Biology subjects

Johnson, S.

Publications and source records attributed to Johnson, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

SAMHD1 controls nucleoside-based cancer therapeutics, deoxyguanosine toxicity, and inflammation

SAMHD1 is a key regulator of dNTP metabolism, being activated by dGTP and converting dNTPs into their respective nucleosides. As a result, it plays a significant role in several distinct diseases. Not only is SAMHD1 a restriction factor for HIV-1 replication, which requires dNTPs for replication, but it is also required for genome stability, is mutated in multiple cancers, and loss of function mutations cause Acardi-Goutieres Syndrome, an early-onset inherited encephalopathy. In this paper we knock out the SAMHD1 gene in pluripotent stem cells, and show that it also affects nucleoside analogue sensitivity of dividing cells and deoxyguanosine toxicity in a cell cycle-independent manner. Comparing wild type and gene-edited macrophages, we also show that deoxyguanosine toxicity is associated with pro-inflammatory cell death, modifications in uric acid production and the formation of monosodium urate crystals within the macrophages. These novel features of the SAMHD1-/- phenotype provide unique insights into the aetiology of Acardi-Goutieres Syndrome, and have implications for certain chemotherapeutic agents.

pharmacology and toxicology↗

bioRxiv2022

Sex-specific T cell exhaustion drives differential immune responses in glioblastoma

Sex differences in glioblastoma (GBM) incidence and outcome are well recognized, and emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunological sex differences are not fully understood. Using GBM models, we demonstrate that T cells play a critical role in driving GBM sex differences. Male mice exhibited accelerated tumor growth, with decreased T cell infiltration and increased T cell exhaustion. Furthermore, a higher frequency of progenitor exhausted T cells was found in males, with improved responsiveness to anti-PD1 treatment. Bone marrow chimera and adoptive transfer models indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, which was further corroborated by in vitro exhaustion assays. Moreover, increased T cell exhaustion was observed in male GBM patients. These findings demonstrate sex-specific pre-determined behavior of T cells is critical in inducing sex differences in GBM progression and immunotherapy response. Statement of significanceImmunotherapies in GBM patients have been unsuccessful due to a variety of factors including the highly immunosuppressive tumor microenvironment in GBM. This study demonstrates that sex-specific T cell behaviors are predominantly intrinsically regulated, further suggesting sex-specific approaches can be leveraged to potentially improve therapeutic efficacy of immunotherapy in GBM.

immunology↗

bioRxiv2022

Tween-20 induces the structural remodelling of single lipid vesicles

The interaction of Tween-20 with lipid membranes is crucial for a number of biotechnological applications including viral inactivation and membrane protein extraction, but the underlying mechanistic details have remained elusive. Evidence from ensemble assays supports a global model of Tween-20 induced membrane disruption that broadly encompasses association of the surfactant with the membrane surface, membrane fragmentation and the release of mixed micelles to solution, but whether this process involves intermediate and dynamic transitions between regimes is an open question. In search of the mechanistic origins of membrane disruption, increasing focus is put on identifying Tween-20 interactions with highly controllable model membranes. In light of this, and to unveil quantitative mechanistic details, we employed highly interdisciplinary biophysical approaches, including quartz-crystal microbalance with dissipation monitoring, steady-state and time-resolved fluorescence and FRET spectroscopy, dynamic light scattering, fluorescence correlation spectroscopy, wide-field single-vesicle imaging and scanning electron microscopy, to interrogate the interactions between Tween-20 and both freely-diffusing and surface-immobilized model-membrane vesicles. Using ultrasensitive sensing approaches, we discovered that Tween-20 leads to a stepwise and phase-dependent structural remodelling of sub-micron sized vesicles that includes permeabilization and swelling, even at detergent concentrations below the critical micellar concentration. These insights into the structural perturbation of lipid vesicles upon Tween-20 interaction highlight the impact on vesicle conformation prior to complete solubilization, and the tools presented may have general relevance for probing the interaction between lipid vesicles and a wide variety of disruptive agents.

biophysics↗

bioRxiv2022

Discovery of a SARS-CoV-2 Broadly-Acting Neutralizing Antibody with Activity against Omicron and Omicron + R346K Variants

The continual emergence of SARS-CoV-2 variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant, has rendered ineffective a number of previously EUA approved SARS-CoV-2 neutralizing antibody therapies. Furthermore, even those approved antibodies with neutralizing activity against Omicron are reportedly ineffective against the subset of Omicron variants that contain a R346K substitution, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. Following a campaign of antibody discovery based on the vaccination of Harbour H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of Spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against the Omicron and Omicron + R346K variants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for use in human clinical trials.

