Nuclear PARPs and genome integrity.

Effective maintenance and stability of our genomes is essential for normal cell division, tissue homeostasis, and cellular and organismal fitness. The processes of chromosome replication and segregation require continual surveillance to insure fidelity. Accurate and efficient repair of DNA damage preserves genome integrity, which if lost can lead to multiple diseases, including cancer. Poly(ADP-ribose) a dynamic and reversible posttranslational modification and the enzymes that catalyze it (PARP1, PARP2, tankyrase 1, and tankyrase 2) function to maintain genome stability through diverse mechanisms. Here we review the role of these enzymes and the modification in genome repair, replication, and resolution in human cells.

Resolution of human ribosomal DNA occurs in anaphase, dependent on tankyrase 1, condensin II, and topoisomerase IIα.

Formation of individualized sister chromatids is essential for their accurate segregation. In budding yeast, while most of the genome segregates at the metaphase to anaphase transition, resolution of the ribosomal DNA (rDNA) repeats is delayed. The timing and mechanism in human cells is unknown. Here we show that resolution of human rDNA occurs in anaphase after the bulk of the genome, dependent on tankyrase 1, condensin II, and topoisomerase IIα. Defective resolution leads to rDNA bridges, rDNA damage, and aneuploidy of an rDNA-containing acrocentric chromosome. Thus, temporal regulation of rDNA segregation is conserved between yeast and man and is essential for genome integrity.

Telomerase can't handle the stress.

Telomerase counteracts the telomere shortening that occurs with each round of cell division. In normal human cells, telomerase is repressed, leading to telomere shortening that triggers replicative senescence. However, in most tumors, telomerase is up-regulated and is essential for telomere maintenance and tumor cell growth. Although long considered a viable target for tumor therapy, successful inhibition of telomerase in cancer therapy remains to be described. In this issue of Genes & Development, Ahmed and Lingner (pp. 658-669) uncover a vulnerability in telomerase upon exposure of cancer cells to oxidative stress. It has long been known that telomeres are sensitive to damage by reactive oxygen species (ROS), but the impact of oxidation on telomerase function in living cells was not known. Using gene knockouts in colon cancer cells, the investigators demonstrate that the antioxidant enzyme peroxiredoxin 1 (PRDX1) and the nudix phosphohydrolase superfamily enzyme (MTH1) cooperate to retain, upon oxidative stress, telomeres in a telomerase-extendible state. Considering that cancer cells are more vulnerable to ROS than noncancer cells, this work may open new avenues targeting telomeres and telomerase in tumor cells.

An HTLV-1 envelope mRNA vaccine is immunogenic and protective in New Zealand rabbits.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma, a severe and fatal CD4+ T-cell malignancy. Additionally, HTLV-1 can lead to a chronic progressive neurodegenerative disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis. Unfortunately, the prognosis for HTLV-1-related diseases is generally poor, and effective treatment options are limited. In this study, we designed and synthesized a codon optimized HTLV-1 envelope (Env) mRNA encapsulated in a lipid nanoparticle (LNP) and evaluated its efficacy as a vaccine candidate in an established rabbit model of HTLV-1 infection and persistence. Immunization regimens included a prime/boost protocol using Env mRNA-LNP or control green fluorescent protein (GFP) mRNA-LNP. After immunization, rabbits were challenged by intravenous injection with irradiated HTLV-1 producing cells. Three rabbits were partially protected and three rabbits were completely protected against HTLV-1 challenge. These rabbits were then rechallenged 15 weeks later, and two rabbits maintained sterilizing immunity. In Env mRNA-LNP immunized rabbits, proviral load and viral gene expression were significantly lower. After viral challenge in the Env mRNA-LNP vaccinated rabbits, an increase in both CD4+/IFN-γ+ and CD8+/IFN-γ+ T-cells was detected when stimulating with overlapping Env peptides. Env mRNA-LNP elicited a detectable anti-Env antibody response after prime/boost vaccination in all animals and significantly higher levels of neutralizing antibody activity. Neutralizing antibody activity was correlated with a reduction in proviral load. These findings hold promise for the development of preventive strategies and therapeutic interventions against HTLV-1 infection and its associated diseases.IMPORTANCEmRNA vaccine technology has proven to be a viable approach for effectively triggering immune responses that protect against or limit viral infections and disease. In our study, we synthesized a codon optimized human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) mRNA that can be delivered in a lipid nanoparticle (LNP) vaccine approach. The HTLV-1 Env mRNA-LNP produced protective immune responses against viral challenge in a preclinical rabbit model. HTLV-1 is primarily transmitted through direct cell-to-cell contact, and the protection offered by mRNA vaccines in our rabbit model could have significant implications for optimizing the development of other viral vaccine candidates. This is particularly important in addressing the challenge of enhancing protection against infections that rely on cell-to-cell transmission.

