Biology subjects

Walsh, E.

Publications and source records attributed to Walsh, E..

15 recordsLinked to original sources

bioRxiv2026

Bravo! A Mosquito Antiviral Protein that Restricts Arboviruses through an RRM3-Dependent Nuclear Mechanism

Culex quinquefasciatus mosquitoes are vectors for several medically important viruses, yet their antiviral immune responses remain understudied compared to Aedes mosquitoes. The aedine broadly antiviral protein (aBravo) is an RNA-binding protein that was found to inhibit arbovirus replication in Aedes aegypti, but the function of the Cx. quinquefasciatus Bravo ortholog (CqBravo) remains unknown. Here, we examined the role of CqBravo in antiviral immunity in Cx. quinquefasciatus-derived Hsu cells and mosquitoes. We found that CqBravo is also broadly antiviral in Cx. quinquefasciatus, reducing replication of La Crosse virus (LACV) and Usutu virus (USUV) in Hsu cells and Sindbis virus (SINV) in vivo. CqBravo localized to the nucleus, and truncation and mutational analyses identified an RNA recognition motif (RRM3) critical for nuclear localization and antiviral activity. Quantitative proteomic analysis revealed that full-length CqBravo expression was associated with distinct changes in host protein abundance that were lost following removal of RRM3. Proteins altered by CqBravo expression were enriched for pathways involved in genome maintenance, including DNA repair, DNA metabolism, and ATP-dependent DNA processing. Overall, we demonstrate that CqBravo is an important antiviral factor in Cx. quinquefasciatus mosquitoes and may play a broader role in mosquito antiviral immunity across species.

microbiology↗

bioRxiv2026

Host-secreted lactate during respiratory viral infection diminishes macrophage antibacterial activity through metabolic reprogramming

In polymicrobial infections, how the host recognizes and responds to pathogens influences which species will persist to cause chronic infections. The human respiratory tract is a common anatomical site for viral-bacterial co-infections, where primary viral infections predispose to secondary bacterial infections, leading to increased morbidity and mortality. Additionally, co-infections are disproportionately prevalent in people with chronic lung diseases, such as chronic obstructive pulmonary disease and cystic fibrosis. We previously reported that primary viral infections and antiviral interferon (IFN) signaling stimulate Pseudomonas aeruginosa (PA) biofilm formation on airway epithelial cells (AECs). IFN signaling induces aerobic glycolysis in AECs and generates lactate as a cellular byproduct. Given that innate immune systems play an integral role in co-infection dynamics, we investigated the role of host-secreted metabolites (i.e. lactate) on innate immune cell activity during respiratory co-infections. We found that exposure to the apical secretions from IFN{beta}-treated AECs significantly compromised macrophage antibacterial activity, with the soluble metabolite lactate playing an important role. Macrophages used monocarboxylate transporters and G-protein receptors to transport and/or sense lactate, respectively, and this exposure to lactate diminished their bacterial-killing activity in a time-exposure dependent manner. Lactate exposure particularly reprogrammed macrophage cellular metabolism towards an anti-inflammatory state by increasing oxidative phosphorylation and fatty acid oxidation. Collectively, these findings provide insight into metabolites as complex regulators of trans-kingdom interactions and epithelial-macrophage crosstalk during respiratory co-infections.

immunology↗

bioRxiv2026

Regulation of midzone microtubule dynamics and abscission in human cells by CAMSAP2 and Kif2a

The spindle midzone, an array of overlapping, antiparallel microtubules, contributes to chromosome segregation and cytokinesis. As cells exit mitosis, midzone microtubules reorganize to form the midbody, the location of cell abscission. The mechanisms governing microtubule dynamics during this transition remain incompletely understood. The microtubule depolymerase, Kif2a, has been shown to contribute to midzone microtubule length control (Uehara et al., 2013), but how the depolymerase is regulated is not understood. Since CAMSAPs govern minus-end microtubule dynamics, we examined their role in midzone microtubule behavior. CAMSAP2, the major CAMSAP in HeLa cells, localized to the minus-ends of midzone microtubules and cells depleted of CAMSAP2, showed similar phenotypes as cells depleted of Kif2a, including elongated and bent midzones and enlarged asters. Next, we localized Kif2a in CAMSAP2-depleted cells and vice versa. CAMSAP2 remained present and extended along elongated midzone microtubules in Kif2a-depleted cells. In contrast Kif2a localization was no longer present at microtubule minus-ends but retained at plus-ends in CAMSAP2-depleted cells. In long-term live-cell movies of CAMSAP2-depleted cells abscission at the midbody was not detected, although two daughter cells formed. Markers for abscission including ESCRT-III component CHMP2A and Spastin were mislocalized, and midzone overlap zones, marked by PRC1, were extended. Together, our results demonstrate that CAMSAP2 is essential for midzone microtubule organization and dynamics, ultimately impacting cell abscission.