microbiology↗

bioRxiv2021

Oral and intranasal Ad5 SARS-CoV-2 vaccines decrease disease and viral transmission in a golden hamster model

Transmission-blocking strategies that slow the spread of SARS-CoV-2 and protect against COVID-19 are needed. We have developed a shelf-stable, orally-delivered Ad5-vectored SARS-CoV-2 vaccine candidate that expresses the spike protein. Here we demonstrated that oral and intranasal SARS-CoV-2 vaccination of this candidate protected against disease in index hamsters, and decreased aerosol transmission to unvaccinated, naive hamsters. We confirmed that mucosally-vaccinated hamsters had robust antibody responses. We then induced a post-vaccination infection by inoculating vaccinated index hamsters with SARS-CoV-2. Oral and IN-vaccinated hamsters had decreased viral RNA and infectious virus in the nose and lungs and experienced less lung pathology compared to mock-vaccinated hamsters post challenge. Naive hamsters exposed in a unidirectional air flow chamber to mucosally-vaccinated, SARS-CoV-2-infected hamsters had lower nasal swab viral RNA and exhibited less clinical symptoms of disease than control animals. Our data demonstrate that oral immunization is a viable strategy to decrease SARS-CoV-2 disease and aerosol transmission.

immunology↗

bioRxiv2021

Distinct cell adhesion signature defines glioblastoma myeloid-derived suppressor cell subsets

Increased myeloid-derived suppressor cell (MDSC) frequency is associated with worse outcomes and poor therapeutic response in glioblastoma (GBM). Monocytic (m) MDSCs represent the predominant subset in the GBM microenvironment. However, the molecular basis of mMDSC enrichment in the tumor microenvironment compared to granulocytic (g) MDSCs has yet to be determined. Here, we report that mMDSCs and gMDSCs display differences in their tumoraccelerating ability, with mMDSCs driving tumor growth in GBM models. Epigenetic assessments indicate enhanced gene accessibility for cell adhesion programs in mMDSCs and higher cellsurface integrin expression in mouse and human mMDSCs. Integrin {beta}1 blockage abrogated the tumor-promoting phenotype of mMDSCs and altered the immune profile in the tumor microenvironment. These findings suggest that integrin {beta}1 expression underlies the enrichment of mMDSCs in tumors and represents a putative immunotherapy target to attenuate myeloid cell-driven immune suppression in GBM. SummaryMyeloid-derived suppressor cells (MDSCs) drive glioblastoma growth; however, the function of specific MDSCs subsets is unclear. Bayik et al. demonstrate that adhesion programs are enhanced in monocytic MDSCs and responsible for their GBM-promoting function.

immunology↗

bioRxiv2021

The WRAD complex represents a therapeutically exploitable target for cancer stem cells in glioblastoma

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1-family-mediated Lys4 methylation of histone H3, associated with positive regulation of transcription. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment diminished reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. SignificanceIn this study, we perform an epigenetic-focused functional genomics screen in glioblastoma organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/461125v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@bc5c5eorg.highwire.dtl.DTLVardef@545a2borg.highwire.dtl.DTLVardef@1ebd3c9org.highwire.dtl.DTLVardef@19d5d30_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Targeting the Cx26/NANOG/Focal Adhesion Kinase Complex via Cell Penetrating Peptides in Triple Negative Breast Cancers

Triple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype with limited treatment options. We previously identified a protein complex unique to TNBC cancer stem cells composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of Cx26 designed to target the complex attenuated tumor growth in pre-clinical models. Histological assessment was employed to verify expression of complex members. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Peptides with high affinity were engineered with a cell-penetrating sequence and assessed in functional assays including cell proliferation, self-renewal, and in vivo tumor growth, and downstream signaling changes were measured. Binding studies revealed that the Cx26 C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar-to-micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. The Cx26 C-terminal tail was tagged with an antennapedia cell-penetrating peptide sequence and intracellular localization was confirmed. The cell-penetrating Cx26 peptide (aCx26-pep) disrupted self-renewal as assessed by tumorsphere formation assay while reducing nuclear FAK and NANOG and inhibiting NANOG target gene expression in TNBC cells but not luminal mammary epithelial cells. In vivo, aCx26-pep reduced tumor growth and proliferation and induced cell death. We provide proof-of-concept that a Cx26 peptide-based strategy inhibits growth and alters NANOG activity in TNBC.

cancer biology↗

bioRxiv2021

A Panel of Diverse Pseudomonas aeruginosa Clinical Isolates for Research and Development