HTLV-1 Hbz protein, but not hbz mRNA secondary structure, is critical for viral persistence and disease development.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic cause of adult T-cell leukemia/lymphoma (ATL) and encodes a viral oncoprotein (Hbz) that is consistently expressed in asymptomatic carriers and ATL patients, suggesting its importance in the development and maintenance of HTLV-1 leukemic cells. Our previous work found Hbz protein is dispensable for virus-mediated T-cell immortalization but enhances viral persistence. We and others have also shown that hbz mRNA promotes T-cell proliferation. In our current studies, we evaluated the role of hbz mRNA on HTLV-1-mediated immortalization in vitro as well as in vivo persistence and disease development. We generated mutant proviral clones to examine the individual contributions of hbz mRNA, hbz mRNA secondary structure (stem-loop), and Hbz protein. Wild-type (WT) and all mutant viruses produced virions and immortalized T-cells in vitro. Viral persistence and disease development were also evaluated in vivo by infection of a rabbit model and humanized immune system (HIS) mice, respectively. Proviral load and sense and antisense viral gene expression were significantly lower in rabbits infected with mutant viruses lacking Hbz protein compared to WT or virus with an altered hbz mRNA stem-loop (M3 mutant). HIS mice infected with Hbz protein-deficient viruses showed significantly increased survival times compared to animals infected with WT or M3 mutant virus. Altered hbz mRNA secondary structure, or loss of hbz mRNA or protein, has no significant effect on T-cell immortalization induced by HTLV-1 in vitro; however, the Hbz protein plays a critical role in establishing viral persistence and leukemogenesis in vivo.

Joint Effects of Indoor Air Pollution and Maternal Psychosocial Factors During Pregnancy on Trajectories of Early Childhood Psychopathology.

BACKGROUND: Prenatal indoor air pollution and maternal psychosocial factors have been associated with adverse psychopathology. We used environmental exposure mixture methodology to investigate joint effects of both exposure classes on child behavior trajectories. METHODS: For 360 children from the South African Drakenstein Child Health Study, we created trajectories of Child Behavior Checklist scores (24, 42, 60 months) using latent class linear mixed effects models. Indoor air pollutants and psychosocial factors were measured during pregnancy (2nd trimester). After adjusting for confounding, single-exposure effects (per natural log-1 unit increase) were assessed using polytomous logistic regression models; joint effects using self-organizing maps (SOM), and principal component (PC) analysis. RESULTS: Three trajectories were chosen for both internalizing and externalizing problems, with "high" (externalizing) or "increasing" (internalizing) being the most adverse trajectories. High externalizing trajectory was associated with increased particulate matter (PM10) exposure (OR [95%-CI]: 1.25 [1.01,1.55]) and SOM exposure profile most associated with smoking (2.67 [1.14,6.27]). Medium internalizing trajectory was associated with increased emotional intimate partner violence (2.66 [1.17,5.57]), increasing trajectory with increased benzene (1.24 [1.02,1.51]) and toluene (1.21 [1.02,1.44]) and the PC most correlated with benzene and toluene (1.25 [1.02, 1.54]). CONCLUSIONS: Prenatal exposure to environmental pollutants and psychosocial factors was associated with internalizing and externalizing child behavior trajectories. Understanding joint effects of adverse exposure mixtures will facilitate targeted interventions to prevent childhood psychopathology.

Alcohol blunts pregnancy-mediated insulin resistance and reduces fetal brain glucose despite elevated fetal gluconeogenesis, and these changes associate with fetal weight outcomes.

