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Summary: The study of biological form is a vital goal of evolutionary biology and functional morphology. We review an emerging set of methods that allow scientists to create and study accurate 3D models of living organisms and animate those models for biomechanical and fluid dynamic analyses. The methods for creating such models include 3D photogrammetry, laser and CT scanning, and 3D software. New multi-camera devices can be used to create accurate 3D models of living animals in the wild and captivity. New websites and virtual reality/augmented reality devices now enable the visualization and sharing of these data. We provide examples of these approaches for animals ranging from large whales to lizards and show applications for several areas: Natural history collections; body condition/scaling, bioinspired robotics, computational fluids dynamics (CFD), machine learning, and education. We provide two datasets to demonstrate the efficacy of CFD and machine learning approaches and conclude with a prospectus.

Summary: We describe the creation of an isogenic cell line panel representing common cancer pathways, with features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cell lines were generated using CRISPR technologies. Surprisingly, most of these lines did not result in complete gene inactivation despite integration of sgRNA at the desired genomic site. A subset of the lines harbored biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterized lines covering 19 frequently lost cancer pathways. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

Abstract Deer louse flies (Lipoptena spp.) are hematophagous ectoparasites of cervids. The genus Lipoptena comprises 32 species, some of which are of veterinary importance as vectors of various pathogens, and are also known to attack human hosts. Recently, deer louse flies have been observed during annual checkups of captive cervids at Khon Kaen Zoo in Khon Kaen, Thailand. However, data on their specific identity and prevalence remain limited. This study aims to identify louse fly samples from captive cervids at Khon Kaen Zoo using morphological and molecular analyses. A total of 60 louse flies were collected from 17 captive cervids and identified based on their morphology. Major morphological characteristics, including mesothoracic bristle patterns, abdominal tergal plate bristles, and terminalia structure indicated that the Khon Kaen louse fly is Lipoptena axis Maa, 1965. Phylogenetic analysis of sequences from a portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene was performed, which confirmed that L. axis of this study belongs to the cervi group, which is distinct from other groups of Lipoptena species. This study represents the first report of L. axis in Thailand. We provide an updated taxonomic key for the identification of Lipoptena species in the cervi group.

Active-learning pedagogies have been repeatedly demonstrated to produce superior learning gains with large effect sizes compared with lecture-based pedagogies. Shifting large numbers of college science, technology, engineering, and mathematics (STEM) faculty to include any active learning in their teaching may retain and more effectively educate far more students than having a few faculty completely transform their teaching, but the extent to which STEM faculty are changing their teaching methods is unclear. Here, we describe the development and application of the machine-learning-derived algorithm Decibel Analysis for Research in Teaching (DART), which can analyze thousands of hours of STEM course audio recordings quickly, with minimal costs, and without need for human observers. DART analyzes the volume and variance of classroom recordings to predict the quantity of time spent on single voice (e.g., lecture), multiple voice (e.g., pair discussion), and no voice (e.g., clicker question thinking) activities. Applying DART to 1,486 recordings of class sessions from 67 courses, a total of 1,720 h of audio, revealed varied patterns of lecture (single voice) and nonlecture activity (multiple and no voice) use. We also found that there was significantly more use of multiple and no voice strategies in courses for STEM majors compared with courses for non-STEM majors, indicating that DART can be used to compare teaching strategies in different types of courses. Therefore, DART has the potential to systematically inventory the presence of active learning with ∼90% accuracy across thousands of courses in diverse settings with minimal effort.

Abstract Compact cell type-specific promoters are important tools for basic and preclinical research and clinical delivery of gene therapy. In this work, we designed novel MiniPromoters to target D1 and D2 type dopaminoceptive medium spiny neurons in the striatum by manually identifying candidate regulatory regions or employing the OnTarget webserver. We then empirically tested the designs in rAAV-PHP.B for specificity and robustness in three systems: intravenous injection in mice, intracerebroventricular injection in mice, and intracerebroventricular injection in non-human primates. Twelve MiniPromoters were designed from eight genes: seven manually and five using OnTarget. When delivered intravenously in mice, three MiniPromoters demonstrated highly selective expression in the striatum, with Ple389 (ADORA2A) showing high levels of dopamine D2-receptor cell co-localization. The same three MiniPromoters also displayed enriched expression in the striatum when delivered intracerebroventricularly in mice with high levels of DARPP32 co-localization. Finally, Ple389 (ADORA2A) was intracerebroventricularly injected in non-human primates and showed enriched expression in the striatum as in the mouse. Ple389 (ADORA2A) demonstrated expression in the medium spiny neurons in all three systems tested and exhibited the highest level of D2-MSNs and DARPP32 co-labeling in mice, demonstrating its potential as a tool for gene therapy approaches for Parkinson and Huntington disease treatment.

Abstract Accelerating text input in augmentative and alternative communication (AAC) is a long-standing area of research with bearings on the quality of life in individuals with profound motor impairments. Recent advances in large language models (LLMs) pose opportunities for re-thinking strategies for enhanced text entry in AAC. In this paper, we present SpeakFaster, consisting of an LLM-powered user interface for text entry in a highly-abbreviated form, saving 57% more motor actions than traditional predictive keyboards in offline simulation. A pilot study on a mobile device with 19 non-AAC participants demonstrated motor savings in line with simulation and relatively small changes in typing speed. Lab and field testing on two eye-gaze AAC users with amyotrophic lateral sclerosis demonstrated text-entry rates 29–60% above baselines, due to significant saving of expensive keystrokes based on LLM predictions. These findings form a foundation for further exploration of LLM-assisted text entry in AAC and other user interfaces.

