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Bennett, C.

Publications and source records attributed to Bennett, C..

At least 19 recordsLinked to original sources

Megakaryocytic proteomic signatures define myeloproliferative neoplasms

The classical Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal stem cell disorders of the bone marrow with significant niche dysregulation driven in part by abnormal megakaryocytes. The three classical MPNs: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), display established megakaryocytic morphological differences which aid in their diagnostic classification. The paucity of megakaryocytes in the bone marrow aspirate has previously limited a proteomic analysis of megakaryocytes in clinical samples. We aimed to undertake an unbiased proteomic analysis of in situ megakaryocytes from the bone marrow of patients with PV, ET, and PMF. Using a novel, deep visual proteomics strategy, we dissected and analyzed megakaryocytes from 60 MPN patients and 20 controls (patients with limited stage non-Hodgkin lymphoma who had uninvolved bone marrow). We reveal that megakaryocyte proteomics from MPN patients are distinct to those from control patients, and further, that the megakaryocytes from the three MPNs have unique, disease-specific signatures. The disease-specific megakaryocyte proteome highlights novel pathways of disease.

cancer biology↗

Task Engagement Gates Interareal Communication Geometry in the Mouse Thalamocortical-Midbrain Visual Circuit

Visual processing unfolds across hierarchically organized brain circuits. Existing theories largely explain changes in population geometry through local shifts in gain, firing-rate statistics, or recurrent dynamics, yet do not account for how interareal coordination interacts with local population geometry to constrain downstream population states. We used task engagement, compared to a passive condition, to probe this coordination in Neuropixels recordings spanning the mouse visual thalamocortical-midbrain circuit. Engagement reduced network activity, response participation, and dimensionality across the hierarchy. To account for this circuit-level organization, we developed a theoretical framework in which afferent population geometry interacts with local recurrent dynamics to constrain the accessible dynamics of downstream populations. Across the thalamocortical stages, population-wide afferent statistics predicted downstream activity and dimensionality. At the cortex--midbrain interface, engagement instead reorganized interareal communication geometry. Together, these results identify interareal input geometry as a key constraint on neural population dynamics and uncover a general principle by which behavioral engagement constrains neural state spaces across distributed visual circuits.

neuroscience↗

Regulation of Human Erythroferrone Expression

Erythroferrone (ERFE) is an erythroblast-secreted hormone that suppresses hepatic hepcidin expression to increase iron availability for erythropoiesis, ensuring recovery from anaemia. ERFE excess drives iron overload in disorders of ineffective erythropoiesis. Despite its pivotal role in systemic iron homeostasis and diseases of erythropoiesis, ERFEs molecular regulation has remained undefined. Here, we applied a genomic approach to characterise the molecular mechanisms governing ERFE expression. Using the HUDEP-2 human erythroid progenitor model, integrative ATAC-seq, CUT&RUN and micro capture-C analysis we identified a stage-specific accessible chromatin region within the ERFE 3 UTR that interacts with the promotor. We also identified enhancer-associated chromatin marks including H3K4me1 and H3K27ac in this region, and demonstrate that this cis-regulatory element is bound by key erythroid transcription factors KLF1, GATA1, TAL1 and STAT5. Functional dissection using CRISPR-Cas9-mediated deletion of the central 3 UTR enhancer element led to marked reduction in ERFE mRNA expression, and we show a corresponding reduction in nascent mRNA, confirming a key role for this region in transcriptional regulation. We define the transcriptional regulatory mechanism by which maturing human erythroblasts activate ERFE, the endocrine signal that coordinates erythropoietic demand with systemic iron mobilisation.

molecular biology↗

An amplicon panel for high-throughput and low-cost genotyping of Yesso scallop Mizuhopecten yessoensis

The Yesso scallop Mizuhopecten yessoensis was imported from Japan to western Canada in the late 1980s to establish an economically viable scallop aquaculture industry. Since this time, the industry in Canada has operated with existing genetic diversity within the broodstock, which is considerably limited relative to wild populations. The sector has not been able to realise its full potential in part due to idiopathic hatchery failures and farm stock collapses due to disease outbreaks associated with the intracellular bacterial pathogen Francisella halioticida. To support Yesso scallop production and breeding, here we generate a low-density, genotyping-by-sequencing amplicon panel using single nucleotide polymorphism (SNP) markers that are evenly spaced across the M. yessoensis genome and that show high heterozygosity in Canada and Japan. The panel can also exploit the high genetic polymorphism of the M. yessoensis genome, with de novo SNP calling identifying over 2,500 high quality SNPs within the 579 sequenced amplicons. We demonstrate the utility and versatility of this new genotyping tool for breeding applications including parentage assignment, low density family-based genome-wide association study, trait heritability evaluation to determine potential for genomic selection, and species differentiation (against the weathervane scallop Patinopecten caurinus). We did not find any genomic regions significantly associated with F. halioticida resistance but did identify potential for genomic selection. We could separate the two species based on genotypes, and we did not see evidence of a past M. yessoensis x P. caurinus hybridization event within the M. yessoensis breeding population at Vancouver Island University. This low-cost genotyping panel is expected to accelerate selective breeding improvements for M. yessoensis in Canada and elsewhere.