Prenatal alcohol exposure (PAE) impairs fetal growth and neurodevelopment. Although alcohol is well known to alter metabolism, its impact on these processes during pregnancy is largely unexplored. Here, we investigate how alcohol affects maternal-fetal glucose metabolism using our established mouse binge model of PAE. In the dam, alcohol reduces the hepatic abundance of glucose and glycolytic intermediates, and the gluconeogenic enzymes glucose-6-phosphtase and phosphoenolpyruvate carboxykinase. Fasting blood glucose is also reduced. In a healthy pregnancy, elevated maternal gluconeogenesis and insulin resistance ensures glucose availability for the fetus. Glucose and insulin tolerance tests reveal that alcohol impairs the dam's ability to acquire insulin resistance. Alcohol-exposed dams have enhanced glucose clearance (p < .05) in early gestation, after just two days of alcohol, and this persists through late term when fetal glucose needs are maximal. However, maternal plasma insulin levels, hepatic insulin signaling, and the abundance of glucose transporter proteins remain unchanged. In the PAE fetus, the expression of hepatic gluconeogenic genes is elevated, and there is a trend for elevated blood and liver glucose levels. In contrast, fetal brain and placental glucose levels remain low. This reduced maternal fasting glucose, reduced hepatic glucose, and elevated glucose clearance inversely correlated with fetal body and brain weight. Taken together, these data suggest that alcohol blunts the adaptive changes in maternal glucose metabolism that otherwise enhance fetal glucose availability. Compensatory attempts by the fetus to increase glucose pools via gluconeogenesis do not normalize brain glucose. These metabolic changes may contribute to the impaired fetal growth and brain development that typifies PAE.

Meeting the need for effective and standardized neonatology training: a pan-European Master's Curriculum.

Neonatology is a pediatric sub-discipline focused on providing care for newborn infants, including healthy newborns, those born prematurely, and those who present with illnesses or malformations requiring medical care. The European Training Requirements (ETR) in Neonatology provide a framework for standardized quality and recognition of equality of training throughout Europe. The latest ETR version was approved by the Union of European Medical Specialists (UEMS) in April 2021. Here, we present the curriculum of the European School of Neonatology Master of Advanced Studies (ESN MAS), which is based on the ETR in Neonatology and aims to provide a model for effective and standardized training and education in neonatal medicine. We review the history and theory that form the foundation of contemporary medical education and training, provide a literature review on best practices for medical training, pediatric training, and neonatology training specifically, including educational frameworks and evidence-based systems of evaluation. The ESN MAS Curriculum is then evaluated in light of these best practices to define its role in meeting the need for a standardized empirically supported neonatology training curriculum for physicians, and in the future for nurses, to improve the quality of neonatal care for all infants. IMPACT STATEMENT: A review of the neonatology training literature was conducted, which concluded that there is a need for standardized neonatology training across international contexts to keep pace with growth in the field and rapidly advancing technology. This article presents the European School of Neonatology Master of Advanced Studies in Neonatology, which is intended to provide a standardized training curriculum for pediatricians and nurses seeking sub-specialization in neonatology. The curriculum is evaluated in light of best practices in medical education, neonatology training, and adult learning theory.

Joint effects of prenatal exposure to indoor air pollution and psychosocial factors on early life inflammation.

It is hypothesized that air pollution and stress impact the central nervous system through neuroinflammatory pathways Despite this, the association between prenatal exposure to indoor air pollution and psychosocial factors on inflammatory markers in infancy has been underexplored in epidemiology studies. This study investigates the individual and joint effects of prenatal exposure to indoor air pollution and psychosocial factors on early life inflammation (interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)). We analyzed data from the South African Drakenstein Child Health Study (N = 225). Indoor air pollution and psychosocial factor measurements were taken in the 2nd trimester of pregnancy. Circulating inflammatory markers (IL-1ß, Il-6, and TNF-α) were measured in serum in the infants at 6 weeks postnatal. Linear regression models were used to investigate associations between individual exposures and inflammatory markers. To investigate joint effects of environmental and psychosocial factors, Self-Organizing Maps (SOM) were used to create exposure profile clusters. These clusters were added to linear regression models to investigate the associations between exposure profiles and inflammatory markers. All models were adjusted for maternal age, maternal HIV status, and ancestry to control for confounding. Most indoor air pollutants were positively associated with inflammatory markers, particularly benzene and TNF-α in single pollutant models. No consistent patterns were found for psychosocial factors in single-exposure linear regression models. In joint effects analyses, the SOM profile with high indoor air pollution, low SES, and high maternal depressive symptoms were associated with higher inflammation. Indoor air pollutants were consistently associated with increased inflammation in both individual and joint effects models, particularly in combination with low SES and maternal depressive symptoms. The trend for individual psychosocial factors was not as clear, with mainly null associations. As we have observed pro- and anti-inflammatory effects, future research should investigate joint effects of these exposures on inflammation and their health effects.

