Biology subjects

Smith, A.

Publications and source records attributed to Smith, A..

At least 145 records · Page 8Linked to original sources

bioRxiv2020

Improved daylight vision following AAV-mediated expression of R9AP in murine rod photoreceptors

Cone photoreceptors mediate daylight vision and are the primary cells responsible for vision in humans. Cone dysfunction leads to poor quality daylight vision because rod photoreceptors become saturated and non-functional at high light levels. Here we demonstrate that in mice lacking cone function, AAV-mediated over-expression of Rgs9-anchor protein (R9AP), a critical component of the GTPase complex that mediates the deactivation of the phototransduction cascade, results in desensitization of rod function and a "photopic shift" of the rod-driven electroretinogram. This treatment enables rods to respond to brighter light (up to [~]2.0 log) with increased visually-evoked cortical responses to high intensity stimulation. These results suggest that AAV-mediated transfer of R9ap into rods might be used to improve daylight vision in humans visually handicapped by cone dysfunction.

bioengineering↗

bioRxiv2020

Microfluidic platform for live cell imaging of 3D cultures with clone retrieval

Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. These combined methods are of particular interest when studying highly dynamic or transient, asynchronous or heterogeneous cell biological and developmental processes. Here we present a method to encapsulate live cells in a 3D hydrogel matrix, via droplet compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem (ES) cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we are able to retain or extract individual bead/droplets by media flow only, obviating the need for enzymatic cell removal from the platform. We show that we can differentiate mES cells, monitor reporter expression by live imaging, and retrieve individual droplets for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays.

bioengineering↗

bioRxiv2020

Short chain fatty acid butyrate promotes virus infection by repressing interferon stimulated genes

Butyrate is an abundant metabolite produced by the gut microbiota and is known to modulate multiple immune system pathways and inflammatory diseases. However, studies of its effects on virus infection of cells are limited and enigmatic. We found that butyrate increases cellular infection and virus replication in influenza virus, reovirus, and human immunodeficiency virus infections. Further exploring this phenomenon, we found that addition of butyrate to cells deficient in type I interferon (IFN) signaling did not increase susceptibility to virus infection. Accordingly, we discovered that butyrate suppressed levels of specific IFN stimulated gene (ISG) products in human and mouse cells. Butyrate did not inhibit IFN-induced phosphorylation of transcription factors STAT1 and STAT2 or their translocation to the nucleus, indicating that IFN signaling was not disrupted. Rather, our data are suggestive of a role for inhibition of histone deacetylase activity by butyrate in limiting ISG induction. Global transcript analysis revealed that butyrate increases expression of more than 800 cellular genes, but represses IFN-induced expression of 60% of ISGs. Overall, we identify a new mechanism by which butyrate promotes virus infection via repression of ISGs. Our findings also add to the growing body of evidence showing that individual ISGs respond differently to type I IFN induction depending on the cellular environment, including the presence of butyrate. ImportanceButyrate is a lipid produced by intestinal bacteria that can regulate inflammation throughout the body. Here we show for the first time that butyrate influences the innate antiviral immune response mediated by type I IFNs. A majority of antiviral genes induced by type I IFNs were repressed in the presence of butyrate, resulting in increased virus infection and replication in cells. This suggests that butyrate could be broadly used as a tool to increase growth of virus stocks for research and for the generation of vaccines. Our research also indicates that metabolites produced by the gut microbiome can have complex effects on cellular physiology as demonstrated by the dampening of an inflammatory innate immune pathway by butyrate resulting in a pro-viral cellular environment.

microbiology↗

bioRxiv2020

Trophectoderm Potency is Retained Exclusively in Human Naïve Cells

Classical mouse embryology has established a paradigm of early development driven by sequential lineage bifurcations. Accordingly, mouse embryonic stem cells derived from early epiblast have lost the potency to produce extraembryonic trophectoderm. We show in contrast that human naive epiblast cells readily make trophectoderm. Inhibition of ERK signalling, instrumental in naive stem cell propagation, unexpectedly potentiates trophectoderm formation, an effect enhanced by Nodal inhibition. Transcriptome analyses authenticate conversion into trophectoderm with subsequent production of syncitiotrophoblast, cytotrophoblast and trophoblast stem cells. Genetic perturbations indicate that NANOG suppresses and TFAP2C enables trophectoderm induction. Consistent with post-implantation progression, trophectoderm potential is extinguished in conventional human pluripotent stem cells, which instead make amnion. Finally, human embryo epiblasts from late blastocysts efficiently generate trophectoderm and differentiated trophoblast. Thus, pluripotent cells in the human embryo retain extraembryonic lineage plasticity and regenerative potential until implantation. Harnessing this unanticipated regulative capacity may be beneficial for assisted reproduction technology.

