Biology subjects

Phillips, M.

Publications and source records attributed to Phillips, M..

29 records · Page 2Linked to original sources

bioRxiv2021

Hematopoietic stem cell requirement for macrophage regeneration is tissue-specific

Tissue-resident macrophages (TRM{Phi}) are important immune sentinels responsible for maintaining tissue and immune homeostasis within their specific niche. Recently, the origins of TRM{Phi} have undergone intense scrutiny where now most TRM{Phi} are thought to originate early during embryonic development independent of hematopoietic stem cells (HSCs). We previously characterized two distinct subsets of mouse peritoneal cavity macrophages (Large and Small Peritoneal Macrophages; LPM and SPM, respectively) whose origins and relationship to both fetal and adult long-term (LT)-HSCs have not been fully investigated. Here we employ highly purified LT-HSC transplantation and in vivo lineage tracing to show a dual ontogeny for LPM and SPM, where the initial wave of peritoneal macrophages is seeded from yolk sac-derived precursors, which later require LT-HSCs for regeneration. In contrast, transplanted fetal and adult LT-HSCs are not able to regenerate brain-resident microglia. Thus, we demonstrate that LT-HSCs retain the potential to develop into TRM{Phi}, but their requirement is tissue-specific.

immunology↗

bioRxiv2021

The genetic basis of Drosophila melanogaster defense against Beauveria bassiana explored through evolve and resequence and quantitative trait locus mapping

Many of the molecular mechanisms for antifungal immunity in Drosophila melanogaster have been defined, but relatively little is known about the genetic basis for variation in antifungal immunity in natural populations. Using two population genetic approaches, Quantitative Trait Locus (QTL) Mapping and Evolve and Resequence (E&R), we explored the genetics underlying D. melanogaster immune defense against infection with the fungus Beauveria bassiana. Immune defense was highly variable both in the recombinant inbred lines from the Drosophila Synthetic Population Resource used for our QTL Mapping and in the synthetic outbred populations used in our E&R study. Survivorship of infection improved dramatically over just 10 generations in the E&R study, and continued to increase for an additional 9 generations, revealing a trade-off with uninfected longevity. Populations selected for increased defense against B. bassiana evolved cross resistance to a second, distinct B. bassiana strain but not to bacterial pathogens. The QTL mapping study revealed that sexual dimorphism in defense depends on host genotype, and the E&R study indicated that dimorphism also depends on the specific pathogen to which the host is exposed. Both the QTL Mapping and E&R experiments generated lists of potentially causal candidate genes, although these lists were non-overlapping.

genomics↗

bioRxiv2021

Structural Characteristics and Proton Conductivity of the Gel Within the Electrosensory Organs of Cartilaginous Fishes

The Ampullae of Lorenzini (AoL) of cartilaginous fishes are sensory organs used to detect environmental electric fields. The proximal ends of the organs are externally visible as pores in the skin that lead into gel-filled tubular canals which terminate in rounded chambers filled with highly specialized electrosensory cells. The viscoelastic gel that fills the organs is composed of proteins and polysaccharides that are not yet completely characterized but are thought to play a critical role in the electrosensing mechanism. Although recent studies have identified various components of AoL gel, it has remained unclear how the component molecules are structurally arranged and how their structure influences the overall function of the AoL. Here we present the first microscopic descriptions and x-ray scattering data from AoL gel extracted from spotted ratfish (Hydrolagus colliei). Our results suggest that AoL gel is colloidal in nature and composed of spherical globules that are approximately 10-100 nm in size. We investigated the structural influence of the protein components of the gel specifically by analyzing gel that had been digested in situ via enzymatic proteolysis. By comparing gel before and after digestion using microscopy, x-ray scattering analyses, and proton conductivity measurements, we directly observed the structural and functional influence of proteins in AoL gel. The findings described here represent the first detailed structural analysis of AoL gel and lay the groundwork for more detailed studies into the specific interactions of molecules inside AoL gel at the nanoscale, with particular reference to their mechanistic role in electrosensing.

biophysics↗

bioRxiv2020

Conjugates of neuroprotective chaperone L-PGDS provide MRI contrast for detection of amyloid β-rich regions in live Alzheimer's Disease mouse model brain

