Biology subjects

Miller, D.

Publications and source records attributed to Miller, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Parkinson's Paradox: Alpha-synuclein's Selective Strike on SNc Dopamine Neurons over VTA

In synucleinopathies, including Parkinsons disease (PD), dopamine neurons in the substantia nigra pars compacta (SNc) exhibit greater vulnerability to degeneration than those in the ventral tegmental area (VTA). While -synuclein (Syn) pathology is implicated in nigral dopamine neuron loss, the mechanisms by which Syn affects neuronal activity and midbrain dopamine network connectivity prior to cell death remain unclear. This study tested the hypothesis that elevated Syn expression induces pathophysiological changes in firing activity and disrupts network connectivity dynamics of dopamine neurons before neuronal loss. We employed two mouse models of synucleinopathy: preformed Syn fibril (PFF) injection and AAV-mediated expression of human Syn (hSyn) under the control of the tyrosine hydroxylase (TH) promoter, both targeting the VTA and SNc. Four weeks post-injection, brain sections underwent histological, electrophysiological, and network analyses. Immunohistochemistry for TH, hSyn, and phospho-Ser129 Syn assessed Syn expression and dopaminergic neuron alterations. Neuronal viability was evaluated using two complementary approaches: quantification of TH+ or FOX3+ and TUNEL labeling. Importantly, these analyses revealed no significant changes in neuronal counts or TUNEL+ cells at this time point, confirming that subsequent functional assessments captured pre-neurodegenerative, Syn-induced alterations rather than late-stage neurodegeneration. Electrophysiological recordings revealed a differential effect of hSyn expression. SNc dopamine neurons exhibited significantly increased baseline firing rates, whereas VTA dopamine neurons remained unchanged. These findings indicate a region-specific vulnerability to Syn-induced hyperactivity of dopamine neurons. Further analysis revealed impaired homeostatic firing rate regulation in SNc, but not VTA, dopamine neurons, demonstrated by a reduced capacity to recover baseline firing following hyperpolarization. Collectively, our results demonstrate that, prior to neurodegeneration, elevated Syn expression differentially disrupts both basal firing activity and network stability of SNc dopamine neurons, while sparing VTA dopamine neurons. By identifying neurophysiological changes preceding dopaminergic neuron loss, these findings provide critical insights into the pathophysiological mechanisms predisposing SNc neurons to degeneration in Parkinsons disease. Significance StatementA central question in Parkinsons disease research is why dopamine neurons in the substantia nigra pars compacta (SNc) are more vulnerable than those in the ventral tegmental area (VTA). This study reveals that alpha-synuclein (Syn) pathology differentially impacts dopamine neuronal activity and network connectivity, causing changes in the SNc before neuronal loss occurs, but not in the VTA. These findings provide a mechanism to explain the differential resilience of these neighboring dopamine neuron populations and provide insights into Parkinsons disease progression. The methodologies developed in this study establish a foundation for investigating network topology in deep brain structures and its role in neurodegenerative disorders.

neuroscience↗

bioRxiv2025

The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming

Acute myocardial infarction (MI) is a leading cause of morbidity and mortality, and therapeutic options remain limited. Endogenously generated nitric oxide (NO) is highly cardioprotective, but protection is not replicated by nitroso-vasodilators (e.g., nitrates, nitroprusside) used in clinical practice, highlighting specificity in NO-based signaling and untapped therapeutic potential. Signaling by NO is mediated largely by S-nitrosylation, entailing specific enzymes that form and degrade S-nitrosothiols in proteins (SNO-proteins), termed nitrosylases and denitrosylases, respectively. SNO-CoA Reductase 2 (SCoR2; product of the Akr1a1 gene) is a recently discovered protein denitrosylase. Genetic variants in SCoR2 have been associated with cardiovascular disease, but its function is unknown. Here we show that mice lacking SCoR2/Akr1a1 exhibit robust protection in an animal model of MI. SCoR2 regulates ketolytic energy availability, antioxidant levels and polyol homeostasis via S-nitrosylation of key metabolic effectors. Human cardiomyopathy shows reduced SCoR2 expression and an S-nitrosylation signature of metabolic reprogramming, mirroring SCoR2-/- mice. Deletion of SCoR2 thus coordinately reprograms multiple metabolic pathways--ketone body utilization, glycolysis, pentose phosphate shunt and polyol metabolism--to limit infarct size, establishing SCoR2 as a novel regulator in the injured myocardium and a potential drug target. Impact statementMice lacking the denitrosylase enzyme SCoR2 demonstrate robust cardioprotection resulting from coordinate reprogramming of multiple metabolic pathways. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/642752v2_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@72bfbborg.highwire.dtl.DTLVardef@189cbc4org.highwire.dtl.DTLVardef@1a0cc51org.highwire.dtl.DTLVardef@1c61004_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Functional Specialization of S-Adenosylmethionine Synthases Links Phosphatidylcholine to Mitochondrial Function and Stress Survival