cell biology↗

bioRxiv2025

CTCF maintains pericentromere function and mitotic fidelity

In mitosis the duplicated genome is aligned and accurately segregated between daughter nuclei. CTCF is a chromatin looping protein in interphase with an unknown role in mitosis. We previously published data showing that CTCF constitutive knockdown causes mitotic failure, but the mechanism remains unknown. To determine the role of CTCF in mitosis, we used a CRISPR CTCF auxin inducible degron cell line for rapid degradation. CTCF degradation for 3 days resulted in increased failure of mitosis and decreased circularity in post-mitotic nuclei. Upon CTCF degradation CENP-E is still recruited to the kinetochore and there is a low incidence of polar chromosomes which occur upon CENP-E inhibition. Instead, immunofluorescence imaging of mitotic spindles reveals that CTCF degradation causes increased intercentromere distances and a wider and more disorganized metaphase plate, a disruption of key functions of the pericentromere. These results are similar to partial loss of cohesin, an established component of the pericentromere. Thus, we reveal that CTCF is a key maintenance factor of pericentromere function, successful mitosis, and post-mitotic nuclear shape.

cell biology↗

bioRxiv2025

Lamin B loss in nuclear blebs is rupture dependent while increased DNA damage is rupture independent

The nucleus houses genetic information and functions separate from the rest of the cell. Loss of nuclear shape results in nuclear ruptures. Nuclear blebs are deformations identified by decreased DNA density, while lamin B levels vary drastically. To determine if decreased lamin B levels are due to nuclear rupture, we used immunofluorescence to measure levels of lamin B and emerin, a nuclear envelope protein that enriches to sites of nuclear rupture. We observed that cell types that exhibit decreased levels of lamin B also show an enrichment of emerin in nuclear blebs. Oppositely, in other cell types, nuclear blebs display maintained levels of lamin B1 and showed no emerin enrichment. To determine how nuclear rupture affects DNA damage, we time lapse imaged nuclear rupture dynamics then fixed the same cells to conduct immunofluorescence of {gamma}H2AX and emerin. We find that DNA damage levels are higher in blebbed nuclei independent of nuclear rupture. Thus, we confirm that lamin B1 loss in nuclear blebs is due to nuclear rupture and blebbed nuclei have increased DNA damage that is independent of rupture. Summary statement (180-200 characters): We measured lamin B and DNA damage in blebbed nuclei to determine the effect of nuclear rupture. We find that nuclear rupture causes loss of lamin B in nuclear blebs but that increased DNA damage in blebbed nuclei is independent of rupture.

cell biology↗

bioRxiv2023

A fluid-walled microfluidic platform for human neuron microcircuits and directed axotomy

In our brains, different neurons make appropriate connections; however, there remain few in vitro models of such circuits. We use an open microfluidic approach to build and study neuronal circuits in vitro in ways that fit easily into existing bio-medical workflows. Dumbbell-shaped circuits are built in minutes in standard Petri dishes; the aqueous phase is confined by fluid walls - interfaces between cell-growth medium and an immiscible fluorocarbon, FC40. Conditions are established that ensure post-mitotic neurons derived from human induced pluripotent stem cells (iPSCs) plated in one chamber of a dumbbell remain where deposited. After seeding cortical neurons on one side, axons grow through the connecting conduit to ramify amongst striatal neurons on the other - an arrangement mimicking unidirectional cortico-striatal connectivity. We also develop a moderate-throughput non-contact axotomy assay. Cortical axons in conduits are severed by a media jet; then, brain-derived neurotrophic factor and striatal neurons in distal chambers promote axon regeneration. As additional conduits and chambers are easily added, this opens up the possibility of mimicking complex neuronal networks, and screening drugs for their effects on connectivity.

