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Justin Hall’s conversation with Hillary Chute describes the evolution of his 2012 anthology No Straight Lines: Four Decades of Queer Comics out of the world’s first museum show of LGBTQ+ comics held at the Cartoon Art Museum in San Francisco in 2006, including its inspiration for a feature-length documentary, directed and produced by Peabody Award-winning and Emmy-nominated filmmaker Vivian Kleiman. Cultivating and catalyzing the field of queer comics, Hall stands out as much for his own creations as for celebrating and drawing attention to other queer artists’ achievements, demonstrating the value of extended family-building and the satisfaction and ongoing discovery of LGBTQ+ recognition as core features of queer life.

BackgroundExcessive use of CT pulmonary angiography (CTPA) to investigate pulmonary embolism (PE) in the emergency department (ED) contributes to adverse patient outcomes. Non-invasive D-dimer testing, in the context of a clinical algorithm, may help decrease unnecessary imaging but this has not been widely implemented in Canadian EDs.AimTo improve the diagnostic yield of CTPA for PE by 5% (absolute) within 12 months of implementing the YEARS algorithm.Measures and designSingle centre study of all ED patients >18 years investigated for PE with D-dimer and/or CTPA between February 2021 and January 2022. Primary and secondary outcomes were the diagnostic yield of CTPA and frequency of CTPA ordered compared with baseline. Process measures included the percentage of D-dimer tests ordered with CTPA and CTPAs ordered with D-dimers ResultsOver 12 months, 2695 patients were investigated for PE, of which 942 had a CTPA. Compared with baseline, the CTPA yield increased by 2.9% (12.6% vs 15.5%, 95% CI −0.06% to 5.9%) and the proportion of patients that underwent CTPA decreased by 11.4% (46.4% vs 35%, 95% CI −14.1% to −8.8%). The percentage of CTPAs ordered with a D-dimer increased by 26.3% (30.7% vs 57%, 95% CI 22.2% 30.3%) and there were two missed PE (2/2695, 0.07%).ImpactImplementing the YEARS criteria may safely improve the diagnostic yield of CTPAs and reduce the number of CTPAs completed without an associated increase in missed clinically significant PEs. This project provides a model for optimising the use of CTPA in the ED.

DEL selections are binding assays conducted with mixtures of chemically diverse DNA-tagged ligands and a screening target. DEL selections use DNA sequence counts to measure target binding, where ideally higher affinity ligands will have higher counts than weaker affinity ligands. However, there is not always a clear relationship between DNA sequence count (assay signal) and binding affinity. This disconnect may be due to the fidelity of library chemistry, where reactions often do not go to completion, and also to repetitive rounds of binding and elution that are standard practice in most DEL selection experiments. We describe here a strategy that addresses both of these issues and provides a means to calculate ligand affinity from primary selection data. The reaction yields of selected compounds during DEL library synthesis can also be predicted with this method.

Injuries to peripheral nerves are frequent, yet no drug therapies are available for effective nerve repair. The slow growth rate of axons and inadequate access to growth factors challenge natural repair of nerves. A better understanding of the molecules that can promote the rate of axon growth may reveal therapeutic opportunities. Molecular profiling of injured neurons at early intervals of injury, when regeneration is at the maximum, has been the gold standard for exploring growth promoters. A complementaryin vitroregenerative priming model was recently shown to induce enhanced outgrowth in adult sensory neurons. In this work, we exploited thein vitropriming model to reveal novel candidates for adult nerve regeneration. We performed a whole-tissue proteomics analysis of thein vitroprimed dorsal root ganglia (DRGs) from adult SD rats and compared their molecular profile with that of thein vivoprimed, and control DRGs. The proteomics data generated are available via ProteomeXchange with identifier PXD031927. From the follow-up analysis, Bioinformatics interventions, and literature curation, we identified several molecules that were differentially expressed in the primed DRGs with a potential to modulate adult nerve regrowth. We then validated the growth promoting roles of mesencephalic astrocyte-derived neurotrophic factor (MANF), one of the hits we identified, in adult rat sensory neurons. Overall, in this study, we explored two growth priming paradigm and shortlisted several candidates, and validated MANF, as potential targets for adult nerve regeneration. We also demonstrate that thein vitropriming model is a valid tool for adult nerve regeneration studies.

