Biology subjects

Skinner, J.

Publications and source records attributed to Skinner, J..

12 recordsLinked to original sources

bioRxiv2025

Human resistin is critical to activation of the NLRP3 inflammasome in macrophages

Elevated levels of human resistin (hResistin) have been associated with diverse inflammatory diseases, but the precise mechanisms through which hResistins many inflammatory effects contribute to the progression of these diseases remain poorly understood. NLRP3 inflammasome activation is essential in many of these inflammatory conditions; however, there is an unmet explanation connecting hResistin with the NLRP3 inflammatory pathway. Here we describe a unique role of hResistin and its rodent homolog, resistin-like molecule alpha (RELM) in priming and activating the NLRP3 inflammasome. Through qPCR and western blot analysis, we found that hResistin-dependent expression and secretion of high mobility group box 1 (HMGB1) in human macrophages primes the expression of NLRP3, pro-capsase-1, pro-interleukin(IL)-1{beta}, and pro-IL-18. Co-immunoprecipitation showed that hResistin binds to Brutons tyrosine kinase (BTK), which causes the kinase to autophosphorylate, allowing BTK to phosphorylate NLRP3, leading to its assembly and activation with subsequent cleavage of pro-caspase-1, pro-IL-1{beta}, and pro-IL-18, causing initiation of the inflammasome cascade. The hResistin-dependent activation and secretion of IL-1{beta} and IL-18 was critical to the proliferation of human pulmonary vascular smooth muscle cells. For confirmation in vivo, we studied rodent and human pulmonary hypertension (PH). Chronic hypoxia-induced PH in wild-type and RELM KO mice showed RELM-dependent upregulation of HMGB1, BTK, and NLRP3 in mouse lung and was linked to vascular remodeling pathways. Immunohistochemistry revealed that the majority of NLRP3-expressing cells were macrophages and the colocalization of hResistin, BTK, and NLRP3 in macrophages was increased in PH patients lungs. Our work reveals a novel immune mechanism demonstrating hResistin is essential to the priming and activation of NLRP3. Inhibiting NLRP3 activation by blocking hResistin with a human monoclonal antibody suggests a likely therapeutic pathway for NLRP3-driven inflammatory diseases.

immunology↗

bioRxiv2024

Metabolic and neuroactivity imbalances in plasma from aniridia patients with PAX6 haploinsufficiency

PAX6 is a transcription factor crucial for the development of the eye, pancreas, and brain. Heterozygous variants resulting in PAX6 haploinsufficiency are the main genetic cause of congenital aniridia, characterized by both anterior and posterior ocular defects and sight loss. The extra-ocular features of PAX6 haploinsufficiency are becoming more widely recognised, with systemic manifestations like obesity, diabetes, and neurological/behavioural disorders being reported. In this study, we uncovered the metabolomic profile of the blood plasma from 25 PAX6-related aniridia patients compared to gender and age-matched controls. We found significant disruptions in lipid and energy metabolism, increased oxidative stress and neurotransmitters imbalances, as well as alterations linked to the gut microbiome. This study identified novel metabolic changes associated with PAX6 haploinsufficiency, providing evidence for the systemic aetiology of congenital aniridia and emphasizing the need for multidisciplinary management and further exploration into ocular and systemic therapeutic approaches.

genetics↗

bioRxiv2024

MAARU: Multichannel Acoustic Autonomous Recording Unit for spatial ecosystem monitoring.

