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Abstract Light sheet fluorescence microscopy (LSFM) is a transformative imaging method that enables the visualization of non-dissected specimen in real-time 3D. Optical clearing of tissues is essential for LSFM, typically employing toxic solvents. Here, we test the applicability of a non-hazardous alternative, ethyl cinnamate (ECi). We comprehensively characterized autofluorescence (AF) spectra in diverse murine tissues—ocular globe, knee, and liver—employing LSFM under various excitation wavelengths (405–785 nm) to test the feasibility of unstained samples for diagnostic purposes, in particular regarding percutaneous biopsies, as they constitute to most harvested type of tissue sample in clinical routine. Ocular globe structures were best discerned with 640 nm excitation. Knee tissue showed complex variation in AF spectra variation influenced by tissue depth and structure. Liver exhibited a unique AF pattern, likely linked to vasculature. Hepatic tissue samples were used to demonstrate the compatibility of our protocol for antibody staining. Furthermore, we employed machine learning to augment raw images and segment liver structures based on AF spectra. Radiologists rated representative samples transferred to the clinical assessment software. Learning-generated images scored highest in quality. Additionally, we investigated an actual murine biopsy. Our study pioneers the application of AF spectra for tissue characterization and diagnostic potential of optically cleared unstained percutaneous biopsies, contributing to the clinical translation of LSFM.

Abstract Massive rotator cuff (RC) tendon tears are associated with progressive fibro-adipogenesis and muscle atrophy that altogether cause shoulder muscle wasting. Platelet derived growth factor β (PDGFRβ) lineage cells, that co-express PDGFRα have previously been shown to directly contribute to scar formation and fat accumulation in a mouse model of irreversible tendon and nerve transection (TTDN). Conversely, PDGFRβ+ lineage cells have also been shown to be myogenic in cultures and in other models of skeletal muscle injury. We therefore hypothesized that PDGFRβ demarcates two distinct RC residing subpopulations, fibro-adipogenic and myogenic, and aimed to elucidate the identity of the PDGFRβ myogenic precursors and evaluate their contribution, if any, to RC myo-regeneration. Lineage tracing revealed increasing contribution of PDGFRβ+ myo-progenitors to the formation of GFP+ myofibers, which were the most abundant myofiber type in regenerated muscle at 2 weeks post-TTDN. Muscle regeneration preceded muscle atrophy and both advanced from the lateral site of tendon transection to the farthest medial region. GFP+/PDGFRβ+Sca-1−lin−CXCR4+Integrin-β1+ marked a novel subset of satellite cells with confirmed myogenic properties. Further studies are warranted to identify the existence of PDGFRβ+ satellite cells in human and other mouse muscles and to define their myo-regenerative potential following acute and chronic muscle injury.

Abstract Adults with sickle cell disease bear a mutation in the β-globin gene, leading to the expression of sickle hemoglobin (HbS; α2βS 2). Adults also possess the gene for γ-globin, which is a component of fetal hemoglobin (HbF, α2γ2); however, γ-chain expression normally ceases after birth. As HbF does not form the fibers that cause the disease, pharmacological and gene-modifying interventions have attempted to either reactivate expression of the γ chain or introduce a gene encoding a modified β chain having γ-like character. Here, we show that a single-site modification on the α chain, αPro114Arg, retards fiber formation as effectively as HbF. Because this addition to the repertoire of anti-sickling approaches acts independently of other modifications, it could be coupled with other therapies to significantly enhance their effectiveness.

Abstract In recent years, computer vision has contributed significantly to the study of farm animal behavior. In complex environments such as commercial farms, however, the automated detection of social behavior and specific interactions between animals can be improved. The present study addresses the automated detection of agonistic interactions between caged animals in a complex environment, relying solely on computer vision. An automated pipeline including group-level temporal action segmentation, object detection, object tracking and rule-based action classification for the detection of agonistic interactions was developed and extensively validated at a level unique in the field. Comparing with observations made by human observers, our pipeline reaches 77% precision and 85% recall using a 5-min tolerance interval for the detection of agonistic interactions. Results obtained using this pipeline allow to construct time-dependent socio-matrices of a group of animals and derive metrics on the dominance hierarchy in a semi-automated manner. Group-housed breeding rabbits (does) with their litters in commercial farms are the main use-case in this work, but the idea is probably also applicable to other social farm animals.

