Biology subjects

Smith, C.

Publications and source records attributed to Smith, C..

At least 163 records · Page 9Linked to original sources

bioRxiv2021

Efficacy of a Broadly Neutralizing SARS-CoV-2 Ferritin Nanoparticle Vaccine in Nonhuman Primates

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates (NHPs). High-dose (50 {micro}g) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response upon high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. SpFNs potent and broad immunogenicity profile and resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses. One-Sentence SummaryA SARS-CoV-2 Spike protein ferritin nanoparticle vaccine, co-formulated with a liposomal adjuvant, elicits broad neutralizing antibody responses that exceed those observed for other major vaccines and rapidly protects against respiratory infection and disease in the upper and lower airways and lung tissue of nonhuman primates.

immunology↗

bioRxiv2021

Expression of CD49f defines subsets of human regulatory T cells with divergent transcriptional landscape and function that correlate with ulcerative colitis disease activity

ObjectiveAdoptive regulatory T cell (Treg) therapy is being trialled for treatment of different autoimmune disorders, including inflammatory bowel diseases (IBD). In-depth understanding of the biological variability of Treg in the human blood may be required to improve IBD immune monitoring and treatment strategies. MethodsThrough a combination of quantitative proteomic, multiparametric flow cytometry, RNA-sequencing data analysis and functional assays on Treg enriched from the blood of ulcerative colitis (UC) patients and healthy controls, we investigated the association between CD49f expression, Treg phenotype and function, and UC disease activity. ResultsHigh-dimensional analysis and filtering defined two distinct subsets of human Treg based on the presence or absence of CD49f with divergent transcriptional landscape and functional activities. CD49f negative Treg are enriched for functional Treg markers and present significantly increased suppressive capacity. In contrast, CD49f high Treg display a pro-inflammatory Th17-like phenotype and accumulate in the blood of UC patients. Dysregulation on CD49f Treg subsets in UC patients correlate with disease activity. ConclusionOverall our findings uncover the importance of CD49f expression on Treg in physiological immunity and in pathological autoimmunity.

immunology↗

bioRxiv2021

TMEM97 increases in synapses and is a potential synaptic Aβ binding partner in human Alzheimer's disease

Synapse loss correlates with cognitive decline in Alzheimers disease, and soluble oligomeric amyloid beta is implicated in synaptic dysfunction and loss. An important knowledge gap is the lack of understanding of how amyloid beta leads to synapse degeneration. In particular, there has been difficulty in determining whether there is a synaptic receptor that binds amyloid beta and mediates toxicity. While many candidates have been observed in model systems, their relevance to human AD brain remains unknown. This is in part due to methodological limitations preventing visualization of amyloid beta binding at individual synapses. To overcome this limitation, we combined two high resolution microscopy techniques: array tomography and Forster resonance energy transfer (FRET) to image over 1 million individual synaptic terminals in temporal cortex from AD (n=9) and age matched control cases (n=6). Within postsynaptic densities, amyloid beta generates a FRET signal with transmembrane protein 97, cellular prion protein, and postsynaptic density 95. Transmembrane protein 97 is also present in a higher proportion of postsynapses in Alzheimers brain compared to controls. Further, we inhibited amyloid beta / transmembrane protein 97 interaction in a mouse model of amyloidopathy by treating with the an allosteric modulator CT1812 or vehicle. CT1812 drug concentration correlated negatively with synaptic FRET signal between transmembrane protein 97 and amyloid beta. In human induced pluripotent stem cell derived neurons challenged with human Alzheimers brain homogenate, transmembrane protein 97 and amyloid beta are present in synapses. Transcriptional changes are induced by A{beta} including changes in genes involved in neurodegeneration and neuroinflammation. CT1812 treatment of these neurons caused changes in gene sets involved in synaptic function. These data support a role for transmembrane protein 97 in the synaptic binding of amyloid beta in human Alzheimers disease brain where it may mediate synaptotoxicity.

neuroscience↗

bioRxiv2020

A comparison of blood and brain-derived ageing and inflammation-related DNA methylation signatures and their association with microglial burdens

