Biology subjects

Robinson, K.

Publications and source records attributed to Robinson, K..

29 records · Page 2Linked to original sources

bioRxiv2021

RAGE antagonist peptide mitigates AGE-mediated endothelial hyperpermeability and accumulation of glycoxidation products in human ascending aortas and in a murine model of aortic aneurysm

BackgroundAortic dissection and aneurysm are the result of altered biomechanical forces associated with structural weakening of the aortic wall caused by genetic or acquired factors. Current guidelines recommend replacement of the ascending aorta when the diameter is >5.5 cm in tricuspid aortic valve patients. Aortopathies are associated with altered wall stress and stiffness as well as endothelial cell dysfunction and synthetic vascular smooth muscle cell (VSMC) phenotype. We reported that these mechanisms are mediated by glycoxidation products [Reactive oxygen species (ROS) and Advance Glycation End products (AGE)]. This study addresses the role of glycoxidation on endothelial function and AGE-mediated aortic stiffness. Hypothesis and aimsHere we investigate how circulating glycation products infiltrate the aortic wall via AGE-mediated endothelial hyperpermeability and contribute to both VSMC synthetic phenotype and extracellular matrix (ECM) remodeling in vivo and ex vivo. We also study how RAGE antagonist peptide (RAP) can rescue the effect of AGEs in vitro and in vivo in eNOS-/- vs WT mice. Methods and resultsHuman ascending aortas (n=30) were analyzed for AGE, ROS, and ECM markers. In vitro glycation was obtained by treating VSMC or human and murine aortas with glyoxal. Endothelial permeability was measured under glycation treatment. Vascular stiffness was measured by a pressure myograph comparing wild-type mice {+/-} glyoxal. eNOS-/- mice, a model of increased endothelial permeability, were treated for 28 days with hyperlipidemic diet {+/-} Angiotensin II (1000ng/kg/min) with or without anti-glycation treatment (RAP 20mg/kg). Echo data of aortic diameter were collected. Murine vascular stiffness was measured by a pressure myograph (n=5/group). Glycoxidation products were detected in all human aortas independently of aortic diameter, with stronger accumulation on the lumen and the adventitia layer. AGEs increased endothelial permeability, induce synthetic phenotypic switch in human VSMCs, and inhibit cell migration. RAP pre-treatment rescue the effect of glyoxal on endothelial cells. Ex vivo glycation treatment of murine arteries impacted on ECM and increased stiffness. Aortic stiffness was higher in eNOS-/- vs WT mice. Ang II-mediated aortopathies results in aortic dilation, and AGE/ROS accumulation, which is rescued by RAGE antagonist peptide treatment of eNOS-/- mice. ConclusionsGlycoxidation reaction mediate EC permeability, VSMCs phenotype, and ECM remodeling leading to dysfunctional microstructure of the ascending aorta, altered vascular stiffness and increasing aortic susceptibility to dilation and rupture. Moreover, we show that RAP can mitigate AGE-mediated endothelial hyper-permeability in vitro and impact on ascending aneurysm in vivo

physiology↗

bioRxiv2021

Overground Gait Kinematics and Muscle Activation Patterns in the Yucatan Mini Pig

A growing number of spinal cord injury, neuromodulation, and cell therapy studies on porcine models, especially the Yucatan minipigs (YMPs), have been recently reported. This is due to the large similarities between human and porcine neuroanatomy and biomechanics. To assess treatment modalities and locomotor recovery in this model, there is an obvious need for detailed characterization of normative overground gait in neurologically intact YMPs. The objective of this study was to assess gait biomechanics and the effect of overground walking speed on gait parameters, kinematics, and electromyographic (EMG) activity in the hindlimb muscles of YMPs. Nine neurologically-intact adult YMPs were trained to walk overground in a straight line. Whole-body kinematics and EMG activity of hindlimb muscles were recorded and analyzed at 6 different speed ranges (0.4-0.59, 0.6-0.79, 0.8-0.99, 1.0-1.19, 1.2-1.39, and 1.4-1.6 m/s). A MATLAB program was developed to detect strides and gait events automatically from motion-captured data. Significant decreases in stride duration, stance and swing times and an increase in stride length were observed with increasing speed. A transition in gait pattern occurred at the 1.0m/s walking speed. Significant increases in the range of motion of the knee and ankle joints were observed at higher speeds. Also, the points of minimum and maximum knee and ankle joint angles occurred earlier in the gait cycle at higher speeds. The onset of EMG activity in the biceps femoris muscle occurred significantly earlier in the gait cycle with increasing speed. A comprehensive characterization of overground walking in neurologically-intact YMPs is provided. These normative measures set the basis against which the effects of future interventions on locomotor capacity in YMPs can be compared.