Abstract Tuberculosis (TB) is a prevalent disease causing an estimated 1.6 million deaths and 10.6 million new cases annually. Discriminating TB disease from differential diagnoses can be complex, particularly in the field. Increased levels of complement component C1q in serum have been identified as a specific and accessible biomarker for TB disease but the source of C1q in circulation has not been identified. Here, data and samples previously collected from human cohorts, a clinical trial and a non-human primate study were used to identify cells producing C1q in circulation. Cell subset frequencies were correlated with serum C1q levels and combined with single cell RNA sequencing and flow cytometry analyses. This identified monocytes as C1q producers in circulation, with a pronounced expression of C1q in classical and intermediate monocytes and variable expression in non-classical monocytes.

Abstract Prior research has revealed a native-accent advantage, whereby nonnative-accented speech is more difficult to process than native-accented speech. Nonnative-accented speakers also experience more negative social judgments. In the current study, we asked three questions. First, does exposure to nonnative-accented speech increase speech intelligibility or decrease listening effort, thereby narrowing the native-accent advantage? Second, does lower intelligibility or higher listening effort contribute to listeners’ negative social judgments of speakers? Third and finally, does increased intelligibility or decreased listening effort with exposure to speech bring about more positive social judgments of speakers? To address these questions, normal-hearing adults listened to a block of English sentences with a native accent and a block with nonnative accent. We found that once participants were accustomed to the task, intelligibility was greater for nonnative-accented speech and increased similarly with exposure for both accents. However, listening effort decreased only for nonnative-accented speech, soon reaching the level of native-accented speech. In addition, lower intelligibility and higher listening effort was associated with lower ratings of speaker warmth, speaker competence, and willingness to interact with the speaker. Finally, competence ratings increased over time to a similar extent for both accents, with this relationship fully mediated by intelligibility and listening effort. These results offer insight into how listeners process and judge unfamiliar speakers.

Abstract We aimed to investigate structural retinal changes in malarial retinopathy (MR) using hand-held optical coherence tomography (HH-OCT) to assess its diagnostic potential. Children with MR (n = 43) underwent ophthalmoscopy, fluorescein angiography and HH-OCT during admission, 1-month (n = 31) and 1-year (n = 8) post-discharge. Controls were comatose patients without malaria (n = 6) and age/sex-matched healthy children (n = 43). OCT changes and retinal layer thicknesses were compared. On HH-OCT, hyper-reflective areas (HRAs) were seen in the inner retina of 81% of MR patients, corresponding to ischaemic retinal whitening on fundus photography. Cotton wool spots were present in 37% and abnormal hyper-reflective dots, co-localized to capillary plexus, in 93%. Hyper-reflective vessel walls were present in 84%, and intra-retinal cysts in 9%. Vascular changes and cysts resolved within 48 h. HRAs developed into retinal thinning at 1 month (p = 0.027) which was more pronounced after 1 year (p = 0.009). Ischaemic retinal whitening is located within inner retinal layers, distinguishing it from cotton wool spots. Vascular hyper-reflectivity may represent the sequestration of parasitized erythrocytes in vessels, a key CM feature. The mechanisms of post-ischemic retinal atrophy and cerebral atrophy with cognitive impairment may be similar in CM survivors. HH-OCT has the potential for monitoring patients, treatment response and predicting neurological deficits.