S-adenosylmethionine (SAM), produced by SAM synthases, is critical for various cellular regulatory pathways and the synthesis of diverse metabolites. Studies have often equated the effects of knocking down one synthase with broader SAM-dependent outcomes such as histone methylation or phosphatidylcholine (PC) production. Humans and many other organisms express multiple SAM synthases. Evidence in Caenorhabditis elegans, which possesses four SAM synthase genes, suggest that the enzymatic source of SAM impacts its function. For instance, loss of sams-1 leads to enhanced heat shock survival and increased lifespan, whereas reducing sams-4 adversely affects heat stress survival. Here, we show that SAMS-1 contributes to a variety of intermediary metabolic pathways, whereas SAMS-4 is more important to generate SAM for methylation reactions. We demonstrate that loss of sams-1 exerts age-dependent effects on nuclear-encoded mitochondrial gene expression, mitochondrial metabolites, and may induce mitophagy. We propose a mechanistic model where reduced SAM from SAMS-1 acts through PC to impact mitochondria, thereby enhancing survival during heat stress.

cell biology↗

bioRxiv2024

RNA editing is a molecular clock in unmodified human cells

Despite major advances in spatial RNA sequencing, the ability to extract temporal information in RNA sequencing experiments is still limited. Here, we describe Transcriptome Timestamping (T2), a system which harnesses naturally occurring A-to-I editing of RNA transcripts in unmodified human cells to infer transcriptional history. T2 provides age estimates for individual RNA transcripts, and serves as an endogenous molecular recorder, differentiating between complex transcriptional programs. We show that T2 can identify transient and transitional transcriptional programs in primary differentiating monocytes that are not apparent from gene expression analysis alone, including a regulatory module in the monocyte-to-macrophage transition that, to our knowledge, has not yet been described in humans. Finally, we show that T2 can also be applied to single cell data, allowing us to identify transcriptional programs in heterogeneous populations, such as asynchronously dividing cells. T2 is a scalable approach to temporal transcriptomics that can be applied to track the activity of thousands of genes in unmodified, primary human cells and tissues, with no genetic engineering.

genomics↗

bioRxiv2024

High-throughput DNA engineering by mating bacteria

To reduce the operational friction and scale DNA engineering, we report here an in vivo DNA assembly technology platform called SCRIVENER (Sequential Conjugation and Recombination for In Vivo Elongation of Nucleotides with low ERrors). SCRIVENER combines bacterial conjugation, in vivo DNA cutting, and in vivo homologous recombination to seamlessly stitch blocks of DNA together by mating E. coli in large arrays or pools. This workflow is simpler, cheaper, and higher throughput than current DNA assembly approaches that require DNA to be moved in and out of cells at different procedural steps. We perform over 5,000 assemblies with two to 13 DNA blocks that range from 240 bp to 8 kb and show that SCRIVENER is capable of assembling constructs as long as 23 kb at relatively high throughput and fidelity. Most SCRIVENER errors are deletions between long interspersed repeats. However, SCRIVENER can overcome these errors by enabling assembly and sequence verification at high replication at a nominal additional cost per replicate. We show that SCRIVENER can be used to build combinatorial libraries in arrays or pools, and that DNA blocks onboarded into the platform can be repurposed and reused with any other DNA block in high throughput without a PCR step. Because of these features, DNA engineering with SCRIVENER has the potential to accelerate design-build-test-learn cycles of DNA products.

synthetic biology↗

bioRxiv2024

BCL2L13 Influences Autophagy and Ceramide Metabolism without Affecting Temozolomide Resistance in Glioblastoma