Efficacy of a dialogic book-sharing intervention in a South African birth cohort: A randomized controlled trial.

OBJECTIVE: Evidence shows that dialogic book-sharing improves language development in young children in low-middle income countries (LMICs), particularly receptive and expressive language. It is unclear whether this intervention also boosts development of other neurocognitive and socio-emotional domains in children. Using a randomized controlled trial (RCT) nested in the Drakenstein Child Health Study (DCHS), a book-sharing intervention was implemented in caregivers of 3.5-year-old preschool children living in low-income South African communities. METHODS: 122 Caregivers and their children (mean age 3.5 years) were randomly assigned to an intervention group (n = 61) or waitlist control group (n = 61). A neurocognitive battery determined baseline receptive and expressive language, executive function, theory of mind, and behavior scores. RESULTS: No differences were observed between intervention and control groups on receptive and expressive language, or any of the neurocognitive or socio-emotional measures from baseline (3.5 years) to 4 months post-intervention administration (4 years). CONCLUSION: The benefits noted in prior literature of book-sharing in infants did not appear to be demonstrated at 4 months post-intervention, in children from 3.5 to 4 years of age. This suggests the importance of early intervention and emphasizes the need for further research on adaptation of book-sharing for older participants in a South African context. TRIAL REGISTRATION: retrospectively registered on 03/04/2022 PACTR202204697674974.

Prevalence of loneliness and associations with health behaviours and body mass index in 5835 people living with and beyond cancer: a cross-sectional study.

BACKGROUND: A cancer diagnosis and its treatment may be an especially isolating experience. Despite evidence that positive health behaviours can improve outcomes for people living with and beyond cancer (LWBC), no studies have examined associations between loneliness and different health behaviours in this population. This study aimed to describe the prevalence of loneliness in a large sample of UK adults LWBC and to explore whether loneliness was associated with multiple health behaviours. METHODS: Participants were adults (aged ≥ 18 years) diagnosed with breast, prostate or colorectal cancer who completed the Health and Lifestyle After Cancer Survey. Loneliness was reported using the UCLA loneliness score, dichotomised into higher (≥ 6) versus lower (< 6) loneliness. Engagement in moderate-to-vigorous physical activity, dietary intake, smoking status, alcohol use, and self-reported height and weight were recorded. Behaviours were coded to reflect meeting or not meeting the World Cancer Research Fund recommendations for people LWBC. Logistic regression analyses explored associations between loneliness and health behaviours. Covariates were age, sex, ethnicity, education, marital status, living situation, cancer type, spread and treatment, time since treatment, time since diagnosis and number of comorbid conditions. Multiple imputation was used to account for missing data. RESULTS: 5835 participants, mean age 67.4 (standard deviation = 11.8) years, completed the survey. 56% were female (n = 3266) and 44% (n = 2553) male, and 48% (n = 2786) were living with or beyond breast cancer, 32% (n = 1839) prostate, and 21% (n = 1210) colorectal. Of 5485 who completed the loneliness scale, 81% (n = 4423) of participants reported lower and 19% (n = 1035) higher loneliness. After adjustment for confounders, those reporting higher levels of loneliness had lower odds of meeting the WCRF recommendations for moderate-to-vigorous physical activity (Odds Ratio [OR] 0.78, 95% Confidence Internal [CI], 0.67, 0.97, p =.028), fruit and vegetable intake (OR 0.81, CI 0.67, 1.00, p =.046), and smoking (OR 0.62, 0.46, 0.84, p =.003). No association was observed between loneliness and the other dietary behaviours, alcohol, or body mass index. CONCLUSIONS: Loneliness is relatively common in people LWBC and may represent an unmet need. People LWBC who experience higher levels of loneliness may need additional support to improve their health behaviours.

Reimagining Ambulatory Care in Urology: Conversion of the Urology Clinic into a Procedure Center Improves Patient's Experience.

