PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens.

Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service. PinAPL-Py implements state-of-the-art tools and statistical models, assembled in a comprehensive workflow covering sequence quality control, automated sgRNA sequence extraction, alignment, sgRNA enrichment/depletion analysis and gene ranking. The workflow is set up to use a variety of popular sgRNA libraries as well as custom libraries that can be easily uploaded. Various analysis options are offered, suitable to analyze a large variety of CRISPR/Cas9 screening experiments. Analysis output includes ranked lists of sgRNAs and genes, and publication-ready plots. PinAPL-Py helps to advance genome-wide screening efforts by combining comprehensive functionality with user-friendly implementation. PinAPL-Py is freely accessible at http://pinapl-py.ucsd.edu with instructions and test datasets

Suturing Workshop for Third-Year Medical Students: A Modern Educational Approach

Background: This study sought to determine if developing suturing workshops based on modern educational theory would lead to a significant increase in third-year medical students’ confidence and preparedness as compared to before the workshop. Methods: A group of pre-clinical, third-year medical students (n = 20) were voluntarily recruited. The workshop consisted of an interactive didactic session, a hands-on suturing session, and a question-answer session with surgeons. The nine-point Likert scale surveys were given pre-and post-workshop to 17 participants. Total scores of “confidence” and “preparedness” were analyzed using the Student t-test. Results of Q-Q plot and normality tests were used to validate the normality assumption. All analysis was conducted using SAS Software 9.4 (Cary, North Carolina). Results: A statistically significant increase in both confidence and preparedness was found between results of pre- and post-workshop surveys. Average total scores in confidence increased by 19.7 points, from 19.3 to 39 (95% CI: 15.0-24.4; P value \u3c 0.001). For scores in preparedness, the total score increased by an average of 18.4 points, from 22.8 to 41.2 (95% CI: 14.1-22.8; P value \u3c 0.001). Conclusions: These findings suggest that a structured course based on modern educational theory can increase both the confidence and preparedness of third-year medical students who are matriculating into their hospitalbased clerkships

The fracture toughness of small animal cortical bone measured using arc-shaped tension specimens: Effects of bisphosphonate and deproteinization treatments

Small animal models, and especially transgenic models, have become widespread in the study of bone mechanobiology and metabolic bone disease, but test methods for measuring fracture toughness on multiple replicates or at multiple locations within a single small animal bone are lacking. Therefore, the objective of this study was to develop a method to measure cortical bone fracture toughness in multiple specimens and locations along the diaphysis of small animal bones. Arc-shaped tension specimens were prepared from the mid-diaphysis of rabbit ulnae and loaded to failure to measure the radial fracture toughness in multiple replicates per bone. The test specimen dimensions, crack length, and maximum load met requirements for measuring the plane strain fracture toughness. Experimental groups included a control group, bisphosphonate treatment group, and an ex vivo deproteinization treatment following bisphosphonate treatment (5 rabbits/group and 15 specimens/group). The fracture toughness of ulnar cortical bone from rabbits treated with zoledronic acid for six months exhibited no difference compared with the control group. Partially deproteinized specimens exhibited significantly lower fracture toughness compared with both the control and bisphosphonate treatment groups. The deproteinization treatment increased tissue mineral density (TMD) and resulted in a negative linear correlation between the measured fracture toughness and TMD. Fracture toughness measurements were repeatable with a coefficient of variation of 12–16% within experimental groups. Retrospective power analysis of the control and deproteinization treatment groups indicated a minimum detectable difference of 0.1 MPa·m1/2. Therefore, the overall results of this study suggest that arc-shaped tension specimens offer an advantageous new method for measuring the fracture toughness in small animal bones

Trust perceptions of metadata in open-source software: The role of performance and reputation

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Open-source software (OSS) is a key aspect of software creation. However, little is known about programmers’ decisions to trust software from OSS websites. The current study emulated OSS websites and manipulated reputation and performance factors in the stimuli according to the heuristic-systematic processing model. We sampled professional programmers—with a minimum experience of three years—from Amazon Mechanical Turk (N = 38). We used a 3 × 3 within-subjects design to investigate the relationship between OSS reputation and performance on users’ time spent on code, the number of interface clicks, trustworthiness perceptions, and willingness to use OSS code. We found that participants spent more time on and clicked the interface more often for code that was high in reputation. Meta-information included with OSS tools was found to affect the degree to which computer programmers interact with and perceive online code repositories. Furthermore, participants reported higher levels of perceived trustworthiness in and trust toward highly reputable OSS code. Notably, we observed fewer significant main effects for the performance manipulation, which may correspond to participants considering performance attributes mainly within the context of reputation-relevant information. That is, the degree to which programmers investigate and then trust OSS code may depend on the initial reputation ratings