1.1 - Acoustic localisation, which relies on simultaneous multi-microphone recording, adds spatial information to recorded audio and has been used in ecosystem monitoring to count individuals to improve abundance estimates, locate illegal activities such as logging/poaching, and monitor behaviour such as habitat use or species interactions. Studies have shown many advantages of acoustic localisation, but uptake remains limited as the equipment is often expensive, inaccessible, or only suitable for short-term deployments. 2 -Here, we present a low-cost, open-source, 6-channel recorder built entirely from commercially available components which can be integrated into a solar-powered, networked system. The MAARU (Multichannel Acoustic Autonomous Recording Unit) works in long-term autonomous, passive, and ad-hoc deployments. We introduce MAARUs hardware and software and present the results of lab and field tests investigating the devices durability, localisation accuracy, and other applications. 3 -MAARU provides multichannel data with similar costs and power demands to equivalent omnidirectional recorders. MAARU devices have been deployed in the UK and Brazil, where we have shown MAARUs can accurately localise pure tones up to 6kHz and 65dB bird calls as far as 8m away ({+/-}10{degrees} range, 100% and >60% of signals respectively), louder calls may have even further detection radii. We also show how beamforming can be used on MAARU devices to improve species ID confidence scores by 20%+ and recall by 10%+ when using the BirdNET automated bird identification algorithms. 4 -MAARU is an accessible, low-cost option for those looking to explore spatial soundscape ecology accurately and easily. Ultimately, the added directional element of the multichannel recording provided by MAARU allows for a new type of exploration into sonic environments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/576628v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1078827org.highwire.dtl.DTLVardef@17bd5b1org.highwire.dtl.DTLVardef@10a6696org.highwire.dtl.DTLVardef@1d518f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2022

Broadly neutralizing antibodies target the coronavirus fusion peptide

The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2 cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2 cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development. One-Sentence SummaryRare monoclonal antibodies from COVID-19 convalescent individuals broadly neutralize coronaviruses by targeting the fusion peptide.

immunology↗

bioRxiv2021

Atypical B cells upregulate co-stimulatory molecules during malaria and secrete antibodies with T follicular helper cell support

Several infectious and autoimmune diseases are associated with an expansion of CD21-CD27- atypical B cells (atBCs). The function of atBCs remains unclear and few studies have investigated the biology of pathogen-specific atBCs during acute infection. Here, we performed longitudinal RNA-sequencing and flow cytometry analyses of Plasmodium falciparum (Pf)-specific B cells before and shortly after febrile malaria, with simultaneous analysis of influenza hemagglutinin (HA)-specific B cells as a comparator. B cell receptor-sequencing showed that Pf-specific atBCs, activated B cells (actBCs) and classical memory B cells share clonality and have comparable somatic hypermutation. In response to malaria, Pf-specific atBCs and actBCs expanded and upregulated molecules that mediate B-T cell interactions, suggesting that atBCs respond to T follicular helper (Tfh) cells. Indeed, in the presence of Tfh cells and Staphylococcal enterotoxin B, atBCs of malaria-exposed individuals differentiated into CD38+ antibody-secreting cells in vitro, suggesting that atBCs may actively contribute to humoral immunity to infectious pathogens. One Sentence SummaryThis study shows that atypical B cells actively respond to acute malaria and have the capacity to produce antibodies with T cell help.

immunology↗

bioRxiv2021

Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

immunology↗

bioRxiv2021

Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice

BackgroundRigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART)-cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII). MethodsWe first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T-cells and evaluated the function of the CART-cells both in vitro and in vivo. In order to inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208). ResultsBoth RV- and Tg-CART-cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART-cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28. ConclusionOur new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies. Importance of studyThe majority of preclinical studies evaluating CART therapy for GBM have utilized xenografts implanted into immunocompromised mice. Because the successful development of these strategies will depend on the understanding of critical interactions between therapeutic cells and the endogenous immune environment, it is essential to develop a novel immunocompetent system which allows us to study these interactions in a robust and reproducible manner. To this end, we created a Tg mouse strain in which all T-cells express a murinized EGFRvIII-CAR. T-cells derived from these mice demonstrated consistent CAR expression and EGFRvIII-specific cytotoxicity while traditional transduction with a CAR vector showed batch-to-batch variability. The syngeneic system also gave us the opportunity to evaluate a combination regimen with blockade of myeloid-derived suppressor cells. The Tg-CART mice represent a novel system for robust, and reproducible preclinical investigations.

immunology↗

bioRxiv2020

Plasmodium falciparum malaria drives epigenetic reprogramming of human monocytes toward a regulatory phenotype