developmental biology↗

bioRxiv2019

Bionic 3D printed corals

Symbiotic corals have evolved as a highly optimised photon augmentation system leading to space-efficient microalgal growth and photosynthetic quantum efficiencies that approach theoretical limits1-3. Corals are characterized by an elastic animal tissue hosting microalgae and a light scattering calcium carbonate skeleton that maximizes light delivery towards otherwise shaded algal-containing tissues4,5. Rapid light attenuation due to algal self-shading is a key limiting factor for the upscaling of microalgal cultivation6,7. Coral-inspired light management systems could overcome this limitation and facilitate scalable bioenergy and bioproduct generation8,9. Here, we developed 3D printed bionic corals capable of growing various types of microalgae with cell densities approaching 109 cells mL-1, up to 100 times greater than in liquid culture. The hybrid photosynthetic biomaterials are produced with a new 3D bioprinting platform which mimics morphological features of living coral tissue and the underlying skeleton with micron resolution, including their optical and mechanical properties. The programmable synthetic microenvironment thus allows for replicating both structural and functional traits of the coral-algal symbiosis. Our work defines a new class of bionic materials capable of interacting with living organisms, that can be exploited for the design of next generation photobioreactors7 and disruptive approaches for coral reef conservation10.

bioengineering↗

bioRxiv2019

The use of CRISPR for variant specificity in the genetic diagnosis of primary immunodeficiency disease (PID)

The functional validation of genetic variants of uncertain significance (VUS) found in PID patients by next-generation sequencing has traditionally been carried out in model systems that are susceptible to artefact. We use CRISPR correction of primary human T lymphocytes to demonstrate that a specific variant in an IL-6R deficient patient is causative for their condition. This methodology can be adapted and used for variant assessment of the heterogeneous genetic defects that affect T lymphocytes in PID.

immunology↗

bioRxiv2019

Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2, provides photoreceptor neuroprotection

Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.

molecular biology↗

bioRxiv2019

Hexokinase 2 is dispensable for photoreceptor development but is required for survival during aging and outer retinal stress

Photoreceptor death is the ultimate cause of vision loss in many retinal degenerative conditions. Identifying novel therapeutic avenues for prolonging photoreceptor health and function has the potential to improve vision and quality of life for patients suffering from degenerative retinal disorders. Photoreceptors are metabolically unique among other neurons in that they process the majority of their glucose via aerobic glycolysis. One of the main regulators of aerobic glycolysis is hexokinase 2 (HK2). Beyond its enzymatic function of phosphorylating glucose to glucose-6-phosphate, HK2 has additional non-enzymatic roles, including the regulation of apoptotic signaling via AKT signaling. Determining the role of HK2 in photoreceptor homeostasis may identify novel signaling pathways that can be targeted with neuroprotective agents to boost photoreceptor survival during metabolic stress. Here we show that following experimental retinal detachment, p-AKT is upregulated and HK2 translocates to mitochondria. Inhibition of AKT phosphorylation in 661W photoreceptor-like cells results in translocation of mitochondrial HK2 to the cytoplasm, increased caspase activity, and decreased cell viability. Rod-photoreceptors lacking HK2 upregulate HK1 and appear to develop normally. Interestingly, we found that HK2-deficient photoreceptors are more susceptible to acute nutrient deprivation in the experimental retinal detachment model. Additionally, HK2 appears to be important for preserving photoreceptors during aging. We show that retinal glucose metabolism is largely unchanged after HK2 deletion, suggesting that the non-enzymatic role of HK2 is important for maintaining photoreceptor health. These results suggest that HK2 expression is critical for preserving photoreceptors during acute nutrient stress and aging. More specifically, p-AKT mediated translocation of HK2 to the mitochondrial surface may be critical for protecting photoreceptors from acute and chronic stress.

neuroscience↗

bioRxiv2019

Genetic correlation between background EEG oscillations and genetic generalized epilepsy