A diaryl ketone series was identified as vanin-1 inhibitors from a high-throughput screening campaign. While this novel scaffold provided valuable probe

Chest X-rays play a significant role in diagnosing pneumonia due to the technology's cost-effectiveness and rapid development times. Detecting pneumonia in chest Xrays is a challenging process that relies heavily upon the availability of trained radiologists and high-quality imagery. Training qualified interpreters require significant resources, while medical imaging remains prone to a wide variety of deficiencies. Therefore, an automated system for pneumonia diagnosis consisting of three phases is proposed. An initial sorting phase consisting of a trained ResNet-18 convolutional neural network separates the dataset according to the interpretive quality of the images, creating a high and low-quality class. The unique image translation capabilities of the CycleGAN network are leveraged in the enhancement phase to translate low-quality images into improved versions. A final ResNet-18 network serves to classify pneumonia in the diagnosis phase. The enhancement system improved mixed quality diagnosis accuracy by 12.1% to 86.7%, with training sets composed of enhanced images achieving an accuracy 15.8% higher than their low-quality counterparts. The system's generalized method for image augmentation successfully mitigates the deficiencies of low-quality data, allowing for a higher accuracy diagnosis than otherwise possible.

Outdoor adventure education (OAE) organizations continually struggle with voluntary turnover of OAE field staff. The unconventional work environment of this unique population creates challenges and difficulties that lead to turnover. The purpose of this study was to explore the relationships between turnover predictor variables and intent to turnover (IT) of OAE field staff. The five predictor variables chosen were (a) tenure, (b) career development opportunities, (c) sense of community, (d) compensation satisfaction, and (e) burnout. Results suggest that compensation satisfaction’s pay subscale and sense of community were significant negative predictors of IT. These findings provide evidence for turnover predictors unique to OAE field staff. Additionally, these findings can aid OAE administrators in prioritizing time and resources when combating employee turnover.Subscribe to JOREL

DNA-encoded libraries (DELs) can contain billions of unique chemical species; selecting against such large inputs, it is typical to find more candidate binders than is reasonable to pursue for follow-up synthesis and testing. Given this wealth of choices, common practice is to limit synthesis to only those compounds estimated to have the greatest chance of being high-affinity binders; of the many potential factors contributing to this estimation, the strength of the selection signal of a candidate binder is always important. We define here methods and equations which relate the theoretical selection signal of a compound to its affinity and chemical yield. Tests using known binders of BRD4 and ROCK2 support the theory backing these equations and suggest they should be of use for prospectively determining affinity and chemical yield from primary DEL selection data.

Current high-resolution camera and video systems require expensively complex equipment and an excessive amount of digital storage to function, effectively limiting their practicality and availability. The research seeks to address this issue by optimizing Generative Adversarial Networks (GANs) for image super-resolution using an evolutionary-based scheme for successive network modification. The capability of GANs to selectively enhance the resolution of a desired image without increasing costs is extremely significant for a substantial range of industries and practices. The network’s capabilities are expressed in terms of produced image quality and network metrics, with preference given to increases in image quality. Using the DIV2K dataset as common input, the highest performing network achieved a comparative increase of 2.22 dB PSNR (29.1%) over the base model, the Super-Resolution Generative Adversarial Network (SRGAN). The most notable contribution to network enhancement with regards to image quality was caused by removing the batch normalization layers from the Generator network. Network performance with regards to subjective image quality was most affected by the inclusion of a second convolutional layer in each residual block of the modified SRGAN Generator. Possible applications of the improved system include enhancing images of license plates for traffic monitoring systems and improving still images from body camera footage to reveal crucial details. Beyond the scope of surveillance, resolution-enhancing GANs may be applied to develop media content or national defense capabilities.

Cyclic GMP-AMP synthase (cGAS) is activated by ds-DNA binding to produce the secondary messenger 2ʹ,3ʹ-cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds-DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2ʹ,3ʹ-cGAMP formation, and interestingly, describe a catalytic mechanism where 2ʹ,3ʹ-cGAMP may be a minor product of cGAS compared to linear nucleotides. This article is protected by copyright. All rights reserved.