In malaria-naive children and adults, Plasmodium falciparum-infected red blood cells (Pf-iRBCs) trigger fever and other symptoms of systemic inflammation. However, in endemic areas where individuals experience repeated Pf infections over many years, the risk of Pf-iRBC-triggered inflammatory symptoms decreases with cumulative Pf exposure. The molecular mechanisms underlying these clinical observations remain unclear. Age-stratified analyses of monocytes collected from uninfected, asymptomatic Malian individuals before the malaria season revealed an inverse relationship between age and Pf-iRBC-inducible inflammatory cytokine (IL-1{beta}, IL-6 and TNF) production, whereas Malian infants and malaria-naive U.S. adults produced similarly high levels of inflammatory cytokines. Accordingly, monocytes of Malian adults produced more IL-10 and expressed higher levels of the regulatory molecules CD163, CD206, Arginase-1 and TGM2. These observations were recapitulated in an in vitro system of monocyte to macrophage differentiation wherein macrophages re-exposed to Pf-iRBCs exhibited attenuated inflammatory cytokine responses and a corresponding decrease in the epigenetic marker of active gene transcription, H3K4me3, at inflammatory cytokine gene loci. Together these data indicate that Pf induces epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype that attenuates inflammatory responses during subsequent Pf exposure. These findings also suggest that past malaria exposure could mitigate monocyte-associated immunopathology induced by other pathogens such as SARS-CoV-2. Author SummaryThe malaria parasite is mosquito-transmitted and causes fever and other inflammatory symptoms while circulating in the bloodstream. However, in regions of high malaria transmission the parasite is less likely to cause fever as children age and enter adulthood, even though adults commonly have malaria parasites in their blood. Monocytes are cells of the innate immune system that secrete molecules that cause fever and inflammation when encountering microorganisms like malaria. Although inflammation is critical to initiating normal immune responses, too much inflammation can harm infected individuals. In Mali, we conducted a study of a malaria-exposed population from infants to adults and found that participants monocytes produced less inflammation as age increases, whereas monocytes of Malian infants and U.S. adults, who had never been exposed to malaria, both produced high levels of inflammatory molecules. Accordingly, monocytes exposed to malaria in the laboratory became less inflammatory when re-exposed to malaria again later, and these monocytes turned down their inflammatory genes. This study helps us understand how people become immune to inflammatory symptoms of malaria and may also help explain why people in malaria-endemic areas appear to be less susceptible to the harmful effects of inflammation caused by other pathogens such as SARS-CoV-2.

immunology↗

bioRxiv2020

Plasmodium falciparum-specific IgM B cells dominate in children, expand with malaria and produce parasite inhibitory IgM

IgG antibodies are known to play a central role in naturally acquired immunity to blood-stage malaria in humans, but little is known about the IgM response to blood-stage malaria, the mechanisms by which IgM may protect, or the underlying biology of Plasmodium falciparum (Pf)-specific IgM B cells. In a Mali cohort spanning infants to adults we conducted a longitudinal analysis of B cells specific for the Pf blood-stage antigens AMA1 and MSP1, as well as the comparator antigen influenza hemagglutinin (HA). At the uninfected baseline, before the malaria season, Pf-specific memory B cells (MBCs) in children are disproportionally IgM+ and only gradually shift to IgG+ with age, in contrast to HA-specific MBCs that are predominantly IgG+ from infancy to adulthood. In response to acute febrile malaria, Pf-specific IgM B cells increase in frequency and upregulate activation and co-stimulatory markers. B cell receptor (BCR) analysis showed that Pf-specific IgM B cells are somatically hypermutated at levels comparable to HA-specific IgG B cells. Finally, IgM antibodies from the plasma of malaria-exposed individuals were comparable to IgG in inhibiting Pf blood-stage growth in vitro, and significantly better at enhancing phagocytosis of Pf merozoites, suggesting that IgM may protect through both direct neutralization and opsonization. Thus, somatically hypermutated Pf-specific IgM MBCs dominate in early life, are activated and expand during acute malaria and are associated with plasma IgM that inhibits parasite growth in vitro.