Temozolomide (TMZ) resistance in glioblastoma (GBM) arises through metabolic rewiring that links mitochondrial function, autophagy balance, and sphingolipid metabolism. TMZ resistant (R) U251 cells exhibited suppressed apoptosis and complete blockade of autophagy flux, evidenced by LC3II and p62 accumulation and insensitivity to Bafilomycin A1. BCL2L13, strongly upregulated in R cells, emerged as a dual regulator of mitophagy and ceramide metabolism. BCL2L13 knockdown (KD) produced opposite effects in TMZ sensitive (NR) and resistant cells: in NR cells, KD elevated LC3II, reduced respiratory reserve, and triggered compensatory lipid synthesis; in R cells, KD decreased LC3II without restoring flux or TMZ sensitivity. Lipidomic profiling revealed that BCL2L13 loss reactivated CerS6 in NR cells, increasing C16:0 and mid-chain ceramides, while relieving CerS2 inhibition in R cells, elevating very long chain (C22 to C24) and glycosylated ceramides. These distinct sphingolipid signatures were confirmed by PLS-DA and KEGG enrichment, which highlighted steroid hormone, arachidonic, and linoleic acid metabolism in NR KD cells versus neuroactive ligand-receptor and signaling pathways in R KD cells. Together, these findings position BCL2L13 as a molecular integrator of mitochondrial respiration, autophagy flux, and CerS-dependent lipid remodeling, unveiling a context-specific metabolic mechanism that supports GBM cell survival under chemotherapeutic stress.

cancer biology↗

bioRxiv2024

IGF-1 Acts through Kiss1-expressing Cells to Influence Metabolism and Reproduction

ObjectiveKisspeptin, encoded by the Kiss1 gene, ties puberty and fertility to energy status; however, the metabolic factors that control Kiss1-expressing cells need to be clarified. MethodsTo evaluate the impact of IGF-1 on the metabolic and reproductive functions of kisspeptin producing cells, we created mice with IGF-1 receptor deletion driven by the Kiss1 promoter (IGF1RKiss1 mice). Previous studies have shown IGF-1 and insulin can bind to each others receptor, permitting IGF-1 signaling in the absence of IGF1R. Therefore, we also generated mice with simultaneous deletion of the IGF1R and insulin receptor (IR) in Kiss1-expressing cells (IGF1R/IRKiss1 mice). ResultsLoss of IGF1R in Kiss1 cells caused stunted body length. In addition, female IGF1RKiss1 mice displayed lower body weight and food intake plus higher energy expenditure and physical activity. This phenotype was linked to higher proopiomelanocortin (POMC) expression and heightened brown adipose tissue (BAT) thermogenesis. Male IGF1RKiss1 mice had mild changes in metabolic functions. Moreover, IGF1RKiss1 mice of both sexes experienced delayed puberty. Notably, male IGF1RKiss1 mice had impaired adulthood fertility accompanied by lower gonadotropin and testosterone levels. Thus, IGF1R in Kiss1-expressing cells impacts metabolism and reproduction in a sex-specific manner. IGF1R/IRKiss1 mice had higher fat mass and glucose intolerance, suggesting IGF1R and IR in Kiss1-expressing cells together regulate body composition and glucose homeostasis. ConclusionsOverall, our study shows that IGF1R and IR in Kiss1 have cooperative roles in body length, metabolism, and reproduction.