neuroscience↗

bioRxiv2021

Pipeline for generating stable large genomic deletions in zebrafish, from small domains to whole gene excisions

Here we describe a short feasibility study and methodological framework for the production of stable, CRISPR/Cas9-based, large genomic deletions in zebrafish, ranging from several base pairs (bp) to hundreds of kilobases (kb). Using a cocktail of four sgRNAs targeting a single genomic region mixed with a marker-sgRNA against the pigmentation gene tyrosinase (tyr), we demonstrate that one can easily and accurately excise genomic regions such as promoters, protein domains, specific exons or whole genes. We exemplify this technique with a complex gene family, neurexins, composed of three duplicated genes with multiple promoters and intricate splicing processes leading to thousands of isoforms. We precisely deleted small regions such as their transmembrane domains (150bp deletion in average) to their entire genomic locus (300kb deletion for nrxn1A for instance). We find that both the concentration and ratio of Cas9/sgRNAs are critical for the successful generation of these large deletions and, interestingly, that their transmission frequency does not decrease with increasing distance between sgRNA target sites. Considering the growing reports and debate about genetically compensated small indel mutants, the use of large-deletion approaches is likely to be widely adopted in studies of gene function. This strategy will also be key to the study of non-coding genomic regions.

genetics↗

bioRxiv2020

Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

Viruses pose a constant threat to human health. As a result our innate immune system has evolved multiple strategies to detect the presence of intracellular viral pathogen-associated molecular patterns (PAMPs) (1). The full repertoire of human immune sensors and their PAMP ligands are not completely understood. Here we report that human NLRP1 senses and is activated by 3C proteases (3Cpros) of enteroviruses. Mechanistically, 3Cpros cleave human NLRP1 at a single site immediately after its primate-specific PYRIN domain, leading to oligomerization of its C-terminal fragment. Expression of 3Cpros in primary human cells cause NLRP1-dependent ASC oligomerization, pyroptotic cell death and IL-1 secretion. Consistent with our observation that NLRP1 is the predominant endogenous inflammasome sensor in human airway epithelium, we find that its genetic deletion, or that of ASC, abrogates IL-18 secretion from rhinovirus (HRV)-infected primary human bronchial epithelial cells. Our findings identify the first cognate PAMP ligand for human NLRP1 and assign a new function for the NLRP1 inflammasome in human antiviral immunity and airway inflammation. These results challenge the widely held notion that viral proteases largely serve to disable host immune sensing, and suggest that the human NLRP1 inflammasome may be a therapeutic target to treat inflammatory airway diseases including asthma. One Sentence SummaryHuman NLRP1 is activated by enteroviral 3C proteases

immunology↗

bioRxiv2020

Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related NLR proteins, NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, the molecular mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to trigger the assembly of distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core composed of oligomerized CARD domains and the outer layer consisting of FIINDUPA rings. Biochemically, oligomerized NLRP1-CARD is sufficient to drive ASC speck formation in cultured human cells via filament formation-a process that is greatly enhanced by NLRP1-FIINDUPA, which forms ring-like oligomers in vitro. In addition, we report the cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments at 3.7 [A], which uncovers unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide unique structural insight into the mechanisms of activation for human NLRP1 and CARD8, uncovering an unexpected level of specificity in inflammasome signaling mediated by heterotypic CARD domain interactions.

immunology↗

bioRxiv2020

Canine cognitive dysfunction (CCD) patients have reduced total hippocampal volumes compared with aging control dogs: a comparative MRI study