genomics↗

Map of spiking activity underlying change detection in the mouse visual system

Visual behavior requires coordinated activity across hierarchically organized brain circuits. Understanding this complexity demands datasets that are both large-scale (sampling many areas) and dense (recording many neurons in each area). Here we present a database of spiking activity across the mouse visual system--including thalamus, cortex, and midbrain--while mice perform an image change detection task. Using Neuropixels probes, we record from >75,000 high-quality units in 54 mice, mapping area-, cortical layer-, and cell type-specific coding of sensory and motor information. Modulation by task-engagement increased across the thalamocortical hierarchy but was strongest in the midbrain. Novel images modulated cortical (but not thalamic) responses through delayed recurrent activity. Population decoding and optogenetics identified a critical decision window for change detection and revealed that mice use an adaptation-based rather than image-comparison strategy. This comprehensive resource provides a valuable substrate for understanding sensorimotor computations in neural networks.

neuroscience↗

Host iron deficiency protects against Plasmodium infection and drives parasite molecular reprofiling.

Iron deficiency, anemia and Plasmodium infection represent significant global health challenges with overlapping geographical distributions, particularly affecting pregnant women in Africa, yet the mechanisms underlying their interactions remain poorly understood. We employed a multilayered approach combining clinical data from Malawian pregnant women (n=711) in the REVAMP trial, a genetic mouse model (Tmprss6-knockout), and in vitro P. falciparum cultures to clarify associations between iron status and malaria susceptibility. Iron deficiency was associated with 50% reduced P. falciparum parasitemia in pregnant women (95% CI [30%-64%], p<0.0001), while iron-deficient mice exhibited significantly improved survival against P. berghei (median 15.5 days vs. 7.0 days for WT mice) and protection from cerebral malaria (83% vs 17% survival). Iron chelation induced substantial transcriptomic and proteomic changes in cultured parasites, affecting host cell invasion and nutrient acquisition processes. Importantly, intravenous iron supplementation did not increase subsequent parasitemia when coupled with malaria prevention. These findings demonstrate that iron deficiency protects against Plasmodium infection and support WHO recommendations for iron supplementation in malaria-endemic regions when combined with adequate malaria prevention strategies.

cell biology↗

Structural basis of respiratory complexes adaptation to cold temperatures

In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain (1, 2). Yet, the structural basis of respiratory complex adaptation to cold remains elusive. Herein we combined thermoregulatory physiology and cryo-EM to study endogenous respiratory supercomplexes exposed to different temperatures. A cold-induced conformation of CI:III2 (termed type 2) was identified with a [~]25{degrees} rotation of CIII2 around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting different catalytic states which favor electron transfer. Large-scale supercomplex simulations in lipid membrane reveal how unique lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations and biochemical analyses unveil the mechanisms and dynamics of respiratory adaptation at the structural and energetic level.

cell biology↗

Spatial analysis of Hofbauer cell transcriptome, distribution and morphology in placentas exposed to Plasmodium falciparum