neuroscience↗

bioRxiv2024

Integrated Movement Models for Individual Tracking and Species Distribution Data

O_LIWhile the quantity, quality, and variety of movement data has increased, methods that jointly allow for population- and species-level movement parameters to be estimated are still needed. We present a formal data integration approach to combine individual-level movement and population-level distribution data. We show how formal data integration can be used to improve precision of individual and population level movement parameters and allow additional population level metrics (e.g., connectivity) to be formally quantified. C_LIO_LIWe describe three components needed for an Integrated Movement Model (IMM): a model for individual movement, a model for among-individual heterogeneity, and a model to quantify changes in species distribution. We outline a general IMM framework and develop and apply a specific stochastic differential equation model to a case study of telemetry and species distribution data for golden eagles in western North American during spring migration. C_LIO_LIWe estimate eagle movements during spring migration from data collected between 2011 and 2019. Individual heterogeneity in migration behavior was modeled for two sub-populations, individuals that make significant northward migrations and those that remained in the southern Rocky Mountain region through the summer. As is the case with most tracking studies, the sample population of individual telemetered birds is not representative of the population, and underrepresents the proportion of long-distance migrants in. The IMM was able to provide a more biological accurate subpopulation structure by jointly estimating the structure using the species distribution data. In addition, the integrated approach a) improves accuracy of other estimated movement parameters, b) allows us to estimate the proportion of migratory and non-migratory birds in a given location and time, and c) estimate future spatio-temporal distributions of birds given a wintering location, which provide estimates of seasonal connectivity and migratory routes. C_LIO_LIWe demonstrate how IMMs can be successfully used to address the challenge of estimating accurate population level movement parameters. Our approach can be generalized to a broad range of available movement models and data types, allowing us to significantly improve our knowledge of migration ecology across taxonomic groups, and address population and continental level information needs for conservation and management. C_LI

ecology↗

bioRxiv2024

The Updated Mouse Universal Genotyping Array Bioinformatic Pipeline Improves Genetic QC in Laboratory Mice

The MiniMUGA genotyping array is a popular tool for genetic QC of laboratory mice and genotyping of samples from most types of experimental crosses involving laboratory strains, particularly for reduced complexity crosses. The content of the production version of the MiniMUGA array is fixed; however, there is the opportunity to improve arrays performance and the associated reports usefulness by leveraging thousands of samples genotyped since the initial description of MiniMUGA in 2020. Here we report our efforts to update and improve marker annotation, increase the number and the reliability of the consensus genotypes for inbred strains and increase the number of constructs that can reliably be detected with MiniMUGA. In addition, we have implemented key changes in the informatics pipeline to identify and quantify the contribution of specific genetic backgrounds to the makeup of a given sample, remove arbitrary thresholds, include the Y Chromosome and mitochondrial genome in the ideogram, and improve robust detection of the presence of commercially available substrains based on diagnostic alleles. Finally, we have made changes to the layout of the report, to simplify the interpretation and completeness of the analysis and added a table summarizing the ideogram. We believe that these changes will be of general interest to the mouse research community and will be instrumental in our goal of improving the rigor and reproducibility of mouse-based biomedical research.

genetics↗

bioRxiv2024

Pooled PPIseq: screening the SARS-CoV-2 and human interface with a scalable multiplexed protein-protein interaction assay platform

Protein-Protein Interactions (PPIs) are a key interface between virus and host, and these interactions are important to both viral reprogramming of the host and to host restriction of viral infection. In particular, viral-host PPI networks can be used to further our understanding of the molecular mechanisms of tissue specificity, host range, and virulence. At higher scales, viral-host PPI screening could also be used to screen for small-molecule antivirals that interfere with essential viral-host interactions, or to explore how the PPI networks between interacting viral and host genomes co-evolve. Current high-throughput PPI assays have screened entire viral-host PPI networks. However, these studies are time consuming, often require specialized equipment, and are difficult to further scale. Here, we develop methods that make larger-scale viral-host PPI screening more accessible. This approach combines the mDHFR split-tag reporter with the iSeq2 interaction-barcoding system to permit massively-multiplexed PPI quantification by simple pooled engineering of barcoded constructs, integration of these constructs into budding yeast, and fitness measurements by pooled cell competitions and barcode-sequencing. We applied this method to screen for PPIs between SARS-CoV-2 proteins and human proteins, screening in triplicate >180,000 ORF-ORF combinations represented by >1,000,000 barcoded lineages. Our results complement previous screens by identifying 74 putative PPIs, including interactions between ORF7A with the taste receptors TAS2R41 and TAS2R7, and between NSP4 with the transmembrane KDELR2 and KDELR3. We show that this PPI screening method is highly scalable, enabling larger studies aimed at generating a broad understanding of how viral effector proteins converge on cellular targets to effect replication.

systems biology↗

bioRxiv2024

A Novel Imaging Mass Spectrometry Computational Tool for Biomedical Discovery: Untargeted pixel-by-pixel imaging of Metabolite Ratio Pairs

