Biology subjects

Thomas, G.

Publications and source records attributed to Thomas, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Micro-Scale Control of Oligodendrocyte Morphology and Myelination by the Intellectual Disability-Linked Protein Acyltransferase ZDHHC9

Mutations in the X-linked ZDHHC9 gene cause cognitive deficits in humans, with a subset of patients suffering from epilepsy. X-linked intellectual disability (XLID) is often ascribed to neuronal deficits, but here we report that expression of human and mouse ZDHHC9 orthologs is far higher in myelinating oligodendrocytes (OLs) than in other CNS cell types. ZDHHC9 codes for a protein acyltransferase (PAT), and we found that ZDHHC9 is the most highly expressed PAT in OLs. Wild type ZDHHC9 localizes to Golgi outposts in OL processes, but other PATs and XLID mutant forms of ZDHHC9 are restricted to OL cell bodies. Using genetic tools for OL progenitor fate tracing and sparse cell labeling, we show that mice lacking Zdhhc9 have grossly normal OL development but display extensive morphological and structural myelin abnormalities. Consistent with the hypothesis that these deficits are OL-autonomous, they are broadly phenocopied by acute Zdhhc9 knockdown in cultured conditions. Finally, we found that ZDHHC9 palmitoylates Myelin Basic Protein (MBP) in heterologous cells, and that palmitoylation of MBP is impaired in the Zdhhc9 knockout brain. Our findings provide critical insights into the mechanisms of ZDHHC9-associated XLID and shed new light on the palmitoylation-dependent control of myelination.

neuroscience↗

bioRxiv2023

Biofilm formation in Zymoseptoria tritici

Zymoseptoria tritici is an economically damaging fungal pathogen of wheat which is able to survive for long periods on the leaf surface. In this environment, the fungus is exposed to many stresses, including fluctuations in temperature, UV radiation, drying, and foliar fungicide applications. We observed biofilm-like cell aggregations on the surface of wheat leaves infected with Z. tritici in both compatible and non-compatible interactions. A literature search revealed few examples of plant pathogenic fungi forming biofilms, but demonstrated that such biofilms have a number of key characteristics, which are shared with other biofilmforming microbes. These include adhesion of cells to the substrate, production of extracellular matrix, altered gene expression and increased tolerance to various stresses. We used a combination of microscopy, qRT-PCR and stress tolerance assays to determine whether putative Z. tritici biofilms possessed these diagnostic characteristics. We show that Z. tritici biofilms resemble in form and function those formed by other filamentous and dimorphic fungi, producing extra-cellular matrix in which cells become embedded, creating layers of hyphal bundles and blastospores. These biofilms show increased tolerance to drying and high temperature. There is also isolate-dependent resistance to reactive oxygen stress and the fungicide carboxin. Taken together, these findings demonstrate that Z. tritici is indeed able to produce genuine biofilms.

microbiology↗

bioRxiv2023

High-resolution molecular atlas of a lung tumor in 3D

Cells live and interact in three-dimensional (3D) cellular neighborhoods. However, histology and spatial omics methods mostly focus on 2D tissue sections. Here we present a 3D spatial atlas of a routine clinical sample, an aggressive human lung carcinoma, by combining in situ quantification of 960 cancer-related genes across [~]340,000 cells with measurements of tissue-mechanical components. 3D cellular neighborhoods subdivided the tumor microenvironment into tumor, stromal, and immune multicellular niches. Interestingly, pseudotime analysis suggested that pro-invasive epithelial-to-mesenchymal transition (EMT), detected in stroma-infiltrating tumor cells, already occurred in one region at the tumor surface. There, myofibroblasts and macrophages specifically co-localized with pre-invasive tumor cells and their multicellular molecular signature identified patients with shorter survival. Moreover, cytotoxic T-cells did not infiltrate this niche but colocalized with inhibitory dendritic and regulatory T cells. Importantly, systematic scoring of cell-cell interactions in 3D neighborhoods highlighted niche-specific signaling networks accompanying tumor invasion and immune escape. Compared to 2D, 3D neighborhoods improved the characterization of immune niches by identifying dendritic niches, capturing the 3D extension of T-cell niches and boosting the quantification of niche-specific cell-cell interactions, including druggable immune checkpoints. We believe that 3D communication analyses can improve the design of clinical studies investigating personalized, combination immuno-oncology therapies.