ObjectiveParoxysmal epileptiform abnormalities on electroencephalography (EEG) are the hallmark of epilepsies, but it is uncertain to what extent epilepsy and background EEG oscillations share neurobiological underpinnings. Here, we aimed to assess the genetic correlation between epilepsy and background EEG oscillations. MethodsConfounding factors, including the heterogeneous etiology of epilepsies and medication effects hamper studies on background brain activity in people with epilepsy. To overcome this limitation, we compared genetic data from a GWAS on epilepsy (n=12,803 people with epilepsy and 24,218 controls) with that from a GWAS on background EEG (n=8,425 subjects without epilepsy), in which background EEG oscillation power was quantified in four different frequency bands: alpha, beta, delta and theta. We replicated our findings in an independent epilepsy replication dataset (n=4851 people with epilepsy and 20,428 controls). To assess the genetic overlap between these phenotypes, we performed genetic correlation analyses using linkage disequilibrium score regression, polygenic risk scores and Mendelian randomization analyses. ResultsOur analyses show strong genetic correlations between genetic generalized epilepsy (GGE) with background EEG oscillations, primarily with the beta frequency band. Furthermore, we show that subjects with higher beta and theta polygenic risk scores have a significantly higher risk of having generalized epilepsy. Mendelian randomization analyses suggest a causal effect of GGE genetic liability on beta oscillations. SignificanceOur results point to shared biological mechanisms underlying background EEG oscillations and the susceptibility for GGE, opening avenues to investigate the clinical utility of background EEG oscillations in the diagnostic work-up of epilepsy.

genetics↗

bioRxiv2019

Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

Nuclear pore complexes (NPCs) are important for many processes beyond nucleocytoplasmic trafficking, including protein modification, chromatin remodeling, transcription, mRNA processing and mRNA export. The multi-faceted nature of NPCs and the slow turnover of their components has made it difficult to understand the role of basket nucleoporins (Nup153, Nup50 and Tpr) in these diverse processes. To address this question, we used an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins: Loss of individual nucleoporins caused distinct alteration in patterns of nucleocytoplasmic trafficking and gene expression. Importantly, Tpr elimination caused rapid and pronounced changes in transcriptomic profiles within two hours of auxin addition. These changes were dissimilar to shifts observed after loss of Nup153 or Nup50, but closely related to changes after depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, GANP association to NPCs was specifically disrupted upon TPR depletion. Together, our findings demonstrate a unique and pivotal role of Tpr in regulating gene expression through GANP- and/or NXF1-dependent mRNA nuclear export.

cell biology↗

bioRxiv2019

NMDA receptor in vasopressin 1b neurons is not required for short-term social memory, object memory or aggression.

The vasopressin 1b receptor (Avpr1b) plays an important role in social behaviors including aggression, social learning and memory. Genetic removal of Avpr1b from mouse models results in deficits in aggression and short-term social recognition in adults. Avpr1b gene expression is highly enriched in the pyramidal neurons of the hippocampal cornu ammonis 2 (CA2) region. Activity of the hippocampal CA2 has been shown to be required for normal short-term social recognition and aggressive behaviors. Vasopressin acts to enhance synaptic responses of CA2 neurons through a NMDA-receptor dependent mechanism. Genetic removal of the obligatory subunit of the NMDA receptor (Grin1) within distinct hippocampal regions impairs non-social learning and memory. However, the question of a direct role for NMDA receptor activity in Avpr1b neurons to modulate social behavior remains unclear. To answer this question, we first created a novel transgenic mouse line with Cre recombinase knocked into the Avpr1b coding region to genetically target Avpr1b neurons. We confirmed this line has dense Cre expression throughout the dorsal and ventral CA2 regions of the hippocampus, along with scattered expression within the caudate-putamen and olfactory bulb. Conditional removal of the NMDA receptor was achieved by crossing our line to an available floxed Grin1 line. The resulting mice were measured on a battery of social and memory behavioral tests. Surprisingly, we did not observe any differences between Avpr1b-Grin1 knockout mice and their wildtype siblings. We conclude that mice without typical NMDA receptor function in Avpr1b neurons can develop normal aggression as well as short-term social and object memory performance.\n\nSignificance StatementActivity of neurons that express vasopressin 1b receptor are essential for aggressive and social recognition behaviors. We created a novel transgenic mouse to allow selective targeting of vasopressin 1b neurons. Our studies indicate that NMDA receptor expression in vasopressin 1b neurons (including most CA2 neurons) are not required for development of the typical expression of aggression or recognition memory. Thus, CA2 neurons may have a unique way of incorporating novel stimuli into memory that deserves further investigation.

