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

Aligning Practice to Policies: Changing the Culture to Recognize and Reward Teaching at Research Universities

Recent calls for improvement in undergraduate education within STEM (science, technology, engineering, and mathematics) disciplines are hampered by the methods used to evaluate teaching effectiveness. Faculty members at research universities are commonly assessed and promoted mainly on the basis of research success. To improve the quality of undergraduate teaching across all disciplines, not only STEM fields, requires creating an environment wherein continuous improvement of teaching is valued, assessed, and rewarded at various stages of a faculty member’s career. This requires consistent application of policies that reflect well-established best practices for evaluating teaching at the department, college, and university levels. Evidence shows most teaching evaluation practices do not reflect stated policies, even when the policies specifically espouse teaching as a value. Thus, alignment of practice to policy is a major barrier to establishing a culture in which teaching is valued. Situated in the context of current national efforts to improve undergraduate STEM education, including the Association of American Universities Undergraduate STEM Education Initiative, this essay discusses four guiding principles for aligning practice with stated priorities in formal policies: 1) enhancing the role of deans and chairs; 2) effectively using the hiring process; 3) improving communication; and 4) improving the understanding of teaching as a scholarly activity. In addition, three specific examples of efforts to improve the practice of evaluating teaching are presented as examples: 1) Three Bucket Model of merit review at the University of California, Irvine; (2) Evaluation of Teaching Rubric, University of Kansas; and (3) Teaching Quality Framework, University of Colorado, Boulder. These examples provide flexible criteria to holistically evaluate and improve the quality of teaching across the diverse institutions comprising modern higher education

Development of a Protocol for Obtaining Biological Samples for Genetic Testing from Remote Individuals

Pharmacogenomic sequencing allows individuals to learn more about how they will respond to certain medications but requires shipping of a biological sample. One complication of sending biological samples to remote laboratories is stability. Blood generally yields sufficient quantities of high-quality DNA but requires a clinic visit. Saliva and buccal swabs are routinely used for DNA extractions, but the DNA quality is notoriously low due to the presence of bacteria in the mouth. Additionally, elderly individuals have difficulty producing enough saliva for testing, and the tubes contain several milliliters of liquid and shipping requires special considerations. Dried blood spot cards, which serve as an alternative to saliva and buccal swabs, yield high-quality DNA and ship easily, but may produce a lower yield. This project aims to determine which biological sample methods can reasonably be obtained from remote individuals

DNA Extraction Method Development for Solid Tissues

Although germline variation testing is traditionally performed using DNA obtained from blood or other liquid samples, determining somatic variation in cancer samples requires DNA extraction directly from tissues. Additionally, epigenetic markers, such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are tissue-specific and change in selected disease states. However, several substances present in tissues are known to inhibit downstream reactions, including polymerase chain reaction PCR). For this project, we are assessing the quantity and quality of DNA obtained from extractions of various vital organs using 30 different commercially available DNA extraction kits to determine optimal kits for each tissue

White Matter Microstructure and Atypical Visual Orienting in 7-Month-Olds at Risk for Autism

The authors sought to determine whether specific patterns of oculomotor functioning and visual orienting characterize 7-month-old infants who later meet criteria for an autism spectrum disorder (ASD) and to identify the neural correlates of these behaviors

The benefit of directly comparing autism and schizophrenia for revealing mechanisms of social cognitive impairment

Autism and schizophrenia share a history of diagnostic conflation that was not definitively resolved until the publication of the DSM-III in 1980. Though now recognized as heterogeneous disorders with distinct developmental trajectories and dissociative features, much of the early nosological confusion stemmed from apparent overlap in certain areas of social dysfunction. In more recent years, separate but substantial literatures have accumulated for autism and schizophrenia demonstrating that abnormalities in social cognition directly contribute to the characteristic social deficits of both disorders. The current paper argues that direct comparison of social cognitive impairment can highlight shared and divergent mechanisms underlying pathways to social dysfunction, a process that can provide significant clinical benefit by informing the development of tailored treatment efforts. Thus, while the history of diagnostic conflation between autism and schizophrenia may have originated in similarities in social dysfunction, the goal of direct comparisons is not to conflate them once again but rather to reveal distinctions that illuminate disorder-specific mechanisms and pathways that contribute to social cognitive impairment

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

A Phase 2, Double-Blind, Randomized, Dose-Ranging Trial Of Reldesemtiv In Patients With ALS

To evaluate safety, dose response, and preliminary efficacy of reldesemtiv over 12 weeks in patients with amyotrophic lateral sclerosis (ALS). Methods: Patients (≤2 years since diagnosis) with slow upright vital capacity (SVC) of ≥60% were randomized 1:1:1:1 to reldesemtiv 150, 300, or 450 mg twice daily (bid) or placebo; active treatment was 12 weeks with 4-week follow-up. Primary endpoint was change in percent predicted SVC at 12 weeks; secondary measures included ALS Functional Rating Scale-Revised (ALSFRS-R) and muscle strength mega-score. Results: Patients (N = 458) were enrolled; 85% completed 12-week treatment. The primary analysis failed to reach statistical significance (p = 0.11); secondary endpoints showed no statistically significant effects (ALSFRS-R, p = 0.09; muscle strength mega-score, p = 0.31). Post hoc analyses pooling all active reldesemtiv-treated patients compared against placebo showed trends toward benefit in all endpoints (progression rate for SVC, ALSFRS-R, and muscle strength mega-score (nominal p values of 0.10, 0.01 and 0.20 respectively)). Reldesemtiv was well tolerated, with nausea and fatigue being the most common side effects. A dose-dependent decrease in estimated glomerular filtration rate was noted, and transaminase elevations were seen in approximately 5% of patients. Both hepatic and renal abnormalities trended toward resolution after study drug discontinuation. Conclusions: Although the primary efficacy analysis did not demonstrate statistical significance, there were trends favoring reldesemtiv for all three endpoints, with effect sizes generally regarded as clinically important. Tolerability was good; modest hepatic and renal abnormalities were reversible. The impact of reldesemtiv on patients with ALS should be assessed in a pivotal Phase 3 trial. (ClinicalTrials.gov Identifier: NCT03160898)