Biology subjects

Watts, A.

Publications and source records attributed to Watts, A..

11 recordsLinked to original sources

bioRxiv2026

A nuclear targeting approach enables efficient transgenesis in the milkweed bug Oncopeltus fasciatus

Insects show extreme diversity and have long intrigued biologists. Recent technological advancements, such as gene editing and transgenesis, should in principle enable the use of almost any insect species for biological research. However, in practice, species-specific challenges remain and conditions must be optimized carefully. Here, using the milkweed bug Oncopeltus fasciatus, we first generate a useful eye- and body-color mutant strain by using CRISPR/Cas9-mediated genome editing. Then we use this strain to develop an efficient piggyBac-mediated transgenesis system using a nuclear targeting approach. We show that incorporating 1x and 3x nuclear localization signals (NLS) into piggyBac mRNA substantially enhances overall transformation efficiency in O. fasciatus. Taking advantage of both the useful mutant strain and the efficient transgenesis system, we successfully integrated multiple expression cassettes ranging from 1.8 to 7.2 kb, including attP strains for phiC31-mediated site-specific integration and histone-labelled strains for live fluorescence imaging. We further provide evidence that the Q system, a binary expression system, is functional in this species, paving the way for future sophisticated genetic manipulations including functional assays for cis-regulatory elements. Together, our results not only expand the genetic toolkit of O. fasciatus as a comparative model insect, but also provide a practical framework for developing efficient transgenesis in other non-traditional model organisms.

developmental biology↗

bioRxiv2022

Timing and Delivery route effects of Cecal Microbiome transplants on Salmonella Typhimurium infections in Chickens

BackgroundExposure to microbes early in life has long-lasting effects on microbial community structure and function of the microbiome. However, in commercial poultry settings chicks are reared as a single-age cohort with no exposure to adult birds which can have profound effects on microbiota development and subsequent pathogen challenge. Microbiota manipulation is a proven and promising strategy to help reduce pathogen load and transmission within broiler flocks. ResultsManipulation of the microbiota between 4 and 72 hours of hatch markedly reduces faecal shedding and colonisation with the foodborne pathogen Salmonella enterica serovar Typhimurium (ST4/74). Administration route has minimal effect on the protection conferred with fewer birds in transplant groups shown to shed ST4/74 in the faeces compared to PBS-gavaged control birds. Analysis of the microbiome following transplantation demonstrated that the relative abundance of the anti-inflammatory bacterium Faecalibacterium prausnitzii was significantly higher in CMT groups compared to PBS controls. The presence of F. prausnitzii was also shown to increase in PBS-challenged birds compared to unchallenged birds potentially indicating a role of this bacterium in limiting Salmonella infections. ConclusionsThis study highlights the efficacy of using microbiome transplants as a means to reduce colonisation and shedding of Salmonella in chickens. Effective protection can be conferred during the first few days of a chicks life regardless of time point and traditional hatchery delivery systems are sufficient to alter the microbiome and transfer donor material. Early microbiota intervention in chickens is a promising route of pathogen control in broiler flocks in the fight to control food-borne outbreaks in the human population.

microbiology↗

bioRxiv2021

In vivo amyloid-like fibrils produced under stress

The participation of amyloids in neurodegenerative diseases and functional processes has triggered the quest for methods allowing their direct detection in vivo. Despite the plethora of data, those methods are still lacking. The autofluorescence from the extended {beta}-sheets of amyloids is here used to follow fibrillation of S. cerevisiae Golgi Reassembly and Stacking Protein (Grh1). Grh1 has been implicated in starvation-triggered unconventional protein secretion (UPS), and here its participation also in heat shock response (HSR) is suggested. Fluorescence Lifetime Imaging (FLIM) is used to detect fibril autofluorescence in cells (E. coli and yeast) under stress (starvation and higher temperature). The formation of Grh1 large complexes under stress is further supported by size exclusion chromatography and ultracentrifugation. The data show for the first time in vivo detection of amyloids without the use of extrinsic probes as well as bring new perspectives on the participation of Grh1 in UPS and HSR.

biophysics↗

bioRxiv2020

A High Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury during the COVID-19 pandemic

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.

cell biology↗

bioRxiv2020

Physicochemical characterization, toxicity and in vivo biodistribution studies of a discoidal, lipid-based drug delivery vehicle: Lipodisq nanoparticles containing doxorubicin

Many promising pharmaceutically active compounds have low solubility in aqueous environments and their encapsulation into efficient drug delivery vehicles is crucial to increase their bioavailability. Lipodisq nanoparticles are approximately 10 nm in diameter and consist of a circular phospholipid bilayer, stabilized by an annulus of SMA (a hydrolysed copolymer of styrene and maleic anhydride). SMA is used extensively in structural biology to extract and stabilize integral membrane proteins for biophysical studies. Here, we assess the potential of these nanoparticles as drug delivery vehicles, determining their cytotoxicity and the in vivo excretion pathways of their polymer and lipid components. Doxorubicin-loaded Lipodisqs were cytotoxic across a panel of cancer cell lines, whereas nanoparticles without the drug had no effect on cell proliferation. Intracellular doxorubicin release from Lipodisqs in HeLa cells occurred in the low-pH environment of the endolysosomal system, consistent with the breakdown of the discoidal structure as the carboxylate groups of the SMA polymer become protonated. Biodistribution studies in mice showed that, unlike other nanoparticles injected intravenously, most of the Lipodisq components were recovered in the colon, consistent with rapid uptake by hepatocytes and excretion into bile. These data suggest that Lipodisqs have the potential to act as delivery vehicles for drugs and contrast agents.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsDLPE1,2-dilauroyl-sn-glycero-3-phosphoethanolamineDLPE-Cy5Cy5 dye covalently conjugated to 1,2-dilauroyl-sn-glycero-3-phosphoethanolamineDLSdynamic light scatteringDMPC1,2-dimyristoyl-sn-glycero-3-phosphocholineDMPG1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol)DOXDoxorubicinEPRenhanced permeability and retention effectLipodisqLQNMRnuclear magnetic resonanceNPnanoparticleRTroom temperatureSMAhydrolysed co-polymer of styrene and maleic anhydrideSMA-Cy5Cy5 dye covalently conjugated to a thiol derivative of SMASMAnhnon-hydrolysed co-polymer of styrene and maleic anhydrideSMAnh-SHthiol derivative of SMAnhSMA-SHthiol derivative of SMAView Full Text