Introduction: The coronavirus disease 2019 (COVID-19) pandemic made it necessary to practice social distancing and limited in-person encounters in health care. These restrictions created alternative opportunities to enhance patient access to care in the ambulatory setting. We hypothesized that by transforming clinics into centers that prioritize procedures and transitioning ambulatory appointments to telehealth, we could establish a secure, streamlined, and productive method for providing patient care. Methods: Clinic templates were restructured to allow the use of the physical space to perform procedure-based clinics exclusively, while switching to virtual telemedicine for all nonprocedural encounters. Staff members were given specific roles to support one of the patient care modalities for a given day (Procedures vs. Telehealth). Performance and patient satisfaction metrics were collected between two periods of time defined as P1 (February-June 2019) and P2 Post-COVID (February-June 2020) and compared. These served as proxies of periods when the clinic workflow and templates were structured in the traditional versus the emerging way. Statistical analysis was performed using bivariate analyses. Results: The percentage of procedures performed among all in-person visits were higher in P2 compared to P1 (45% vs. 29%, p < 0.001). Although total charges and relative value units were lower in P2, the overall revenue generated was higher compared to P1 ($4,597,846 vs. $4,517,427$, respectively). This increase in revenue was mainly driven by the higher relative income generated by procedures. Patient experience, reflected through patient-reported outcomes, was more favorable in P2 where patients seemed more likely to "Recommend this provider office" (90% vs. 85.7%, p = 0.01), report improved "Access overall" (56% vs. 49%, p = 0.02), and felt they were "Moving through your visit overall" (59% vs. 51%, p = 0.007). Conclusions: Our data suggest that reorganizing urology clinics into a space that is centered around outpatient procedures can represent a model that improves the patient's access to care and clinical experience, while simultaneously improving operational financial strength. This efficient care model could be considered for many practice settings and drive high-value outpatient care.

Implementation of Patient Blood Management in Orthotopic Heart Transplants: A Single Centre Retrospective Observational Review.

BACKGROUND: Blood transfusion in the perioperative cardiothoracic setting has accepted risks including deep sternal wound infection, increased intensive care unit length of stay, lung injury, and cost. It has an immunomodulatory effect which may cause allo-immunisation. This may influence long-term survival through immune-mediated factors. Targeting coagulation defects to reduce unnecessary or inappropriate transfusions may reduce these complications. METHODS: In 2012, an institution-wide patient blood management evidence-based algorithmic bleeding management protocol was implemented at The Prince Charles Hospital, Brisbane, Australia. The benefit of this has been previously reported in our lung transplant and cardiac surgery (excluding transplants) cohorts. This study aimed to investigate the effect of this on our orthotopic heart transplant recipients. RESULTS: After the implementation of the protocol, despite no difference in preoperative haemoglobin levels and higher risk patients (EuroSCORE 20 vs 26; p=0.013), the use of packed red blood cells (13.0 U vs 4.4 U; p=0.046) was significantly lower postoperatively and fresh frozen plasma was significantly lower both intra- and postoperatively (7.4 U vs 0.6 U; p<0.001, and 3.3 U vs 0.6 U; p=0.011 respectively). Concurrently, the use of prothrombin complex concentrate (33% vs 78%; p<0.001) and desmopressin (5% vs 22%; p=0.0028) was significantly higher in the post-protocol group, while there was less use of recombinant factor VIIa (15% vs 4%; p=0.058). Intraoperative units of cryoprecipitate also rose from 0.9 to 2.0 (p=0.006). CONCLUSIONS: We have demonstrated that a targeted patient blood management protocol with point-of-care testing for heart transplant recipients is correlated with fewer blood products used postoperatively, with some increase in haemostatic products and no evidence of increased adverse events.

Measuring dispositional empathy in South African children.