Hippocampal atrophy is a key pathologic and MRI feature of human Alzheimers disease (AD). Hippocampal atrophy has not been documented via MRI in canine cognitive dysfunction (CCD), which is considered the dog model of human AD. The purpose of this retrospective comparative volumetric MRI study was to compare total hippocampal volumes between successfully aging (control) dogs and dogs diagnosed with CCD. Mimics(R) software was used to derive total hippocampal volumes and total brain volumes from the MRI studies of 42 aging dogs ([≥] 9 years): 16 dogs diagnosed with CCD and 26 successfully aging controls. Total hippocampal volume normalized to total brain volume was significantly less for CCD patients compared with control dogs (p=0.04). The results of this study suggest that-similar to human AD-hippocampal atrophy is a pathological feature of CCD. This finding has potential importance for both investigating disease mechanisms related to dementia as well as future hippocampal-targeted therapies.

neuroscience↗

bioRxiv2019

Characterization of GRK5 as a novel regulator of rhabdomyosarcoma tumor cell growth and self-renewal

Rhabdomyosarcoma (RMS) is the most common soft-tissue pediatric sarcoma. Treatment options remain limited, presenting an urgent need for novel therapeutic targets. Using a high-throughput siRNA screen against the human kinome, we identified GRK5, a G-protein receptor kinase, as a novel regulator of RMS tumor cell growth and self-renewal. Through functional assays in vitro and in vivo, we show that GRK5 regulates cell cycling in a kinase-independent manner to promote RMS tumor cell growth. GRK5 interacts with NFAT to facilitate autoregulation of NFAT1 expression in a kinase independent manner, and loss of NFAT1 phenocopies GRK5 loss-of-function effects on cell cycle arrest. Self-renewal of RMS, required for recapitulation of tumor heterogeneity, is significantly reduced with loss of GRK5 due to increased cell death. Treatment of human RMS xenografts in mice with CCG-215022, a GRK5-selective inhibitor, reduces tumor growth of RMS. GRK5 represents a novel therapeutic target for the treatment of RMS. Statement of SignificanceGRK5 promotes growth and self-renewal of RMS, thereby representing a novel therapeutic target for improving survival outcomes of RMS patients. GRK5 regulates RMS tumor cell growth in a kinase-independent manner through direct interaction with NFAT1. This finding promises novel drug design, targeting non-kinase domains of GRK5.

cancer biology↗

bioRxiv2019

HDAC6 promotes self-renewal and migration/invasion of rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a devastating pediatric sarcoma. The survival outcomes remain poor for patients with relapsed or metastatic disease. Effective targeted therapy is lacking due to our limited knowledge of the underlying cellular and molecular mechanisms leading to disease progression. In this study, we used functional assays in vitro and in vivo (zebrafish and xenograft mouse models) to demonstrate the crucial role of HDAC6, a cytoplasmic histone deacetylase, in driving RMS tumor growth, self-renewal and migration/invasion. Treatment with the HDAC6-selective inhibitor, Tubastatin A, recapitulates the HDAC6 loss-of-function phenotypes. HDAC6 regulates cytoskeletal dynamics to promote tumor cell migration and invasion. RAC1, a Rho family GTPase, is an essential mediator of HDAC6 function, and is necessary and sufficient for RMS cell migration and invasion. High expression of RAC1 correlates with poor clinical prognosis in RMS patients. Targeting the HDAC6-RAC1 axis represents a promising therapeutic option for improving survival outcomes of RMS patients.

cancer biology↗

bioRxiv2018

Macronutrients modulate resistance to infection and immunity in Drosophila

Immunity and nutrition are two essential modulators of individual fitness However, while the implications of immune function and nutrition on an individual s lifespan and reproduction are known, the interplay between feeding behaviour, infection, and immune function, remains poorly understood. In this study, we used the fruit fly, Drosophila melanogaster, to investigate how infection through septic injury modulates nutritional intake, and how macronutrient balance affects survival to infection by the pathogenic Gram-positive bacterium Micrococcus luteus. Our results show that infected flies maintain carbohydrate intake, but reduce protein intake, thereby shifting from a protein-to-carbohydrate (P:C) ratio of ~1:4 to ~1:10 relative to non-infected and sham-infected flies. Strikingly, we found that the proportion of flies dying after M. luteus infection was significantly lower when flies were fed a low-P high-C diet, revealing that flies shift their macronutrient intake as means of nutritional self-medication against bacterial infection. This is likely due to the effects of macronutrient balance on the regulation of the constitutive expression of innate immune genes, as a low-P high-C diet was linked to an up-regulation in the expression of key antimicrobial peptides. Together, our results reveal the intricate relationship between macronutrient intake and resistance to infection, and integrate the molecular cross-talk between metabolic and immune pathways into the framework of nutritional immunology.