Abstract Shell ring archaeological sites are one of the most visible site types along the lower South Atlantic Coast of the United States. These cultural sites are large, circular to arcuate piles of mollusk shells with some reaching over three meters in elevation and over 100 m in diameter. They are comprised largely of mollusk shells (e.g., Eastern oyster, Crassostrea virginica), but also contain early pottery, nonhuman faunal remains, and other artifacts. Our work establishes that they represent the earliest widespread Native American villages occupied year-round in the Eastern Woodlands of North America. Significantly, our results from sea-level modelling and isotope geochemistry on mollusks establish that the inhabitants of these earliest villages (ca. 5000–3800 yrs. BP) lived within a fluctuating coastal environment, harvested certain resources year-round, and targeted diverse habitats across the estuaries. Both the growth and decline of these earliest villages are associated with a concomitant rise and lowering of sea level that impacted the productivity of the oyster reef fishery along the South Atlantic Coast. Despite these large-scale environmental changes, this research indicates that Native American fishing villages persisted along the coast for over 1000 years.

Abstract Melioidosis caused by Burkholderia pseudomallei (Bp) is a public health threat. Genomic-epidemiology research on this deadly disease is scarce. We investigated whole-genome sequences of Bp isolates in relation to environmental source and drug susceptibility. In total, 563 Bp isolates were collected from 11 Northeast Thai provinces during the period 2004–2021. Patients (n = 530 isolates), infected animals (n = 8), and environmental sources (n = 25) provided samples. Phylogenetic analysis revealed genetic diversity among the Bp isolates, including numerous well-supported clusters of varying sizes. Through in-depth analysis of 38 monophyletic clades (MCs), we found eleven associated with province of origin (p-value

Abstract Autism spectrum disorder (ASD) is diagnosed using comprehensive behavioral information. Neuroimaging offers additional information but lacks clinical utility for diagnosis. This study investigates whether multi-forms of magnetic resonance imaging (MRI) contrast can be used individually and in combination to produce a categorical classification of young individuals with ASD. MRI data were accessed from the Autism Brain Imaging Data Exchange (ABIDE). Young participants (ages 2–30) were selected, and two group cohorts consisted of 702 participants: 351 ASD and 351 controls. Image-based classification was performed using one-channel and two-channel inputs to 3D-DenseNet deep learning networks. The models were trained and tested using tenfold cross-validation. Two-channel models were twinned with combinations of structural MRI (sMRI) maps and amplitude of low-frequency fluctuations (ALFF) or fractional ALFF (fALFF) maps from resting-state functional MRI (rs-fMRI). All models produced classification accuracy that exceeded 65.1%. The two-channel ALFF-sMRI model achieved the highest mean accuracy of 76.9% ± 2.34. The one-channel ALFF-based model alone had mean accuracy of 72% ± 3.1. This study leveraged the ABIDE dataset to produce ASD classification results that are comparable and/or exceed literature values. The deep learning approach was conducive to diverse neuroimaging inputs. Findings reveal that the ALFF-sMRI two-channel model outperformed all others.

Abstract Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are manifestations of the post-acute sequelae of SARS-CoV-2. Our understanding of metabolic decline after acute COVID-19 remains unclear due to the lack of animal models. Here, we report a non-human primate model of metabolic post-acute sequelae of SARS-CoV-2 using SARS-CoV-2 infected African green monkeys. Using this model, we identify a dysregulated blood chemokine signature during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. Hyperglycemia also correlates with liver glycogen levels, but there is no evidence of substantial long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the African green monkey model exhibits important similarities to humans and can be utilized to assess therapeutic candidates to combat COVID-related metabolic defects.

Worldwide, enhancement of oyster populations is undertaken to achieve a variety of goals including support of food production, local economies, water quality, coastal habitat, biodiversity, and cultural heritage. Although numerous strategies for improving oyster stocks exist, enhancement efforts can be thwarted by long-standing conflict among community groups about which strategies to implement, where efforts should be focused, and how much funding should be allocated to each strategy. The objective of this paper is to compare two engagement approaches that resulted in recommendations for multi-benefit enhancements to oyster populations and the oyster industry in Maryland, U.S.A., using the Consensus Solutions process with collaborative simulation modeling. These recommendations were put forward by the OysterFutures Workgroup in 2018 and the Maryland Oyster Advisory Commission (OAC) in 2021. Notable similarities between the efforts were the basic principles of the Consensus Solutions process: neutral facilitation, a 75% agreement threshold, the presence of management agency leadership at the meetings, a scientific support team that created a management scenario model in collaboration with community group representatives, numerous opportunities for representatives to listen to each other, and a structured consensus building process for idea generation, rating, and approval of management options. To ensure meaningful representation by the most affected user groups, the goal for membership composition was 60% from industry and 40% from advocacy, agency, and academic groups in both processes. Important differences between the processes included the impetus for the process (a research program versus a legislatively-mandated process), the size of the groups, the structure of the meetings, and the clear and pervasive impact of the COVID-19 pandemic on the ability of OAC members to interact. Despite differences and challenges, both groups were able to agree on a package of recommendations, indicating that consensus-based processes with collaborative modeling offer viable paths toward coordinated cross-sector natural resource decisions with scientific basis and community support. In addition, collaborative modeling resulted in ‘myth busting’ findings that allowed participants to reassess and realign their thinking about how the coupled human-oyster system would respond to management changes.