bioengineering↗

bioRxiv2023

Modulation of Retinal Inflammation Delays Degeneration in a Mouse Model of Geographic Atrophy

The advanced form of AMD, geographic atrophy, is associated with increased RPE oxidative stress and chronic inflammation. Here we evaluated the effects of delivering an anti-inflammatory viral gene by an AAV-vector in a mouse model of geographic atrophy. We measured changes in retinal function, structure, and morphology over nine months with electroretinography, optical coherence tomography, and fundoscopy, respectively. In addition, we used retinal tissue to quantify changes in markers of inflammation by multiplex ELISA, RT-qPCR, and immunofluorescence staining. Our AAV significantly delayed the loss of retinal function and structure and decreased retinal inflammation compared to the control AAV treatment. Our results suggest that modulating retinal inflammation could significantly slow the progression of geographic atrophy.

neuroscience↗

bioRxiv2023

Spatial transcriptomics reveals unique gene expression changes in different brain regions after sleep deprivation

Sleep deprivation has far-reaching consequences on the brain and behavior, impacting memory, attention, and metabolism. Previous research has focused on gene expression changes in individual brain regions, such as the hippocampus or cortex. Therefore, it is unclear how uniformly or heterogeneously sleep loss affects the brain. Here, we use spatial transcriptomics to define the impact of a brief period of sleep deprivation across the brain. We find that sleep deprivation induced pronounced differences in gene expression across the brain, with the greatest changes in the hippocampus, neocortex, hypothalamus, and thalamus. Both the differentially expressed genes and the direction of regulation differed markedly across regions. Importantly, we developed bioinformatic tools to register tissue sections and gene expression data into a common anatomical space, allowing a brain-wide comparison of gene expression patterns between samples. Our results suggest that distinct molecular mechanisms acting in discrete brain regions underlie the biological effects of sleep deprivation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/524406v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@86d135org.highwire.dtl.DTLVardef@1110670org.highwire.dtl.DTLVardef@bdaddeorg.highwire.dtl.DTLVardef@e1d268_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISpatial transcriptomics using the Visium platform reveals the transcriptional signature across the brain, recapitulating the anatomy of the mouse brain C_LIO_LISleep deprivation induces transcriptomic changes unique to each brain region C_LIO_LIThe hippocampus is the brain region impacted the most by acute sleep deprivation, with most differentially regulated genes significantly downregulated C_LIO_LIThe neocortex exhibits layer-specific changes in gene expression, with most differentially regulated genes significantly upregulated C_LIO_LIRegistration of spatial transcriptomic data to a common anatomical reference space (Allen Common Coordinate Framework) allows statistical analysis of gene expression across regions of the brain and for multi-sample analysis C_LI

neuroscience↗

bioRxiv2022

Genetic Variation and Disease Severity of Respiratory Syncytial Viruses

Respiratory Syncytial Virus (RSV) disease in newborns ranges from mild symptoms to severe disease requiring hospitalization. RSV is classified into two subtypes (RSVA and RSVB) based on antigenic and genetic differences. The role these genomic variations play in disease severity remains unknown. Genome sequences were obtained using next-generation RNA sequencing on archived frozen nasal swabs of young children (< 8 months-old) infected with RSV in Rochester, NY between 1977-1998. Samples were chosen from both children hospitalized with severe RSV disease (inpatient) and those presenting with mild symptoms (outpatient) during their first cold-season. Both A and B subtypes demonstrated significant differences in the phylogeny and primary-protein structure during this time period. We found a significant association between RSV phylogeny over this time period and disease severity. For both subtypes, the G-protein demonstrated the greatest amino acid substitutions, although the number of amino acid substitutions was higher in the RSVA subtype. We found a significant association between G-protein variation and disease severity for RSVA, but not RSVB. For both subtypes, variation in the M2-2 protein was significantly associated with disease severity. These results suggest that the genetic variability of RSV proteins may contribute to disease severity in humans. ImportanceEach cold-season Respiratory Syncytial Virus (RSV) infects thousands of children in the US. Some will display mild cold symptoms while others develop severe disease, sometimes resulting in lifelong lung problems or fatality. RSV initiates infection and replicates in the nasopharynx. Substitutions in the RSV genome can be found in clinically isolated nasal-swab samples of RSV infected children. Whether these genome variations contribute to severe disease is unknown. Here we found a statistically significant association between RSV phylogeny and disease severity. Furthermore, we found specific RSV proteins (G and M2-2) whose amino acid variation was statistically associated with severe disease, although which protein was associated depended on subtype. Taken together, our results suggest that RSV genotype contributed to disease severity over this time period.