Placental infection remains a significant health burden for mothers and their babies in low-income countries, especially in sub-Saharan Africa, where malaria transmission is intense. An increase in inflammatory biomarkers and poor vascularisation are characteristics of placentas infected with malaria. Hofbauer cells (HBCs) - placental villous macrophages of fetal origin - are one of the most abundant immune cells in the placenta. HBCs are thought to have roles in angiogenic processes and have been linked with the pathophysiology of several infections and inflammatory conditions during pregnancy, including malaria (caused by Plasmodium falciparum). However, there is limited in situ data on the transcriptional, proteomic or morphologic profile of these cells either during or following clearance of P. falciparum infection. We leveraged placental samples prospectively collected at delivery from 610 Malawian women enduring a high burden of malaria and other infections and nutritional deficiencies. We profiled placentas through spatial transcriptomic and proteomic platforms to discern in situ HBC features that could distinguish placentas with or without evidence of past malaria. In this cohort, past placental infection was common and was associated with lower birth weight babies (adjusted effect [95% confidence interval], -80.9 [-165.9, -3.7] g, P= 0.040). However, at term, HBC numbers, abundance, and transcriptional profiles from placentas with evidence of past infection were similar to those of placentas without malaria. HBCs may recover post-infection back to a basal state or may be replaced in the tissue over the course of pregnancy. Placentas with evidence of past malaria did show evidence of reduced fetal vessel development (mean area difference: -22.8% [-37.6, -7.9], P=0.003). Reduced vascular development following infection early in pregnancy may reflect disturbances to the normal vasculogenic and angiogenic processes, of which HBCs are an integral part.

immunology↗

SHIELD: Skull-shaped hemispheric implants enabling large-scale-electrophysiology datasets in the mouse brain

To understand the neural basis of behavior, it is essential to measure spiking dynamics across many interacting brain regions. While new technologies, such as Neuropixels probes, facilitate multi-regional recordings, significant surgical and procedural hurdles remain for these experiments to achieve their full potential. Here, we describe a novel 3D-printed cranial-replacement implant (SHIELD) enabling electrophysiological recordings from distributed areas of the mouse brain. This skull-shaped implant is designed with customizable insertion holes, allowing dozens of cortical and subcortical structures to be recorded in a single mouse using repeated multi-probe insertions over many days. We demonstrate the procedures high success rate, biocompatibility, lack of adverse effects on behavior, and compatibility with imaging and optogenetics. To showcase the scientific utility of the SHIELD implant, we use multi-probe recordings to reveal novel insights into how alpha rhythms organize spiking activity across visual and sensorimotor networks. Overall, this method enables powerful large-scale electrophysiological measurements for the study of distributed brain computation.

neuroscience↗

Coordinated changes in a cortical circuit sculpt effects of novelty on neural dynamics

Recent studies have found dramatic cell-type specific responses to stimulus novelty, highlighting the importance of analyzing the cortical circuitry at the cell-type specific level of granularity to understand brain function. Although initial work classified and characterized activity for each cell type, the specific alterations in cortical circuitry--particularly when multiple novelty effects interact--remain unclear. To address this gap, we employed a large-scale public dataset of electrophysiological recordings in the visual cortex of awake, behaving mice using Neuropixels probes and designed population network models to investigate the observed changes in neural dynamics in response to a combination of distinct forms of novelty. The model parameters were rigorously constrained by publicly available structural datasets, including multi-patch synaptic physiology and electron microscopy data. Our systematic optimization approach identified tens of thousands of model parameter sets that replicate the observed neural activity. Analysis of these solutions revealed generally weaker connections under novel stimuli, as well as a shift in the balance e between SST and VIP populations. Along with this, PV and SST populations experienced overall more excitatory influences compared to excitatory and VIP populations. Our results also highlight the role of VIP neurons in multiple aspects of visual stimulus processing and altering gain and saturation dynamics under novel conditions. In sum, our findings provide a systematic characterization of how the cortical circuit adapts to stimulus novelty by combining multiple rich public datasets.

neuroscience↗

Serum iron and hepatic transferrin receptor expression are circadian-regulated

Serum iron has long been thought to exhibit diurnal variation and is subsequently considered an unreliable biomarker of systemic iron status. Circadian regulation (endogenous [~]24-hour periodic oscillation of a biologic function) governs many critical physiologic processes. It is unknown whether serum iron levels are regulated by circadian machinery; likewise, the circadian nature of key players of iron homeostasis is unstudied. Here we show that serum iron, hepatic transferrin receptor (TFR1) gene (Tfrc) expression and erythropoietic activity exhibit circadian rhythms. Daily oscillations of serum iron, hepatic Tfrc expression and erythropoietic activity are maintained in mice housed in constant darkness, where oscillation reflects an endogenous circadian period. Oscillations of serum iron, hepatic Tfrc and erythropoietic activity were ablated when circadian machinery was disrupted in Bmal1 knockout mice. Interestingly, we find that circadian oscillations of erythropoietic activity and hepatic Tfrc expression are maintained in opposing phase, likely allowing for optimised usage and storage of serum iron whilst maintaining adequate serum levels. This study provides the first confirmatory evidence that serum iron is circadian regulated and uncovers liver-specific circadian regulation of TFR1, a major player in cellular iron uptake.