Abstract Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. Quantification of the contractile function in single hiPSC-CMs at high-throughput and over time is essential to disentangle how cellular mechanisms affect heart function. Here, we present CONTRAX, an open-access, versatile, and streamlined pipeline for quantitative tracking of the contractile dynamics of single hiPSC-CMs over time. Three software modules enable: parameter-based identification of single hiPSC-CMs; automated video acquisition of >200 cells/hour; and contractility measurements via traction force microscopy. We analyze >4,500 hiPSC-CMs over time in the same cells under orthogonal conditions of culture media and substrate stiffnesses; +/− drug treatment; +/− cardiac mutations. Using undirected clustering, we reveal converging maturation patterns, quantifiable drug response to Mavacamten and significant deficiencies in hiPSC-CMs with disease mutations. CONTRAX empowers researchers with a potent quantitative approach to develop cardiac therapies.

Abstract Our limited understanding of the pathophysiological mechanisms that operate during sepsis is an obstacle to rational treatment and clinical trial design. There is a critical lack of data from low- and middle-income countries where the sepsis burden is increased which inhibits generalized strategies for therapeutic intervention. Here we perform RNA sequencing of whole blood to investigate longitudinal host response to sepsis in a Ghanaian cohort. Data dimensional reduction reveals dynamic gene expression patterns that describe cell type-specific molecular phenotypes including a dysregulated myeloid compartment shared between sepsis and COVID-19. The gene expression signatures reported here define a landscape of host response to sepsis that supports interventions via targeting immunophenotypes to improve outcomes.

Abstract T cells recirculate through tissues and lymphatic organs to scan for their cognate antigen. Radiation therapy provides site-specific cytotoxicity to kill cancer cells but also has the potential to eliminate the tumor-specific T cells in field. To dynamically study the effect of radiation on CD8 T cell recirculation, we used the Kaede mouse model to photoconvert tumor-infiltrating cells and monitor their movement out of the field of radiation. We demonstrate that radiation results in loss of CD8 T cell recirculation from the tumor to the lymph node and to distant sites. Using scRNASeq, we see decreased proliferating CD8 T cells in the tumor following radiation therapy resulting in a proportional enrichment in exhausted phenotypes. By contrast, 5 days following radiation increased recirculation of T cells from the tumor to the tumor draining lymph node corresponds with increased immunosurveillance of the treated tumor. These data demonstrate that tumor radiation therapy transiently impairs systemic T cell recirculation from the treatment site to the draining lymph node and distant untreated tumors. This may inform timing therapies to improve systemic T cell-mediated tumor immunity.

Abstract Despite the high prevalence of low birth weight infants in sub-Saharan Africa and the associated poor outcomes, weight change during the newborn period has not been well characterized for this population. We prospectively assessed growth over the first 30 days among 120 infants born

Abstract Pancreatic ductal adenocarcinoma (PDAC) is considered the third leading cause of cancer mortality in the western world, offering advanced stage patients with few viable treatment options. Consequently, there remains an urgent unmet need to develop novel therapeutic strategies that can effectively inhibit pro-oncogenic molecular targets underpinning PDACs pathogenesis and progression. One such target is c-RAF, a downstream effector of RAS that is considered essential for the oncogenic growth and survival of mutant RAS-driven cancers (including KRASMT PDAC). Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF–PDE8A protein–protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF–cAMP/PKA signaling axes and treating KRAS–c-RAF dependent PDAC. Through disrupting the c-RAF–PDE8A protein complex, DRx-170 promotes the inactivation of c-RAF through an allosteric mechanism, dependent upon inactivating PKA phosphorylation. DRx-170 inhibits cell proliferation, adhesion and migration of a KRASMT PDAC cell line (PANC1), independent of ERK1/2 activity. Moreover, combining DRx-170 with afatinib significantly enhances PANC1 growth inhibition in both 2D and 3D cellular models. DRx-170 sensitivity appears to correlate with c-RAF dependency. This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS–c-RAF dependent PDAC.

Abstract Friedreich’s ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN. However, unlike endogenous hFXN, endogenous mFXN is further processed into N-terminally truncated, extra-mitochondrial mFXN forms of unknown function. This study assessed mature exogenous hFXN expression levels in the heart and liver of C57Bl/6 mice 7–10 months after intravenous administration of a recombinant adeno-associated virus encoding hFXN (AAVrh.10hFXN) and examined the potential for hFXN truncation in mice. AAVrh.10hFXN induced dose-dependent expression of hFXN in the heart and liver. Interestingly, hFXN was processed into truncated forms, but found at lower levels than mature hFXN. However, the truncations were at different positions than mFXN. AAVrh.10hFXN induced mature hFXN expression in mouse heart and liver at levels that approximated endogenous mFXN levels. These results suggest that AAVrh.10hFXN can likely induce expression of therapeutic levels of mature hFXN in mice.