Pseudomonas aeruginosa is a leading cause of community-acquired and hospital-acquired infections. Successful treatment is hampered by its remarkable ability to rapidly develop resistance to antimicrobial agents, mostly through mutation. In response, the World Health Organization listed carbapenem-resistant P. aeruginosa as a Priority 1 (Critical) pathogen for research and development of new treatments. A key resource in developing effective countermeasures is access to diverse and clinically relevant strains for testing. Herein we describe a panel of 100 diverse P. aeruginosa strains to support this endeavor. Whole genome sequencing was performed on 3,785 P. aeruginosa housed in our repository. Isolates were cultured from clinical samples collected from healthcare facilities around the world between 2003 and 2017. Core-genome multi-locus sequence typing and high-resolution SNP-based phylogenetic analyses were used to select a panel of 100 strains that captured the genetic diversity of this collection. Comprehensive antibiotic susceptibility testing was also performed using 14 clinically relevant antibiotics. This 100-strain diversity panel contained representative strains from 91 different sequence-types, including genetically distinct isolates from major epidemic clones ST-111, ST-235, ST-244, and ST-253. Seventy-one distinct antibiotic susceptibility profiles were identified ranging from pan-sensitive to pan-resistant. Known resistance alleles as well as the most prevalent mutations underlying the antibiotic susceptibilities were characterized for all isolates. This panel provides a diverse and comprehensive set of P. aeruginosa strains for use in developing solutions to antibiotic resistance. The isolates, and all available meta-data including genome sequences, are available to industry, academic institutions, federal and other laboratories at no additional cost. ImportancePseudomonas aeruginosa is one of the most important human pathogens and a leading target in the development of new drugs and therapeutics. This species displays a remarkable level of diversity and any potential therapeutic must contend with this characteristic to ensure it retains efficacy across different strains. To date, only limited panels of P. aeruginosa are available for testing, and none have been designed to capture the genetic diversity of the species. The panel described herein has been designed to address this shortcoming by providing a set of 100 distinct strains that greatly captures the diversity of the species. This panel will be of significant value to research groups working on this important pathogen, both for research purposes and in the development of new diagnostics and countermeasures.

microbiology↗

bioRxiv2021

Targeting properdin - Structure and function of a novel family of tick-derived complement inhibitors

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. We have identified a novel family of alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. For the first time this study provides a full functional and structural characterization of a properdin inhibitor, opening avenues for future therapeutic approaches.

immunology↗

bioRxiv2021

Delta C2 Domain β1-2 loop contributes to robust Notch signalling

Accurate Notch signalling is critical for organism development and homeostasis. Fine-tuning of Notch-ligand interactions have substantial impact on signalling-outputs. Recent structural studies identified a conserved N-terminal C2 domain in human Notch ligands which conferred phospholipid binding in vitro. Here we show that Drosophila ligands adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called {beta}1-2 loop that has been associated with phospholipid binding. Mutations in the {beta} 1-2 loop of Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo we deleted five residues within the {beta} 1-2 loop of endogenous Delta by CRISPR/Cas9 gene editing. Strikingly, this change compromised ligand function. The modified Delta enhanced phenotypes produced by Delta loss of function alleles and suppressed that of Notch alleles. As the modified protein was present on the cell surface in normal amounts, these results argue that C2 domain phospholipid-binding is necessary for robust signalling in vivo where the balance of cis and trans ligand-receptor interactions is finely tuned.

developmental biology↗

bioRxiv2020

Paternal hypoxia exposure primes offspring for increased hypoxia resistance

In a time of rapid environmental change, understanding how the challenges experienced by one generation can influence the fitness of future generations is critically needed. Using tolerance assays, transcriptomic and methylome approaches, we use zebrafish as a model to investigate transgenerational acclimation to hypoxia. We show that short-term paternal exposure to hypoxia endows offspring with greater tolerance to acute hypoxia. We detected two hemoglobin genes that are significantly upregulated by more than 7-fold in the offspring of hypoxia exposed males. Moreover, the offspring which maintained equilibrium the longest showed greatest upregulation in hemoglobin expression. We did not detect differential methylation at any of the differentially expressed genes, suggesting that another epigenetic mechanism is responsible for alterations in gene expression. Overall, our findings suggest that a memory of past hypoxia exposure is maintained and that this environmentally induced information is transferred to subsequent generations, pre-acclimating progeny to cope with hypoxic conditions.