Abstract Infantile nystagmus (IN) may result from aetiologies including albinism and FRMD7 mutations. IN has low prevalence, and twins with IN are rare. Whilst discordant presentation has been previously reported for IN, we present for the first time the comprehensive assessment of diagnostically discordant monozygotic twins. From a cohort of over 2000 patients, we identified twins and triplets discordant for nystagmus. Using next-generation sequencing, high-resolution infra-red pupil tracking and optical coherence tomography, we characterised differences in genotype and phenotype. Monozygotic twins (n = 1), dizygotic twins (n = 3) and triplets (n = 1) were included. The monozygotic twins had concordant TYR variants. No causative variants were identified in the triplets. Dizygotic twins had discordant variants in TYR, OCA2 and FRMD7. One unaffected co-twin demonstrated sub-clinical nystagmus. Foveal hypoplasia (FH) was noted in four of five probands. Both co-twins of the monozygotic pair and triplets displayed FH. In three families, at least one parent had FH without nystagmus. FH alone may be insufficient to develop nystagmus. Whilst arrested optokinetic reflex pathway development is implicated in IN, discordant twins raise questions regarding where differences in development have arisen. In unaffected monozygotes therefore, genetic variants may predispose to oculomotor instability, with variable expressivity possibly responsible for the discordance observed.

Abstract The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis.

In cancer research, RAS biology has been focused on only a handful of tumor types. While RAS genes have long been suspected as common contributors to a wide spectrum of cancer types, robust evidence is required to firmly establish their critical oncogenic significance. We present a data mining study using DepMap genome-wide CRISPR screening data, which provide substantial evidence to support the prominent pervasive oncogenic role and tissue-specific permissiveness of RAS gene mutations. Differential analysis of CRISPR effect scores identifies K- or N-RAS genes as the most differential gene in contrasts of (K-, N-, combined) RAS mutant versus wild-type cell lines across multiple tissue types. The distinguished tissue-specific pattern of KRAS vs. NRAS as top differential genes in subsets of tissue types and evidence from genome data supported the idea of KRAS- and NRAS-engaged tissue types. To our knowledge, this is the first report of prominent pervasive oncogenic role of RAS mutations revealed by gene dependency data that is beyond the current understanding of the oncogenic role of RAS genes and their well-known involved tissue types. Our findings strongly support RAS mutations as primary oncogenic drivers beyond traditionally recognized cancer types and offer insights into their tissue-specific permissiveness.

Investigators in neuroscience have turned to Big Data to address replication and reliability issues by increasing sample size. These efforts unveil new questions about how to integrate data across distinct sources and instruments. The goal of this study was to link scores across common auditory verbal learning tasks (AVLTs). This international secondary analysis aggregated multisite raw data for AVLTs across 53 studies totaling 10,505 individuals. Using the ComBat-GAM algorithm, we isolated and removed the component of memory scores associated with site effects while preserving instrumental effects. After adjustment, a continuous item response theory model used multiple memory items of varying difficulty to estimate each individual's latent verbal learning ability on a single scale. Equivalent raw scores across AVLTs were then found by linking individuals through the ability scale. Harmonization reduced total cross-site score variance by 37% while preserving meaningful memory effects. Age had the largest impact on scores overall (- 11.4%), while race/ethnicity variable was not significant (p > 0.05). The resulting tools were validated on dually administered tests. The conversion tool is available online so researchers and clinicians can convert memory scores across instruments. This work demonstrates that global harmonization initiatives can address reproducibility challenges across the behavioral sciences.

Abstract Cytosolic Malic Enzyme (ME1) provides reduced NADP for anabolism and maintenance of redox status. To examine the role of ME1 in tumor genesis of the gastrointestinal tract, we crossed mice having augmented intestinal epithelial expression of ME1 (ME1-Tg mice) with ApcMin/+ mice to obtain male ApcMin/+/ME1-Tg mice. ME1 protein levels were significantly greater within gut epithelium and adenomas of male ApcMin/+/ME1-Tg than ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice had larger and greater numbers of adenomas in the small intestine (jejunum and ileum) than male ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice exhibited greater small intestine crypt depth and villus length in non-adenoma regions, correspondent with increased KLF9 protein abundance in crypts and lamina propria. Small intestines of male ApcMin/+/ME1-Tg mice also had enhanced levels of Sp5 mRNA, suggesting Wnt/β-catenin pathway activation. A small molecule inhibitor of ME1 suppressed growth of human CRC cells in vitro, but had little effect on normal rat intestinal epithelial cells. Targeting of ME1 may add to the armentarium of therapies for cancers of the gastrointestinal tract.