Both the metabolic theory of ecology and dynamic energy budget theory predict that climate influences body size through its effects on first-order determinants of energetics: reactive temperatures, carbon resources and oxygen availability. Although oxygen is seldom limiting in terrestrial systems, temperature and resources vary spatially. We used redundancy analyses and variation partitioning to evaluate the influence of climatic temperature, precipitation and their seasonalities on multivariate body size across the distributions of four species of the western rattlesnake group in North America (Crotalus pyrrhus, C. scutulatus, C. oreganus and C. viridis). Most species showed a pattern of increased body size in cooler, mesic climates and decreased body size in warmer, xeric climates. Exceptions to the pattern provided additional context through climatic idiosyncrasies in the distributions of each species. For example, the general pattern of a negative influence of temperature on body size was not apparent for C. oreganus, which ranges across the mildest climates overall among the four species. In contrast to previous studies, we found that seasonality had negligible effects on body size. We suggest that precipitation gradients correlate positively with resource availability in driving intraspecific body size and that temperature compounds this gradient by increasing baseline metabolic demands and restricting activity in particularly warm or otherwise extreme climates.

Abstract Manufacturing custom three-dimensional (3D) carbon functional materials is of utmost importance for applications ranging from electronics and energy devices to medicine, and beyond. In lieu of viable eco-friendly synthesis pathways, conventional methods of carbon growth involve energy-intensive processes with inherent limitations of substrate compatibility. The yearning to produce complex structures, with ultra-high aspect ratios, further impedes the quest for eco-friendly and scalable paths toward 3D carbon-based materials patterning. Here, we demonstrate a facile process for carbon 3D printing at room temperature, using low-power visible light and a metal-free catalyst. Within seconds to minutes, this one-step photocatalytic growth yields rod-shaped microstructures with aspect ratios up to ~500 and diameters below 10 μm. The approach enables the rapid patterning of centimeter-size arrays of rods with tunable height and pitch, and of custom complex 3D structures. The patterned structures exhibit appealing luminescence properties and ohmic behavior, with great potential for optoelectronics and sensing applications, including those interfacing with biological systems.

Imaging is increasingly used to capture information on the marine environment thanks to the improvements in imaging equipment, devices for carrying cameras and data storage in recent years. In that context, biologists, geologists, computer specialists and end-users must gather to discuss the methods and procedures for optimising the quality and quantity of data collected from images. The 4th Marine Imaging Workshop was organised from 3-6 October 2022 in Brest (France) in a hybrid mode. More than a hundred participants were welcomed in person and about 80 people attended the online sessions. The workshop was organised in a single plenary session of presentations followed by discussion sessions. These were based on dynamic polls and open questions that allowed recording of the imaging community’s current and future ideas. In addition, a whole day was dedicated to practical sessions on image analysis, data standardisation and communication tools. The format of this edition allowed the participation of a wider community, including lower-income countries, early career scientists, all working on laboratory, benthic and pelagic imaging.This article summarises the topics addressed during the workshop, particularly the outcomes of the discussion sessions for future reference and to make the workshop results available to the open public.

Abstract We present new chronologies that inform the timing and tempo of shell ring and shell mound construction on the South Atlantic Bight. Our project combines recently acquired dates with legacy radiocarbon dates from over 25 rings and mounds to provide a higher-resolution chronology regarding the occupation and formation of this larger landscape of the earliest fishing villages along the East Coast of the United States. We resolve the ordering and timing of occupation of these rings and mounds through Bayesian statistical modeling. These new models historicize and contextualize these shell rings in ways previously impossible. Specifically, our new chronologies of these villages indicate that the earliest villages were established prior to the invention of pottery. The early period of village establishment evidences isolated village rings, whereas later periods seem to have more villages, but these appear to have been relocated to other areas and/or islands over time. Shell mounds are fewer in number, are spread throughout the time period, and may represent special purpose sites compared to shell-rings. Once villages spread, they quickly adopted new technologies (i.e., pottery) and created new institutions and practiced village relocation, which allowed this way of life to persist for more than a thousand years.

Abstract Previous studies have reported increased retinal venous oxygen saturation and decreased retinal blood flow and oxygen metabolism in non-proliferative diabetic retinopathy (NPDR). The current study aimed to determine alterations in both inner retinal oxygen delivery (DO2) and metabolism (MO2) in proliferative DR (PDR) as well as at stages of NPDR. A total of 123 subjects participated in the study and were categorized into five groups: non-diabetic control (N = 32), diabetic with no diabetic retinopathy (NDR, N = 34), mild NPDR (N = 31), moderate to severe NPDR (N = 17), or PDR (N = 9). Multi-modal imaging was performed to measure oxygen saturation and blood flow, which were used for derivation of DO2 and MO2. There were significant associations of groups with DO2 and MO2. DO2 was lower in PDR and not significantly different in NDR and NPDR stages as compared to the non-diabetic control group. MO2 was decreased in PDR and moderate to severe NPDR as compared to the control group, and not significantly reduced in NDR and mild NPDR. The findings demonstrate reductions in both DO2 and MO2 in PDR and MO2 in moderate to severe NPDR, suggesting their potential as biomarkers for monitoring progression and treatment of DR.

Abstract Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations, maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here, we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model, we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity.

Summary: AML is characterized by mutations in genes associated with growth regulation such as internal tandem duplications (ITD) in the receptor kinase FLT3. Inhibitors targeting FLT3 (FLT3i) are being used to treat patients with FLT3-ITD+ but most relapse and become resistant. To elucidate the resistance mechanism, we compared the gene regulatory networks (GRNs) of leukemic cells from patients before and after relapse, which revealed that the GRNs of drug-responsive patients were altered by rewiring their AP-1-RUNX1 axis. Moreover, FLT3i induces the upregulation of signaling genes, and we show that multiple cytokines, including interleukin-3 (IL-3), can overcome FLT3 inhibition and send cells back into cycle. FLT3i leads to loss of AP-1 and RUNX1 chromatin binding, which is counteracted by IL-3. However, cytokine-mediated drug resistance can be overcome by a pan-RAS inhibitor. We show that cytokines instruct AML growth via the transcriptional regulators AP-1 and RUNX1 and that pan-RAS drugs bypass this barrier.