microbiology↗

bioRxiv2021

Creating wounds in cell monolayers using micro-jets

Many wound-healing assays are used in cell biology and biomedicine; they are often labor intensive and/or require specialized and costly equipment. We describe a contactless method to create wounds with any imaginable 2D pattern in cell monolayers using micro-jets of either media or an immiscible and biocompatible fluorocarbon (i.e., FC40). We also combine this with another method that allows automation and multiplexing using standard Petri dishes. A dish is filled with a thin film of media overlaid with FC40, and the two liquids reshaped into an array of microchambers in minutes. Each chamber in such a grid is isolated from others by fluid walls of FC40. Cells are now added, allowed to grow into a monolayer, and wounds created using the microjets; then, healing is monitored by microscopy. As arrays of chambers can be made using the media and Petri dishes familiar to biologists, and as dishes fit seamlessly into their incubators, microscopes, and workflows, we anticipate this assay will find wide application in biomedicine.

bioengineering↗

bioRxiv2020

A systems genomics approach uncovers molecular associates of RSV severity

Globally, RSV infection results in millions of hospitalizations and thousands of deaths each year. Specific factors that contribute to disease severity, such as premature birth and certain comorbidities, are well established. Additionally, genetic variants resulting in alterations in the adaptive and innate immune response appear to be associated with RSV severity. While previous studies focused on a single aspect of the disease, we jointly modeled the association of disparate data modalities with RSV severity. To investigate the host response to respiratory syncytial virus (RSV) infection in infants, we performed a systems-level study of RSV pathophysiology, incorporating high-throughput measurements of the peripheral innate and adaptive immune systems and the airway epithelium and microbiota. Specifically, we developed and employed a novel multi-omic data integration method based on multilayered principal component analysis, penalized regression, and feature weight back-propagation. Our novel approach enabled us to identify cell type specific and shared cellular pathways associated with RSV severity. Of particular interest was the association between RSV severity, activation of pathways controlling Th17 and acute phase response signaling, and inhibition of B cell receptor signaling, which were present in both airway and immune cells. These data identify specific aspects of dysregulation between the humoral and mucosal response to RSV that may play a critical role in determining illness severity.

genomics↗

bioRxiv2020

CX3CR1 Engagement by Respiratory Syncytial Virus Leads to Induction of Nucleolin and Dysregulation of Cilia-related Genes

