Cataract-associated P23T γ 3D-crystallin retains a native-like fold in amorphous-looking aggregates formed at physiological pH

Cataracts cause vision loss through the large-scale aggregation of eye lens proteins as a result of ageing or congenital mutations. The development of new treatments is hindered by uncertainty about the nature of the aggregates and their mechanism of formation. We describe the structure and morphology of aggregates formed by the P23T human Î 3D-crystallin mutant associated with congenital cataracts. At physiological pH, the protein forms aggregates that look amorphous and disordered by electron microscopy, reminiscent of the reported formation of amorphous deposits by other crystallin mutants. Surprisingly, solid-state NMR reveals that these amorphous deposits have a high degree of structural homogeneity at the atomic level and that the aggregated protein retains a native-like conformation, with no evidence for large-scale misfolding. Non-physiological destabilizing conditions used in many in vitro aggregation studies are shown to yield qualitatively different, highly misfolded amyloid-like fibrils

Improved microarray gene expression profiling of virus-infected cells after removal of viral RNA

<p>Abstract</p> <p>Background</p> <p>Sensitivity and accuracy are key points when using microarrays to detect alterations in gene expression under different conditions. Critical to the acquisition of reliable results is the preparation of the RNA. In the field of virology, when analyzing the host cell's reaction to infection, the often high representation of viral RNA (vRNA) within total RNA preparations from infected cells is likely to interfere with microarray analysis. Yet, this effect has not been investigated despite the many reports that describe gene expression profiling of virus-infected cells using microarrays.</p> <p>Results</p> <p>In this study we used coronaviruses as a model to show that vRNA indeed interferes with microarray analysis, decreasing both sensitivity and accuracy. We also demonstrate that the removal of vRNA from total RNA samples, by means of virus-specific oligonucleotide capturing, significantly reduced the number of false-positive hits and increased the sensitivity of the method as tested on different array platforms.</p> <p>Conclusion</p> <p>We therefore recommend the specific removal of vRNA, or of any other abundant 'contaminating' RNAs, from total RNA samples to improve the quality and reliability of microarray analyses.</p

Interleukin-33 rescues perivascular adipose tissue anticontractile function in obesity

Perivascular adipose tissue (PVAT) depots are metabolically active and play a major vasodilator role in healthy lean individuals. In obesity, they become inflamed and eosinophil-depleted and the anticontractile function is lost with the development of diabetes and hypertension. Moreover, eosinophil-deficient ΔdblGATA-1 mice lack PVAT anticontractile function and exhibit hypertension. Here, we have investigated the effects of inducing eosinophilia on PVAT function in health and obesity. Control, obese, and ΔdblGATA-1 mice were administered intraperitoneal injections of interleukin-33 (IL-33) for 5 days. Conscious restrained blood pressure was measured, and blood was collected for glucose and plasma measurements. Wire myography was used to assess the contractility of mesenteric resistance arteries. IL-33 injections induced a hypereosinophilic phenotype. Obese animals had significant elevations in blood pressure, blood glucose, and plasma insulin, which were normalized with IL-33. Blood glucose and insulin levels were also lowered in lean treated mice. In arteries from control mice, PVAT exerted an anticontractile effect on the vessels, which was enhanced with IL-33 treatment. In obese mice, loss of PVAT anticontractile function was rescued by IL-33. Exogenous application of IL-33 to isolated arteries induced a rapidly decaying endothelium-dependent vasodilation. The therapeutic effects were not seen in IL-33-treated ΔdblGATA-1 mice, thereby confirming that the eosinophil is crucial. In conclusion, IL-33 treatment restored PVAT anticontractile function in obesity and reversed development of hypertension, hyperglycemia, and hyperinsulinemia. These data suggest that targeting eosinophil numbers in PVAT offers a novel approach to the treatment of hypertension and type 2 diabetes in obesity

Addressing Recruitment Challenges in the Engage-HU Trial in Young Children with Sickle Cell Disease