Seaweed, often considered an overlooked resource, are marine algae with many applications in various industries. The many species of seaweed possess a range of nutritional values, environmental benefits, and their versatility make them an attractive subject of research and development. One promising method for preserving and utilising seaweed effectively is freeze drying. This review explores the world of seaweed, its properties, applications, and the potential of freeze-drying techniques in harnessing the above benefits. The review examines the freeze-drying process and discusses its applicability and advantages in preserving seaweed as a sustainable resource. The discussion also includes the diverse uses of seaweed, from culinary applications to pharmaceuticals and beyond, highlighting the potential of freeze-drying to unlock seaweed’s full potential

Abstract Educational policies such as the No Child Left Behind Act in 2001 and Every Student Succeeds Act in 2015 have emphasized the need for hard evidence such as standardized test data to make educational decisions. Therefore, teachers have been expected to use student assessment data to make informed curricular decisions and adjustments in teaching practice. This has led school districts to turn to data-based decision-making (DBDM) to create an effective process of student assessment data analysis, and reflection during professional learning communities (PLCs). The study explored teachers’ collaboration on the DBDM when teachers were analyzing student assessment data from common, district, and state assessments, as well as its influence on their instructional decisions. Participating teachers’ department and PLC meetings were recorded, and individual teachers’ classrooms were also observed. Teacher interview data and their lesson plans were also included and analyzed qualitatively. Results revealed that the purpose of collaboration determined types of data used, modes of analysis, and the impact on their teaching. Furthermore, teachers used varied assessment data to plan and adjust their instruction. Their motivation to enhance their teaching practices was also increased by exchanging ideas and insights rooted in the data.

Abstract Citrullinated vimentin has been linked to several chronic and autoimmune diseases, but how citrullinated vimentin is associated with disease prevalence and genetic variants in a clinical setting remains unknown. The aim of this study was to obtain a better understanding of the genetic variants and pathologies associated with citrullinated and MMP-degraded vimentin. Patient Registry data, serum samples and genotypes were collected for a total of 4369 Danish post-menopausal women enrolled in the Prospective Epidemiologic and Risk Factor study (PERF). Circulating citrullinated and MMP-degraded vimentin (VICM) was measured. Genome-wide association studies (GWAS) and phenome wide association studies (PheWAS) with levels of VICM were performed. High levels of VICM were significantly associated with the prevalence of chronic pulmonary diseases and death from respiratory and cardiovascular diseases (CVD). GWAS identified 33 single nucleotide polymorphisms (SNPs) with a significant association with VICM. These variants were in the peptidylarginine deiminase 3/4 (PADI3/PADI4) and Complement Factor H (CFH)/KCNT2 gene loci on chromosome 1. Serum levels of VICM, a marker of citrullinated and MMP-degraded vimentin, were associated with chronic pulmonary diseases and genetic variance in PADI3/PADI4 and CFH/ KCNT2. This points to the potential for VICM to be used as an activity marker of both citrullination and inflammation, identifying responders to targeted treatment and patients likely to experience disease progression.

Abstract Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.

Abstract Vascular pulsation at the optic nerve head (ONH) reflects vessel properties. Reduction in the stimulated retinal vasodilatory capacity has been reported in diabetes, but its relation with vascular pulsation is unknown. Here we report a new retinal imaging system for correlative assessment of ONH vascular pulsation and stimulated retinal vasodilation. Retinal reflectance images were acquired before and during light flicker stimulation to quantify arterial and venous vasodilation (DAR, DVR) in subjects with and without diabetic retinopathy (N = 25). ONH vascular pulsation amplitude and frequency (PA, PF), were quantified by curve fitting of periodic intensity waveforms acquired in retinal vasculature (RV) and ONH tissue (ONHT) regions. The relationships between pulsation metrics, heart rate (HR), intraocular pressure (IOP), and vasodilatory responses were evaluated. Pulsation metrics were not significantly different between regions (p ≥ 0.70). In RV, inter-image variabilities of PA and PF were 10% and 6%, whereas inter-observer variabilities were 7% and 2% respectively. In both regions, PF was correlated with HR (p ≤ 0.001). PA was associated with DAR in both regions (p ≤ 0.03), but only with DVR in RV (p ≤ 0.05). Overall, ONH vascular pulsation was associated with stimulated retinal vasodilation, suggesting diabetes may have concomitant effects on retinal vasculature compliance and neurovascular coupling.

Abstract Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the interactions between oncogenes and tumor suppressor genes that shape this landscape remain poorly resolved and cannot be revealed by human cancer genomics alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. We show that the fitness landscape is rugged—the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context—and shows no evidence of diminishing-returns epistasis within variants of the same oncogene. These findings argue against a simple linear signaling relationship amongst these three oncogenes and imply a critical role for off-axis signaling in determining the fitness effects of inactivating tumor suppressors.

Abstract All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA’s anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA’s anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice—a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.