systems biology↗

bioRxiv2023

SARS-CoV-2 Spike Protein Reduces Burst Activities in Neurons Measured by Micro-Electrode Arrays

SARS-CoV-2 caused a large-scale global pandemic between 2020 and 2022. Despite efforts to understand its biology and mechanisms of pathogenicity, the viral impact on the neurological systems remains unclear. The main goal of this study was to quantify the neurological phenotypes induced by SARS-CoV-2 spike protein in neurons, measured by in-vitro multi-well micro-electrode arrays (MEAs). We extracted the whole-brain neurons from the newborn P1 mice and plated them on multi-well micro-electrode arrays and administered purified recombinant spike proteins (S1 and S2 subunits respectively) from the SARS-CoV-2 virus. The signals from the MEAs were transmitted from an amplifier to a high-performance computer for recording and analysis. We used an in-house developed algorithm to quantify neuronal phenotypes. Among all the phenotypic features analyzed, we discovered that the S1 protein of SARS-CoV-2 decreased the mean burst numbers observed on each electrode; This effect was not observed for the spike 2 protein (S2) and could be rescued by an anti-S1 antibody. Finally, our data strongly suggest that the receptor binding domain (RBD) of S1 is responsible for the reduction of burst activities in neurons. Overall, our results strongly indicate that spike proteins may play an important role in altering neuronal phenotypes, specifically the burst patterns, when neurons are exposed during early development.

microbiology↗

bioRxiv2023

miR-7 controls glutamatergic transmission and neuronal connectivity in a Cdr1as-dependent manner

The circular RNA (circRNA) Cdr1as is conserved across mammals and highly expressed in neurons, where it directly interacts with microRNA miR-7. However, the biological function of this interaction is unknown. Here, using primary forebrain murine neurons, we demonstrate that stimulating neurons by sustained depolarization rapidly induced two-fold transcriptional up-regulation of Cdr1as and strong post-transcriptional stabilization of miR-7. Cdr1as loss caused doubling of glutamate release from stimulated synapses and increased frequency and duration of local neuronal bursts. Moreover, periodicity of neuronal networks was increased and synchronicity was impaired. Strikingly, these effects were reverted by sustained expression of miR-7 which also cleared Cdr1as molecules from neuronal projections. Consistently, without Cdr1as, transcriptomic changes caused by miR-7 overexpression were stronger (including miR-7-targets down-regulation) and enriched in secretion/synaptic plasticity pathways. Altogether, our results suggest that in forebrain neurons Cdr1as buffers miR-7 activity to control glutamatergic excitatory transmission and neuronal connectivity important for long-lasting synaptic adaptations.

molecular biology↗

bioRxiv2023

Ethanolaminephosphate cytidyltransferase is essential for survival, lipid homeostasis and stress tolerance in Leishmania major