molecular biology↗

Staphylococcus aureus biofilm secreted factors cause mucosal damage, mast cell infiltration and goblet cell hyperplasia in a rat rhinosinusitis model

Chronic Rhinosinusitis (CRS) is an inflammatory condition of the paranasal sinus mucosa. Despite being a common health issue, the exact cause of CRS is yet to be understood. However, research suggests that Staphylococcus aureus, particularly in the biofilm form, drives the disease. This study aimed to investigate the impact of long-term exposure to secreted factors of Staphylococcus aureus biofilm (SABSF), harvested from clinical isolates of non-CRS carriers and CRS patients, on the nasal mucosa in a rat model. Wistar rats were randomised (n=5/group) to receive daily intranasal instillations of 40 L (200 g/L) SABSF for 28 days or vehicle control with S. aureus isolated from the sinuses of a non-CRS carrier, a type 2 endotype CRS with nasal polyps (CRSwNP) patient, and a non-type 2 endotype CRS without nasal polyps (CRSsNP) patient. The sinonasal samples of the rats were then analysed through histopathology and transcriptome profiling. The results showed that all three intervention groups displayed significant lymphocytic infiltration (p[&le;]0.05). However, only the SABSF collected from the CRSwNP patient caused significant mucosal damage, mast cell infiltration, and goblet cell hyperplasia compared to the control. The transcriptomics results indicated that SABSF significantly enriched multiple inflammatory pathways and showed distinct transcriptional expression differences between the control group and the SABSF collected from CRS patients (p[&le;]0.05). Additionally, the SABSF challenges induced the expression of IgA and IgG but not IgE. In conclusion, this in vivo study indicates that long-term exposure to SABSF leads to an inflammatory response in the nasal mucosa with increased severity for S. aureus isolated from a CRSwNP patient. The findings of this study shed light on the role of S. aureus in the development of CRS and could inform future research and treatment efforts.

immunology↗

Stimulus novelty uncovers coding diversity in visual cortical circuits

Detecting novel stimuli in the environment is critical for learning and survival, yet the neural basis of novelty processing is not understood. To characterize cell type-specific novelty processing, we surveyed the activity of [~]15,000 excitatory and inhibitory neurons in mice performing a visual task with novel and familiar stimuli. Clustering revealed a dozen functional neuron types defined by experience-dependent encoding. Vasoactive-intestinal-peptide (Vip) expressing inhibitory neurons were diverse, encoding novel stimuli, omissions of familiar stimuli, or behavioral features. Distinct Somatostatin (Sst) expressing inhibitory neurons encoded either familiar or novel stimuli. Subsets of excitatory neurons co-clustered with specific Vip or Sst subpopulations, while Sst and Vip inhibitory clusters were non-overlapping. This study establishes that novelty processing is mediated by diverse functional neuron types in the visual cortex.

neuroscience↗

Maternal dietary deficiencies in folates or choline during pregnancy and lactation worsen stroke outcome in 3-month-old male and female mouse offspring

Maternal one-carbon (1C) metabolism plays an important role in early life programming. There is a well-established connection between the fetal environment and the health status of the offspring. However, there is a knowledge gap on how maternal nutrition impacts stroke outcome in offspring. The aim of our study was to investigate the role of maternal dietary deficiencies in folic acid or choline on stroke outcome in 3-month-old offspring. Adult female mice were fed a folic acid deficient diet (FADD), choline deficient diet (ChDD), or control diet (CD) prior to pregnancy. They were continued on diets during pregnancy and lactation. Male and female offspring were weaned onto a CD and at 2 months of age were subject to ischemic stroke within the sensorimotor cortex via photothrombosis damage. At 3-months-of-age, motor function was measured in offspring and tissue was collected for analysis. Mothers maintained on either a FADD or ChDD had reduced levels of S-adenosylmethionine in liver and S-adenosylhomocysteine in plasma. After ischemic stroke, motor function was impaired in 3-month-old offspring from deficient mothers compared to CD animals. In brain tissue, there was no difference in ischemic damage volume. When protein levels were assessed in brain tissue, there were lower levels of neurodegeneration in males compared to females and betaine levels were reduced in offspring from ChDD mothers. Our results demonstrate that a deficient maternal diet during critical timepoints in neurodevelopment results in worse stroke outcomes. This study emphasizes the importance of maternal diet and the impact it can have on offspring health.

