Biology subjects

Fox, J.

Publications and source records attributed to Fox, J..

30 records · Page 2Linked to original sources

bioRxiv2022

Bacterial Microbiome of Foreskin Tissue: Impact of Short-course PrEP and Host Inflammation

As part of the CHAPS randomized clinical trial, we sequenced a segment of the bacterial 16S rRNA gene from foreskin tissue of 144 adolescents from South Africa and Uganda collected during surgical penile circumcision after receipt of 1 to 2 doses of placebo, emtricitabine with tenofovir disoproxil fumarate, or emtricitabine with tenofovir alafenamide. We found a large proportion of Corynebacterium in addition to other anaerobic species. Cutibacterium acnes was more abundant among participants from South Africa than Uganda, though this made no difference in surgical recovery. We did not find a difference in bacterial populations by treatment received nor bacterial taxa that were differentially abundant between participants who received placebo versus active drug. Using RNAseq libraries from foreskin tissue of the same participants, we found negative correlations between the relative abundance of bacterial taxa and the expression of genes downstream of the innate response to bacteria and regulation of the inflammatory response. When participants were divided into clusters based on bacterial community composition, two main clusters emerged which were distinguished by high and low bacterial diversity. Random forest classification showed higher expression of NFATC3 and SELENOS and lower expression of STAP1 and NLRP6 in the higher diversity group compared to the lower. Our results show no difference in the tissue microbiome of the foreskin with short-course PrEP but that bacterial taxa were largely inversely correlated with gene expression, consistent with non-inflammatory colonization. Author SummaryWe investigated the bacterial community of the foreskin of the penis. Previous studies found increased inflammation with certain anaerobic bacteria from swabs taken under the foreskin, but we found that higher relative abundances of the bacteria were correlated with lower expression of inflammatory genes. We did not find different bacteria in participants who received medicine to prevent HIV. Understanding the relationship between bacteria and inflammation in the penis will help us to understand how interventions like penile circumcision reduce the risk of acquiring sexually transmitted infections such as HIV.

microbiology↗

bioRxiv2022

Allosteric Inhibition of PTP1B by a Nonpolar Terpenoid

Protein tyrosine phosphatases (PTPs) are promising drug targets for treating a wide range of diseases such as diabetes, cancer, and neurological disorders, but their conserved active sites have complicated the design of selective therapeutics. This study examines the allosteric inhibition of PTP1B by amorphadiene (AD), a terpenoid hydrocarbon that is an unusually selective inhibitor. Molecular dynamics (MD) simulations carried out in this study suggest that AD can stably sample multiple neighboring sites on the allosterically influential C-terminus of the catalytic domain. Binding to these sites requires a disordered 7 helix, which stabilizes the PTP1B-AD complex and may contribute to the selectivity of AD for PTP1B over TCPTP. Intriguingly, the binding mode of AD differs from that of the most well-studied allosteric inhibitor of PTP1B. Indeed, biophysical measurements and MD simulations indicate that the two molecules can bind simultaneously. Upon binding, both inhibitors destabilize the 7 helix and disrupt hydrogen bonds that facilitate closure of the catalytically essential WPD loop. These findings indicate that AD is a promising scaffold for building allosteric inhibitors of PTP1B and illustrate, more broadly, how unfunctionalized terpenoids can engage in specific interactions with protein surfaces.

biochemistry↗

bioRxiv2021

ChAdOx1 nCoV-19 (AZD1222) vaccine elicits monoclonal antibodies with potent cross-neutralizing activity against SARS-CoV-2 viral variants

Although the antibody response to COVID-19 vaccination has been studied extensively at the polyclonal level using immune sera, little has been reported on the antibody response at the monoclonal level. Here we isolate a panel of 44 anti-SARS-CoV-2 monoclonal antibodies (mAbs) from an individual who received two doses of the ChAdOx1 nCoV-19 (AZD1222) vaccine at a 12-week interval. We show that despite a relatively low serum neutralization titre, mAbs with potent neutralizing activity against the current SARS-CoV-2 variants of concern (B.1.1.7, P.1, B.1.351 and B.1.617.2) were obtained. The vaccine elicited neutralizing mAbs form 8 distinct competition groups and bind epitopes overlapping with neutralizing mAbs elicited following SARS-CoV-2 infection. AZD1222 elicited mAbs are more mutated than mAbs isolated from convalescent donors 1-2 months post infection. Spike reactive IgG+ B cells were still detectable 9-months post boost. These findings give molecular insights into AZD1222 elicited antibody response.