Glycerophospholipids including phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are vital components of biological membranes. Trypanosomatid parasites of the genus Leishmania can acquire PE and PC via de novo synthesis and the uptake/remodeling of host lipids. In this study, we investigated the ethanolaminephosphate cytidyltransferase (EPCT) in Leishmania major, which is the causative agent for cutaneous leishmaniasis. EPCT is a key enzyme in the ethanolamine branch of the Kennedy pathway which is responsible for the de novo synthesis of PE. Our results demonstrate that L. major EPCT is a cytosolic protein capable of catalyzing the formation of CDP-ethanolamine from ethanolamine-phosphate and cytidine triphosphate. Genetic manipulation experiments indicate that EPCT is essential in both the promastigote and amastigote stages of L. major as the chromosomal null mutants cannot survive without the episomal expression of EPCT. This differs from our previous findings on the choline branch of the Kennedy pathway (responsible for PC synthesis) which is required only in promastigotes but not amastigotes. While episomal EPCT expression does not affect promastigote proliferation under normal conditions, it leads to reduced production of ethanolamine plasmalogen or plasmenylethanolamine, the dominant PE subtype in Leishmania. In addition, parasites with epsiomal EPCT exhibit heightened sensitivity to acidic pH and starvation stress, and significant reduction in virulence. In summary, our investigation demonstrates that proper regulation of EPCT expression is crucial for PE synthesis, stress response, and survival of Leishmania parasites throughout their life cycle. AUTHOR SUMMARYIn nature, Leishmania parasites alternate between fast replicating, extracellular promastigotes in sand fly gut and slow growing, intracellular amastigotes in macrophages. Previous studies suggest that promastigotes acquire most of their lipids via de novo synthesis whereas amastigotes rely on the uptake and remodeling of host lipids. Here we investigated the function of ethanolaminephosphate cytidyltransferase (EPCT) which catalyzes a key step in the de novo synthesis of phosphatidylethanolamine (PE) in Leishmania major. Results showed that EPCT is indispensable for both promastigotes and amastigotes, indicating that de novo PE synthesis is still needed at certain capacity for the intracellular form of Leishmania parasites. In addition, elevated EPCT expression alters overall PE synthesis and compromises parasites tolerance to adverse conditions and is deleterious to the growth of intracellular amastigotes. These findings provide new insight into how Leishmania acquire essential phospholipids and how disturbance of lipid metabolism can impact parasite fitness.

microbiology↗

bioRxiv2022

PhyloAcc-GT: A Bayesian method for inferringpatterns of substitution rate shifts and associations with binary traits under gene tree discordance

An important goal of evolutionary genomics is to identify genomic regions whose substitution rates differ among lineages. For example, genomic regions experiencing accelerated molecular evolution in some lineages may provide insight into links between genotype to phenotype. Several comparative genomics methods have been developed to identify genomic accelerations between species, including a Bayesian method called PhyloAcc, which models shifts in substitution rate in multiple target lineages on a phylogeny. However, few methods consider the possibility of discordance between the trees of individual loci and the species tree due to incomplete lineage sorting, which might cause false positives. Here we present PhyloAcc-GT, which extends PhyloAcc by modeling gene tree heterogeneity to detect rate shifts across genomic regions. Given a species tree, we adopt the multispecies coalescent model as the prior distribution of gene trees, use Markov chain Monte Carlo (MCMC) for inference, and design novel MCMC moves to sample gene trees efficiently. Through extensive simulations, we show that PhyloAcc-GT outperforms PhyloAcc and other methods in identifying target-lineage-specific accelerations and detecting complex patterns of rate shifts, and is robust to specification of population size parameters. We apply PhyloAcc-GT to two examples of convergent evolution: flightlessness in ratites and marine mammal adaptations. PhyloAcc-GT is usually more conservative than PhyloAcc in calling convergent rate shifts because it identifies more accelerations on ancestral than on terminal branches. In summary, PhyloAcc-GT is a useful tool to identify shifts in substitution rate associated with specific target lineages while accounting for incomplete lineage sorting.

evolutionary biology↗

bioRxiv2022

Brain-wide oscillatory network selectively encodes aggression

Aggression is a psychological state characterized by intent to harm oneself or others, and often manifests as hostile, harmful, or violent action. Social aggression can aid an organism in securing access to resources1,2, or it can impair group function and survival in behavioral pathology3-5. Since many brain regions contribute to multiple social behaviors6-8, expanded knowledge of how the brain distinguishes between social states would enable the development of interventions that suppress violent action, while leaving other social behaviors intact. Here we showed that a murine aggressive internal state is encoded by a widespread network. This network is organized by prominent and synchronized theta (4-11 Hz) and beta (14-30 Hz) oscillations that relay through the medial prefrontal cortex, and couples to widespread cellular firing. Strikingly, network activity during social isolation was found to encode the trait aggressiveness of mice, and causal cellular manipulations known to impact aggression were found to modulate the networks activity. We next established that this network mediates aggression using closed-loop stimulation of medial prefrontal cortex and causal mediation analysis. Finally, we deployed Long-term integration of Circuits using connexins (LinCx) to selectively edit a key circuit within the network from the medial prefrontal cortex to nucleus accumbens. Editing this circuit chronically suppressed violent action while leaving non-aggressive social behavior intact.