Background: Sickle cell disease (SCD) is a genetic disorder that causes significant medical and neurologic morbidity in children. Hydroxyurea (HU) is the primary medication used to prevent these complications. National Heart, Lung, and Blood Institute (NHLBI) guidelines recommend offering HU to children as young as 9 months of age with SCD (HbSS or HbSB0 thalassemia) using a shared decision-making approach. Although HU has proven efficacious it remains underutilized and caregivers report that they are not always actively involved in the decision to initiate this therapy. Reasons for limited HU uptake likely include lack of clinician knowledge and training and negative caregiver perceptions. Thus, we developed the Engage-HU trial as a novel approach to address HU utilization barriers. A critical consideration for this trial was that SCD primarily affects individuals of African and Hispanic/Latino descent. In these minority populations, intervention trials are sometimes terminated early because of recruitment difficulties related to mistrust of research, caregiver burden, and transportation issues. As such, the Engage-HU trial design included best-practice strategies for recruiting people of color in research. This study describes these strategies, the initial recruitment plan, preliminary recruitment outcomes and strategies, and our procedural adaptations. Study Design and Methods: Engage-HU is a randomized control trial (NCT03442114) to assess how clinicians can engage caregivers in a shared discussion that considers their values and preferences and includes evidence that supports HU. Engage-HU compares two dissemination methods for clinicians to facilitate shared decision-making with caregivers of young children with SCD: 1) the American Society of Hematology Pocket Guide, and 2) the HU Shared-Decision Making (H-SDM) Toolkit. The study aims to recruit 174 caregivers and evaluate the effectiveness of the dissemination methods on patient-centered outcomes (caregiver confidence in decision-making and perceptions of experiencing shared decision-making) as well as HU uptake and child health outcomes. Eligible children are aged 0 to 5 years, candidates for HU, and their caregiver has not made a decision about HU in the past 3 months. The trial is being conducted at 9 sites in the United States and uses a stepped-wedge design. Data will be analyzed based on the intent-to-treat principle. All participants will remain in the arm of the study to which they were randomized, regardless of whether or not they receive the assigned dissemination method. The primary endpoints are caregiver decisional uncertainty and caregiver perception of shared decision-making measured using validated tools. Data will be analyzed using a linear mixed effects regression model with a robust variance estimator and maximum likelihood estimation with observations clustered within site. The Engage-HU trial includes adaptations to increase recruitment such as tailored messaging, a relational recruitment approach, streamlined data collection, and a Stakeholder Advisory Committee. However, even with these adaptations, the first 6-months of the trial yielded lower than anticipated recruitment. Rather than terminate the trial or accept low enrollment, the research team implemented a series of recruitment strategies to address barriers including helping to improve research coordinator knowledge of the study purpose and adjusting no-show and follow-up procedures (e.g., calls to families after missed appointments and reminder calls before appointments). Site clinicians and clinic staff were provided with additional training so they could give more context about Engage-HU to caregivers and the study principal investigator led monthly "all coordinator" calls to provide support by sharing updates and experiences about successful recruitment. Implementation of these strategies resulted in triple the number of enrollments over the next 7-months compared to the previous 6-months (Table 1). Our goal in sharing this information is to provide lessons learned that can be implemented in future trials with the systematically underserved SCD population. It is also anticipated that methods described here may also inform clinical approaches to better engage caregivers of young children around critical clinical conversations, such as initiating medications like HU. Disclosures King: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioline: Consultancy; RiverVest: Consultancy; Novimmune: Research Funding; Celgene: Consultancy; Tioma Therapuetics: Consultancy; Amphivena Therapeutics: Research Funding; WUGEN: Current equity holder in private company; Cell Works: Consultancy; Incyte: Consultancy. Smith-Whitley:Prime: Other: Education material; Celgene: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Neumayr:Emmaus: Consultancy; Bayer: Consultancy; CTD Holdings: Consultancy; Pfizer: Consultancy; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Micelle: Other: Site principal investigator; GBT: Other: Site principal investigator; PCORI: Other: site principal investigator; Novartis: Other: co-investigator; Bluebird Bio: Other: co-investigator; Sangamo Therapeutics: Other; Silarus: Other; Celgene: Other; La Jolla Pharmaceuticals: Other; Forma: Other; Imara: Other; National Heart, Lung, and Blood Institute: Other; Health Resources and Services Administration: Other; Centers for Disease Control and Prevention: Other; Seattle Children's Research: Other. Yates:Novartis: Research Funding. Thompson:Novartis: Consultancy, Honoraria, Research Funding; CRISPR/Vertex: Research Funding; BMS: Consultancy, Research Funding; Baxalta: Research Funding; Biomarin: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding. </jats:sec