Mass spectrometry imaging (MSI) is a powerful technology used to define the spatial distribution and relative abundance of metabolites across tissue cryosections. While software packages exist for pixel-by-pixel individual metabolite and limited target pairs of ratio imaging, the research community lacks an easy computing and application tool that images any metabolite abundance ratio pairs. Importantly, recognition of correlated metabolite pairs may contribute to the discovery of unanticipated molecules in shared metabolic pathways. Here, we describe the development and implementation of an untargeted R package workflow for pixel-by-pixel ratio imaging of all metabolites detected in an MSI experiment. Considering untargeted MSI studies of murine brain and embryogenesis, we demonstrate that ratio imaging minimizes systematic data variation introduced by sample handling, markedly enhances spatial image contrast, and reveals previously unrecognized metabotype-distinct tissue regions. Furthermore, ratio imaging facilitates identification of novel regional biomarkers and provides anatomical information regarding spatial distribution of metabolite-linked biochemical pathways. The algorithm described herein is applicable to any MSI dataset containing spatial information for metabolites, peptides or proteins, offering a potent hypothesis generation tool to enhance knowledge obtained from current spatial metabolite profiling technologies.

bioinformatics↗

bioRxiv2023

Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

synthetic biology↗

bioRxiv2023

M2-polarized macrophages prevent preterm birth and improve neonatal survival and immunity

Preterm birth (PTB), often preceded by preterm labor, is a major cause of neonatal morbidity and mortality worldwide. Most PTB cases involve intra-amniotic inflammation without detectable microorganisms, termed in utero sterile inflammation, for which there is no established treatment. Here, we propose homeostatic macrophages to prevent PTB and adverse neonatal outcomes caused by in utero sterile inflammation. Single-cell atlases of the maternal-fetal interface revealed that homeostatic maternal macrophages are reduced with human labor. M2 macrophage treatment prevented PTB and reduced adverse neonatal outcomes in mice with in utero sterile inflammation. Specifically, M2 macrophages halted premature labor by suppressing inflammatory responses in the amniotic cavity, including inflammasome activation, and mitigated placental and offspring lung inflammation. Moreover, M2 macrophages restored neonatal gut homeostasis and enhanced resistance to systemic bacterial infection. Our findings show that M2 macrophages are a promising strategy to mitigate PTB and improve neonatal outcomes from in utero sterile inflammation.

immunology↗

bioRxiv2023

Using big sequencing data to identify chronic SARS-Coronavirus-2 infections

The evolution of SARS-Coronavirus-2 (SARS-CoV-2) has been characterized by the periodic emergence of highly divergent variants, many of which may have arisen during chronic infections of immunocompromised individuals. Here, we harness a global phylogeny of [~]11.7 million SARS-CoV-2 genomes and search for clades composed of sequences with identical metadata (location, age, and sex) spanning more than 21 days. We postulate that such clades represent repeated sampling from the same chronically infected individual. A set of 271 such chronic-like clades was inferred, and displayed signatures of an elevated rate of adaptive evolution, in line with validated chronic infections. More than 70% of adaptive mutations present in currently circulating variants are found in BA.1 chronic-like clades that predate the circulating variants by months, demonstrating the predictive nature of such clades. We find that in chronic-like clades the probability of observing adaptive mutations is approximately 10-20 higher than that in global transmission chains. We next employ language models to find mutations most predictive of chronic infections and use them to infer hundreds of additional chronic-like clades in the absence of metadata and phylogenetic information. Our proposed approach presents an innovative method for mining extensive sequencing data and providing valuable insights into future evolutionary patterns.

evolutionary biology↗

bioRxiv2023

Mutant Huntingtin impairs neurodevelopment in human brain organoids through CHCHD2-mediated neurometabolic failure

Expansion of the glutamine tract (poly-Q) in the protein Huntingtin (HTT) causes the neurodegenerative disorder Huntingtons disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. 70Q introduction caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics and elevated resting energy expenditure. Elimination of the poly-Q tract of HTT normalized CHCHD2 expression and mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early intervention target for HD.

neuroscience↗

bioRxiv2023

Aryl Hydrocarbon Receptor Suppresses Eosinophilic Esophagitis Responses through OVOL1 and SPINK7

