DNA Extraction Method Development for Ocular Tissues

Purpose: DNA extraction kits are traditionally developed to work with liquid tissues such as blood, saliva, and swabs, but some have been proposed to work with solid tissues. Somatic variation in cancers can be important for tumor subtyping and treatment guidance, including ocular tumors. Additionally, epigenetic marks such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are tissue-specific and change in disease states, particularly evident in diabetic retinopathy and age-related macular degeneration. Commercial DNA extraction kits are available from several vendors, but the various kits have different strengths and weaknesses, and the removal of PCR inhibitors will vary with each kit. This project investigates the yield and purity of DNA from ocular tissues using commercial DNA extraction kits. Methods: Cornea, neural retina, RPE/choroid layer, optic nerve, and capsular bag were collected and aliquoted into 15 mg aliquots. Extractions were performed using the following kits: DNEasy Blood and Tissue Kit (Qiagen;), GeneJET Genomic DNA Purification Kit (ThermoFisher Scientific), Monarch HMW DNA Extraction Kit for Tissue (New England Biosciences), and genomicPrep Mini Spin Kit (Cytiva). DNA was quantified using the Qubit Fluorometer and molecular weight was checked by agarose gel. Several more kits are currently being tested. Results: All four kits yielded high molecular weight DNA (above 20 kbp). The Monarch HMW kit yielded DNA with significantly higher molecular weights. The DNA yields per milligram of tissue were highest using the DNEasy Blood and Tissue Kit for optic nerve, neural retina, and RPE/choroid. The yield was highest for the cornea using the genomicPrep Mini Spin Kit. Only the genomicPrep Mini Spin Kit yielded sufficient DNA for quantification from the capsular bag, and total yields were minimal (600 ng or less). Additional kits are currently being tested, but initial results indicate that several commercial kits will be sufficient for DNA extraction of ocular tissues. Further work is needed to purify epithelial cells and stem cells from the intraocular lens. Conclusions: Of the kits tested, all are sufficient to obtain significant amounts of DNA from all ocular tissues aside from the capsular bag. The Monarch HMW yielded the highest molecular weight, but significantly lower quantities of DNA than the other kits, indicating that it may not be ideal for most purposes. Protocol development for the capsular bag is still underway

Rheumatoid arthritis - treatment: 180. Utility of Body Weight Classified Low-Dose Leflunomide in Japanese Rheumatoid Arthritis

Background: In Japan, more than 20 rheumatoid arthritis (RA) patients died of interstitial pneumonia (IP) caused by leflunomide (LEF) were reported, but many of them were considered as the victims of opportunistic infection currently. In this paper, efficacy and safety of low-dose LEF classified by body weight (BW) were studied. Methods: Fifty-nine RA patients were started to administrate LEF from July 2007 to July 2009. Among them, 25 patients were excluded because of the combination with tacrolimus, and medication modification within 3 months before LEF. Remaining 34 RA patients administered 20 to 50 mg/week of LEF were followed up for 1 year and enrolled in this study. Dose of LEF was classified by BW (50 mg/week for over 50 kg, 40 mg/week for 40 to 50 kg and 20 to 30 mg/week for under 40 kg). The average age and RA duration of enrolled patients were 55.5 years old and 10.2 years. Prednisolone (PSL), methotrexate (MTX) and etanercept were used in 23, 28 and 2 patients, respectively. In case of insufficient response or adverse effect, dosage change or discontinuance of LEF were considered. Failure was defined as dosages up of PSL and MTX, or dosages down or discontinuance of LEF. Last observation carried forward method was used for the evaluation of failed patients at 1 year. Results: At 1 year after LEF start, good/ moderate/ no response assessed by the European League Against Rheumatism (EULAR) response criteria using Disease Activity Score, including a 28-joint count (DAS28)-C reactive protein (CRP) were showed in 14/ 10/ 10 patients, respectively. The dosage changes of LEF at 1 year were dosage up: 10, same dosage: 5, dosage down: 8 and discontinuance: 11 patients. The survival rate of patients in this study was 23.5% (24 patients failed) but actual LEF continuous rate was 67.6% (11 patients discontinued) at 1 year. The major reason of failure was liver dysfunction, and pneumocystis pneumonia was occurred in 1 patient resulted in full recovery. One patient died of sepsis caused by decubitus ulcer infection. DAS28-CRP score was decreased from 3.9 to 2.7 significantly. Although CRP was decreased from 1.50 to 0.93 mg/dl, it wasn't significant. Matrix metalloproteinase (MMP)-3 was decreased from 220.0 to 174.2 ng/ml significantly. Glutamate pyruvate transaminase (GPT) was increased from 19 to 35 U/l and number of leukocyte was decreased from 7832 to 6271 significantly. DAS28-CRP, CRP, and MMP-3 were improved significantly with MTX, although they weren't without MTX. Increase of GPT and leukopenia were seen significantly with MTX, although they weren't without MTX. Conclusions: It was reported that the risks of IP caused by LEF in Japanese RA patients were past IP history, loading dose administration and low BW. Addition of low-dose LEF is a potent safe alternative for the patients showing unsatisfactory response to current medicines, but need to pay attention for liver function and infection caused by leukopenia, especially with MTX. Disclosure statement: The authors have declared no conflicts of interes

Crowdsourcing hypothesis tests: Making transparent how design choices shape research results