neuroscience↗

Novavax NVX-COV2373 triggers potent neutralization of Omicron sub-lineages

The SARS-CoV-2 Omicron (B.1.1.529) Variant of Concern (VOC) and its sub-lineages (including BA.2, BA.4/5, BA.2.12.1) contain spike mutations that confer high level resistance to neutralizing antibodies. The NVX-CoV2373 vaccine, a protein nanoparticle vaccine, has value in countries with constrained cold-chain requirements. Here we report neutralizing titers following two or three doses of NVX-CoV2373. We show that after two doses, Omicron sub-lineages BA.1 and BA.4 were resistant to neutralization by 72% (21/29) and 59% (17/29) of samples. However, after a third dose of NVX-CoV2373, we observed high titers against Omicron BA.1 (GMT: 1,197) and BA.4 (GMT: 582), with responses similar in magnitude to those triggered by three doses of an mRNA vaccine. These data are of particular relevance as BA.4 is emerging to become the dominant strain in many locations, and highlight the potential utility of the NVX-CoV2373 vaccine as a booster in resource-limited environments.

immunology↗

Iron homeostasis governs erythroid phenotype in Polycythemia Vera

Polycythemia Vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients hematocrit and hemoglobin concentration, placing them at risk of life-threatening thrombotic events. Our GWAS of 440 PV cases and 403,351 controls utilising UK Biobank data found that SNPs in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed over-representation of homozygous HFE mutations in PV patients. HFE influences expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of PV mouse models, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Further, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signalling via GP130 coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.

cancer biology↗

A survey of neurophysiological differentiation across mouse visual brain areas and timescales

Neurophysiological differentiation (ND), a metric that quantifies the number of distinct activity states that the brain or its part visits over a period of time, has been used as a correlate of meaningfulness or subjective perception of visual stimuli. ND has largely been studied in non-invasive human whole-brain recordings where spatial resolution is limited. However, it is likely that perception is supported by discrete populations of spiking neurons rather than the whole brain. Therefore, in this study, we use Neuropixels recordings from the mouse brain to characterize the ND metric within neural populations recorded at single-cell resolution in localized regions. Using the spiking activity of thousands of simultaneously recorded neurons spanning 6 visual cortical areas as well as the visual thalamus, we show that the ND of stimulus-evoked activity of the entire visual cortex is higher for naturalistic stimuli relative to artificial ones. This finding holds in most individual areas throughout the visual hierarchy as well. For animals performing an image change detection task, ND of the entire visual cortex (though not individual areas) is higher for successful detection compared to failed trials, consistent with the assumed perception of the stimulus. Analysis of spiking activity allows us to characterize the ND metric across a wide range of timescales from 10s of milliseconds to a few seconds. This analysis reveals that although ND of activity of single neurons is often maximized at an optimal timescale around 100 ms, the optimum shifts to under 5 ms for ND of neuronal ensembles. Finally, we find that the ND of activations in convolutional neural networks (CNNs) trained on an image classification task shows distinct trends relative to the mouse visual system: ND is often higher for less naturalistic stimuli and varies by orders of magnitude across the hierarchy, compared to modest variation in the mouse brain. Together, these results suggest that ND computed on cellular-level neural recordings can be a useful tool highlighting cell populations that may be involved in subjective perception. SummaryAdvances in our understanding on neural coding has revealed that information about visual stimuli is represented across several brain regions. However, availability of information does not imply that it is necessarily utilized by the brain, much less that it is subjectively perceived. Since percepts originate in neural activity, distinct percepts must be associated with distinct states of neural activity, at least within the brain region that supports the percepts. Thus, one approach developed in this direction is to quantify the number of distinct states that the activity of the brain goes through, called neurophysiological differentiation (ND). ND of the entire brain has been shown to reflect subjective reports of visual stimulus meaningfulness. But what specific subpopulations within the brain could be supporting conscious perception, and what is the correct timescale on which states should be quantified? In this study, we analyze ND of spiking neural activity in the mouse visual cortex recorded using Neuropixels probes, allowing us to characterize the ND metric across a wide range of timescales all the way down from 5 ms to a few seconds. It also allows us to understand the ND of neural activity of different ensembles of neurons, from individual thalamic or cortical ensembles to those spanning across multiple visual areas in the mouse brain.

neuroscience↗

Transgenic Drosophila lines for LexA-dependent gene and growth regulation

Conditional expression of short hairpin RNAs (shRNAs) with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell-cell communication in vivo benefits from simultaneous use of two independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-shRNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based shRNA expression vector to achieve shRNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.

genetics↗