Respiratory syncytial virus (RSV) contains a conserved CX3C motif on the ectodomain of the G-protein. The motif has been indicated as facilitating attachment of the virus to the host initiating infection via the human CX3CR1 receptor. The natural CX3CR1 ligand, CX3CL1, has been shown to induce signaling pathways resulting in transcriptional changes in the host cells. We hypothesize that binding of RSV to CX3CR1 via CX3C leads to transcriptional changes in host epithelial cells. Using transcriptomic analysis, the effect of CX3CR1 engagement by RSV was investigated. Normal human bronchial epithelial (NHBE) cells were infected with RSV virus containing either wildtype G-protein, or a mutant virus containing a CX4C mutation in the G-protein. RNA sequencing was performed on mock and 4-days-post-infected cultures. NHBE cultures were also treated with purified recombinant wild-type A2 G-protein. Here we report that RSV infection resulted in significant changes in the levels 766 transcripts. Many nuclear associated proteins were upregulated in the WT group, including Nucleolin. Alternatively, cilia-associated genes, including CC2D2A and CFAP221 (PCDP1), were downregulated. The addition of recombinant G-protein to the culture lead to the suppression of cilia-related genes while also inducing Nucleolin. Mutation of the CX3C motif (CX4C) reversed these effects on transcription decreasing nucleolin induction and lessening the suppression of cilia-related transcripts in culture. Furthermore, immunohistochemical staining demonstrated decreases in in ciliated cells and altered morphology. Therefore, it appears that engagement of CX3CR1 leads to induction of genes necessary for RSV entry as well as dysregulation of genes associated with cilia function. ImportanceRespiratory Syncytial Virus (RSV) has an enormous impact on infants and the elderly including increased fatality rates and potential for causing lifelong lung problems. Humans become infected with RSV through the inhalation of viral particles exhaled from an infected individual. These virus particles contain specific proteins that the virus uses to attach to human ciliated lung epithelial cells, initiating infection. Two viral proteins, G-protein and F-protein, have been shown to bind to human CX3CR1and Nucleolin, respectively. Here we show that the G-protein induces Nucleolin and suppresses gene transcripts specific to ciliated cells. Furthermore, we show that mutation of the CX3C-motif on the G-protein, CX4C, reverses these transcriptional changes.

microbiology↗

bioRxiv2020

Pygmaeomycetaceae, a new root associated family in Mucoromycotina from the pygmy pine plains

A new genus, Pygmaeomyces, and two new species are described based on phylogenetic analyses, phenotypic and ecological characters. The species delimitation was based on concordance of gene genealogies. The Pygmaeomyces cultures were isolated from the roots of mountain laurel (Kalmia latifolia) and pitch pine (Pinus rigida) from the acidic and oligotrophic New Jersey Pygmy Pine Plains; however, they likely have a broader distribution because their internal transcribed spacer (ITS) sequences have high similarity with a number of environmental sequences from multiple independent studies. Based on the phylogeny and phenotypical characters, a new family Pygmaeomycetaceae is proposed to accommodate this new lineage in Mucoromycotina. Pygmaeomycetaceae corresponds to Clade GS23, which was identified based on a sequence-only soil fungal survey and was believed to be a distinct new class. Compared to the culture-based methods, we observed that sequence-only analyses tend to over-estimate the taxonomic level. Results from this work will facilitate ecological and evolutionary studies on root-associated fungi.

microbiology↗

bioRxiv2020

Jet-printing microfluidic devices on demand

There is an unmet demand for microfluidics in biomedicine. We describe contactless fabrication of microfluidic circuits on standard Petri dishes using just a dispensing needle, syringe pump, 3-way traverse, cell-culture media, and an immiscible fluorocarbon (FC40). A submerged micro-jet of FC40 is projected through FC40 and media on to the bottom of a dish, where it washes media away to leave liquid fluorocarbon walls pinned to the substrate by interfacial forces. Such fluid walls can be built into almost any imaginable 2D circuit in minutes, which we exploit to clone cells using limiting dilution in a way that beats the Poisson limit, sub-culture adherent cells, and feed arrays of cells continuously for a week. This general method should have wide application in biomedicine. One sentence summaryIn the everyday world, we cannot build complex structures out of liquids as they collapse into puddles; in the microworld we can.

bioengineering↗

bioRxiv2019

Opponent neurochemical and functional processing in NREM and REM sleep in visual learning

Sleep is beneficial for learning. However, whether NREM or REM sleep facilitates learning, whether the learning facilitation results from plasticity increases or stabilization and whether the facilitation results from learning-specific processing are all controversial. Here, after training on a visual task we measured the excitatory and inhibitory neurochemical (E/I) balance, an index of plasticity in human visual areas, for the first time, while subjects slept. Off-line performance gains of presleep learning were associated with the E/I balance increase during NREM sleep, which also occurred without presleep training. In contrast, increased stabilization was associated with decreased E/I balance during REM sleep only after presleep training. These indicate that the above-mentioned issues are not matters of controversy but reflect opposite neurochemical processing for different roles in learning during different sleep stages: NREM sleep increases plasticity leading to performance gains independently of learning, while REM sleep decreases plasticity to stabilize learning in a learning-specific manner.

neuroscience↗