Eosinophilic esophagitis (EoE) is a type 2 allergic disease characterized by esophageal inflammation and epithelial cell dysfunction. Acquired loss of the anti-serine protease of kazal type 7 (SPINK7) in the squamous epithelium of the esophagus has a causal role in EoE pathogenesis. Yet there is a limited understanding of the factors that regulate its expression and responsiveness to inflammatory stimuli. Herein, we identified the transcription factor, ovo like transcriptional repressor 1 (OVOL1) as an esophageal selective gene product that regulates SPINK7 promoter activity. Overexpression of OVOL1 increased SPINK7 expression, whereas, its depletion decreased SPINK7 expression, impaired epithelial barrier and increased production of the pro-atopy cytokine thymic stromal lymphopoietin (TSLP). Mechanistically, ligands of AHR induced nuclear translocation of OVOL1 which in turn promoted epithelial cell differentiation, barrier function and SPINK7 expression. Interleukin (IL)-4 and IL-13 abolished AHR ligand-induced OVOL1 nuclear translocation. Stimulation with IL-13 abrogated the nuclear translocation of OVOL1 and promoted enhanced degradation of OVOL1 protein. This effect of IL-13 was dependent on the esophageal specific cysteine protease calpain-14. Translational studies demonstrated loss of OVOL1 protein expression in patients with EoE. In summary, AHR mediates its action via OVOL1-induced SPINK7 transcription, and IL-4 and IL-13 repress this pathway in EoE. As such, activation of the AHR pathway is a potential intervention strategy for reversing EoE. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/541192v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1e946a1org.highwire.dtl.DTLVardef@1c087forg.highwire.dtl.DTLVardef@71ecc8org.highwire.dtl.DTLVardef@17044da_HPS_FORMAT_FIGEXP M_FIG C_FIG The influence of the exposome on regulatory networks in EoE pathogenesis. AHR is activated and influenced by diet nutrients, environmental toxicants, microbiome composition, tryptophan metabolites, and drugs. When AHR is activated, it promotes translocation of OVOL1 to the nucleus, which in turn promotes expression of epithelial genes including SPINK7. SPINK7 expression promotes epithelial differentiation, barrier function, decreased proteolytic activity, and decreased TSLP production. IL-4 and IL-13 inhibit OVOL1 nuclear translocation and therefore, repress SPINK7 expression. IL-13-stimulated CAPN14 expression decreases OVOL1 protein expression and SPINK7 transcription.

immunology↗

bioRxiv2023

Visible-Light Optical Coherence Tomography Fibergraphy of the Tree Shrew Retinal Ganglion Cell Axon Bundles

We seek to develop techniques for high-resolution imaging of the tree shrew retina for visualizing and parameterizing retinal ganglion cell (RGC) axon bundles in vivo. We applied visible-light optical coherence tomography fibergraphy (vis-OCTF) and temporal speckle averaging (TSA) to visualize individual RGC axon bundles in the tree shrew retina. For the first time, we quantified individual RGC bundle width, height, and cross-sectional area and applied vis-OCT angiography (vis-OCTA) to visualize the retinal microvasculature in tree shrews. Throughout the retina, as the distance from the optic nerve head (ONH) increased from 0.5 mm to 2.5 mm, bundle width increased by 30%, height decreased by 67%, and cross-sectional area decreased by 36%. We also showed that axon bundles become vertically elongated as they converge toward the ONH. Ex vivo confocal microscopy of retinal flat-mounts immunostained with Tuj1 confirmed our in vivo vis-OCTF findings.

bioengineering↗

bioRxiv2023

Genome-scale analysis of interactions between genetic perturbations and natural variation

Interactions between genetic perturbations and segregating loci can cause perturbations to show different phenotypic effects across genetically distinct individuals. To study these interactions on a genome scale in many individuals, we used combinatorial DNA barcode sequencing to measure the fitness effects of 7,700 CRISPRi perturbations targeting 1,712 distinct genes in 169 yeast cross progeny (or segregants). We identified 460 genes whose perturbation has different effects across segregants. Several factors caused perturbations to show variable effects, including baseline segregant fitness, the mean effect of a perturbation across segregants, and interacting loci. We mapped 234 interacting loci and found four hub loci that interact with many different perturbations. Perturbations that interact with a given hub exhibit similar epistatic relationships with the hub and show enrichment for cellular processes that may mediate these interactions. These results suggest that an individuals response to perturbations is shaped by a network of perturbation-locus interactions that cannot be measured by approaches that examine perturbations or natural variation alone.

genetics↗