An Evaluation of Studies on the Potential Threats Contributing to the Decline of Eastern Migratory North American Monarch Butterflies (Danaus plexippus)

The migratory monarch butterflies (Danaus plexippus) of eastern North America have undergone large-scale declines, which may be attributable to a variety of underlying causes. The uncertainty about the primary cause of declines and whether individual threats are likely to increase in the future presents challenges for developing effective conservation management and policy initiatives that aim to improve population viability. This paper identifies five potential threats and classifies these threats according to the types of studies (observational, experimental, simulation/models) and their current impact and anticipated risk. Broadly, the threats can be classified into five categories: (1) change in suitable abiotic environmental conditions; (2) deforestation in the overwintering range; (3) exposure to contaminants including the bacteria Bacillus thuringiensis, herbicides, and insecticides; (4) loss of breeding habitat; and (5) predation, parasitism, and species-specific pathogens. The vast distribution of the monarch butterfly makes it likely that population declines are attributed to a suite of interacting factors that vary spatially and temporally in their contribution. Nonetheless, the published papers we reviewed suggest the decline in suitable environmental conditions in addition to overwintering (i.e., deforestation) and breeding habitat loss are the most likely threats to continue to affect the population viability of monarch butterflies

(3α,5α)3-Hydroxypregnan-20-one (3α,5α-THP) Regulation of the HPA Axis in the Context of Different Stressors and Sex

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3α,5α-THP regulation of stress-induced hypothalamic CRF, CRF receptor type 1 (CRFR1), CRF binding protein (CRFBP), pro-opiomelanocortin (POMC), and glucocorticoid receptor (GR) by western blot and circulating corticosterone (CORT) by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague Dawley rats. Tissue was collected after rats were injected with 3α,5α-THP (15 mg/kg, IP) or vehicle 15 min prior to 30 min of restraint stress (RS), or 10 min of forced swim stress (FSS) and 20 min recovery. The initial exposure to a stress stimulus increased circulating CORT levels in both males and females, but 3α,5α-THP attenuated the CORT response only in females after RS. 3α,5α-THP reduced GR levels in male and females, but differently between stressors. 3α,5α-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3α,5α-THP reduced the CRFR1, CRFBP, and POMC increases after RS and FSS in males, but in females only after FSS. Our results showed different stress responses following different types of stressors: 3α,5α-THP regulated the HPA axis at different levels, depending on sex

Spontaneous Isomerization of Long-Lived Proteins Provides a Molecular Mechanism for the Lysosomal Failure Observed in Alzheimer's Disease.

Proteinaceous aggregation is a well-known observable in Alzheimer's disease (AD), but failure and storage of lysosomal bodies within neurons is equally ubiquitous and actually precedes bulk accumulation of extracellular amyloid plaque. In fact, AD shares many similarities with certain lysosomal storage disorders though establishing a biochemical connection has proven difficult. Herein, we demonstrate that isomerization and epimerization, which are spontaneous chemical modifications that occur in long-lived proteins, prevent digestion by the proteases in the lysosome (namely, the cathepsins). For example, isomerization of aspartic acid into l-isoAsp prevents digestion of the N-terminal portion of Aβ by cathepsin L, one of the most aggressive lysosomal proteases. Similar results were obtained after examination of various target peptides with a full series of cathepsins, including endo-, amino-, and carboxy-peptidases. In all cases peptide fragments too long for transporter recognition or release from the lysosome persisted after treatment, providing a mechanism for eventual lysosomal storage and bridging the gap between AD and lysosomal storage disorders. Additional experiments with microglial cells confirmed that isomerization disrupts proteolysis in active lysosomes. These results are easily rationalized in terms of protease active sites, which are engineered to precisely orient the peptide backbone and cannot accommodate the backbone shift caused by isoaspartic acid or side chain dislocation resulting from epimerization. Although Aβ is known to be isomerized and epimerized in plaques present in AD brains, we further establish that the rates of modification for aspartic acid in positions 1 and 7 are fast and could accrue prior to plaque formation. Spontaneous chemistry can therefore provide modified substrates capable of inducing gradual lysosomal failure, which may play an important role in the cascade of events leading to the disrupted proteostasis, amyloid formation, and tauopathies associated with AD