Abstract Genes act in concert with each other in specific contexts to perform their functions. Determining how these genes influence complex traits requires a mechanistic understanding of expression regulation across different conditions. It has been shown that this insight is critical for developing new therapies. Transcriptome-wide association studies have helped uncover the role of individual genes in disease-relevant mechanisms. However, modern models of the architecture of complex traits predict that gene-gene interactions play a crucial role in disease origin and progression. Here we introduce PhenoPLIER, a computational approach that maps gene-trait associations and pharmacological perturbation data into a common latent representation for a joint analysis. This representation is based on modules of genes with similar expression patterns across the same conditions. We observe that diseases are significantly associated with gene modules expressed in relevant cell types, and our approach is accurate in predicting known drug-disease pairs and inferring mechanisms of action. Furthermore, using a CRISPR screen to analyze lipid regulation, we find that functionally important players lack associations but are prioritized in trait-associated modules by PhenoPLIER. By incorporating groups of co-expressed genes, PhenoPLIER can contextualize genetic associations and reveal potential targets missed by single-gene strategies.

Abstract Multiplexed DNA fluorescence in situ hybridization (FISH) imaging technologies have been developed to map the folding of chromatin fibers at tens of nanometers and up to several kilobases in resolution in single cells. However, computational methods to reliably identify chromatin loops from such imaging datasets are still lacking. Here we present a Single-Nucleus Analysis Pipeline for multiplexed DNA FISH (SnapFISH), to process the multiplexed DNA FISH data and identify chromatin loops. SnapFISH can identify known chromatin loops from mouse embryonic stem cells with high sensitivity and accuracy. In addition, SnapFISH obtains comparable results of chromatin loops across datasets generated from diverse imaging technologies. SnapFISH is freely available at https://github.com/HuMingLab/SnapFISH .

The cerebellum has been implicated in the regulation of social behavior. Its influence is thought to arise from communication, via the thalamus, to forebrain regions integral in the expression of social interactions, including the anterior cingulate cortex (ACC). However, the signals encoded or the nature of the communication between the cerebellum and these brain regions is poorly understood. Here, we describe an approach that overcomes technical challenges in exploring the coordination of distant brain regions at high temporal and spatial resolution during social behavior. We developed the E-Scope, an electrophysiology-integrated miniature microscope, to synchronously measure extracellular electrical activity in the cerebellum along with calcium imaging of the ACC. This single coaxial cable device combined these data streams to provide a powerful tool to monitor the activity of distant brain regions in freely behaving animals. During social behavior, we recorded the spike timing of multiple single units in cerebellar right Crus I (RCrus I) Purkinje cells (PCs) or dentate nucleus (DN) neurons while synchronously imaging calcium transients in contralateral ACC neurons. We found that during social interactions a significant subpopulation of cerebellar PCs were robustly inhibited, while most modulated neurons in the DN were activated, and their activity was correlated with positively modulated ACC neurons. These distinctions largely disappeared when only non-social epochs were analyzed suggesting that cerebellar-cortical interactions were behaviorally specific. Our work provides new insights into the complexity of cerebellar activation and co-modulation of the ACC during social behavior and a valuable open-source tool for simultaneous, multimodal recordings in freely behaving mice.

[This corrects the article DOI: 10.1371/journal.pone.0281172.].

BackgroundThe Australian National Perinatal Data Collection collates all live and stillbirths from States and Territories in Australia. In that database, maternal cigarette smoking is noted twice (smoking 20 weeks gestation). Cannabis use and other forms of nicotine use, for example vaping and nicotine replacement therapy, are nor reported. The 2021 report shows the rate of smoking for Australian Indigenous mothers was 42% compared with 11% for Australian non-Indigenous mothers. Evidence shows that Indigenous babies exposed to maternal smoking have a higher rate of adverse outcomes compared to non-Indigenous babies exposed to maternal smoking (S1 File).ObjectivesThe reasons for the differences in health outcome between Indigenous and non-Indigenous pregnancies exposed to tobacco and nicotine is unknown but will be explored in this project through a number of activities. Firstly, the patterns of parental and household tobacco, nicotine and cannabis use and exposure will be mapped during pregnancy. Secondly, a range of biological samples will be collected to enable the first determination of Australian Indigenous people's nicotine and cannabis metabolism during pregnancy; this assessment will be informed by pharmacogenomic analysis. Thirdly, the pharmacokinetic and pharmacogenomic findings will be considered against maternal, placental, foetal and neonatal outcomes. Lastly, an assessment of population health literacy and risk perception related to tobacco, nicotine and cannabis products peri-pregnancy will be undertaken.MethodsThis is a community-driven, co-designed, prospective, mixed-method observational study with regional Queensland parents expecting an Australian Indigenous baby and their close house-hold contacts during the peri-gestational period. The research utilises a multi-pronged and multi-disciplinary approach to explore interlinked objectives.ResultsA sample of 80 mothers expecting an Australian Indigenous baby will be recruited. This sample size will allow estimation of at least 90% sensitivity and specificity for the screening tool which maps the patterns of tobacco and nicotine use and exposure versus urinary cotinine with 95% CI within ±7% of the point estimate. The sample size required for other aspects of the research is less (pharmacokinetic and genomic n = 50, and the placental aspects n = 40), however from all 80 mothers, all samples will be collected.ConclusionsResults will be reported using the STROBE guidelines for observational studies.ForwardWe acknowledge the Traditional Custodians, the Butchulla people, of the lands and waters upon which this research is conducted. We acknowledge their continuing connections to country and pay our respects to Elders past, present and emerging. Notation: In this document, the terms Aboriginal and Torres Strait Islander and Indigenous are used interchangeably for Australia's First Nations People. No disrespect is intended, and we acknowledge the rich cultural diversity of the groups of peoples that are the Traditional Custodians of the land with which they identify and with whom they share a connection and ancestry.