neuroscience↗

bioRxiv2019

Label-free single-cell microfluidics reveals antibiotic transport kinetics in Gram-negative bacteria

The double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to intracellular antibiotic accumulation. A quantitative understanding of antibiotic transport in these cells is crucial for drug development, but this has proved elusive due to the complexity of the problem and a dearth of suitable investigative techniques. Here we combine microfluidics and time-lapse auto-fluorescence microscopy to quantify antibiotic uptake label-free in hundreds of individual Escherichia coli cells. By manipulating the microenvironment, we showed that drug (ofloxacin) accumulation is higher in growing versus non-growing cells. Using genetic knockouts, we provide the first direct evidence that growth phase is more important for drug accumulation than the presence or absence of individual transport pathways. We use our experimental results to inform a mathematical model that predicts drug accumulation kinetics in subcellular compartments. These novel experimental and theoretical results pave the way for the rational design of new Gram-negative antibiotics.

biophysics↗

bioRxiv2019

Efficient RNA-mediated reprogramming of human somatic cells to naive pluripotency facilitated by tankyrase inhibition

In contrast to conventional human pluripotent stem cells (hPSC) that are related to post-implantation embryo stages, naive hPSC exhibit features of pre-implantation epiblast. Naive hPSC are established by resetting conventional hPSC, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that tankyrase inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSC can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene free naive hPSC offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSC.

developmental biology↗

bioRxiv2019

Increasing objective cardiometabolic burden associated with attenuations in the P3b event-related potential component in older adults

Cardiometabolic diseases and risk factors increase the risk of late-life cognitive impairment and dementia, and have also been associated with detrimental grey and white matter changes. However the functional brain changes associated with cardiometabolic health in late-life are unclear. We sought to characterise these functional changes by recording event-related potentials (ERPs) during a n-back working memory task (0, 1 and 2 back) in 85 adults (60% female) between 50 and 80 years of age. Due to a stratified recruitment approach, participants varied widely regarding cognitive function and cardiometabolic health. Standard and objective cut-offs for high blood glucose, waist to hip ratio (i.e. obesity), high blood cholesterol, and hypertension were employed to generate a summative score for cardiometabolic burden (none, one, or two or more above cut-off). Mixed effects modelling (covarying for age and gender) revealed no statistically significant associations between cardiometabolic burden and visual P1 and N1 component amplitudes. There was a significant effect for the P3b component: as cardiometabolic burden increased, P3b amplitude decreased. We show that cardiometabolic factors related to the development of cognitive impairment and dementia in late-life associate with functional brain activity, as recorded via ERPs. Findings have relevance for the monitoring of lifestyle interventions (typically targeting cardiometabolic factors) in ageing, as ERPs may provide a more sensitive measure of change than cognitive performance. Further, our results raise questions related to the findings of a broad range of ERP studies where the groups compared may differ in their cardiometabolic health status (not only in psychological symptomatology).

neuroscience↗

bioRxiv2019

Comparative analysis of diverse cell states establishes an epigenetic basis for inferring regulatory genes governing cell identity

Determining genes orchestrating cell differentiation in development and disease remains a fundamental goal of cell biology. This study establishes a genome-wide metric based on the gene-repressive tri-methylation of histone 3 lysine 27 (H3K27me3) across hundreds of diverse cell types to identify genetic regulators of cell differentiation. We introduce a computational method, TRIAGE, that uses discordance between gene-repressive tendency and expression to identify genetic drivers of cell identity. We apply TRIAGE to millions of genome-wide single-cell transcriptomes, diverse omics platforms, and eukaryotic cells and tissue types. Using a wide range of data, we validate TRIAGEs performance for identifying cell-type specific regulatory factors across diverse species including human, mouse, boar, bird, fish, and tunicate. Using CRISPR gene editing, we use TRIAGE to experimentally validate RNF220 as a regulator of Ciona cardiopharyngeal development and SIX3 as required for differentiation of endoderm in human pluripotent stem cells. A record of this papers Transparent Peer Review process is included in the Supplemental Information.

