Biology subjects

Butler, M.

Publications and source records attributed to Butler, M..

10 recordsLinked to original sources

bioRxiv2026

Arp2/3-mediated turnover of large clathrin lattices is regulated through the tyrosine kinase ACK

Clathrin mediated endocytosis (CME) is a vital cellular process that mediates cell signaling by controlling the internalization of extracellular cargo and activated receptors. Arp2/3-branched actin provides force to assist in membrane invagination and scission in CME. Loss of Arp2/3-branched actin in conditional Arpc2 KO fibroblasts results in an increase of large arrested clathrin lattices (LACLs) visualized by live-cell TIRF imaging. Additional structural details of LACLs were revealed using electron microscopy and include an increase in arrested clathrin lattices of various curvatures. Numerous CME proteins have heightened levels of tyrosine phosphorylation at these LACLs in Arpc2 KO cells. We identified the non-receptor tyrosine kinase ACK (Activated Cdc42-Associated Kinase) as a key upstream regulator of LACL turnover. CRISPR KO of ACK abrogates LACL tyrosine phosphorylation and impairs cells capacity to resolve LACLs. Our results support a model where ACK recruitment and activation at LACLs drives branched actin formation to help resolve large accumulations of arrested CME structures.

Cell Biology↗

bioRxiv2026

Immobilized neuroglobin scavenges carbon monoxide from circulating carboxyhemoglobin

Carbon monoxide (CO) poisoning is responsible for around 50,000 emergency department visits per year in the U.S. alone. With the present standard of care, persistent neurological sequelae occur in [~]30-40% of severe CO poisoning cases. Currently, there is no available targeted molecular antidote for CO poisoning. In previous work, we have developed an antidotal therapy for CO poisoning based on an engineered hemeprotein, human neuroglobin (Ngb-H64Q-CCC). Intravenous infusion of Ngb-H64Q-CCC removes CO from the circulating red blood cells and improves survival in a lethal CO-poisoning mouse model. However, the infusion of heme-containing proteins has inherent heme toxicity risks that may limit the dose that can be used safely without liver or kidney toxicity. In order to overcome these problems, we have investigated the development of immobilized Ngb in a solid matrix. This approach allows for the development of a CO removal system using an extracorporeal blood circulating system coupled with a stationary matrix with immobilized Ngb-H64Q-CCC. Such system avoids drug infusion and possible organ injury, allows for antidote recycling, and provides advantages for storage and handling of the antidote. By assessing the efficacy of Ngb-H64Q-CCC immobilized through different linkage strategies, we have identified N-hydroxysuccinimide agarose resin as a viable stationary phase. The immobilized protein shows preserved heme redox activity, can be chemically reduced/oxidized for activation/CO release purposes, and retains its CO removal capacity after successive regeneration cycles. We expect that this novel approach will advance the development of new scavenger-based therapies for CO poisoning.

bioengineering↗

bioRxiv2026

Exploiting HLA-II Promiscuity via Peptide Terminal Overhang Recognition for Pan-Allelic and Tumor-Selective AML Immunotherapy

Antibodies targeting peptides presented by human leukocyte antigen (HLA) molecules expand the therapeutic landscape by enabling recognition of intracellular antigens. While most efforts have focused on allele-restricted peptides presented by HLA class I (HLA-I), HLA class II (HLA-II) epitopes remain underexplored despite their potential for promiscuous presentation. Acute myeloid leukemia (AML) is characterized by high expression of both HLA-II and the myeloid lineage antigen myeloperoxidase (MPO). Here, we identified an MPO-derived epitope (MPO100-132) that is promiscuously presented by multiple HLA-II molecules. We generated a MPO100-132-specific antibody (146D5) that recognizes the N-terminal overhang of this peptide independent of specific HLA contacts, enabling pan-allelic recognition. Engineered into bispecific T cell engagers (BiTEs), this antibody mediated robust cytotoxicity against primary AML samples across diverse HLA-II backgrounds. Crucially, 146D5-based BiTEs selectively spared normal myeloid cells, indicating that the MPO100-132 peptide, derived from the MPO propeptide, was functionally undetectable in normal myeloid cells, providing a significant safety window. In vivo, the MPO-targeting BiTE demonstrated potent antitumor activity and prolonged survival in AML xenograft models. Our findings identify peptide terminal overhangs as an actionable class of antibody targets and introduce a strategy to exploit HLA-II promiscuity for broadly applicable HLA-dependent but allele-agnostic immunotherapies.

immunology↗

bioRxiv2025

Matrix Metalloproteinase (MMP) inhibition rescues endothelial glycocalyx damage and reduces neutrophil infiltration in sepsis-associated acute kidney injury