Engaging Caregivers and Providers of Children With Sickle Cell Anemia in Shared Decision Making for Hydroxyurea: Protocol for a Multicenter Randomized Controlled Trial

BACKGROUND: Sickle cell anemia (SCA) is a genetic blood disorder that puts children at a risk of serious medical complications, early morbidity and mortality, and high health care utilization. Until recently, hydroxyurea was the only disease-modifying treatment for this life-threatening disease and has remained the only option for children younger than 5 years. Evidence-based guidelines recommend using a shared decision-making (SDM) approach for offering hydroxyurea to children with SCA (HbSS or HbS/β0 thalassemia) aged as early as 9 months. However, the uptake remains suboptimal, likely because caregivers lack information about hydroxyurea and have concerns about its safety and potential long-term side effects. Moreover, clinicians do not routinely receive training or tools, especially those that provide medical evidence and consider caregivers' preferences and values, to facilitate a shared discussion with caregivers. OBJECTIVE: The aim of this study is to understand how best to help parents of young children with sickle cell disease and their clinicians have a shared discussion about hydroxyurea (one that considers medical evidence and parent values and preferences). METHODS: We designed our study to compare the effectiveness of two methods for disseminating hydroxyurea guidelines to facilitate SDM: a clinician pocket guide (ie, usual care) and a clinician hydroxyurea SDM toolkit (H-SDM toolkit). Our primary outcomes are caregiver reports of decisional uncertainty and knowledge of hydroxyurea. The study also assesses the number of children (aged 0-5 years) who were offered and prescribed hydroxyurea and the resultant health outcomes. RESULTS: The Ethics Committee of the Cincinnati Children's Hospital Medical Center approved this study in November 2017. As of February 2021, we have enrolled 120 caregiver participants. CONCLUSIONS: The long-term objective of this study is to improve the quality of care for children with SCA. Using multicomponent dissemination methods developed in partnership with key stakeholders and designed to address barriers to high-quality care, caregivers of patients with SCA can make informed and shared decisions about their health. TRIAL REGISTRATION: ClinicalTrials.gov NCT03442114; https://clinicaltrials.gov/ct2/show/NCT03442114. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/27650

The PIKfyve Inhibitor YM201636 Blocks the Continuous Recycling of the Tight Junction Proteins Claudin-1 and Claudin-2 in MDCK cells

Tight junctions mediate the intercellular diffusion barrier found in epithelial tissues but they are not static complexes; instead there is rapid movement of individual proteins within the junctions. In addition some tight junction proteins are continuously being endocytosed and recycled back to the plasma membrane. Understanding the dynamic behaviour of tight junctions is important as they are altered in a range of pathological conditions including cancer and inflammatory bowel disease. In this study we investigate the effect of treating epithelial cells with a small molecule inhibitor (YM201636) of the lipid kinase PIKfyve, a protein which is involved in endocytic trafficking. We show that MDCK cells treated with YM201636 accumulate the tight junction protein claudin-1 intracellularly. In contrast YM201636 did not alter the localization of other junction proteins including ZO-1, occludin and E-cadherin. A biochemical trafficking assay was used to show that YM201636 inhibited the endocytic recycling of claudin-1, providing an explanation for the intracellular accumulation. Claudin-2 was also found to constantly recycle in confluent MDCK cells and treatment with YM201636 blocked this recycling and caused accumulation of intracellular claudin-2. However, claudin-4 showed negligible endocytosis and no detectable intracellular accumulation occurred following treatment with YM201636, suggesting that not all claudins show the same rate of endocytic trafficking. Finally, we show that, consistent with the defects in claudin trafficking, incubation with YM201636 delayed formation of the epithelial permeability barrier. Therefore, YM201636 treatment blocks the continuous recycling of claudin-1/claudin-2 and delays epithelial barrier formation