immunology↗

bioRxiv2021

Surface GluA1 and glutamate transmission are increased in VPS35 D620N neurons and altered by LRRK2 kinase inhibition

Vacuolar protein sorting 35 (VPS35) regulates receptor recycling from endosomes. A missense mutation in VPS35 (D620N) leads to autosomal-dominant, late-onset Parkinsons disease. Here, we use a VPS35 D620N knock-in mouse to study the neurobiology of this mutation. In brain tissue, we confirm previous findings that the mutation results in reduced binding of VPS35 with WASH-complex member FAM21, and robustly elevated phosphorylation of the LRRK2 kinase substrate Rab10. In cultured cortical neurons, the mutation results in increased endosomal recycling protein density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate release, and GluA1 surface expression. LRRK2 kinase inhibition exerted genotype-specific effects on GluA1 surface expression, but did not impact glutamate release phenotypes. These results improve our understanding of the early effects of the D620N mutation on cellular functions that are specific to neurons. These observations provide candidate pathophysiological pathways that may drive eventual transition to late-stage parkinsonism in VPS35 families, and support a synaptopathy model of neurodegeneration.

neuroscience↗

bioRxiv2020

Clinical positioning of the IAP antagonist tolinapant (ASTX660) in colorectal cancer

Cancer cells frequently express elevated levels of Inhibitor of Apoptosis Proteins (IAPs): cIAPI, cIAP2 and XIAP. Elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in microsatellite stable (MSS) stage-III colorectal cancer (CRC) patients treated with adjuvant chemotherapy, suggesting their involvement in promoting resistance. Preclinical analysis of the IAP inhibitor tolinapant in CRC cell lines demonstrated robust on-target effects and caspase-8-dependent apoptosis that was inhibited by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinipant-induced apoptosis was augmented by standard-of-care chemotherapy (FOLFOX) in CRC disease models, due (at least in part) to FOLFOX-induced downregulation of Class-I histone deacetylases, leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, this effect could be phenocopied using a Class-I HDAC inhibitor. Further analyses revealed that caspase-8-knockout RIPK3-positive CRC models were sensitive to tolinostat-induced necroptosis, an effect that could be exploited with the FDA-approved caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX chemotherapy in poor prognosis MSS CRC with elevated cIAP1/2 expression.

cancer biology↗

bioRxiv2019

A data-based guide to the North American ecology faculty job market

Every year for three years (2016 to 2018), I tried to identify every single person hired as a tenure track prof in ecology or an allied field (e.g., fish & wildlife) in N. America. I identified a total of 566 hires. I used public sources to compile various data on the new hires and the institutions that hired them (e.g., number of publications, teaching experience, hiring institution Carnegie class). I also compiled data provided by anonymous ecology faculty job seekers on ecoevojobs.net (e.g., number of positions applied for, number of publications, numbers of interviews and offers). And I polled readers of the Dynamic Ecology blog to get information about applicant and search committee behavior (e.g., regarding customization of applications to the hiring institution). These data address some widespread anxieties and misunderstandings about the ecology faculty job market, and also speak to gender diversity and equity in recent ecology faculty hiring. They complement, and in some cases improve on, other sources of information, such as anecdotal personal experiences.

scientific communication and education↗

bioRxiv2019

Regulation of FLIP(L) and TRAIL-R2 signalling by the SCFSkp2 Ubiquitin Ligase Complex