Abstract We applied two state-of-the-art, knowledge independent data-mining methods – Dynamic Quantum Clustering (DQC) and t-Distributed Stochastic Neighbor Embedding (t-SNE) – to data from The Cancer Genome Atlas (TCGA). We showed that the RNA expression patterns for a mixture of 2,016 samples from five tumor types can sort the tumors into groups enriched for relevant annotations including tumor type, gender, tumor stage, and ethnicity. DQC feature selection analysis discovered 48 core biomarker transcripts that clustered tumors by tumor type. When these transcripts were removed, the geometry of tumor relationships changed, but it was still possible to classify the tumors using the RNA expression profiles of the remaining transcripts. We continued to remove the top biomarkers for several iterations and performed cluster analysis. Even though the most informative transcripts were removed from the cluster analysis, the sorting ability of remaining transcripts remained strong after each iteration. Further, in some iterations we detected a repeating pattern of biological function that wasn’t detectable with the core biomarker transcripts present. This suggests the existence of a “background classification” potential in which the pattern of gene expression after continued removal of “biomarker” transcripts could still classify tumors in agreement with the tumor type.

The quantitative characterization and prediction of localized severe weather events that emerge as coherences generated by the highly non-linear interacting multivariate dynamics of global weather systems poses a significant challenge whose solution is increasingly important in the face of climate change where weather extremes are on the rise. As weather measurement systems (multiband satellite, radar, etc) continue to dramatically improve, increasingly complex time-dependent multivariate 3D datasets offer the potential to inform such problems but pose an increasingly daunting computational challenge. Here we describe the application to global weather systems of a novel computational method called the Entropy Field Decomposition (EFD) capable of efficiently characterizing coherent spatiotemporal structures in non-linear multivariate interacting physical systems. Using the EFD derived system configurations, we demonstrate the application of a second novel computational method called Space-Time Information Trajectories (STITs) that reveal how spatiotemporal coherences are dynamically connected. The method is demonstrated on the specific phenomenon known as atmospheric rivers (ARs) which are a prime example of a highly coherent, in both space and time, severe weather phenomenon whose generation and persistence are influenced by weather dynamics on a wide range of spatial and temporal scales. The EFD reveals how the interacting wind vector field and humidity scalar field couple to produce ARs, while the resulting STITS reveal the linkage between ARs and large-scale planetary circulations. The focus on ARs is also motivated by their devastating social and economic effects that have made them the subject of increasing scientific investigation to which the EFD may offer new insights. The application of EFD and STITs to the broader range of severe weather events is discussed.

Abstract Shift work increases the risk for human errors, such that drowsiness due to shift work has contributed to major industrial disasters, including Space Shuttle Challenger, Chernobyl and Alaska Oil Spill disasters, with extraordinary socio-economical costs. Overnight operations pose a challenge because our circadian biology inhibits cognitive performance at night. Yet how the circadian system modulates cognition over multiple days under realistic shift work conditions remains to be established. Importantly, because task-specific cognitive brain regions show different 24-h circadian dynamics, we hypothesize that circadian misalignment impacts cognition task-dependently. Using a biologically-driven paradigm mimicking night shift work, with a randomized, cross-over design, we show that misalignment between the circadian pacemaker and behavioral/environmental cycles increases cognitive vulnerability on sustained attention, cognitive throughput, information processing and visual-motor performance over multiple days, compared to circadian alignment (day shifts). Circadian misalignment effects are task-dependent: while they acutely impair sustained attention with recovery after 3-days, they progressively hinder daily learning. Individuals felt sleepier during circadian misalignment, but they did not rate their performance as worse. Furthermore, circadian misalignment effects on sustained attention depended on prior sleep history. Collectively, daily circadian misalignment may provide an important biological framework for developing countermeasures against adverse cognitive effects in shift workers.