Inflammation and ageing-related DNA methylation patterns in the blood have been linked to a variety of morbidities, including cognitive decline and neurodegenerative disease. However, it is unclear how these blood-based patterns relate to patterns within the brain, and how each associates with central cellular profiles. In this study, we profiled DNA methylation in both the blood and in five post-mortem brain regions (BA17, BA20/21, BA24, BA46 and hippocampus) in 14 individuals from the Lothian Birth Cohort 1936. Microglial burdens were additionally quantified in the same brain regions. DNA methylation signatures of five epigenetic ageing biomarkers ( epigenetic clocks), and two inflammatory biomarkers (DNA methylation proxies for C-reactive protein and interleukin-6) were compared across tissues and regions. Divergent correlations between the inflammation and ageing signatures in the blood and brain were identified, depending on region assessed. Four out of the five assessed epigenetic age acceleration measures were found to be highest in the hippocampus ({beta} range=0.83-1.14, p[≤]0.02). The inflammation-related DNA methylation signatures showed no clear variation across brain regions. Reactive microglial burdens were found to be highest in the hippocampus ({beta}=1.32, p=5x10-4); however, the only association identified between the blood- and brain-based methylation signatures and microglia was a significant positive association with acceleration of one epigenetic clock (termed DNAm PhenoAge) averaged over all five brain regions ({beta}=0.40, p=0.002). This work highlights a potential vulnerability of the hippocampus to epigenetic ageing and provides preliminary evidence of a relationship between DNA methylation signatures in the brain and differences in microglial burdens.

neuroscience↗

bioRxiv2020

Therapeutic doses of paracetamol with co-administration of cysteine and mannitol during early development result in long term behavioral changes in laboratory rats.

Based on several lines of evidence, numerous investigators have suggested that paracetamol exposure during early development can induce neurological disorders. We had previously postulated that paracetamol exposure early in life, if combined with antioxidants that prevent accumulation of NAPQI, the toxic metabolite of paracetamol, might be innocuous. In this study, we administered paracetamol at or below the currently recommended therapeutic dose to male laboratory rat pups aged 4-10 days. The antioxidants cysteine and mannitol were included to prevent accumulation of NAPQI. In addition, animals were exposed to a cassette of common stress factors: an inflammatory diet, psychological stress, antibiotics, and mock infections using killed bacteria. At age 37-49 days, observation during introduction to a novel conspecific revealed increased rearing behavior, an asocial behavior, in animals treated with paracetamol plus antioxidants, regardless of their exposure to oxidative stress factors (2-way ANOVA; P < 0.0001). This observation would suggest that the initial hypothesis is incorrect, and that oxidative stress mediators do not entirely eliminate the effects of paracetamol on neurodevelopment. This study provides additional cause for caution when considering the use of paracetamol in the pediatric population, and provides evidence that the effects of paracetamol on neurodevelopment need to be considered both in the presence and in the absence of oxidative stress.

animal behavior and cognition↗

bioRxiv2020

Elevated temperatures drive the evolution of armor loss in the threespine stickleback Gasterosteus aculeatus

While there is evidence of genetic and phenotypic responses to climate change, few studies have demonstrated change in functional traits with a known genetic basis. Here we present evidence for an evolutionary adaptive response to elevated temperatures in freshwater populations of the threespine stickleback (Gasterosteus aculeatus). Using a unique set of historical data and museum specimens, in combination with contemporary samples, we fitted a Bayesian spatial model to identify a population-level decline in the number of lateral bony plates, comprising anti-predator armor, in multiple populations of sticklebacks over the last 90 years in Poland. Armor loss was predicted by elevated temperatures and is proposed to be a correlated response to selection for reduced body size. This study demonstrates a change in a functional trait of known genetic basis in response to elevated temperature, and illustrates the utility of the threespine stickleback as a model for measuring the evolutionary and ecological impacts of environmental change across the northern hemisphere.

ecology↗

bioRxiv2020

Effects of NT5C2 germline variants on 6-mecaptopurine metabolism in children with acute lymphoblastic leukemia

