Biology subjects

Fitzpatrick, J. A.

Publications and source records attributed to Fitzpatrick, J. A..

7 recordsLinked to original sources

bioRxiv2025

Backyard proteomics: A case study with the black widow spider

Nearly all methods of mass spectrometry-based proteomics rely on knowing the proteome of the species. In less studied organisms without annotated genomes, it can seem impossible to perform proteomic analysis. In this study we sought to answer the question - does enough information exist to do proteomics on any organism we want? As a case study we started with material available due to an infestation of a home with black widow spiders. Thanks to the recent publication of an annotated genome for one species of black widow spider, we were able to identify 5502 protein groups and assign putative annotations using ortholog mapping. We also demonstrate that had we not had this resource, over 2000 proteins could be identified using other available spider genome annotations, despite their unrelatedness. Moreover, regardless of spider proteome used, proteins annotated as toxins were almost exclusively observed in the main body of the mature female black widow spider. Overall, these results provide a draft proteome map for the black widow spider and valuable data for validating machine learning models, while also suggesting that the door to insightful quantitative proteomics may already be open for millions of less studied organisms. All raw and processed proteomic data are available through the ProteomeXchange repository as accession PXD051601. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/648442v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@7e4c7eorg.highwire.dtl.DTLVardef@1cfeba1org.highwire.dtl.DTLVardef@1769a02org.highwire.dtl.DTLVardef@1790450_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Endogenous recapitulation of Alzheimers disease neuropathology through human 3D direct neuronal reprogramming

Alzheimers disease (AD) is a neurodegenerative disorder that primarily affects elderly individuals, and is characterized by hallmark neuronal pathologies including extracellular amyloid-{beta} (A{beta}) plaque deposition, intracellular tau tangles, and neuronal death. However, recapitulating these age-associated neuronal pathologies in patient-derived neurons has remained a significant challenge, especially for late-onset AD (LOAD), the most common form of the disorder. Here, we applied the high efficiency microRNA-mediated direct neuronal reprogramming of fibroblasts from AD patients to generate cortical neurons in three-dimensional (3D) Matrigel and self-assembled neuronal spheroids. Our findings indicate that neurons and spheroids reprogrammed from both autosomal dominant AD (ADAD) and LOAD patients exhibited AD-like phenotypes linked to neurons, including extracellular A{beta} deposition, dystrophic neurites with hyperphosphorylated, K63-ubiquitin-positive, seed-competent tau, and spontaneous neuronal death in culture. Moreover, treatment with {beta}- or {gamma}-secretase inhibitors in LOAD patient-derived neurons and spheroids before A{beta} deposit formation significantly lowered A{beta} deposition, as well as tauopathy and neurodegeneration. However, the same treatment after the cells already formed A{beta} deposits only had a mild effect. Additionally, inhibiting the synthesis of age-associated retrotransposable elements (RTEs) by treating LOAD neurons and spheroids with the reverse transcriptase inhibitor, lamivudine, alleviated AD neuropathology. Overall, our results demonstrate that direct neuronal reprogramming of AD patient fibroblasts in a 3D environment can capture age-related neuropathology and reflect the interplay between A{beta} accumulation, tau dysregulation, and neuronal death. Moreover, miRNA-based 3D neuronal conversion provides a human-relevant AD model that can be used to identify compounds that can potentially ameliorate AD-associated pathologies and neurodegeneration.

neuroscience↗

bioRxiv2022

Structural mechanism of leaflet-specific phospholipid modulation of a pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To elucidate the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open conformation. The structure shows a bound phospholipid in an outer leaflet site, and conformational changes in the phospholipid binding site unique to the open state. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the open state of a pLGIC by specific, state-dependent binding to this site.

biophysics↗

bioRxiv2021

Global cerebrospinal fluid circulation mapping using gold nanoparticle enhanced X-ray microtomography reveals region-specific brain and spinal cord CSF pathways

Cerebrospinal fluid (CSF) movement within the brain interstitium is essential for the development and functioning of the brain. However, the interstitium has largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with CSF. Here, we developed a novel technique for CSF tracking, gold nanoparticle enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF pathways during development. Using this method and subsequent histological analysis, we map global CSF pathways and present novel particle size-dependent circulation patterns through the CNS. We identify an intraparenchymal CSF circulation that targets stem cell-rich and cholinergic neuronal populations. CSF solute distribution to these areas is mediated by CSF flow along projections from the basal cisterns which is altered in posthemorrhagic hydrocephalus. Our study uncovers region-specific patterns in a biologically driven CSF circulation that has implications for normal brain development and the pathophysiology of hydrocephalus and neurodegenerative disorders.

neuroscience↗

bioRxiv2020

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis

Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.

immunology↗

bioRxiv2018

PolyA tracks and poly-lysine repeats are the Achilles heel of Plasmodium falciparum

Plasmodium falciparum, the causative agent of human malaria, is an apicomplexan parasite with a complex, multi-host life cycle. Sixty percent of transcripts from its extreme AT-rich (81%) genome possess coding polyadenosine (polyA) runs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate that transcripts with polyA runs encoding poly-lysine are hot spots for ribosome stalling and frameshifting, eliciting mRNA surveillance pathways and attenuating protein synthesis in the majority of prokaryotic and eukaryotic organisms. Here, we show that the P. falciparum translational machinery is paradigm-breaking. Using bioinformatic and biochemical approaches, we demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately transcribed and translated in P. falciparum cells. Translation of polyA tracks in the parasite does not elicit any response from mRNA surveillance pathways usually seen in host human cells or organisms with similar AT content. The translation efficiency and accuracy of the parasite protein synthesis machinery reveals a unique role of ribosomes in the evolution and adaptation of P. falciparum to an AU-rich transcriptome and polybasic amino sequences. Finally, we show that the ability of P. falciparum to synthesize long poly-lysine repeats has given this parasite a unique protein exportome and an advantage in infectivity that can be suppressed by addition of exogenous poly-basic polymers.

microbiology↗

bioRxiv2017

HID-1 controls cargo sorting and dense core formation by influencing trans- Golgi network acidification in neuroendocrine cells

Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. They form at the trans-Golgi network (TGN) where their soluble content aggregates to form a dense core, but the mechanisms controlling biogenesis are still not completely understood. Recent studies have implicated the peripheral membrane protein HID-1 in neuropeptide sorting and insulin secretion. Using CRISPR/Cas9, we generated HID-1 KO rat neuroendocrine cells, and show that the absence of HID-1 results in specific defects in peptide hormone and monoamine storage and regulated secretion. Loss of HID-1 causes a reduction in the number of LDCVs and affects their morphology and biochemical properties due to impaired cargo sorting and dense core formation. HID-1 KO cells also exhibit defects in TGN acidification together with mislocalization of the Golgi-enriched vacuolar H+-ATPase subunit isoform a2. We propose that HID-1 influences early steps in LDCV formation by controlling dense core formation at the TGN.

cell biology↗