Endogenous brain proteins can recognize the toxic oligomers of amyloid-{beta} (A{beta}) peptides implicated in Alzheimers disease (AD) and interact with them to prevent their aggregation. Lipocalin-type Prostaglandin D Synthase (L-PGDS) is a major A{beta}-chaperone protein in the human cerebrospinal fluid. Here we demonstrate that L-PGDS detects amyloids in diseased mouse brain. Conjugation of L-PGDS with magnetic nanoparticles enhanced the contrast for magnetic resonance imaging. We conjugated the L-PGDS protein with ferritin nanocages to detect amyloids in the AD mouse model brain. We show here that the conjugates administered through intraventricular injections co-localize with amyloids in the mouse brain. These conjugates can target the brain regions through non-invasive intranasal administration, as shown in healthy mice. These conjugates can inhibit the aggregation of amyloids in vitro and show potential neuroprotective function by breaking down the mature amyloid fibrils.

biochemistry↗

bioRxiv2020

The BRPF1 bromodomain is a molecular reader of di-acetyllysine

Bromodomain-containing proteins are often part of chromatin-modifying complexes, and their activity can lead to altered expression of genes that drive cancer, inflammation and neurological disorders in humans. Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ (monocytic leukemic zinc-finger protein) HAT (histone acetyltransferase) complex, which is associated with chromosomal translocations known to contribute to the development of acute myeloid leukemia (AML). BRPF1 contains a unique combination of chromatin reader domains including two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) domain. BRPF1 is known to recruit the MOZ HAT complex to chromatin by recognizing acetylated lysine residues on the N-terminal histone tail region through its bromodomain. However, histone proteins can contain several acetylation modifications on their N-terminus, and it is unknown how additional marks influence bromodomain recruitment to chromatin. Here, we identify the BRPF1 bromodomain as a selective reader of di-acetyllysine modifications on histone H4. We used ITC assays to characterize the binding of di-acetylated histone ligands to the BRPF1 bromodomain and found that the domain binds preferentially to histone peptides H4K5acK8ac and H4K5acK12ac. Analytical ultracentrifugation (AUC) experiments revealed that the monomeric state of the BRPF1 bromodomain coordinates di-acetylated histone ligands. NMR chemical shift perturbation studies, along with binding and mutational analyses, revealed non-canonical regions of the bromodomain-binding pocket that are important for histone tail recognition. Together, our findings provide critical information on how the combinatorial action of post-translational modifications can modulate BRPF1 bromodomain binding and specificity.

biochemistry↗

bioRxiv2020

Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain

A population of >6 million people worldwide at high risk of Alzheimers disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of {beta}-amyloid-(A{beta})-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar A{beta} deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome-21-gene BACE2, but prevented by combined chemical {beta} and {gamma}-secretase inhibition. We found that T21-organoids secrete increased proportions of A{beta}-preventing (A{beta}1-19) and A{beta}-degradation products (A{beta}1-20 and A{beta}1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1-inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in [~]30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

neuroscience↗

bioRxiv2020

Xenobiotic compounds modulate cytotoxicity of Aβ amyloids and interact with neuroprotective chaperone L-PGDS

A positive association of the exposure to different classes of xenobiotics such as commonly prescribed drugs and polycyclic aromatic hydrocarbons (PAH) typically those found in air pollution-related particulate matter with Alzheimers disease (AD) may point to direct physical interaction of those compounds with the amyloid formation and clearance processes. In this study, for the first time, we provide evidence of such interactions for three representative compounds from prescription drugs and air pollution, e.g. anticholinergic drugs Chlorpheniramine, a common antihistamine, and Trazodone, an antidepressant as well as 9,10-PQ, a common PAH anthraquinone abundantly present in diesel exhaust and associated with AD. We demonstrate that these three compounds bind to the lipophilic compound carrier and neuroprotective amyloid beta (A{beta}) chaperone lipocalin-type prostaglandin D synthase (L-PGDS) with high affinity attenuating its neuroprotective chaperone function with Chlorpheniramine exhibiting markedly stronger inhibitory effects. We also show that these compounds directly interact with A{beta}(1-40) increasing the fibrils yield with altered fibril morphology and increased the cytotoxicity of the resulting fibrils. We propose that exposure to some xenobiotics in the peripheral tissues such as gut and lungs might result in the accumulation of these compounds in the brain facilitated by the carrier function of L-PGDS. This might lead to attenuation of its neuroprotective function and direct modification of A{beta} amyloid morphology and cytotoxicity. This hypothesis might provide a mechanistic link between exposure to xenobiotic compounds and the increased risk of Alzheimers disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/920884v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@14a4d92org.highwire.dtl.DTLVardef@12aadeeorg.highwire.dtl.DTLVardef@100e5e1org.highwire.dtl.DTLVardef@1f44b9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Chitin is present within the Ampullae of Lorenzini of chondrichthyan fishes