Depending on its expression levels, the long splice form of the pseudo-caspase FLIP (FLIP(L)) can act as an inhibitor (high expression) or activator (low expression) of apoptosis induction by the TRAIL-R2 death-inducing signalling complex (DISC); its expression levels are therefore tightly regulated. Here, we demonstrate that the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2) regulates the stability of FLIP(L) (but not the short splice form FLIP(S)), and, unusually, this is mediated by direct binding of FLIP(L) to Cullin-1 rather than via Skp2. By fine mapping the interaction of FLIP(L) with Cullin-1 to the large subunit of its pseudo-caspase domain, we found that the interaction is significantly stronger with FLIP(L)s DISC-processed p43-form. Importantly, this interaction disrupts the ability of p43-FLIP to interact with FADD, caspase-8 and another DISC component, TRAF2. Moreover, we find that SCFSkp2 associates with TRAIL-R2 constitutively and does so independently of FLIP(L) and other canonical DISC components. Inhibition of Cullin-1 expression (using siRNA) or activity (using a NEDDylation inhibitor, MLN4924) enhanced FLIP(L) and TRAF2 levels at the TRAIL-R2 DISC and enhanced caspase-8 processing. This suggests that processing of FLIP(L) to p43-FLIP at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIPs association with the DISC thereby altering caspase-8 processing. These findings provide important new insights into how FLIP(L) expression and TRAIL-R2 signaling is controlled.

molecular biology↗

bioRxiv2019

HIV reservoir size is determined prior to ART initiation and linked to CD8 T cell activation and memory expansion

Initiation of antiretroviral therapy (ART) in early compared with chronic HIV infection is associated with a smaller HIV reservoir. This longitudinal analysis of 63 individuals who commenced ART during primary HIV infection (PHI) investigates which pre-and post-therapy factors associate most closely with reservoir size (HIV DNA) following treatment initiation during PHI. The best predictor of reservoir size at one-year was pre-ART HIV DNA which was in turn significantly associated with CD8 memory differentiation (effector memory, naive and T-betnegEomesneg subsets), CD8 T cell activation (CD38 expression) and PD-1 and Tim-3 expression on memory CD4 T cells. No associations were found for any immunological variables following one-year of ART. HIV reservoir size is determined around the time of ART initiation in individuals treated during PHI. CD8 T cell activation and memory expansion are linked to HIV reservoir size, suggesting the importance of the initial host-viral interplay in eventual reservoir size.

immunology↗

bioRxiv2019

Latent Toxoplasma gondii infection increases soluble mutant huntingtin and promotes neurodegeneration in the YAC128 mouse model of Huntington's disease

Toxoplasma gondii causes a prevalent neuroinvasive protozoal pathogen that in immune competent individuals results in latent infection characterized by intra-cellular parasite cysts in brain. Despite life-long infection, the role of latent toxoplasmosis on chronic neurodegenerative processes is poorly understood. Huntingtons disease (HD) is a progressive neurodegenerative disorder caused by a dominant CAG repeat expansion in the huntingtin gene (HTT) that results in the expression and accumulation of mutant huntingtin protein (mHTT). The mutant HD gene is fully penetrant. However, there is significant variability in disease progression that is in part explained by as yet unidentified environmental factors. The kynurenine pathway of tryptophan metabolism (KP) is an inflammatory pathway and its activation is implicated in HD pathogenesis. KP upregulation also occurs in response to infection with Toxoplasma gondii suggesting that the latent infection may promote HD. We discovered that mice on the FVB/NJ background develop latent toxoplasmosis following infection with the ME49 strain of T. gondii. This finding enabled us to address the hypothesis that latent toxoplasmosis potentiates disease in the YAC128 mouse model of HD, as these mice are maintained on the FVB/NJ background. Wild-type and HD mice were infected at 2-months of age. During the 10-month follow-up, infection had adverse effects on mice of both genotypes. However, YAC128 HD mice demonstrated specific vulnerability to latent toxoplasmosis, as demonstrated by the presence of increased striatal degeneration, high levels of the blood neurodegeneration marker neurofilament light protein, and elevated brain soluble mHTT. Our studies have uncovered a novel HD-infection interaction in mice that provides insights into the large variability of the human HD phenotype.

neuroscience↗

bioRxiv2019

Iron activates microglia and directly stimulates indoleamine-2,3-dioxygenase activity in the N171-82Q mouse model of Huntington's disease