Endothelial glycocalyx (eGlx), a carbohydrate-rich endothelial coat, maintains vascular homeostasis, and its disruption contributes to several vascular diseases. We previously identified matrix metalloproteinase (MMP) 2 and 9-mediated eGlx shedding as a key mechanism in human glomerular endothelial cell (GEnC) damage and kidney dysfunction in diabetes. Here, we sought to determine whether this mechanism contributes to renal and systemic microvascular endothelial cell injury in sepsis-AKI. Sepsis-AKI was induced in mice by lipopolysaccharide (LPS) injection. Confocal microscopy demonstrated glomerular and peritubular eGlx damage, and increased circulating SDC4 demonstrated systemic eGlx shedding. These were associated with enhanced mRNA expression of eGlx components (syndecans 1 and 4) and endothelial inflammatory markers, Intercellular and Vascular Cell Adhesion Molecules (ICAM and VCAM) in the kidney. Additionally, sepsis-AKI markers, serum creatinine and urea, were increased. We found that renal and circulating MMP9, but not MMP2, levels were raised in sepsis-AKI. Moreover, MMP9 staining colocalises with neutrophil, endothelial and mesangial staining in glomeruli in sepsis-AKI. Treatment with MMP2 and 9 inhibitor 1, 1h before LPS injection, prevented glomerular, peritubular and systemic eGlx damage in sepsis-AKI. MMP inhibition also attenuated glomerular leucocyte numbers in sepsis-AKI. We confirmed increased MMP9 activity, shedding of eGlx syndecan 1 and elevated angiopoietin 2 levels, an endothelial damage marker, in sepsis-AKI in humans. Moreover, we used human glomerular endothelial cells (GEnC) in vitro to demonstrate that human sepsis-AKI serum directly damages GEnC eGlx. Our studies confirm that MMP-mediated eGlx damage is a key contributor to renal and systemic microvascular dysfunction in sepsis-AKI and represents a potential therapeutic target for protecting the microvasculature.

cell biology↗

bioRxiv2021

Automated Raman micro-spectroscopy of epithelial cells for the high-throughput classification.

Raman micro-spectroscopy is a powerful technique for the identification and classification of cancer cells and tissues. In recent years, the application of Raman spectroscopy to detect bladder, cervical, and oral cytological samples has been reported to have an accuracy that is greater than standard pathology. However, despite being entirely non-invasive and relatively inexpensive, the slow recording time, and lack of reproducibility, have prevented the clinical adoption of the technology. Here we present an automated Raman cytology system that can facilitate high-throughput screening and improve reproducibility. The proposed system is designed to be integrated directly into the standard pathology clinic, taking into account their methodologies and consumables. The system employs image processing algorithms and integrated hardware/software architectures in order to achieve automation and is tested using the ThinPrep standard, including the use of glass slides, and a number of bladder cancer cell lines. The entire automation process is implemented using the open source Micro-Manager platform, and is made freely available. We believe this code can be readily integrated into existing commercial Raman micro-spectrometers.

pathology↗

bioRxiv2020

Implementation of a pre- and in-scan system to minimize head motion in pediatric participants undergoing fMRI scans

BackgroundPerforming fMRI scans of children can be a difficult task, as participants tend to move while being scanned. Head motion represents a significant confound in functional magnetic resonance imaging (fMRI) connectivity analyses, and methods to limit the impact of movement on data quality are needed. One approach has been to use shorter MRI protocols, though this potentially reduces the reliability of the results. ObjectiveHere we describe steps we have taken to limit head motion in an ongoing fMRI study of children undergoing a 60 minute MRI scan protocol. Specifically, we have used a mock scan protocol that trains participants to lie still while being scanned. We provide a detailed protocol and describe other in-scanner measures we have implemented, including an incentive system and the use of a weighted blanket. Materials and methodsParticipants who received a formal mock scan (n = 12) were compared to participants who had an informal mock scan (n = 7). A replication group of participants (n = 16), including five with autism spectrum disorder, who received a formal mock scan were also compared to the informal mock scan group. The primary measure of interest was the mean frame-to-frame displacement across eight functional runs during the fMRI protocol. ResultsParticipants in the formal mock scan and replication group tended to exhibit more low-motion functional scans than the informal mock scan group (P < 0.05). Across different functional scan conditions (i.e. while watching movie clips, performing an attention task, and during resting-state scans), effect sizes tended to be large (Hedges g > 0.8). ConclusionResults indicate that with appropriate measures, it is possible to achieve low-motion fMRI data in younger participants undergoing a long scan protocol.

neuroscience↗

bioRxiv2019

Pelleted-hay alfalfa feed increases sheep wether weight gain and rumen bacterial richness over loose-hay alfalfa feed.