cell biology↗

bioRxiv2019

An innovative non-invasive technique for subcutaneous tumour measurements

In oncological drug development, animal studies continue to play a central role in which the volume of subcutaneous tumours is monitored to assess the efficacy of new drugs. Tumour volume is currently estimated by measuring length and width with callipers and then estimating the volume of the tumour as if it were a regular spheroid. However, this method is subjective, insufficiently traceable, and is subject to error in the accuracy of volume estimates as tumours frequently are irregular.\n\nThis paper explores the extent of inconsistencies in calliper measurements by conducting a statistical review of a large dataset consisting of 2,500 tumour volume measurements from 1,600 mice by multiple operators across 6 mouse strains and 20 tumour models. We also explore the impact of six different tumour morphologies on volume estimation and the detection of treatment effects using a computational tumour growth model. Finally, we propose an alternative method to callipers for estimating volume - BioVolume, a 3D scanning technique. BioVolume simultaneously captures both stereo RGB (Red, Green and Blue) images from different light sources and infrared thermal images of the tumour. It detects the tumour region automatically and estimates the tumour volume in under a second. BioVolume has been tested on a dataset of 297 scans from over 120 mice collected by four different operators.\n\nThis work demonstrates that it is possible to record tumour measurements in a rapid, minimally invasive, morphology-independent way, and with less human-bias compared to callipers, whilst also improving data traceability. Furthermore, the images collected by BioVolume may be useful, for example, as a source of biomarkers for animal welfare and secondary drug toxicity / efficacy.

cancer biology↗

bioRxiv2019

Methylomic profiles reveal sex-specific shifts in leukocyte composition associated with post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a debilitating mental disorder precipitated by trauma exposure. However, only some persons exposed to trauma develop PTSD. There are sex differences in risk; twice as many women as men, develop a lifetime diagnosis of PTSD. Methylomic profiles derived from peripheral blood are well-suited for investigating PTSD because DNA methylation (DNAm) encodes individual response to trauma and may play a key role in the immune dysregulation characteristic of PTSD pathophysiology. In the current study, we leveraged recent methodological advances to investigate sex-specific shifts in DNAm-based leukocyte composition that are associated with lifetime PTSD. We estimated leukocyte composition on a combined methylation array dataset (483 participants, [~]450k CpG sites) consisting of two civilian cohorts, the Detroit Neighborhood Health Study and Grady Trauma Project. Sex-stratified Mann-Whitney U test and two-way ANCOVA revealed that lifetime PTSD was associated with a small but significant elevation in monocyte proportions in males, but not in females (Holm-adjusted p-val < 0.05). No difference in monocyte proportions was observed between current and remitted PTSD cases in males, suggesting that this sex-specific peak shift reflects a long-standing trait of lifetime history of PTSD, rather than current state of PTSD. Associations with lifetime PTSD or PTSD status were not observed in any other leukocyte subtype and our finding in monocytes was confirmed using cell estimates based on a different deconvolution algorithm, suggesting that our sex-specific findings are robust across cell estimation approaches. Overall, our main finding of elevated monocyte proportions in males, but not in females with lifetime history of PTSD provides evidence for a sex-specific shift in peripheral blood leukocyte composition that is detectable in methylomic profiles and that reflects long-standing changes associated with PTSD diagnosis.

genomics↗

bioRxiv2019

Glycosylation of gigaxonin regulates intermediate filaments: Novel molecular insights into giant axonal neuropathy

Gigaxonin (also known as KLHL16) is an E3 ligase adaptor protein that promotes the ubiquitination and degradation of intermediate filament (IF) proteins. Mutations in human gigaxonin cause the fatal neurodegenerative disease giant axonal neuropathy (GAN), in which IF proteins accumulate and aggregate in axons throughout the nervous system, impairing neuronal function and viability. Despite this pathophysiological significance, the upstream regulation and downstream effects of normal and aberrant gigaxonin function remain incompletely understood. Here, we report that gigaxonin is modified by O-linked-beta-N-acetylglucosamine (O-GlcNAc), a prevalent form of intracellular glycosylation, in a nutrient- and growth factor-dependent manner. Mass spectrometry analyses of human gigaxonin revealed nine candidate sites of O-GlcNAcylation, two of which - serine 272 and threonine 277 - are required for its ability to mediate IF turnover in novel gigaxonin-deficient human cell models that we created. Taken together, these results suggest that nutrient-responsive gigaxonin O-GlcNAcylation forms a regulatory link between metabolism and IF proteostasis. Our work may have significant implications for understanding the non-genetic modifiers of GAN phenotypes and for the optimization of gene therapy for this disease.

cell biology↗