Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome ...sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.
Chronic graft-versus-host disease (cGVHD) remains a major complication following allogeneic bone marrow transplantation (BMT). The discovery of novel therapeutics is dependent on assessment in ...preclinical murine models of cGVHD. Rho-associated kinase 2 (ROCK2) recently was shown to be implicated in regulation of interleukin-21 (IL-21) and IL-17 secretion in mice and humans. Here, we report that the selective ROCK2 inhibitor KD025 effectively ameliorates cGVHD in multiple models: a full major histocompatibility complex (MHC) mismatch model of multiorgan system cGVHD with bronchiolitis obliterans syndrome and a minor MHC mismatch model of sclerodermatous GVHD. Treatment with KD025 resulted in normalization of pathogenic pulmonary function, which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. Spleens of mice treated with KD025 had decreased frequency of T follicular helper cells and increased frequency of T follicular regulatory cells, accompanied by a reduction in signal transducer and activator of transcription 3 (STAT3) and concurrent increase in STAT5 phosphorylation. The critical role of STAT3 in this cGVHD model was confirmed by data showing that mice transplanted with inducible STAT3-deficient T cells had pulmonary function comparable to the healthy negative controls. The therapeutic potential of targeted ROCK2 inhibition in the clinic was solidified further by human data demonstrating the KD025 inhibits the secretion of IL-21, IL-17, and interferon γ along with decreasing phosphorylated STAT3 and reduced protein expression of interferon regulatory factor 4 and B-cell lymphoma 6 (BCL6) in human peripheral blood mononuclear cells purified from active cGVHD patients. Together these data highlight the potential of targeted ROCK2 inhibition for clinical cGVHD therapy.
•The ROCK2 inhibitor, KD025, decreases chronic GVHD pathology in multiple murine models.•KD025 inhibits STAT3 phosphorylation to decrease RORγt and Bcl6 expression in both murine and human cells.
Interdisciplinary collaboration is essential to understand ecological systems at scales critical to human decision making. Current reward structures are problematic for scientists engaged in ...interdisciplinary research, particularly early career researchers, because academic culture tends to value only some research outputs, such as primary-authored publications. Here, we present a framework for the costs and benefits of collaboration, with a focus on early career stages, and show how the implementation of novel measures of success can help defray the costs of collaboration. Success measures at team and individual levels include research outputs other than publications, including educational outcomes, dataset creation, outreach products (eg blogs or social media), and the application of scientific results to policy or management activities. Promotion and adoption of new measures of success will require concerted effort by both collaborators and their institutions. Expanded measures should better reflect and reward the important work of both disciplinary and interdisciplinary teams at all career stages, and help sustain and stimulate a collaborative culture within ecology.
Chronic GVHD (cGVHD) poses a significant risk for HSCT patients. Preclinical development of new therapeutic modalities has been hindered by models with pathologic findings that may not simulate the ...development of human cGVHD. Previously, we have demonstrated that cGVHD induced by allogeneic HSCT after a conditioning regimen of cyclophosphamide and total-body radiation results in pulmonary dysfunction and airway obliteration, which leads to bronchiolitis obliterans (BO), which is pathognomonic for cGVHD of the lung. We now report cGVHD manifestations in a wide spectrum of target organs, including those with mucosal surfaces. Fibrosis was demonstrated in the lung and liver and was associated with CD4+ T cells and B220+ B-cell infiltration and alloantibody deposition. Donor bone marrow obtained from mice incapable of secreting IgG alloantibody resulted in less BO and cGVHD. Robust germinal center reactions were present at the time of cGVHD disease initiation. Blockade of germinal center formation with a lymphotoxin-receptor–immunoglobulin fusion protein suppressed cGVHD and BO. We conclude that cGVHD is caused in part by alloantibody secretion, which is associated with fibrosis and cGVHD manifestations including BO, and that treatment with a lymphotoxin-β receptor–immunoglobulin fusion protein could be beneficial for cGVHD prevention and therapy.
Abnormal cholesterol/lipid homeostasis is linked to neurodegenerative conditions such as age-related macular degeneration (AMD), which is a leading cause of blindness in the elderly. The most ...prevalent form, termed “dry” AMD, is characterized by pathological cholesterol accumulation beneath the retinal pigment epithelial (RPE) cell layer and inflammation-linked degeneration in the retina. We show here that the cholesterol-regulating microRNA miR-33 was elevated in the RPE of aging mice. Expression of the miR-33 target ATP-binding cassette transporter (ABCA1), a cholesterol efflux pump genetically linked to AMD, declined reciprocally in the RPE with age. In accord, miR-33 modulated ABCA1 expression and cholesterol efflux in human RPE cells. Subcutaneous delivery of miR-33 antisense oligonucleotides (ASO) to aging mice and non-human primates fed a Western-type high fat/cholesterol diet resulted in increased ABCA1 expression, decreased cholesterol accumulation, and reduced immune cell infiltration in the RPE cell layer, accompanied by decreased pathological changes to RPE morphology. These findings suggest that miR-33 targeting may decrease cholesterol deposition and ameliorate AMD initiation and progression.
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Age-related macular degeneration (AMD) is an irreversible blinding condition. Currently, no defined therapies are particularly available to treat the dry form of AMD. Here, Gnanaguru and colleagues demonstrate that anti-sense oligonucleotide targeting of microRNA-33a/b isoforms reduces cholesterol accumulation and immune cell infiltration and ameliorates pathologies associated with dry AMD in clinically relevant non-human primates.