6-mercaptopurine (6-MP) is widely used in the treatment of acute lymphoblastic leukemia (ALL), and its cytotoxicity is primarily mediated by thioguanine nucleotide metabolites (TGN). Recent genome-wide association study has identified germline polymorphisms (e.g., rs72846714) in the NT5C2 gene associated with 6-MP metabolism in patients with ALL. However, the full spectrum of genetic variation in NT5C2 is unclear and its impact on 6-MP drug activation has not been comprehensively examined. To this end, we performed targeted sequencing of NT5C2 in 588 children with ALL and identified 121 single nucleotide polymorphisms (SNPs) nominally associated with erythrocyte TGN during 6-MP treatment (P < 0.05). Of these, 61 variants were validated in a replication cohort of 372 children with ALL. After considering linkage disequilibrium and multivariate analysis, we confirmed two clusters of variants, represented by rs72846714 and rs58700372, that independently affected 6-MP metabolism. Functional studies showed that rs58700372 directly altered the activity of an intronic enhancer, with the variant allele linked to higher transcription activity and reduced 6-MP metabolism (lower TGN). By contrast, rs72846714 was not located in a regulatory element and instead its association signal was explained by linkage disequilibrium with a proximal functional variant rs12256506 that activated NT5C2 transcription in-cis. Our results indicated that NT5C2 germline variation significantly contributes to inter-patient variability in thiopurine drug disposition.

genetics↗

bioRxiv2020

CASCADES, a novel SOX2 super-enhancer associated long noncoding RNA, regulates cancer stem cell specification and differentiation in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults, with a median survival of just over one year. The failure of available treatments to achieve remission in patients with GBM has been attributed to the presence of cancer stem cells (CSCs), which are thought to play a central role in tumor development and progression and serve as a treatment-resistant cell repository capable of driving tumor recurrence; in fact, the property of "stemness" itself may be responsible for treatment resistance. In this study, we identify a novel lncRNA, Cancer stem cell associated distal enhancer of SOX2 (CASCADES) that functions as an epigenetic regulator in glioma CSCs (GSCs). CASCADES is expressed in IDH-wild type GBM and significantly enriched in GSCs. Knockdown of CASCADES in GSCs results in differentiation towards a neuronal lineage in a cell- and cancer-specific manner. Bioinformatics analysis reveals that CASCADES functions as a super-enhancer associated lncRNA epigenetic regulator of SOX2. Our findings identify CASCADES as a critical regulator of stemness in GSCs and represent a novel epigenetic and therapeutic target for disrupting the cancer stem cell compartment in GBM.

cancer biology↗

bioRxiv2020

Bioarchaeological analysis of one of the earliest Islamic burials in the Levant

The Middle East plays a central role in human history harbouring a vast diversity of ethnic, cultural and religious groups. However, much remains to be understood about past and present genomic diversity in this region. Here, we present for the first time, a multidisciplinary bioarchaeological analysis of two individuals dated to late 7th and early 8th centuries from Tell Qarassa, an open-air site in modern-day Syria. Radiocarbon dates, religious and cultural burial evidence indicate that this site represents one of the earliest Islamic Arab burials in the Levant during the Late Antiquity period. Interestingly, we found genomic similarity to a genotyped group of modern-day Bedouins and Saudi rather than to most neighbouring Levantine groups. This is highlighted through substantial Neolithic Levant ancestry in our samples, inviting an alternative scenario of long-term continuity in this region. This raises questions about the influence of ancient populations and historical migrations to genetic structure in the Middle East. As our study represents the first genomic analysis of an early Islamic burial in the Levant, we discuss our findings and possible historic scenarios in light of forces such as genetic drift and their possible interaction with religious and cultural processes.

genetics↗

bioRxiv2020

Prophylactic Application of Tailocins Prevents Infection by Pseudomonas syringae

Tailocins are phage-derived bacteriocins that demonstrate great potential as agricultural antimicrobials given their high killing efficiency and their precise strain-specific targeting ability. Our group has recently categorized and characterized tailocins produced by and tailocin sensitivities of the phytopathogen Pseudomonas syringae, and here we extend these experiments to test whether prophylactic tailocin application can prevent infection of Nicotiana benthamiana by P. syringae pv. syringae B728a. Specifically, we demonstrate that multiple strains can produce tailocins that prevent infection by strain B728a and engineer a deletion mutant to prove that tailocin targeting is responsible for this protective effect. Lastly, we provide evidence that heritable resistance mutations do not explain the minority of cases where tailocins fail to prevent infection. Our results extend previous reports of prophylactic use of tailocins against phytopathogens, and establish a model system with which to test and optimize tailocin application for prophylactic treatment to prevent phytopathogen infection.