OBJECTIVE: Empathy is a key factor to examine in development, because of its predictive associations with both aggression and successful prosocial behaviour. However, established measures of empathy for Low-to-Middle Income Countries, including South Africa, are lacking. In children, parent-report measures are key. However, a local study examining empathy and aggression (Malcolm-Smith et al., 2015) found poor psychometric performance for a widely used parent-report measure of dispositional empathy, the Griffith Empathy Measure (GEM). We thus investigated which of two questionnaires measuring dispositional cognitive and affective empathy perform better in this context. METHOD: We contrasted internal consistency reliability of a simplified version of the GEM (SGEM; n = 160) and a parent-report version of the Questionnaire of Cognitive and Affective Empathy (QCAE; n = 440) in a low-mid socio-economic status sample. Convergence between the measures and factor structure were also assessed. RESULTS: The parent-report version of the QCAE performed well as a measure of child dispositional cognitive and affective empathy, with good reliability (overall α = 0.90 vs. SGEM α = .63), and confirmatory factor analysis supporting the two-factor structure. The SGEM's reliability and failure to correlate with QCAE indicated poor psychometric performance. CONCLUSION: This is the first psychometric evaluation of the QCAE as a parent-report measure, and our results indicate that it should prove useful for future assessments of dispositional empathy in children across a variety of contexts.

Associations between birth defects with neural crest cell origins and pediatric embryonal tumors.

BACKGROUND: There are few assessments evaluating associations between birth defects with neural crest cell developmental origins (BDNCOs) and embryonal tumors, which are characterized by undifferentiated cells having a molecular profile similar to neural crest cells. The effect of BDNCOs on embryonal tumors was estimated to explore potential shared etiologic pathways and genetic origins. METHODS: With the use of a multistate, registry-linkage cohort study, BDNCO-embryonal tumor associations were evaluated by generating hazard ratios (HRs) and 95% confidence intervals (CIs) with Cox regression models. BDNCOs consisted of ear, face, and neck defects, Hirschsprung disease, and a selection of congenital heart defects. Embryonal tumors included neuroblastoma, nephroblastoma, and hepatoblastoma. Potential HR modification (HRM) was investigated by infant sex, maternal race/ethnicity, maternal age, and maternal education. RESULTS: The risk of embryonal tumors among those with BDNCOs was 0.09% (co-occurring n = 105) compared to 0.03% (95% CI, 0.03%-0.04%) among those without a birth defect. Children with BDNCOs were 4.2 times (95% CI, 3.5-5.1 times) as likely to be diagnosed with an embryonal tumor compared to children born without a birth defect. BDNCOs were strongly associated with hepatoblastoma (HR, 16.1; 95% CI, 11.3-22.9), and the HRs for neuroblastoma (3.1; 95% CI, 2.3-4.2) and nephroblastoma (2.9; 95% CI, 1.9-4.4) were elevated. There was no notable HRM by the aforementioned factors. CONCLUSIONS: Children with BDNCOs are more likely to develop embryonal tumors compared to children without a birth defect. Disruptions of shared developmental pathways may contribute to both phenotypes, which could inform future genomic assessments and cancer surveillance strategies of these conditions.

Healthcare fragmentation, multimorbidity, potentially inappropriate medication, and mortality: a Danish nationwide cohort study.

BACKGROUND: Patients with multimorbidity are frequent users of healthcare, but fragmented care may lead to suboptimal treatment. Yet, this has never been examined across healthcare sectors on a national scale. We aimed to quantify care fragmentation using various measures and to analyze the associations with patient outcomes. METHODS: We conducted a register-based nationwide cohort study with 4.7 million Danish adult citizens. All healthcare contacts to primary care and hospitals during 2018 were recorded. Clinical fragmentation indicators included number of healthcare contacts, involved providers, provider transitions, and hospital trajectories. Formal fragmentation indices assessed care concentration, dispersion, and contact sequence. The patient outcomes were potentially inappropriate medication and all-cause mortality adjusted for demographics, socioeconomic factors, and morbidity level. RESULTS: The number of involved healthcare providers, provider transitions, and hospital trajectories rose with increasing morbidity levels. Patients with 3 versus 6 conditions had a mean of 4.0 versus 6.9 involved providers and 6.6 versus 13.7 provider transitions. The proportion of contacts to the patient's own general practice remained stable across morbidity levels. High levels of care fragmentation were associated with higher rates of potentially inappropriate medication and increased mortality on all fragmentation measures after adjusting for demographic characteristics, socioeconomic factors, and morbidity. The strongest associations with potentially inappropriate medication and mortality were found for ≥ 20 contacts versus none (incidence rate ratio 2.83, 95% CI 2.77-2.90) and ≥ 20 hospital trajectories versus none (hazard ratio 10.8, 95% CI 9.48-12.4), respectively. Having less than 25% of contacts with your usual provider was associated with an incidence rate ratio of potentially inappropriate medication of 1.49 (95% CI 1.40-1.58) and a mortality hazard ratio of 2.59 (95% CI 2.36-2.84) compared with full continuity. For the associations between fragmentation measures and patient outcomes, there were no clear interactions with number of conditions. CONCLUSIONS: Several clinical indicators of care fragmentation were associated with morbidity level. Care fragmentation was associated with higher rates of potentially inappropriate medication and increased mortality even when adjusting for the most important confounders. Frequent contact to the usual provider, fewer transitions, and better coordination were associated with better patient outcomes regardless of morbidity level.