ABSTRACTChitin is synthesized by a variety of organisms using enzymes called chitin synthases and was recently discovered in a number of aquatic vertebrates. In our ongoing investigations into the presence of vertebrate chitin, we unexpectedly found evidence of the polysaccharide within the electrosensory organs, known as Ampullae of Lorenzini, of diverse chondrichthyan fishes. Experiments with histochemical reagents, chemical analyses, and enzymatic digestions suggested that chitin is a component of the hydrogel filling the structures. Further, in situ hybridization with a sequence from the little skate (Leucoraja erinacea) revealed that chitin synthase expression is localized to cells inside the organs. Collectively, these findings suggest that chondrichthyan fishes endogenously synthesize chitin and beg further investigation into the function of chitin in the electrosensory system.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Regulation of KRAS4A/B splicing in cancer stem cells by the RBM39 splicing complex.

The KRAS oncogene is expressed as major KRAS4B and minor KRAS4A splice isoforms, the distinct functions of which in cancer are unknown. We demonstrate here that KRAS4A is enriched in cancer stem-like cells, and is activated by hypoxia, whereas KRAS4B is more widely expressed and responds to ER stress. Mice completely lacking either isoform are viable but resistant to lung cancer development, as are Kras4A/B double heterozygous mice expressing both isoforms, but in which splice regulation has been uncoupled. Splicing of KRAS4A, but not KRAS4B, in human tumor cells can be inhibited by treatment with the splice inhibitor indisulam, or by CRISPR/Cas inhibition of the RBM39 splicing complex. Our data suggest that control of KRAS4A/B splicing is a targetable vulnerability in KRAS mutant tumors.

cancer biology↗

bioRxiv2019

Comprehensive analysis of the internal structure and firmness in American cranberry (Vaccinium macrocarpon Ait.) fruit

Cranberry (Vaccinium macrocarpon Ait.) fruit quality traits encompass many properties. Among these, fruit firmness has become a quality standard due to the recent demand increase for sweetened and dried cranberries (SDC). Traditionally, this trait has been measured by the cranberry industry using compression tests; however, it is poorly understood how fruit firmness is influenced by other characteristics. In this study, we developed a high-throughput computer-vision method to measure the internal structure of cranberry fruit, which may in turn influence cranberry fruit firmness. We measured the internal structure of 16 cranberry cultivars measured over a 40-day period. Internal structure data paired with fruit firmness values at each evaluation period allowed us to explore the correlations between firmness and internal morphological characteristics.

plant biology↗

bioRxiv2018

Structure and mechanism of TagA, a novel membrane-associated glycosyltransferase that produces wall teichoic acids in pathogenic bacteria

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to {beta}-lactam antibiotics. The membrane-associated TagA glycosyltranserase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzymes lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.\n\nAUTHOR SUMMARYGram-positive bacteria cause thousands of deaths in the United States each year and are a growing health concern because many bacterial strains have become resistant to commonly used antibiotics. One of the most abundant polymers displayed on the surface of Gram-positive bacteria is wall teichoic acid (WTA), a negatively charged carbohydrate polymer that has critical functions in cell division, morphology, adhesion and pathogenesis. The WTA biosynthetic pathway has drawn significant interest as a drug target because clinically important methicillin-resistant S. aureus (MRSA) strains that lack WTA are defective in host colonization and re-sensitized to {beta}-lactam antibiotics. To understand how bacteria produce WTA, we determined the structure and deduced the enzymatic mechanism of TagA, an important enzyme that is required for WTA synthesis. This research reveals a new method for enzyme regulation, whereby peripheral membrane association enables TagA to adopt its active form as a monomer. As TagA enzymes are highly conserved in bacteria, they can be expected to operate through a similar mechanism. The results of this work provide insight into WTA biosynthesis and could lead to innovative approaches to treat infections caused by pathogenic bacteria.

biochemistry↗