microbiology↗

bioRxiv2020

Methods for detecting PER2::LUCIFERASE bioluminescence rhythms in freely moving mice

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of bioluminescence from a freely-moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. We tested delivery of D-luciferin via a subcutaneous minipump and in the drinking water. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than D-luciferin. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

systems biology↗

bioRxiv2020

A warming Southern Ocean may compromise Antarctic blue whale foetus growth

After declining in abundance due to commercial whaling during the 20th Century, populations of the Antarctic blue whale (Balaenoptera musculus intermedia) have failed to recover to pre-exploitation levels. Using historical whaling data from 1926-1954, in combination with temperature data for the Southern Ocean, a gamma GLMM with temporal dependency was fitted to 20,144 records of B. m. intermedia foetus size using Bayesian inference. There was a negative relationship between antecedent winter sea surface temperature (SST) in the Southern Ocean on foetus size. This relationship is proposed as being mediated by a positive effect of the extent of winter sea ice on Antarctic krill (Euphasia superba) abundance on which B. m. intermedia feed. There was also a positive density-dependent effect of a krill surplus at low whale population sizes. However, the positive effect of a krill surplus at low B. m. intermedia population size on foetus growth was reversed at elevated winter SST due to a negative impact on E. superba recruitment. Projected increases in temperature in the Southern Ocean are predicted to compromise the growth rates of B. m. intermedia foetuses, with implications for the capacity of the subspecies to recover from overexploitation.

ecology↗

bioRxiv2020

Acetyl-Leucine slows disease progression in lysosomal storage disorders

Acetyl-DL-leucine (ADLL) is a derivative of the branched chain amino acid leucine. In observational clinical studies ADLL improved symptoms of ataxia, in particular in patients with the lysosomal storage disorder (LSD), Niemann-Pick disease type C 1 (NPC1). Here, we investigated ADLL and its enantiomers acetyl-L-leucine (ALL) and acetyl-D-leucine (ADL) in symptomatic Npc1-/- mice and observed an improvement in ataxia with both enantiomers and ADLL. When ADLL and ALL were administered pre-symptomatically to Npc1-/- mice, both treatments delayed disease progression and extended life span, whereas ADL did not. These data are consistent with ALL being the neuroprotective enantiomer. Altered glucose and antioxidant metabolism were found to be implicated as one of the potential mechanisms of action of the L enantiomer in Npc1-/- mice. When miglustat and ADLL were used in combination significant synergy resulted. In agreement with these pre-clinical data, when NPC1 patients were evaluated after 12 months of ADLL treatment, rates of disease progression were slowed, with stabilisation or improvement in multiple neurological domains. A beneficial effect of ADLL on gait was also observed in this study in a mouse model of GM2 gangliosidosis (Sandhoff disease) and in Tay-Sachs and Sandhoff disease patients in individual-cases of off-label-use. Taken together, we have identified an unanticipated neuroprotective effect of acetyl-L-leucine and underlying mechanisms of action in LSDs, supporting its further evaluation in clinical trials in lysosomal disorders.

neuroscience↗

bioRxiv2020

The Receptor Kinase BRI1 promotes cell proliferation in Arabidopsis by phosphorylation- mediated inhibition of the growth repressing peptidase DA1.

Brassinosteroids (BR) have centrally important functions in plant growth by promoting cell proliferation and cell expansion through phosphorylation-mediated regulatory cascades that are initiated by perception of BR by receptor-like kinases of the BRI1 family. These BR-mediated growth responses have been explained by transcriptional controls mediated by phosphorylation of BES1/BZR1 transcription factors. Here we link BRI1-mediated phosphorylation to another growth regulatory network that directly mediates protein stability. BRI1 and its co-receptor BAK1 phosphorylate and inhibit the activities of the growth repressor DA1 by promoting the formation of high molecular weight complexes. Phospho-mimic forms of DA1 are less active while phospho-dead mutants have increased growth-repressive activity, while their regulatory monoubiquitylation is unaffected. BR inhibition of DA1 activity maintains higher levels of DA1 substrates such as UBP15 that sustain the potential for cell proliferation during leaf growth. Reduced BR levels lead to activation of DA1 peptidase activity and a transition from cell proliferation to cell growth and differentiation. This dual monoubiquitylation-phosphorylation regulation supports the key role of BR levels in maintaining the proliferative potential of cells during organ growth.