Streamlining quantitative joint-wide medial femoro-tibial histopathological scoring of mouse post-traumatic knee osteoarthritis models.

OBJECTIVES: Histological scoring remains the gold-standard for quantifying post-traumatic osteoarthritis (ptOA) in animal models, allowing concurrent evaluation of numerous joint tissues. Available systems require scoring multiple sections/joint making analysis laborious and expensive. We investigated if a single section allowed equivalent quantitation of pathology in different joint tissues and disease stages, in three ptOA models. METHOD: Male 10-12-week-old C57BL/6 mice underwent surgical medial-meniscal-destabilization, anterior-cruciate-ligament (ACL) transection, non-invasive-ACL-rupture, or served as sham-surgical, non-invasive-ACL-strain, or naïve/non-operated controls. Mice (n = 12/group) were harvested 1-, 4-, 8-, and 16-week post-intervention. Serial sagittal toluidine-blue/fast-green stained sections of the medial-femoro-tibial joint (n = 7/joint, 84 µm apart) underwent blinded scoring of 40 histology-outcomes. We evaluated agreement between single-slide versus entire slide-set maximum or median scores (weighted-kappa), and sensitivity/specificity of single-slide versus median/maximum to detect OA pathology. RESULTS: A single optimal mid-sagittal section showed excellent agreement with median (weighted-kappa 0.960) and maximum (weighted-kappa 0.926) scores. Agreement for individual histology-outcomes was high with only 19/240 median and 15/240 maximum scores having a weighted-kappa ≤0.4, the majority of these (16/19 and 11/15) in control groups. Statistically-significant histology-outcome differences between ptOA models and their controls detected with the entire slide-set were reliably reproduced using a single slide (sensitivity >93.15%, specificity >93.10%). The majority of false-negatives with single-slide scoring were meniscal and subchondral bone histology-outcomes (89%) and occurred in weeks 1-4 post-injury (84%). CONCLUSION: A single mid-sagittal slide reduced the time needed to score diverse histopathological changes by 87% without compromising the sensitivity or specificity of the analysis, across a variety of ptOA models and time-points.

Wnt5a Promotes AT1 and Represses AT2 Lineage-Specific Gene Expression in a Cell-Context-Dependent Manner.

Lung maturation is not limited to proper structural development but also includes differentiation and functionality of various highly specialized alveolar cell types. Alveolar type 1 (AT1s) cells occupy nearly 95% of the alveolar surface and are critical for establishing efficient gas exchange in the mature lung. AT1 cells arise from progenitors specified during the embryonic stage as well as alveolar epithelial progenitors expressing surfactant protein C (Sftpcpos cells) during postnatal and adult stages. Previously, we found that Wnt5a, a non-canonical Wnt ligand, is required for differentiation of AT1 cells during the saccular phase of lung development. To further investigate the role of Wnt5a in AT1 cell differentiation, we generated and characterized a conditional Wnt5a gain-of-function mouse model. Neonatal Wnt5a gain-of-function disrupted alveologenesis through inhibition of cell proliferation. In this setting Wnt5a downregulated ß-catenin-dependent canonical Wnt signaling, repressed AT2 (anti-AT2) and promoted AT1 (pro-AT1) lineage-specific gene expression. In addition, we identified 2 subpopulations of Sftpchigh and Sftpclow alveolar epithelial cells. In Sftpclow cells, Wnt5a exhibits pro-AT1 and anti-AT2 effects, concurrent with inhibition of canonical Wnt signaling. Interestingly, in the Sftpchigh subpopulation, although increasing AT1 lineage-specific gene expression, Wnt5a gain-of-function did not change AT2 gene expression, nor inhibit canonical Wnt signaling. Using primary epithelial cells isolated from human fetal lungs, we demonstrate that this property of Wnt5a is evolutionarily conserved. Wnt5a therefore serves as a selective regulator that ensures proper AT1/AT2 balance in the developing lung.