Surface plasmon resonance (SPR) is a standard method for evaluating direct protein-small molecule binding. While studying the catalytic mechanism of cyclic GMP-AMP synthase (cGAS), we developed an SPR-based method to measure steady-state K

Thermal denaturation (Tm) data are easy to obtain; it is a technique that is used by both small labs and large‐scale industrial organizations. The link between ligand affinity (K D) and ΔTm is understood for reversible denaturation; however, there is a gap in our understanding of how to quantitatively interpret ΔTm for the many proteins that irreversibly denature. To better understand the origin, and extent of applicability, of a K D to ΔTm correlate, we define equations relating K D and ΔTm for irreversible protein unfolding, which we test with computational models and experimental data. These results suggest a general relationship exists between K D and ΔTm for irreversible denaturation.

In this paper, an efficient method for classifying and identifying instrumental audio is proposed via utilizing a deep learning image classification algorithm. The method of classification will involve analyzing the visual equivalent of an audio sample with a neural network to identify the generating musical instrument. Audio samples are converted into a logarithmic spectrogram format, which allows visual classifiers to attempt the identification of the audio source. The primary focus is on developing an efficient method for analyzing audio spectrograms using various forms of neural networks and analysis techniques. The use of deep learning convolutional neural networks in analyzing visually formatted audio data provides an enhanced classification method over traditional schemes. A classification accuracy of 73.7% was achieved with a limited data set and minimal manipulation of network architecture.

Surface plasmon resonance (SPR) is a standard method for evaluating direct protein-small molecule binding. While studying the catalytic mechanism of cyclic GMP-AMP synthase (cGAS), we developed an SPR-based method to measure steady-state KM values that complements traditional SPR affinity measurements. The method relies on refractive changes to detect protein interaction with substrates and products, and takes advantage of stimulator of type 1 interferon genes (STING) binding to the cGAS product, 2',3'-cGAMP. The specific method described here uses co-immobilization of cGAS and double-stranded DNA through a biotin tag; it should be generally applicable to other proteins and protein complexes.

The most common mutation in cystic fibrosis (CF) patients is deletion of F508 (ΔF508) in the first nucleotide binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR). ΔF508 causes a decrease in the trafficking of CFTR to the cell surface and reduces the thermal stability of isolated NBD1; it is well established that both of these effects can be rescued by additional revertant mutations in NBD1. The current paradigm in CF small molecule drug discovery is that, like revertant mutations, a path may exist to ΔF508 CFTR correction through a small molecule chaperone binding to NBD1. We, therefore, set out to find small molecule binders of NBD1 and test whether it is possible to develop these molecules into potent binders that increase CFTR trafficking in CF-patient-derived human bronchial epithelial cells. Several fragments were identified that bind NBD1 at either the CFFT-001 site or the BIA site. However, repeated attempts to improve the affinity of these fragments resulted in only modest gains. Although these results cannot prove that there is no possibility of finding a high-affinity small molecule binder of NBD1, they are discouraging and lead us to hypothesize that the nature of these two binding sites, and isolated NBD1 itself, may not contain the features needed to build high-affinity interactions. Future work in this area may, therefore, require constructs including other domains of CFTR in addition to NBD1, if high-affinity small molecule binding is to be achieved.

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations associated with CF cause loss-of-function in CFTR leading to salt imbalance in epithelial tissues. Kalydeco (also called VX-770 or ivacaftor) was approved for CF treatment in 2012 but little is known regarding the compound’s interactions with CFTR including the site of binding or mechanisms of action. In this study we use hydrogen/deuterium exchange (HDX) coupled with mass spectrometry to assess the conformational dynamics of a thermostabilized form of CFTR in apo and ligand-bound states. We observe HDX protection at a known binding site for AMPPNP and significant protection for several regions of CFTR in the presence of Kalydeco. The ligand-induced changes of CFTR in the presence of Kalydeco suggest a potential binding site.

Cell functions depend on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than that of dynein, and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the energetics and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, intermonomer nuclear Overhauser effects assigned to residues expected at interface positions, and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled coil. Secondary chemical shifts and (15)N backbone relaxation identify the boundaries and distinguishing structural features of the coiled coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled coil is critical for bicoid mRNA localization because it favors structural organization of Swallow, which except for the central LC8-promoted coiled coil is primarily disordered.

Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF-β1 and investigated the changes in the cardiac structure and function leading to AF.Transgenic goats with cardiac specific overexpression of constitutively active TGF-β1 were generated by somatic cell nuclear transfer. We examined myocardial tissue, ECGs, echocardiographic data, and AF susceptibility in transgenic and wild-type control goats. Transgenic goats exhibited significant increase in fibrosis and myocyte diameters in the atria compared to controls, but not in the ventricles. P-wave duration was significantly greater in transgenic animals starting at 12 months of age, but no significant chamber enlargement was detected, suggesting conduction slowing in the atria. Furthermore, this transgenic goat model exhibited a significant increase in AF vulnerability. Six of 8 transgenic goats (75%) were susceptible to AF induction and exhibited sustained AF (2 minutes), whereas none of 6 controls displayed sustained AF (P0.01). Length of induced AF episodes was also significantly greater in the transgenic group compared to controls (687 ± 212.02 seconds vs. 2.50 ± 0.88 seconds, P0.0001), but no persistent or permanent AF was observed.A novel transgenic goat model with a substrate for AF was generated. In this model, cardiac overexpression of TGF-β1 led to an increase in fibrosis and myocyte size in the atria, and to progressive P-wave prolongation. We suggest that these factors underlie increased AF susceptibility.

Extracellular vesicles (EVs) present an attractive delivery vehicle for therapeutic proteins. They intrinsically contain many proteins which can provide information to other cells. Advantages include reduced immune reactivity, especially if derived from the same host, stability in biologic fluids and ability to target uptake. Those from mesenchymal stem cells appear to be intrinsically therapeutic, while those from cancer cells promote tumor progression. Therapeutic proteins can be loaded into vesicles by overexpression in the donor cell, with oligomerization and membrane sequences increasing their loading. Examples of protein delivery for therapeutic benefit in pre-clinical models include delivery of: catalase for Parkinson’s disease to reduce oxidative stress and thus help neurons to survive; prodrug activating enzymes which can convert a prodrug which crosses the blood-brain barrier (BBB) into a toxic chemotherapeutic drug for schwannomas and gliomas; and the apoptosis-inducing enzyme, caspase-1 (ICE) under a Schwann cell specific promoter for schwannoma. This therapeutic delivery strategy is novel and being explored for a number of diseases.

Extracellular stimulation of the B cell receptor or mast cell FcεRI receptor activates a cascade of protein kinases, ultimately leading to antigenic or inflammation immune responses, respectively. Syk is a soluble kinase responsible for transmission of the receptor activation signal from the membrane to cytosolic targets. Control of Syk function is, therefore, critical to the human antigenic and inflammation immune response, and an inhibitor of Syk could provide therapy for autoimmune or inflammation diseases. We report here a novel allosteric Syk inhibitor, X1, that is noncompetitive against ATP (K(i) 4 ± 1 μM) and substrate peptide (K(i) 5 ± 1 μM), and competitive against activation of Syk by its upstream regulatory kinase LynB (K(i) 4 ± 1 μM). The inhibition mechanism was interrogated using a combination of structural, biophysical, and kinetic methods, which suggest the compound inhibits Syk by reinforcing the natural regulatory interactions between the SH2 and kinase domains. This novel mode of inhibition provides a new opportunity to improve the selectivity profile of Syk inhibitors for the development of safer drug candidates.

The roadblock/LC7 dynein light chain is a ubiquitous component of all dyneins and is essential for many diverse processes including proper axonal transport and dendrite growth. In addition, LC7 functions in non-dynein transcriptional activation of the transforming growth factor-beta complex. Crystal structures of Drosophila melanogaster LC7 in the apo form and in complex with a segment of the disordered N-terminal domain of dynein intermediate chain (IC) provide the first definitive identification of the IC sequence recognized by LC7. The site, confirmed by isothermal titration calorimetry studies, overlaps the IC sequence considered in the literature to be an IC self-association domain. The IC peptide binds as two amphipathic helices that lie along an extensive hydrophobic cleft on LC7 and ends with a polar side-chain interaction network that includes conserved residues from both proteins. The LC7 recognition sequence on IC and its interface with LC7 are well conserved and are, thus, likely representative of all IC x LC7 structures. Interestingly, the position of bound IC in the IC x LC7 complex mimics a helix that is integrated into the primary structure in distantly related LC7 homologs. The IC x LC7 structure further shows that the naturally occurring robl(Z) deletion mutation contains the majority of the IC binding site and suggests that promotion of IC binding by phosphorylation of LC7 is an indirect effect.