Plasmatic and urinary glycosaminoglycans characterization in mucopolysaccharidosis II Patient treated with enzyme-replacement therapy with Idursulfase

We report the structural characterization of plasmatic and urinary GAGs in a Patient affected by MPS II (Hunter syndrome) before and during the first ten months of enzyme-replacement therapy (ERT). Plasmatic GAGs before ERT were rich in pathological DS consisting of iduronic acid (IdoA) and composed of ~90% \uf044Di4s and trace amounts of disulfated disaccharides. DS was also characterized as the main (~90%) urinary GAG mainly composed of ~90% \uf044Di4s with minor percentages of monosulfated and disulfated disaccharides, in particular \u394Di2,4dis. After 300 days of ERT, plasmatic DS strongly decreased but ~14% of IdoA-rich \uf044Di4s was still detected. Similarly, urinary galactosaminoglycans were mainly composed of 78% \uf044Di4s, ~11% \uf044Di6s and ~4% \uf044Di0s with the persistence of \u394Di2,4dis (~4%). About 40% of IdoA-formed \uf044Di4s were also calculated thus confirming that pathological DS is still present in excreted urinary GAGs during ERT. By considering the % of IdoA, we observed rather similar kinetics of excretion in fluids from the beginning of the treatment. Immediately after the first enzyme infusion, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and this process lasts for about two weeks. After this, the percentage of IdoA-rich material present in biological fluids remains fairly constant over the following nine months of treatment. To date, these are the first data regarding plasmatic and urinary kinetics directly measured on products released by the activity of the recombinant enzyme Idursulfase, iduronate-2-sulfatase, evaluated using specific and sensitive analytical procedures

Seropositivity to Herpes Simplex Virus Antibodies and Risk of Alzheimer's Disease: A Population-Based Cohort Study

International audienceBACKGROUND: Herpes Simplex Virus (HSV) infection has been proposed as a possible risk factor of Alzheimer's Disease (AD) notably because it is neurotropic, ubiquitous in the general population and able to establish lifelong latency in the host. The fact that HSV was present in elderly subjects with AD suggests that the virus could be a co-factor of the disease. We investigated the risk of developing AD in anti-HSV immunoglobulin G (IgG) positive subjects (indicator of a lifelong infection to HSV) and IgM-positive subjects (indicator of primary infection or reactivation of the virus) in a longitudinal population-based cohort of elderly subjects living in the community. METHODS: Cox proportional hazard models were used to study the risk of developing AD according to the presence or not of anti-HSV IgG and IgM antibodies, assessed in the sera of 512 elderly initially free of dementia followed for 14 years. RESULTS: During the follow-up, 77 incident AD cases were diagnosed. Controlled for age, gender, educational level and Apolipoprotein E4 (APOE4) status, IgM-positive subjects showed a significant higher risk of developing AD (HR = 2.55; 95% CI [1.38-4.72]), although no significant increased risk was observed in IgG-positive subjects (HR = 1.67; 95%CI [0.75-3.73]). No modification effect with APOE4 status was found. CONCLUSION: Reactivation of HSV seropositivity is highly correlated with incident AD. HSV chronic infection may therefore be contributive to the progressive brain damage characteristic of AD