Huntingtons disease (HD) is a neurodegenerative disorder caused by a dominant CAG-repeat expansion in the huntingtin gene. Morphologic activation of microglia is a key marker of neuroinflammation that is present before clinical onset in HD patients. The kynurenine pathway of tryptophan degradation is restricted in part to microglia and is activated in HD, where it contributes to disease progression. Indoleamine-2,3-dioxygenase (IDO) is a microglial enzyme that catalyzes the first step in this pathway. HD brain microglial cells also accumulate iron; however, the role of iron in promoting microglial activation and the kynurenine pathway is unclear. Based on analyses of morphological characteristics of microglia, we showed that HD mice demonstrate an activated microglial morphology compared with controls. Neonatal iron supplementation resulted in additional microglial morphology changes compared with HD controls. Increased microglial activation in iron-supplemented HD mice was indicated by increased soma volume and decreased process length. In our assessment of whether iron can affect the kynurenine pathway, iron directly enhanced the activity of human recombinant IDO1 with an EC50 of 1.24 nM. We also detected elevated microglial cytoplasmic labile iron in N171-82Q HD mice, an increase that is consistent with the cellular location of IDO. We further demonstrated that neonatal iron supplementation, a model for studying the role of iron in neurodegeneration, activates IDO directly in the mouse brain and promotes neurodegeneration in HD mice. Kynurenine pathway metabolites were also modified in HD and by iron supplementation in wild-type mice. These findings indicate that iron dysregulation contributes to the activation of microglia and the kynurenine pathway in a mouse model of HD.

neuroscience↗

bioRxiv2019

Mutant huntingtin protein alters the response of microglial cells to inflammatory stimuli

Huntingtons disease (HD) is a progressive neurodegenerative disease that affects the striatum and cerebral cortex. It is caused by a dominant CAG trinucleotide expansion in exon 1 of the HTT gene. Mutant huntingtin protein (mHtt) is expressed in neurons and immune cells. HD patients demonstrate altered blood cytokine profiles and altered responses of peripheral immune cells to inflammatory stimuli. However, the effects of mHtt on microglial immune responses are not fully understood. Herein we discuss the current understanding of how mHtt alters microglial inflammatory responses. Using lentivirus, we expressed the N171 N-terminal fragment of wild-type or mhtt containing 18 and 82 glutamine repeats in cultured EOC-20 microglial cells. We then measured responses to lipopolysaccharide or interleukin-6. Mutant huntingtin-expressing microglial cells produced less interleukin-6 and nitric oxide in response to lipopolysaccharide stimulation than wild-type huntingtin-expressing cells. However, mHtt-expressing microglia stimulated with interleukin-6 produced more nitric oxide than wild-type cells. Mutant huntingtin-expressing cells had higher basal NF-{kappa}B and further elevations of NF-{kappa}B after interleukin-6 but not lipopolysaccharide stimulation. Thus we demonstrate the potential of mHtt to dampen responses to lipopolysaccharide but potentiate responses to interleukin-6. This work adds to the emerging understanding that mHtt alters not only baseline status of cells but may also result in altered immune responses dependent on the nature of the inflammatory stimuli. We also present our perspective that in human HD the extent of inflammation may depend, in part, on altered responses to varied inflammatory stimuli including environmental factors such as infection.

neuroscience↗

bioRxiv2017

Enrichment of the HIV reservoir in CD32+ CD4 T cells occurs early in blood and tissue

The Fc receptor CD32 has been proposed as a marker for CD4 T cells latently infected with HIV. We demonstrate that enrichment for HIV DNA in CD32+ CD4 T cells can be found early in infection in both tissue and blood. However, we find no evidence for a correlation between CD32 expression on CD4 T cells and either HIV DNA levels or time to rebound viraemia following treatment interruption. CD32+ CD4 T cells have a more differentiated memory phenotype, and high levels of expression of immune checkpoint receptors PD-1, Tim-3 and TIGIT as well as the activation marker, HLA DR. There was no difference in the phenotype or frequency of CD32 expressing cells prior to or after the initiation of antiretroviral therapy, or compared with healthy controls, suggesting that preferential infection or survival, rather than up-regulation, may be responsible for the observed enrichment of proviral HIV DNA in CD32+ CD4 T cells.

immunology↗