To what extent are research results influenced by subjective decisions that scientists make as they design studies? Fifteen research teams independently designed studies to answer fiveoriginal research questions related to moral judgments, negotiations, and implicit cognition. Participants from two separate large samples (total N > 15,000) were then randomly assigned to complete one version of each study. Effect sizes varied dramatically across different sets of materials designed to test the same hypothesis: materials from different teams renderedstatistically significant effects in opposite directions for four out of five hypotheses, with the narrowest range in estimates being d = -0.37 to +0.26. Meta-analysis and a Bayesian perspective on the results revealed overall support for two hypotheses, and a lack of support for three hypotheses. Overall, practically none of the variability in effect sizes was attributable to the skill of the research team in designing materials, while considerable variability was attributable to the hypothesis being tested. In a forecasting survey, predictions of other scientists were significantly correlated with study results, both across and within hypotheses. Crowdsourced testing of research hypotheses helps reveal the true consistency of empirical support for a scientific claim.</div

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Myocardial infarction after intravitreal vascular endothelial growth factor inhibitors: a whole population study

Purpose: To determine the risk of thromboembolic and gastrointestinal bleeding events in the 12 months after injections of bevacizumab or ranibizumab compared with photodynamic therapy and a non-treated community sample.Methods: Hospital and death records were examined for 1,267 patients treated with vascular endothelial growth factor inhibitor and 399 patients treated with photodynamic therapy attending Western Australian eye clinics from 2002 to 2008, and 1,763 community controls, aged =50 years. Hospital records from 1995 to 2009 were analyzed for history of myocardial infarction (MI), stroke, and gastrointestinal bleeding before treatment. Records were searched for evidence of these events in the 12 months after treatment.Results: The 12-month MI rate was higher for vascular endothelial growth factor inhibitor patients than photodynamic therapy patients and the community group (1.9/100 vs. 0.8 and 0.7, respectively). No differences were observed between patients treated with bevacizumab and ranibizumab. The adjusted MI rate was 2.3 times greater than the community group (95% confidence interval, 1.2–4.5) and photodynamic therapy rate (95% confidence interval, 0.7–7.7). The 12-month MI risk did not increase with the number of injections administered (hazard ratio, 0.9; 95% confidence interval, 0.5–1.5). Stroke and gastrointestinal bleeding did not differ between any exposure groups.Conclusion: Although all the adverse events examined were rare, patients treated with vascular endothelial growth factor inhibitors were significantly more likely to experience fatal or nonfatal MI than the community group. This increased risk may be related to the underlying age-related macular degeneration or vascular endothelial growth factor inhibitor use itself

User testing of a diagnostic decision support system with machine-assisted chart review to facilitate clinical genomic diagnosis.

OBJECTIVES: There is a need in clinical genomics for systems that assist in clinical diagnosis, analysis of genomic information and periodic reanalysis of results, and can use information from the electronic health record to do so. Such systems should be built using the concepts of human-centred design, fit within clinical workflows and provide solutions to priority problems. METHODS: We adapted a commercially available diagnostic decision support system (DDSS) to use extracted findings from a patient record and combine them with genomic variant information in the DDSS interface. Three representative patient cases were created in a simulated clinical environment for user testing. A semistructured interview guide was created to illuminate factors relevant to human factors in CDS design and organisational implementation. RESULTS: Six individuals completed the user testing process. Tester responses were positive and noted good fit with real-world clinical genetics workflow. Technical issues related to interface, interaction and design were minor and fixable. Testers suggested solving issues related to terminology and usability through training and infobuttons. Time savings was estimated at 30%-50% and additional uses such as in-house clinical variant analysis were suggested for increase fit with workflow and to further address priority problems. CONCLUSION: This study provides preliminary evidence for usability, workflow fit, acceptability and implementation potential of a modified DDSS that includes machine-assisted chart review. Continued development and testing using principles from human-centred design and implementation science are necessary to improve technical functionality and acceptability for multiple stakeholders and organisational implementation potential to improve the genomic diagnosis process

User testing of a diagnostic decision support system with machine-assisted chart review to facilitate clinical genomic diagnosis

Objectives There is a need in clinical genomics for systems that assist in clinical diagnosis, analysis of genomic information and periodic reanalysis of results, and can use information from the electronic health record to do so. Such systems should be built using the concepts of human-centred design, fit within clinical workflows and provide solutions to priority problems.Methods We adapted a commercially available diagnostic decision support system (DDSS) to use extracted findings from a patient record and combine them with genomic variant information in the DDSS interface. Three representative patient cases were created in a simulated clinical environment for user testing. A semistructured interview guide was created to illuminate factors relevant to human factors in CDS design and organisational implementation.Results Six individuals completed the user testing process. Tester responses were positive and noted good fit with real-world clinical genetics workflow. Technical issues related to interface, interaction and design were minor and fixable. Testers suggested solving issues related to terminology and usability through training and infobuttons. Time savings was estimated at 30%–50% and additional uses such as in-house clinical variant analysis were suggested for increase fit with workflow and to further address priority problems.Conclusion This study provides preliminary evidence for usability, workflow fit, acceptability and implementation potential of a modified DDSS that includes machine-assisted chart review. Continued development and testing using principles from human-centred design and implementation science are necessary to improve technical functionality and acceptability for multiple stakeholders and organisational implementation potential to improve the genomic diagnosis process

Too Much Feeling : SE Hinton's The Outsiders (1967), Conflicting Emotions, Identity, and Socialization

This article argues that emotions are utilized for norm breaking, identity formation, and socialization in S.E. Hinton's YA novel The Outsiders (1967). Drawing on the history of emotions studies, it investigates how emotional expressions are utilized to negotiate and contest given emotional norms on the one hand, and Young Adult literary conventions on the other. The point of departure is intersectional and focuses on the relationship between emotion, power, and socialization. In particular, the article considers how intersections of age, gender, and class relate to depictions of feeling and establishing of new emotional norms. The article shows that the feelings that the main character Pony expresses are part of a reiterative process of negotiation of power; they work as instruments for changing emotional norms connected to his age, class, and gender