evolutionary biology↗

bioRxiv2020

Molecular structure of the intact bacterial flagellar basal body

Bacterial flagella self-assemble a strong, multi-component drive shaft that couples rotation in the inner membrane to the microns-long flagellar filament that powers bacterial swimming in viscous fluids. We here present structures of the intact Salmonella flagellar basal body, solved using cryo-electron microscopy to resolutions between 2.2 and 3.7 [A]. The structures reveal molecular details of how 173 protein molecules of 13 different types assemble into a complex spanning two membranes and a cell wall. The helical drive shaft at one end is intricately interwoven with the inner membrane rotor component, and at the other end passes through a molecular bearing that is anchored in the outer membrane via interactions with the lipopolysaccharide. The in situ structure of a protein complex capping the drive shaft provides molecular insight into the assembly process of this molecular machine.

microbiology↗

bioRxiv2020

Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein

Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomers cytoplasmic domain, which stabilises the nonameric ring assembly.

microbiology↗

bioRxiv2020

Molecular basis for B. pertussis interference with complement, coagulation, fibrinolytic and contact activation systems: The cryo-EM structure of the Vag8-C1 inhibitor complex

Complement, contact activation, coagulation, and fibrinolysis are serum protein cascades that need strict regulation to maintain human health. Serum glycoprotein, C1-inhibitor (C1-INH) is a key regulator (inhibitor) of serine proteases of all the above-mentioned pathways. Recently, an autotransporter protein, Virulence Associated Gene 8 (Vag8) produced by the whopping cough causing pathogen, Bordetella pertussis has been shown to bind and interfere with C1-INH function. Here we present the structure of Vag8: C1-INH complex determined using cryo-electron microscopy at 3.6 [A] resolution. The structure shows a unique mechanism of C1-INH inhibition not employed by other pathogens where Vag8 sequesters the Reactive Centre Loop of the C1-INH preventing its interaction with the target proteases. ImportanceThe structure 105 kDa protein complex is one of the smallest to be determined using cryo-electron microscopy at high resolution. The mechanism of disrupting C1-INH revealed by the structure is crucial to understand how pathogens by producing a single virulence factor can disturb several homeostasis pathways. Virulence mechanisms such as the one described here assume more importance given the emerging evidence about dysregulation of contact activation, coagulation and fibrinolysis leading to COVID-19 pneumonia.

microbiology↗

bioRxiv2020

Blood clot fracture properties are dependent on red blood cell and fibrin content

Thrombus fragmentation during endovascular stroke treatment, such as mechanical thrombectomy, leads to downstream emboli, resulting in poor clinical outcomes. Clinical studies suggest that fragmentation risk is dependent on clot composition. This current study presents the first experimental characterization of the fracture properties of blood clots, in addition to the development of a predictive model for blood clot fragmentation. A bespoke experimental test-rig and compact tension specimen fabrication has been developed to measure fracture toughness of thrombus material. Fracture tests are performed on three physiologically relevant clot compositions: a high fibrin 5% H clot, a medium fibrin 20% H clot, a low-fibrin 40% H clot. Fracture toughness is observed to significantly increase with increasing fibrin content, i.e. red blood cell-rich clots are more prone to tear during loading compared to the fibrin-rich clots. Results also reveal that the mechanical behaviour of clot analogues is significantly different in compression and tension. Finite element cohesive zone modelling of clot fracture experiments show that fibrin fibres become highly aligned in the direction perpendicular to crack propagation, providing a significant toughening mechanism. The results presented in this study provide the first characterization of the fracture behaviour of blood clots and are of key importance for development of next-generation thrombectomy devices and clinical strategies.

bioengineering↗

bioRxiv2020

Discovery and Development of Human SARS-CoV-2 Neutralizing Antibodies using an Unbiased Phage Display Library Approach

SARS-CoV-2 neutralizing antibodies represent an important component of the ongoing search for effective treatment of and protection against COVID-19. We report here on the use of a naive phage display antibody library to identify a panel of fully human SARS-CoV-2 neutralizing antibodies. Following functional profiling in vitro against an early pandemic isolate as well as a recently emerged isolate bearing the D614G Spike mutation, the clinical candidate antibody, STI-1499, and the affinity-engineered variant, STI-2020, were evaluated for in vivo efficacy in the Syrian golden hamster model of COVID-19. Both antibodies demonstrated potent protection against the pathogenic effects of the disease and a dose-dependent reduction of virus load in the lungs, reaching undetectable levels following a single dose of 500 micrograms of STI-2020. These data support continued development of these antibodies as therapeutics against COVID-19 and future use of this approach to address novel emerging pandemic disease threats.

microbiology↗

bioRxiv2020

A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.)

A contiguous assembly of the inbred EL10 sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short read error correction. The EL10.1 assembly was 540 Mb, of which 96.7% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNAseq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals drawn from EL10 progeny and three unrelated germplasm accessions, producing estimates from 633 to 875 Mb/1C. Read depth mapping with short-read whole genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

genomics↗