Fetal anemia and elevated hepcidin in a mouse model of fetal alcohol spectrum disorder.

INTRODUCTION: Prenatal alcohol exposure (PAE) impairs offspring growth and cognition, and this is worsened by concurrent iron deficiency. Alcohol disrupts fetal iron metabolism and produces functional iron deficiency, even when maternal iron status is adequate. We used a mouse model of moderate PAE to investigate the mechanisms underlying this dysregulated iron status. METHODS: C57BL/6J female mice received 3 g/kg alcohol daily from embryonic day (E) 8.5-17.5 and were assessed at E17.5. RESULTS: Alcohol reduced fetal hemoglobin, hematocrit, and red blood cell counts, despite elevated erythropoietin production. Alcohol suppressed maternal hepcidin expression and the upstream iron-sensing BMP/SMAD pathway, consistent with its effects in the nonpregnant state. In contrast, alcohol elevated fetal hepcidin, although this was not accompanied by an upregulation of the BMP/SMAD or proinflammatory IL-6/STAT3 pathways. Fetal expression of hepatic genes contributing to hemoglobin synthesis and iron metabolism were unaffected by alcohol, whereas those affecting ribosome biogenesis were suppressed, suggesting a novel candidate effector for this fetal anemia. CONCLUSION: These data confirm and extend prior observations that PAE disrupts maternal and fetal iron metabolism and impairs the fetus's ability to regulate iron status. We propose this dysregulation increases gestational iron needs and represents a conserved response to PAE. IMPACT: Prenatal alcohol exposure causes a functional iron deficiency in a model that also impairs cognition in later life. Prenatal alcohol exposure causes fetal anemia. This fetal anemia is accompanied by elevated hepcidin and erythropoietin. Findings are consistent with prior observations that prenatal alcohol exposure increases maternal-fetal iron requirements during pregnancy.

Strong recommendations from low certainty evidence: a cross-sectional analysis of a suite of national guidelines.

BACKGROUND: Clinical guidelines should be based on a thorough evaluation of the evidence and generally include a rating of the quality of evidence and assign a strength to recommendations. Grading of Recommendations Assessment, Development and Evaluation (GRADE) guidance warns against making strong recommendations when the certainty of the evidence is low or very low, but has identified five paradigmatic situations (e.g. life-threatening situations) where this may be justified. AIMS AND OBJECTIVES: We aimed to characterize the strength of recommendations and certainty of the evidence in Irish National Clinical Guidelines using the GRADE approach. METHODS: All National Clinical Guidelines from the National Clinical Effectiveness Committee (NCEC) website using the GRADE approach (fully or partially) were included. All recommendations and their corresponding certainty of the evidence, strength of recommendations and justifications were extracted. Authors classified instances of strong recommendations with low certainty evidence (referred to as discordant recommendations) into one of the five paradigmatic situations. Descriptive statistics were calculated. RESULTS: From the 29 NCEC Clinical Guidelines available at the time of analysis, we identified 8 guidelines using GRADE with a total of 240 recommendations; 38 recommendations did not use the GRADE approach and were excluded. Half of the included guidelines focused on emergency situations. In the final dataset of 202 recommendations, 151 (74.7%) were classified as strong and 51 (25.3%) as conditional. Of the 151 strong recommendations, 55 (36.4%) were supported by high or moderate certainty evidence and 96 (63.6%) by low or very low certainty evidence and were considered discordant. Of these 96 discordant recommendations, 55 (73.7%) were consistent with one of the five paradigmatic situations. However, none were specifically described as such within the guidelines. CONCLUSIONS: The proportion of discordant recommendations identified in this analysis was higher than some previous international studies (range of all strong recommendations being discordant 30-50%), but similar to other guidelines focused on emergency situations. The majority of discordant recommendations could be mapped to one of the five situations, but no National Clinical Guideline explicitly referenced this. Guideline developers require further guidance to enable greater transparency in the reporting of the reasons for discordant recommendations.