion. These findings demonstrate the effectiveness of CRISPR/SaCas9 delivered by a consolidated AAV delivery system in the correction of DMD in vivo, representing a promising therapeutic approach to correct the genetic causes of DMD.Intravitreal injection is the most widely used injection technique for ocular gene delivery. However, vector diffusion is attenuated by physical barriers and neutralizing antibodies in the vitreous. The 13-lined ground squirrel (13-LGS), as in humans, has a larger relative vitreous body volume than the more common rodent models such as rats and mice, which would further reduce transduction efficiency with the intravitreal injection route. We report here a „pre-retinal” injection approach that leads to detachment of the posterior hyaloid membrane and delivers vector into the space between vitreous and inner retina. Vectors carrying a ubiquitously expressing mCherry reporter were injected into the deep vitreous or pre-retinal space in adult wild-type 13-LGSs. Then, adeno-associated virus (AAV)-mediated mCherry expression was evaluated with non-invasive imaging, immunofluorescence, and flow cytometry. Compared to deep vitreous delivery, pre-retinal administration achieved pan-retinal gene expression with a lower vector dose volume and significantly increased the number of transduced cone photoreceptors. These results suggest that pre-retinal injection is a promising tool in the development of gene therapy strategies in animal models and is a potential approach for use in human research, particularly in younger individuals with an intact posterior hyaloid membrane and stable vitreous.Nucleoside-modified, lipid nanoparticle-encapsulated mRNAs have recently emerged as suitable vaccines for influenza viruses and other pathogens in part because the platform allows delivery of multiple antigens in a single immunization. mRNA vaccines allow for easy antigen modification, enabling rapid iterative design. We studied protein modifications such as mutating functional sites, changing secretion potential, and altering protein conformation, which could improve the safety and/or potency of mRNA-based influenza virus vaccines. Mice were vaccinated intradermally with wild-type or mutant constructs of influenza virus hemagglutinin (HA), neuraminidase (NA), matrix protein 2 (M2), nucleoprotein (NP), or matrix protein 1 (M1). Membrane-bound HA constructs elicited more potent and protective antibody responses than secreted forms. Altering the catalytic site of NA to reduce enzymatic activity decreased reactogenicity while protective immunity was maintained. Disruption of M2 ion channel activity improved immunogenicity and protective efficacy. A comparison of internal proteins NP and M1 revealed the superiority of NP in conferring protection from influenza virus challenge. These findings support the use of the nucleoside-modified mRNA platform for guided antigen design for influenza virus with extension to other pathogens.Hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) requires the collection of a large number of cells. While bone marrow (BM) is the most common source of HSPCs in pediatric donors, the collection of autologous peripheral blood stem cells (PBSCs) is an attractive alternative for GT. We present safety and efficacy data of a 10-year cohort of 45 pediatric patients who underwent PBSC collection for backup and/or purification of CD34+ cells for ex vivo gene transfer. Median age was 3.7 years and median weight 15.8 kg. After mobilization with lenograstim/plerixafor (n = 41) or lenograstim alone (n = 4) and 1-3 cycles of leukapheresis, median collection was 37 × 106 CD34+ cells/kg. The procedures were well tolerated. Patients who collected ≥7 and ≥13 × 106 CD34+ cells/kg in the first cycle had pre-apheresis circulating counts of at ≥42 and ≥86 CD34+ cells/μL, respectively. Weight-adjusted CD34+ cell yield was positively correlated with peripheral CD34+ cell counts and influenced by female gender, disease, and drug dosage. All patients received a GT product above the minimum target, ranging from 4 to 30.9 × 106 CD34+ cells/kg. Pediatric PBSC collection compares well to BM harvest in terms of CD34+ cell yields for the purpose of GT, with a favorable safety profile.Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34+ cells/μL required to initiate apheresis. A median of 21.8 CD34+ cells/μL was reached at the peak of mobilization. Significantly, the oldest patients (15 and 16 years old) were the only ones who did not reach that threshold. A median of 4.27 million CD34+ cells/kg was collected in 2 or 3 aphereses. These numbers were markedly decreased to 1.1 million CD34+ cells/kg after immunoselection, probably because of weak expression of the CD34 antigen. However, these numbers were sufficient to facilitate the engraftment of corrected HSPCs in non-conditioned patients. No procedure-associated serious adverse events were observed. Mobilization of CD34+ cells correlated with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher proportion of CD34+ cells in bone marrow (BM). All these values offer crucial information for the enrollment of FA patients for gene therapy protocols.Although adeno-associated viral (AAV) vector-mediated retinal gene therapies have demonstrated efficacy, the mechanisms underlying dose-dependent retinal inflammation remain poorly understood. Here, we present a quantitative analysis of cellular immune response to subretinal AAV gene therapy in mice using multicolor flow cytometry with a panel of key immune cell markers. A significant increase in CD45+ retinal leukocytes was detected from day 14 post-subretinal injection of an AAV8 vector (1 × 109 genome copies) encoding green fluorescent protein (GFP) driven by a ubiquitous promoter. These predominantly consisted of infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells; no significant change in resident microglia population was detected. This cellular response was persistent at 28 days and suggestive of type 1 cell-mediated effector immunity. High levels (80%) of GFP fluorescence were found in the microglia, implicating their role in viral antigen presentation and peripheral leukocyte recruitment. When compared against AAV.GFP in paired eyes, an equivalent dose of an otherwise identical vector encoding the human therapeutic transgene Rab-escort protein 1 (REP1) elicited a significantly diminished cellular immune response (4.2-fold; p = 0.0221). However, the distribution of immune cell populations remained similar, indicating a common mechanism of AAV-induced immune activation.Neuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin, which regulates neuronal lysosomal function. Absence of progranulin causes cerebellar atrophy, seizures, dementia, and vision loss. As progranulin gene therapies targeting the brain are developed, it is advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development the retina is easily visible through direct imaging, can be assessed through quantitative methods in vivo, and requires smaller amounts of adeno-associated virus (AAV). In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. Mice heterologously expressing progranulin showed a reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV92YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with AAV2.7m8-scCAG-PGRN exhibit no improvement, and mice injected at 12 months of age have thinner retinas than do their controls. Thus, delivery of progranulin proves to be time sensitive and dependent on route of administration, requiring early delivery for optimal therapeutic benefit.Developing robust methodology for the sustainable production of red blood cells in vitro is essential for providing an alternative source of clinical-quality blood, particularly for individuals with rare blood group phenotypes. Immortalized erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34+ cells that had improved differentiation and enucleation potential compared to other lines reported. In this study we show that our immortalization approach is reproducible for erythroid cells differentiated from bone marrow and also from far more accessible peripheral and cord blood CD34+ cells, consistently generating lines with similar improved erythroid performance. Extensive characterization of the lines shows them to accurately recapitulate their primary cell equivalents and provides a molecular signature for immortalization. In addition, we show that only cells at a specific stage of erythropoiesis, predominantly proerythroblasts, are amenable to immortalization. Our methodology provides a step forward in the drive for a sustainable supply of red cells for clinical use and for the generation of model cellular systems for the study of erythropoiesis in health and disease, with the added benefit of an indefinite expansion window for manipulation of molecular targets.