An emerging area in environmental toxicology is the role that chemicals and chemical mixtures have on the cells of the human immune system. This is an important area of research that has been most ...widely pursued in relation to autoimmune diseases and allergy/asthma as opposed to cancer causation. This is despite the well-recognized role that innate and adaptive immunity play as essential factors in tumorigenesis. Here, we review the role that the innate immune cells of inflammatory responses play in tumorigenesis. Focus is placed on the molecules and pathways that have been mechanistically linked with tumor-associated inflammation. Within the context of chemically induced disturbances in immune function as co-factors in carcinogenesis, the evidence linking environmental toxicant exposures with perturbation in the balance between pro- and anti-inflammatory responses is reviewed. Reported effects of bisphenol A, atrazine, phthalates and other common toxicants on molecular and cellular targets involved in tumor-associated inflammation (e.g. cyclooxygenase/prostaglandin E2, nuclear factor kappa B, nitric oxide synthesis, cytokines and chemokines) are presented as example chemically mediated target molecule perturbations relevant to cancer. Commentary on areas of additional research including the need for innovation and integration of systems biology approaches to the study of environmental exposures and cancer causation are presented.
People often prefer evidence-based psychosocial interventions (EBPIs) for mental health care; however, these interventions frequently remain unavailable to people in nonspecialty or integrated ...settings, such as primary care and schools. Previous research has suggested that usability, a concept from human-centered design, could support an understanding of the barriers to and facilitators of the successful adoption of EBPIs and support the redesign of EBPIs and implementation strategies.
This study aimed to identify and categorize usability issues in EBPIs and their implementation strategies.
We adapted a usability issue analysis and reporting format from a human-centered design. A total of 13 projects supported by the National Institute of Mental Health-funded Accelerating the Reach and Impact of Treatments for Youth and Adults with Mental Illness Center at the University of Washington used this format to describe usability issues for EBPIs and implementation strategies with which they were working. Center researchers used iterative affinity diagramming and coding processes to identify usability issue categories. On the basis of these categories and the underlying issues, we propose heuristics for the design or redesign of EBPIs and implementation strategies.
The 13 projects reported a total of 90 usability issues, which we categorized into 12 categories, including complex and/or cognitively overwhelming, required time exceeding available time, incompatibility with interventionist preference or practice, incompatibility with existing workflow, insufficient customization to clients/recipients, intervention buy-in (value), interventionist buy-in (trust), overreliance on technology, requires unavailable infrastructure, inadequate scaffolding for client/recipient, inadequate training and scaffolding for interventionists, and lack of support for necessary communication. These issues range from minor inconveniences that affect a few interventionists or recipients to severe issues that prevent all interventionists or recipients in a setting from completing part or all of the intervention. We propose 12 corresponding heuristics to guide EBPIs and implementation strategy designers in preventing and addressing these usability issues.
Usability issues were prevalent in the studied EBPIs and implementation strategies. We recommend using the lens of usability evaluation to understand and address barriers to the effective use and reach of EBPIs and implementation strategies.
RR2-10.2196/14990.
Hearing aids (HAs) provide the basis for improving audibility and minimizing developmental delays in children with mild to severe hearing loss. Multiple guidelines exist to recommend methods for ...optimizing amplification in children, but few previous studies have reported HA fitting outcomes for a large group of children. The present study sought to evaluate the proximity of the fitting to prescriptive targets and aided audibility of speech, as well as survey data from pediatric audiologists who provided HAs for the children in the present study. Deviations from prescriptive target were predicted to have a negative impact on aided audibility. In addition, children who were fitted using verification with probe microphone measurements were expected to have smaller deviations from prescriptive targets and greater audibility than cohorts fitted without these measures.
HA fitting data from 195 children with mild to severe hearing losses were analyzed as part of a multicenter study evaluating outcomes in children with hearing loss. Proximity of fitting to prescriptive targets was quantified by calculating the average root-mean-square (RMS) error of the fitting compared with Desired Sensation Level prescriptive targets for 500, 1000, 2000, and 4000 Hz. Aided audibility was quantified using the Speech Intelligibility Index. Survey data from the pediatric audiologists who fit amplification for children in the present study were collected to evaluate trends in fitting practices and relate those patterns to proximity of the fitting to prescriptive targets and aided audibility.
More than half (55%) of the children in the study had at least 1 ear that deviated from prescriptive targets by more than 5 dB RMS on average. Deviation from prescriptive target was not predicted by pure-tone average, assessment method, or reliability of assessment. Study location was a significant predictor of proximity to prescriptive target with locations that recruited participants who were fit at multiple clinical locations (University of Iowa and Boys Town National Research Hospital) having larger deviations from target than the location where the participants were recruited primarily from a single, large pediatric audiology clinic (University of North Carolina). Fittings based on average real-ear to coupler differences resulted in larger deviations from prescriptive targets than fittings based on individually measured real-ear to coupler differences. Approximately 26% of the children in the study has aided audibility less than 0.65 on the Speech Intelligibility Index (SII). Aided audibility was significantly predicted by the proximity to prescriptive targets and pure-tone average, but not age in months.
Children in the study had a wide range of fitting outcomes in terms of proximity to prescriptive targets (RMS error) and aided speech audibility (SII). Even when recommended HA verification strategies were reported, fittings often exceeded the criteria for both proximity to the prescriptive target and aided audibility. The implications for optimizing amplification for children are also discussed.
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