Dyadic Developmental Psychotherapy (DDP) is a family-based therapy for adopted children aiming to achieve secure attachment between the child and parent. Due to restrictions under the COVID-19 pandemic, delivery of DDP transitioned from face-to-face to online methods. This study aimed to explore families experience of online DDP compared to face-to-face DDP, looking at the advantages and disadvantages of remote delivery methods and the implications this has on future service delivery for clinicians. Semi-structured interviews with 6 families were conducted online. Analysis of transcripts using Interpretative Phenomenological Analysis (IPA) revealed four superordinate themes: environment and child engagement, non-verbal communication, travel and familiarity with remote interactions. Parents recognised the influence the physical and online environment had on their child's engagement levels, however, varied in their experience and hence preference of delivery method. All families emphasised the importance of non-verbal communication within DDP sessions and majority highlighted this may be lost online. For families who travelled to face-to-face DDP, car journeys provided a unique opportunity to decompress and reflect after sessions. For families where travel is unfeasible, online DDP was a lifeline, demonstrating the ability of remote therapy to widen access to specialist healthcare. Familiarity with online work emerged as a strong indicator of positive attitudes towards remote DDP, especially if the previous experience is positive and the child is confident using technology. Overall, families differed greatly in their experience of remote and face-to-face DDP indicating a new approach must be undertaken with each family beginning therapy, ensuring it is unique and individual to their needs.

Episodic increases in cerebral blood flow (CBF) are thought to contribute to improved cerebrovascular function and health. Head-out water immersion (HOWI) may be a useful modality to increase CBF secondary to the hydrostatic pressure placed on the body. However, it is unclear whether water temperatures common to the general public elicit similar cerebrovascular responses. We tested the hypothesis that mean middle cerebral artery blood velocity (MCAvmean) and cerebrovascular reactivity to CO2 (CVRCO2) would be higher during an acute bout of thermoneutral (TN; 35°C) vs. cool (COOL; 25°C) HOWI. Ten healthy participants (age: 23±3 y; 4 women) completed two randomized HOWI visits. Right MCAvmean, end-tidal CO2 (PETCO2) mean arterial pressure (MAP), and MCA conductance (MCAvmean/MAP) were continuously recorded. CVRCO2 was assessed using a stepped hypercapnia protocol before (PRE), at 30 minutes of HOWI (HOWI), immediately after HOWI (POST-1), and 45 minutes after HOWI (POST-2). Absolute values are reported as mean ± SD. MCAvmean, PETCO2, MAP, and CVRCO2 were not different between conditions at any timepoint (all P≥0.17). In COOL, MCAvmean increased from PRE (61±9 cm/s) during HOWI (68±11 cm/s), at POST-1 (69±11 cm/s), and POST-2 (72±8 cm/s) (all P

Abstract Radiation therapy induces immunogenic cell death in cancer cells, whereby released endogenous adjuvants are sensed by immune cells to direct adaptive immune responses. TLRs expressed on several immune subtypes recognize innate adjuvants to direct downstream inflammatory responses in part via the adapter protein MyD88. We generated Myd88 conditional knockout mice to interrogate its contribution to the immune response to radiation therapy in distinct immune populations in pancreatic cancer. Surprisingly, Myd88 deletion in Itgax (CD11c)-expressing dendritic cells had little discernable effects on response to RT in pancreatic cancer and elicited normal T cell responses using a prime/boost vaccination strategy. Myd88 deletion in Lck-expressing T cells resulted in similar or worsened responses to radiation therapy compared to wild-type mice and lacked antigen-specific CD8+ T cell responses from vaccination, similar to observations in Myd88 −/− mice. Lyz2-specific loss of Myd88 in myeloid populations rendered tumors more susceptible to radiation therapy and elicited normal CD8+ T cell responses to vaccination. scRNAseq in Lyz2-Cre/Myd88 fl/fl mice revealed gene signatures in macrophages and monocytes indicative of enhanced type I and II interferon responses, and improved responses to RT were dependent on CD8+ T cells and IFNAR1. Together, these data implicate MyD88 signaling in myeloid cells as a critical source of immunosuppression that hinders adaptive immune tumor control following radiation therapy.

Abstract Parkinson’s disease (PD) is a complex neurodegenerative disease with etiology rooted in genetic vulnerability and environmental factors. Here we combine quantitative epidemiologic study of pesticide exposures and PD with toxicity screening in dopaminergic neurons derived from PD patient induced pluripotent stem cells (iPSCs) to identify Parkinson’s-relevant pesticides. Agricultural records enable investigation of 288 specific pesticides and PD risk in a comprehensive, pesticide-wide association study. We associate long-term exposure to 53 pesticides with PD and identify co-exposure profiles. We then employ a live-cell imaging screening paradigm exposing dopaminergic neurons to 39 PD-associated pesticides. We find that 10 pesticides are directly toxic to these neurons. Further, we analyze pesticides typically used in combinations in cotton farming, demonstrating that co-exposures result in greater toxicity than any single pesticide. We find trifluralin is a driver of toxicity to dopaminergic neurons and leads to mitochondrial dysfunction. Our paradigm may prove useful to mechanistically dissect pesticide exposures implicated in PD risk and guide agricultural policy.