plant biology↗

bioRxiv2020

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

immunology↗

bioRxiv2020

A Cryptic Site of Vulnerability on the Receptor Binding Domain of the SARS-CoV-2 Spike Glycoprotein

SARS-CoV-2 is a zoonotic virus that has caused a pandemic of severe respiratory disease--COVID-19-- within several months of its initial identification. Comparable to the first SARS-CoV, this novel coronaviruss surface Spike (S) glycoprotein mediates cell entry via the human ACE-2 receptor, and, thus, is the principal target for the development of vaccines and immunotherapeutics. Molecular information on the SARS-CoV-2 S glycoprotein remains limited. Here we report the crystal structure of the SARS-CoV-2 S receptor-binding-domain (RBD) at a the highest resolution to date, of 1.95 [A]. We identified a set of SARS-reactive monoclonal antibodies with cross-reactivity to SARS-CoV-2 RBD and other betacoronavirus S glycoproteins. One of these antibodies, CR3022, was previously shown to synergize with antibodies that target the ACE-2 binding site on the SARS-CoV RBD and reduce viral escape capacity. We determined the structure of CR3022, in complex with the SARS-CoV-2 RBD, and defined a broadly reactive epitope that is highly conserved across betacoronaviruses. This epitope is inaccessible in the "closed" prefusion S structure, but is accessible in "open" conformations. This first-ever resolution of a human antibody in complex with SARS-CoV-2 and the broad reactivity of this set of antibodies to a conserved betacoronavirus epitope will allow antigenic assessment of vaccine candidates, and provide a framework for accelerated vaccine, immunotherapeutic and diagnostic strategies against SARS-CoV-2 and related betacoronaviruses. HIGHLIGHTSHigh resolution structure of the SARS-CoV-2 Receptor-Binding-Domain (RBD). Recognition of the SARS-CoV-2 RBD by SARS-CoV antibodies. Structure of the SARS-COV-2 RBD in complex with antibody CR3022. Identification of a cryptic site of vulnerability on the SARS-CoV-2 Spike.

immunology↗

bioRxiv2020

Non-coding germline GATA3 variants alter chromatin topology and contribute to pathogenesis of acute lymphoblastic leukemia

Inherited non-coding genetic variants confer significant disease susceptibility in many cancers. However, the molecular processes of by which germline variants contribute to somatic lesions are poorly understood. We performed targeted sequencing in 5,008 patients and identified a key regulatory germline variant in GATA3 strongly associated with Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). By creating an isogenic cellular model with CRISPR-Cas9 system, we showed that this variant activated a strong enhancer that significantly upregulated GATA3 transcription, which in turn reshaped the global chromatin accessibility and 3D genome organization. Remarkably, this genotype switch induced a chromatin loop between the CRLF2 oncogene and a distal enhancer, similar to the somatically acquired super-enhancer hijacking event in patients. GATA3 genotype-related alterations in transcriptional control and 3D chromatin organization were further validated in Ph-like ALL patients. Finally, we showed that GATA3 directly regulates CRLF2 and potentiates the oncogenic effects of JAK-STAT signaling in leukemogenesis. Altogether, our results provide evidence for a novel mechanism by which a germline non-coding variant contributes to oncogene activation epigenetic regulation and 3D genome reprogramming.

cancer biology↗

bioRxiv2020

Large extracellular vesicles can be characterised by multiplex labelling using imaging flow cytometry

Extracellular vesicles (EVs) are heterogeneous in size (30nm-10{micro}m), content (lipid, RNA, DNA, protein) and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs and examine EV marker expression and diameter using imaging flow cytometry. MethodsWe describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and TRPS. Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. ResultsUW228-2 cells prolifically release EVs of up to 6 {micro}m. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. ConclusionsWe have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9 and LAMP1) on individual EVs within a widely heterogeneous population. Taken together data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.

cell biology↗