neuroscience↗

bioRxiv2022

Sexual dimorphism versus biomechanical loading in occipital bone morphological variation

We explore the contribution of biological sex and biomechanical activity from subsistence to occipital bone variation. Previous studies have used occipital bone traits to determine biological sex and identify ancestry to differing degrees of success. Biomechanical stress from variation in subsistence and gender-based divisions of labor could perhaps explain some of the noise in the signal for these grouping variables. To explore this possibility, we used metric (foramen magnum length and breadth, external occipital protuberance depth, lambda-inion length, bicondylar breadth) and nonmetric traits (general occipital form, presence of a nuchal crest, and nuchal line count). We collected original data and mined published data for our analysis using skeletal collections of Native American hunter-gatherers and horticulturalists, a historic military site, and contemporary study collections. We find that the foramen magnum area exhibits sexual dimorphism and is not influenced by subsistence but the accuracy of sex estimation is only 71%, suggesting the chance of being correct at slightly more than two-thirds. All traits exhibited sex-based variation but only bicondylar breadth and lambda-inion exhibited subsistence-based variation. Given the limited amount of variance explained by either sex or sex and subsistence, biomechanics may still play a role but not from the influence of subsistence practices. Additional data from a wider array of skeletal samples, perhaps with known occupation, is warranted if we are to understand how occipital variation is shaped. PRACTITIONER POINTSO_LIThe study examines the role of biological sex and biomechanical stress in shaping occipital morphology. C_LIO_LIWe introduce an area variable to describe the foramen magnum and apply ectocranial metric and nonmetric states for the first time to sex estimation. C_LIO_LIWe share methods and R code for cleaning data, testing assumptions, and analysis of research questions. C_LI

physiology↗

bioRxiv2021

Interleukin-1 is overexpressed in injured muscles following spinal cord injury and promotes neurogenic heterotopic ossification

Neurogenic heterotopic ossifications (NHOs) form in periarticular muscles following severe spinal cord (SCI) and traumatic brain injuries. The pathogenesis of NHO is poorly understood with no effective preventive treatment. The only curative treatment remains surgical resection of pathological NHOs. In a mouse model of SCI-induced NHO that involves a transection of the spinal cord combined with a muscle injury, a differential gene expression analysis revealed that genes involved in inflammation such as interleukin-1{beta} (IL-1{beta}) were overexpressed in muscles developing NHO. Using mice knocked-out for the gene encoding IL-1 receptor (IL1R1) and neutralizing antibodies for IL-1 and IL-1{beta}, we show that IL-1 signaling contributes to NHO development following SCI in mice. Interestingly, other proteins involved in inflammation that were also overexpressed in muscles developing NHO, such as colony-stimulating factor-1, tumor necrosis factor or C-C chemokine ligand-2 did not promote NHO development. Finally using NHO biopsies from SCI and TBI patients, we show that IL-1{beta} is expressed by CD68+ macrophages. IL-1 and IL-1{beta} produced by activated human monocytes promote calcium mineralization of fibro-adipogenic progenitors isolated from muscles surrounding NHOs. Altogether these data suggest that interleukin-1 promotes NHO development in both humans and mice.

physiology↗

bioRxiv2021

Long-term precision editing of neural circuits using engineered gap junction hemichannels

The coordination of activity between brain cells is a key determinant of neural circuit function; nevertheless, approaches that selectively regulate communication between two distinct cellular components of a mammalian circuit remain sparse. To address this gap, we developed a novel class of gap junctions by selectively engineering two connexin proteins found in Morone americana (white perch fish): connexin34.7 (Cx34.7) and connexin35 (Cx35). By iteratively exploiting protein mutagenesis, a novel in vitro assay of connexin docking, and computational modeling of connexin hemichannel interactions, we uncovered a pattern of structural motifs that contribute to hemichannel docking compatibility. Targeting these motifs, we designed Cx34.7 and Cx35 hemichannels that dock with each other, but not with themselves, nor with other major connexins expressed in the mammalian central nervous system. We validated these hemichannels in vivo using C. elegans and mice, demonstrating that they can facilitate communication across neural circuits composed of pairs of distinct cell types and modify behavior accordingly. Thus, we establish a potentially translational approach, Long-term integration of Circuits using connexins (LinCx), for context-precise circuit-editing with unprecedented spatiotemporal specificity in mammals.