Abstract To conduct a systematic review of the comparative efficacy of various psychotropic medications for the treatment of disruptive behavior (DBs) in youths. To this aim, we systematically reviewed randomized clinical trials (RCTs) of various psychotropic medications targeting symptoms of DBs and applied network meta-analysis to investigate their relative efficacy. Fifty-five RCTs meeting the inclusion criteria were selected. To predict and interpret relative treatment efficacy, we compared the efficacy of various psychotropic medications prescribed for DB symptoms based on their mechanism of action. Network meta-analysis revealed that for reducing DBs, second-generation antipsychotics, stimulants, and non-stimulant ADHD medications were more efficacious than placebo, and second-generation antipsychotics were the most efficacious. The dopaminergic modulation of top-down inhibitory process by these medications is discussed in this review. This study offers information on the relative efficacy of various psychotropic medications for the treatment of DB, and insight into a potential neurobiological underpinning for those symptoms. It also illustrates the potential utility of these neurobiological mechanisms as a target for future treatment studies.

Abstract Tissue resident memory (Trm) CD8 T cells infiltrating tumors represent an enriched population of tumor antigen-specific T cells, and their presence is associated with improved outcomes in patients. Using genetically engineered mouse pancreatic tumor models we demonstrate that tumor implantation generates a Trm niche that is dependent on direct antigen presentation by cancer cells. However, we observe that initial CCR7-mediated localization of CD8 T cells to tumor draining lymph nodes is required to subsequently generate CD103+ CD8 T cells in tumors. We observe that the formation of CD103+ CD8 T cells in tumors is dependent on CD40L but independent of CD4 T cells, and using mixed chimeras we show that CD8 T cells can provide their own CD40L to permit CD103+ CD8 T cell differentiation. Finally, we show that CD40L is required to provide systemic protection against secondary tumors. These data suggest that CD103+ CD8 T cell formation in tumors can occur independent of the two-factor authentication provided by CD4 T cells and highlight CD103+ CD8 T cells as a distinct differentiation decision from CD4-dependent central memory.

Over the past decade and a half, dynamic functional imaging has revealed low-dimensional brain connectivity measures, identified potential common human spatial connectivity states, tracked the transition patterns of these states, and demonstrated meaningful transition alterations in disorders and over the course of development. Recently, researchers have begun to analyze these data from the perspective of dynamic systems and information theory in the hopes of understanding how these dynamics support less easily quantified processes, such as information processing, cortical hierarchy, and consciousness. Little attention has been paid to the effects of psychiatric disease on these measures, however. We begin to rectify this by examining the complexity of subject trajectories in state space through the lens of information theory. Specifically, we identify a basis for the dynamic functional connectivity state space and track subject trajectories through this space over the course of the scan. The dynamic complexity of these trajectories is assessed along each dimension of the proposed basis space. Using these estimates, we demonstrate that schizophrenia patients display substantially simpler trajectories than demographically matched healthy controls and that this drop in complexity concentrates along specific dimensions. We also demonstrate that entropy generation in at least one of these dimensions is linked to cognitive performance. Overall, the results suggest great value in applying dynamic systems theory to problems of neuroimaging and reveal a substantial drop in the complexity of schizophrenia patients’ brain function.

ABSTRACTMaterial extrusion additive manufacturing is of increased interest in producing materials with very high loadings of particles, specifically through the use of the direct ink write (DIW), or robocasting, technique and the use of highly loaded particle suspensions (HLS). Applications from biomedical composites to solid rocket propellants to powder metallurgy green bodies would benefit from the complex parts enabled by additive manufacturing but require very high particle contents during processing. This leads to very high viscosity fluids and challenges in flowing and curing the inks. In this comprehensive review, we examine the main components of designing an ink formulation and a DIW process: the ink rheology, the print mechanics and the solidification/post-processing. Our expanded discussion of these elements includes an introduction to the basics as well as the latest research in the field, so serves to both introduce a new practitioner and generate new ideas for those already working in the area. We finish with a discussion of two important applications and a perspective on the future directions of DIW for highly loaded particle materials.

The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following β-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.

Patient derived xenograft (PDX) models are regarded as gold standard preclinical models in leukaemia research, especially in testing new drug combinations where typically 45-50 mice are used per assay. 9000 animal experiments are performed annually in the UK in leukaemia research with these expensive procedures being classed as moderate severity, meaning they cause significant pain, suffering and visible distress to animal’s state. Furthermore, not all clinical leukaemia samples engraft and when they do data turnaround time can be between 6-12 months. Heavy dependence on animal models is because clinical leukaemia samples do not proliferate in vitro. Alternative cell line models though popular for drug testing are not biomimetic – they are not dependent on the microenvironment for survival, growth and treatment response and being derived from relapse samples they do not capture the molecular complexity observed at disease presentation. Here we have developed an in vitro platform to rapidly establish co-cultures of patient-derived leukaemia cells with 3D bone marrow mesenchyme spheroids, BM-MSC-spheroids. We optimise protocols for developing MSC-spheroid leukaemia co-culture using clinical samples and deliver drug response data within a week. Using three patient samples representing distinct cytogenetics we show that patient-derived-leukaemia cells show enhanced proliferation when co-cultured with MSC-spheroids. In addition, MSC-spheroids provided improved protection against treatment. This makes our spheroids suitable to model treatment resistance – a major hurdle in current day cancer management Given this 3Rs approach is 12 months faster (in delivering clinical data), is a human cell-based biomimetic model and uses 45-50 fewer animals/drug-response assay the anticipated target end-users would include academia and pharmaceutical industry. This animal replacement prototype would facilitate clinically translatable research to be performed with greater ethical, social and financial sustainability.