Diet composed of smaller particles can improve feed intake, digestibility, and animal growth or health, but in ruminant species can reduce rumination and buffering - the loss of which may inhibit fermentation and digestibility. However, the explicit effect of particle size on the rumen microbiota remains untested, despite their crucial role in digestion. We evaluated the effects of reduced particle size on rumen microbiota by feeding long-stem (loose) alfalfa hay compared to a ground and pelleted version of the same alfalfa in yearling sheep wethers. In situ digestibility of the pelleted diet was greater at 48 h compared with loose hay; however, distribution of residual fecal particle sizes in sheep did not differ between the dietary treatments at any time point. Both average daily gain and feed efficiency were greater for the wethers consuming the pelleted diet. Observed bacterial richness was very low at the end of the adaptation period and increased over the course of the study, suggesting the rumen bacterial community was still in flux after two weeks of adaptation. The pelleted-hay diet group had a greater increase in bacterial richness, including common fibrolytic rumen inhabitants. The pelleted diet was positively associated with several Succiniclasticum, a Prevotella, and uncultured taxa in the Ruminococcaceae and Rickenellaceae families and Bacteroidales order. Pelleting an alfalfa hay diet for sheep does shift the rumen microbiome, though the interplay of diet particle size, retention and GI transit time, microbial fermentative and hydrolytic activity, and host growth or health is still largely unexplored.

microbiology↗

bioRxiv2018

Octapeptin C4 Induces Less Resistance and Novel Mutations in an Epidemic Carbapenemase-producing Klebsiella pneumoniae ST258 Clinical Isolate Compared to Polymyxins

Polymyxin B and E (colistin) have been pivotal in the treatment of extensively drug-resistant (XDR) Gram-negative bacterial infections, with increasing use over the past decade. Unfortunately, resistance to these antibiotics is rapidly emerging. The structurally-related octapeptin C4 (OctC4) has shown significant potency against XDR bacteria, including against polymyxin-resistant (Pmx-R) strains, but its mode of action remains undefined. We sought to compare and contrast the acquisition of XDR Klebsiella pneumoniae (ST258) resistance in vitro with all three lipopeptides to help elucidate the mode of action of the drugs and potential mechanisms of resistance evolution. Strikingly, 20 days of exposure to the polymyxins resulted in a dramatic (1000-fold) increase in the minimum inhibitory concentration (MIC) for the polymyxins, reflecting the evolution of resistance seen in clinical isolates, whereas for OctC4 only a 4-fold increase was witnessed. There was no cross-resistance observed between the polymyxin - and octapeptin-induced resistant strains. Sequencing revealed previously known gene alterations for polymyxin resistance, including crrB, mgrB, pmrB, phoPQ and yciM, and novel mutations in qseC. In contrast, mutations in mlaDF and pqiB, 1genes related to phospholipid transport, were found in octapeptin-resistant isolates. Mutation effects were validated via complementation assays. These genetic variations were reflected in phenotypic changes to lipid A. Pmx-R isolates increased 4-amino-4-deoxy-arabinose fortification to phosphate groups of lipid A, whereas OctC4 induced strains harbored a higher abundance of hydroxymyristate and palmitoylate. The results reveal a differing mode of action compared to polymyxins which provides hope for future therapeutics to combat the increasingly threat of XDR bacteria.

microbiology↗

bioRxiv2018

Spatial and temporal PCP protein dynamics coordinate cell intercalation during neural tube closure

Planar cell polarity (PCP) controls the convergent extension cell movements that drive axis elongation in all vertebrates. Though asymmetric localization of core PCP proteins is central to their function, we currently understand little about PCP protein localization as it relates to the subcellular behaviors that drive convergent extension. Here, we have used high magnification time-lapse imaging to simultaneously monitor cell intercalation behaviors and the localization of the PCP proteins Prickle2 and Vangl2. We observed the expected asymmetric enrichment of PCP proteins, but more interestingly, we also observed tight temporal and spatial correlation of PCP protein enrichment with contractile behavior in cell-cell junctions. These patterns of localization were associated with similar pattern of protein turnover at junctions as assessed by FRAP. In fact, dynamic enrichment of PCP proteins was linked more strongly to junction behavior than to spatial orientation. Finally, recruitment of Prickle2 and Vangl2 to cell-cell junctions was temporally and spatially coordinated with planar polarized oscillations of actomyosin enrichment, and all of these dynamic relationships were disrupted when PCP signaling was manipulated. Together, these results provide a dynamic and quantitative view of PCP protein localization during convergent extension and suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking during cell intercalation of the closing vertebrate neural tube.

developmental biology↗

bioRxiv2017

DNA-Demethylating Agents enhance cytolytic activity of CD8+ T Cells and anti-tumor immunity

Recent studies have shown that DNA methyltransferase inhibitors (DNMTi) can induce IRF7 activation and Type I/III interferon signaling through dsRNA-mediated viral mimicry in cancer cells. By performing a large pan-cancer analysis using TCGA data, we determined that IRF7 activation is associated with higher CD8+ T cell tumor infiltration and higher cytolytic activity across multiple cancer types. Accordingly, we demonstrate that DNMTi treatment results in increased CD8+ T cell tumor infiltration, enhanced cytolytic activity and CD8+ T cell dependent tumor growth inhibition. Finally, we show that DNMTi triggers a process marked by the induction of viral mimicry directly on CD8+ T cells, leading to activation of dsRNA sensing pathway, and up-regulation of T cell activation markers, effector cytokines, and Granzyme B. Taken together, our findings suggest that dsRNA sensing pathway activation in the immune compartment, through pharmacological DNA demethylation, is a viable strategy for boosting anti-tumor immune response.

cancer biology↗