neuroscience↗

bioRxiv2020

JAK1 palmitoylation by ZDHHC3/7 is Essential for Neuropoietic Cytokine Signaling and DRG Neuron Survival

Janus Kinase-1 (JAK1) plays key roles in pro-survival signaling during neurodevelopment and in responses to neuronal injury. JAK1 was identified as a potential palmitoyl-protein in high-throughput studies, but the importance of palmitoylation for JAK1s roles in neurons has not been addressed. Here, we report that JAK1 is endogenously palmitoylated in cultured Dorsal Root Ganglion (DRG) neurons and, using an shRNA knockdown/rescue approach, reveal that JAK1 palmitoylation is important for neuropoietic cytokine-dependent signaling and neuronal survival. We further identify the related palmitoyl acyltransferases (PATs) ZDHHC3 and ZDHHC7 as dominant regulators of JAK1 palmitoylation in transfected non-neuronal cells and endogenously in neurons. At the molecular level, palmitoylation is critical for JAK1s kinase activity in transfected cells and even in vitro, likely because palmitoylation facilitates transphosphorylation of key sites in the JAK1 activation loop. These findings provide new insights into palmitoylation-dependent control of neuronal development and survival.

neuroscience↗

bioRxiv2020

Palmitoylation Couples DLK to JNK3 to Facilitate Pro-degenerative Axon-to-Soma Signaling

Dual Leucine-zipper Kinase (DLK, a MAP3K) mediates neuronal responses to diverse injuries and insults via c-Jun N-terminal Kinase (JNK) family Mitogen-activated Protein Kinases (MAPKs). It is unclear why DLK couples to JNKs in mammalian neurons versus other MAPKs, especially because some invertebrate DLK orthologs couple instead to the related p38 family MAPKs. Here we identify two mechanisms that potentially explain this DLK-JNK coupling. First, neural-specific JNK3, but not p38-MAPK, catalyzes positive feedback phosphorylation of DLK that further activates DLK and locks the DLK-JNK3 module in a highly active state. Furthermore, the pro-degenerative JNK2 and JNK3, but not the related JNK1, are endogenously palmitoylated. Moreover, palmitoylation targets both DLK and JNK3 to the same axonal vesicles and JNK3 palmitoylation is essential for pro-degenerative axonal retrograde signaling in vivo. These findings provide insights into DLK-JNK signaling relevant to multiple neuropathological conditions and answer long-standing questions regarding the selective pro-degenerative roles of JNK2/3.

neuroscience↗

bioRxiv2020

Treating Porcine Abscesses With Histotripsy: A Pilot Study

Infected abscesses are walled-off collections of pus and bacteria. They are a common sequela of complications in the setting of surgery, trauma, systemic infections, and other disease states. Current treatment is typically limited to antibiotics with long-term catheter drainage, or surgical wash-out when inaccessible to percutaneous drainage or unresponsive to initial care efforts. Antibiotic resistance is also a growing concern. Although bacteria can develop drug resistance, they remain susceptible to thermal and mechanical damage. In particular, short pulses of focused ultrasound (i.e., histotripsy) generate mechanical damage through localized cavitation, representing a potential new paradigm for treating abscesses non-invasively, without the need for long-term catheterization and antibiotics. In this pilot study, boiling and cavitation histotripsy treatments were applied to subcutaneous and intramuscular abscesses developed in a novel porcine model. Ultrasound imaging was used to evaluate abscess maturity, for treatment monitoring and assessment of post-treatment outcomes. Disinfection was quantified by counting bacteria colonies from samples aspirated before and after treatment. Histopathological evaluation of the abscesses was performed to identify changes resulting from histotripsy treatment and potential collateral damage. Cavitation histotripsy was more successful in reducing the bacterial load while having a smaller treatment volume compared with boiling histotripsy. The results of this pilot study suggest focused ultrasound may lead to a technology for in situ treatment of acoustically accessible abscesses.