physiology↗

bioRxiv2018

DNA analysis of a large collection of shark fins from a US retail shop: species composition, global extent of trade and conservation - a Technical Report from the Monterey Bay Aquarium

We identified shark fins sampled across the entirety of a shark fin shop that had operated on the west coast of the United States until 2014. From these specimens we obtained 963 species identifications with Cytochrome oxidase (COI) sequencing and 1,720 identifications with control region (CR) sequences. We found 36-39 distinct species with COI and 38-41 with CR. Of the species identified, 16-23 are currently listed as Endangered or Vulnerable on the IUCN Red List, an additional 2 are considered data deficient, and 7 currently listed under CITES Appendix II. Of the 2.5 tonnes of fins from this collection, we estimated 56-66% (CR or COI, respectively) come from CITES-listed species or those the IUCN considers threatened or data deficient. Most of these species occur outside of the United States EEZ, comprising a global set of species that is common in most fin surveys. The principal target shark fishery in the United States (spiny dogfish; Squalus acanthias) has no fins in our collection. Fins seen abundantly in our collection include pelagic species such as thresher, mako, oceanic whitetip, silky, blue and hammerhead sharks, as in previous samples of the shark fin supply chain. However, in addition, we see a large flood of blacktip, dusky, sandbar, and smalltail sharks that are common in shallow coastal waters. This may indicate that the global market for shark fins takes sharks from nearshore coastal zones, all over the world. Abundant species in the fin shop included globally-distributed species such as scalloped hammerheads and shortfin mako sharks, but also regionally-restricted species such as finetooth, blacknose, and Caribbean Reef sharks found only in the western Atlantic or Caribbean. Specimens identified from rare species of particular conservation concern included the wedgefish genus Rhyncobatus and the white shark. Both molecular markers performed well in identifying most fins, achieving a similar degree of taxonomic certainty. The universal primers for COI regularly amplified bacteria in wet fin samples, but the CR primers were able to return shark sequences even from these degraded samples. However, the CR primers amplified a second gene, likely a pseudogene, in some important and abundant species, and seriously underestimated some species of high conversation concern such as the thresher sharks.

genetics↗

bioRxiv2018

DPP9 is an endogenous and direct inhibitor of the NLRP1 inflammasome that guards against human auto-inflammatory diseases

The inflammasome is a critical immune complex that activates IL-1 driven inflammation in response to pathogen- and danger-associated signals. Nod-like receptor protein-1 (NLRP1) is a widely expressed inflammasome sensor. Inherited gain-of-function mutations in NLRP1 cause a spectrum of human Mendelian diseases, including systemic autoimmunity and skin cancer susceptibility. However, its endogenous regulation and its cognate ligands are still unknown. Here we apply a proteomics screen to identify dipeptidyl dipeptidase, DPP9 as a novel interacting partner and a specific endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP9 inhibition via small molecule drugs, targeted mutations in its catalytic site and CRISPR/Cas9-mediated genetic deletion potently and specifically activate the NLRP1 inflammasome leading to pyroptosis and IL-1 processing via ASC and caspase-1. Mechanistically, DPP9 maintains NLRP1 in its monomeric, inactive state by binding to the auto-cleaving FIIND domain. NLRP1-FIIND is a self-sufficient DPP9 binding module and its disruption by a single missense mutation abrogates DPP9 binding and explains the aberrant inflammasome activation in NAIAD patients with arthritis and dyskeratosis. These findings uncover a unique peptidase enzyme-based mechanism of inflammasome regulation, and suggest that the DPP9-NLRP1 complex could be broadly involved in human inflammatory disorders.

immunology↗