immunology↗

bioRxiv2018

Eye-position error influence over \"open-loop\" smooth pursuit initiation

The oculomotor system integrates a variety of visual signals into appropriate motor plans, but such integration can have widely varying time scales. For example, smooth pursuit eye movements to follow a moving target are slower and longer-lasting than saccadic eye movements, and it has been suggested that initiating a smooth pursuit eye movement involves an obligatory open-loop interval, in which new visual motion signals presumably cannot influence the ensuing motor plan for up to 100 ms after movement initiation. However, this view runs directly contrary to the idea that the oculomotor periphery has privileged access to short-latency visual signals. Here we show that smooth pursuit initiation is sensitive to visual inputs, even in \"open-loop\" intervals. We instructed male rhesus macaque monkeys to initiate saccade-free smooth pursuit eye movements, and we injected a transient, instantaneous eye position error signal at different times relative to movement initiation. We found robust short-latency modulations in eye velocity and acceleration, starting only [~]50 ms after transient signal occurrence, and even during \"open-loop\" pursuit initiation. Critically, the spatial direction of the injected position error signal had predictable effects on smooth pursuit initiation, with forward errors increasing eye acceleration and backwards errors reducing it. Catch-up saccade frequencies and amplitudes were also similarly altered [~]50 ms after transient signals, much like well-known effects on microsaccades during fixation. Our results demonstrate that smooth pursuit initiation is highly sensitive to visual signals, and that catch-up saccade generation is reset after a visual transient.

neuroscience↗

bioRxiv2018

The transfer function of the rhesus macaque oculomotor system for small-amplitude slow motion trajectories

Two main types of small eye movements occur during gaze fixation: microsaccades and slow ocular drifts. While microsaccade generation has been relatively well-studied, ocular drift control mechanisms are unknown. Here we explored the degree to which monkey smooth eye movements, on the velocity scale of slow ocular drifts, can be generated systematically. Two male rhesus macaque monkeys tracked a spot moving sinusoidally, but slowly, along the horizontal or vertical directions. Maximum target displacement in the motion trajectory was 30 min arc (0.5 deg), and we varied the temporal frequency of target motion from 0.2 to 5 Hz. We obtained an oculomotor \"transfer function\" by measuring smooth eye velocity gain (relative to target velocity) as a function of frequency, similar to past work with large-amplitude pursuit. Monkey eye velocities as slow as those observed during slow ocular drifts were clearly target-motion driven. Moreover, like with large-amplitude smooth pursuit, eye velocity gain varied with temporal frequency. However, unlike with large-amplitude pursuit, exhibiting low-pass behavior, small-amplitude motion tracking was band-pass with the best ocular movement gain occurring at ~0.8-1 Hz. When oblique directions were tested, we found that the horizontal component of pursuit gain was larger than the vertical component. Our results provide a catalogue of the control abilities of the monkey oculomotor system for slow target motions, and they also support the notion that smooth fixational ocular drifts are controllable. This has implications for neural investigations of drift control and the image-motion consequences of drifts on visual coding in early visual areas.

neuroscience↗

bioRxiv2017

Serial Crystallography with Multi-stage Merging of 1000’s of Images

KAMO and Blend provide particularly effective tools to manage automatically the merging of large numbers of datasets from serial crystallography. The requirement for manual intervention in the process can be reduced by extending Blend to support additional clustering options such as use of more accurate cell distance metrics and use of reflection-intensity correlation coefficients to infer "distances" among sets of reflec- tions. This increases the sensitivity to differences in unit cell parameters and allows for clustering to assemble nearly complete datasets on the basis of intensity or ampli- tude differences. If datasets are already sufficiently complete to permit it, one applies KAMO once and clusters the data using intensities only. If starting from incomplete datasets, one applies KAMO twice, first using cell parameters. In this step we use either the simple cell vector distance of the original Blend, or we use the more sensi- tive NCDist. This step tends to find clusters of sufficient size so that, when merged, each cluster is sufficiently complete to allow reflection intensities or amplitudes to be compared. One then uses KAMO again using the correlation between the reflections having a common hkl to merge clusters in a way sensitive to structural differences that may not have perturbed the cell parameters sufficiently to make meaningful clusters. Many groups have developed effective clustering algorithms that use a measurable physical parameter from each diffraction still or wedge to cluster the data into cate- gories which then can be merged, one hopes, to yield the electron density from a single protein form. Since these physical parameters are often largely independent from one another, it should be possible to greatly improve the efficacy of data clustering software by using a multi-stage partitioning strategy. Here, we have demonstrated one possible approach to multi-stage data clustering. Our strategy is to use unit-cell clustering until merged data is sufficiently complete then to use intensity-based clustering. We have demonstrated that, using this strategy, we are able to accurately cluster datasets from crystals that have subtle differences.

bioinformatics↗