Sexual minority women (SMW; e.g., lesbian, bisexual) are more likely than heterosexual women to be heavy drinkers, with bisexual women showing the highest risk. There is ample literature demonstrating that intimate relationships protect against stress-related health risk behaviors in the general population. However, very little research has focused on SMW’s relationships and far less is known about the relationships of SMW of color. Using intersectionality theory as our framework, we tested two competing models to determine whether the effects of minority sexual identity (lesbian, bisexual) and race/ethnicity (African American, Latinx, White) are 1) additive, or 2) multiplicative in the associations between relationship status and heavy drinking.

Data are from a diverse sample of cisgender sexual minority women (N = 641) interviewed in Wave 3 of the CHLEW study, a 20-year longitudinal study of SMW’s health.

Findings from two- and three-way interactions provide mixed evidence for both the additive and muts of relationships is important. Our findings demonstrate that the protective qualities of intimate relationships among SMW vary based on sexual identity and race/ethnicity – and the intersections between them.

Little research has focused on health within sexual minority women’s relationships, particularly among sexual minority women of color. Given the potential additive or multiplicative effects of multiple sources of oppression such as heterosexism, racism, and sexism, understanding the potential protective effects of relationships is important. Our findings demonstrate that the protective qualities of intimate relationships among SMW vary based on sexual identity and race/ethnicity – and the intersections between them.Oxidation of organic contaminants by sulfate radical (SO4 •-) is becoming more popular for the treatment of hazardous waste sites by in situ chemical oxidation (ISCO) and industrial wastewater by advanced oxidation processes (AOPs). It is well documented that SO4 •- can produce similar oxygen-containing transformation products as hydroxyl radical-based treatment processes, but SO4 •- also has the potential to produce organosulfates by radical addition. Experiments conducted with a suite of 23 aromatic and 5 aliphatic compounds, including several contaminants typically detected at hazardous waste sites, demonstrated the formation of at least one stable sulfate-containing product for 25 of the compounds. These compounds likely exhibit higher mobility in the subsurface due to a lower affinity for surfaces (e.g., aquifer solids, activated carbon) than most other transformation products. Although the health risks associated with organosulfates are still uncertain, some aromatic organosulfates produced in this study (i.e. phenyl sulfate and p-cresyl sulfate) are known to be harmful uremic toxins. Further study of organosulfate formation, fate, and toxicity is needed before SO4 •–based treatment processes are more widely employed.The picture word interference (PWI) paradigm and ERPs were used to investigate whether lexical selection in deaf and hearing ASL-English bilinguals occurs via lexical competition or whether the response exclusion hypothesis (REH) for PWI effects is supported. The REH predicts that semantic interference should not occur for bimodal bilinguals because sign and word responses do not compete within an output buffer. Bimodal bilinguals named pictures in ASL, preceded by either a translation equivalent, semantically-related, or unrelated English written word. In both the translation and semantically-related conditions bimodal bilinguals showed facilitation effects reduced RTs and N400 amplitudes for related compared to unrelated prime conditions. We also observed an unexpected focal left anterior positivity that was stronger in the translation condition, which we speculate may be due to articulatory priming. Overall, the results support the REH and models of bilingual language production that assume lexical selection occurs without competition between languages.Speakers learning a second language show systematic differences from native speakers in the retrieval, planning, and articulation of speech. A key challenge in examining the interrelationship between these differences at various stages of production is the need for manual annotation of fine-grained properties of speech. We introduce a new method for automatically analyzing voice onset time (VOT), a key phonetic feature indexing differences in sound systems cross-linguistically. In contrast to previous approaches, our method allows reliable measurement of prevoicing, a dimension of VOT variation used by many languages. Analysis of VOTs, word durations, and reaction times from German-speaking learners of Spanish (Baus et al., 2013) suggest that while there are links between the factors impacting planning and articulation, these two processes also exhibit some degree of independence. We discuss the implications of these findings for theories of speech production and future research in bilingual language processing.