bioengineering↗

bioRxiv2020

Coupled Control of Distal Axon Integrity and Somal Responses to Axonal Damage by the Palmitoyl Acyltransferase ZDHHC17

After optic nerve crush (ONC), the cell bodies and distal axons of most retinal ganglion cells (RGCs) degenerate. RGC somal and distal axon degeneration were previously thought to be controlled by two distinct pathways, involving activation of the kinase DLK and loss of the axon survival factor NMNAT2, respectively. However, we found that mutual palmitoylation by the palmitoyl acyltransferase ZDHHC17 couples the DLK and NMNAT2 signals, which together form a "trust, but verify system". In healthy optic nerves, ZDHHC17-dependent palmitoylation ensures NMNAT-dependent distal axon integrity, while following ONC, ZDHHC17-dependent palmitoylation is critical for DLK-dependent somal degeneration. We found that ZDHHC17 also controls survival-versus-degeneration decisions in sensory neurons and identified motifs in NMNAT2 and DLK that govern their ZDHHC17-dependent regulation. These findings suggest that the control of somal and distal axon integrity should be considered as a single, holistic process, involving two palmitoylation-dependent pathways acting in concert.

neuroscience↗

bioRxiv2020

Brain-wide electrical dynamics encode an appetitive socioemotional state

Many cortical and subcortical regions contribute to complex social behavior; nevertheless, the network level architecture whereby the brain integrates this information to encode appetitive socioemotional behavior remains unknown. Here we measure electrical activity from eight brain regions as mice engage in a social preference assay. We then use machine learning to discover an explainable brain network that encodes the extent to which mice chose to engage another mouse. This socioemotional network is organized by theta oscillations leading from prelimbic cortex and amygdala that converge on ventral tegmental area, and network activity is synchronized with brain-wide cellular firing. The network generalizes, on a mouse-by-mouse basis, to encode socioemotional behaviors in healthy animals, but fails to encode an appetitive socioemotional state in a high confidence genetic mouse model of autism. Thus, our findings reveal the architecture whereby the brain integrates spatially distributed activity across timescales to encode an appetitive socioemotional brain state in health and disease.

neuroscience↗

bioRxiv2020

Deep sequencing of B cell receptor repertoires from COVID-19 patients reveals strong convergent immune signatures

Deep sequencing of B cell receptor (BCR) heavy chains from a cohort of 19 COVID-19 patients from the UK reveals a stereotypical naive immune response to SARS-CoV-2 which is consistent across patients and may be a positive indicator of disease outcome. Clonal expansion of the B cell memory response is also observed and may be the result of memory bystander effects. There was a strong convergent sequence signature across patients, and we identified 777 clonotypes convergent between at least four of the COVID-19 patients, but not present in healthy controls. A subset of the convergent clonotypes were homologous to known SARS and SARS-CoV-2 spike protein neutralising antibodies. Convergence was also demonstrated across wide geographies by comparison of data sets between patients from UK, USA and China, further validating the disease association and consistency of the stereotypical immune response even at the sequence level. These convergent clonotypes provide a resource to identify potential therapeutic and prophylactic antibodies and demonstrate the potential of BCR profiling as a tool to help understand and predict positive patient responses.

immunology↗

bioRxiv2020

The serine-threonine kinase TAO3 promotes cancer invasion and tumor growth by facilitating trafficking of endosomes containing the invadopodia scaffold TKS5α

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. We used our high-content screening assay to identify kinases impacting invadopodia formation. Among the top hits we selected TAO3, a STE20-like kinase of the GCK subfamily, for further analysis. TAO3 was over-expressed in many human cancers, and regulated invadopodia formation in melanoma, breast and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in 3-dimensional matrices and in vivo. We developed potent catalytic inhibitors of TAO3 that inhibited invadopodia formation and function, and tumor cell extravasation and growth. Using these inhibitors, we determined that TAO3 activity was required for endosomal trafficking of TKS5, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action. SIGNIFICANCETargeting tumor invasive behavior represents an understudied opportunity. We used an unbiased screening approach to identify kinases required for invadopodia formation and function. We validated TAO3, both genetically and with a novel inhibitor, and determined TAO3 function. Our data support clinical development of this class of target.

cancer biology↗