pharmacology and toxicology↗

bioRxiv2020

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a fingerprint of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (g-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

biochemistry↗

bioRxiv2019

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix, which includes the Golgi Reassembly and Stacking Proteins (GRASPs), which participate in cisternae stacking and lateral linkage in vertebrates. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different protein binding partners. The conserved N-terminus of the GRASP family includes two PDZ domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content, however PDZ1 alone mediates nearly all the interactions between GRASPs and their binding partners. In this work, NMR, Synchrotron-Radiation Circular Dichroism and Molecular Dynamics were used to examine the structure, flexibility and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual {beta}31{beta}4{beta}52{beta}6{beta}1{beta}2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable {beta}2-strand and a more flexible and unstable 2-helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using Molecular Dynamics simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.\n\nSignificance StatementGolgi Reassembly and Stacking Proteins (GRASPs) play pivotal roles in the maintenance of Golgi structure as well as in unconventional protein secretion. Their broad network of interactions is mainly sustained by the two-PDZ domains located in the N-terminal portion of the protein. The asymmetry of the PDZ domains in terms of number and diversity of interacting partners has been long recognized, but the molecular determinants of that asymmetry remains largely unknown. The biophysical data presented here provide a firm basis for understanding why PDZ1 behaves differently to PDZ2 in solution, despite their similar 3D structures. Furthermore, we propose that PDZ2 assist ligand binding to PDZ1, by means of conformational stabilization.

biophysics↗

bioRxiv2019

Exploring Conformational Transitions and Free Energy Profiles of Proton Coupled Oligopeptide Transporters

Proteins involved in peptide uptake and transport belong to the proton-coupled oligopeptide transporter (POT) family. Crystal structures of POT family members reveal a common fold consisting of two domains of six transmembrane helices that come together to form a \"V\" shaped transporter with a central substrate binding site. Proton coupled oligopeptide transporters operate through an alternate access mechanism, where the membrane transporter undergoes global conformational changes, alternating between inward-facing (IF), outward-facing (OF) and occluded (OC) states. Conformational transitions are promoted by proton and ligand binding, however, due to the absence of crystallographic models of the outward-open state, the role of H+ and ligands are still not fully understood. To provide a comprehensive picture of the POT conformational equilibrium, conventional and enhanced sampling molecular dynamics simulations of PepTst in the presence or absence of ligand and protonation were performed. Free energy profiles of the conformational variability of PepTst were obtained from microseconds of adaptive biasing force (ABF) simulations. Our results reveal that both, proton and ligand, significantly change the conformational free energy landscape. In the absence of ligand and protonation, only transitions involving IF and OC states are allowed. After protonation of the residue Glu300, the wider free energy well for Glu300 protonated PepTst indicates a greater conformational variability relative to the apo system, and OF conformations becames accessible. For the Glu300 Holo-PepTst, the presence of a second free energy minimum suggests that OF conformations are not only accessible, but also, stable. The differences in the free energy profiles demonstrate that transitions toward outward facing conformation occur only after protonation and, probably, this should be the first step in the mechanism of peptide transport. Our extensive ABF simulations provide a fully atomic description of all states of the transport process, offering a model for the alternating access mechanism and how protonation and ligand control the conformational changes.\n\nGraphical TOC Entry\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/651646v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (21K):\norg.highwire.dtl.DTLVardef@8c8a84org.highwire.dtl.DTLVardef@da15bborg.highwire.dtl.DTLVardef@1c8dab0org.highwire.dtl.DTLVardef@18d87e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2018

International travelers and genomics uncover a ‘hidden’ Zika outbreak

The ongoing Zika epidemic in the Americas has challenged public health surveillance, response, and control systems. Even as the epidemic appears to be near its end in the Americas, it is unclear whether substantial Zika virus transmission may still be ongoing. This issue is exacerbated by large discrepancies in local case reporting and significant delays in detecting outbreaks due to surveillance gaps. To uncover locations with lingering outbreaks in the Americas, we investigated travel-associated Zika cases diagnosed in the United States and Europe to identify signatures of transmission dynamics that were not captured by local reporting. We found that a large and unreported Zika outbreak occurred in Cuba during 2017, a year after peak transmission in neighboring countries, with cases still appearing in 2018. By sequencing Zika virus from infected travelers, we show that the 2017 outbreak in Cuba was sparked by long-lived lineages of Zika virus introduced from multiple places in the Americas a year prior. Our data suggest that while aggressive mosquito control in Cuba may initially have been effective at mitigating Zika virus transmission, in the absence of vaccines, herd immunity, or strong international coordination, such control measures may need to be maintained to be effective. Our study highlights how Zika virus may still be silently spreading in the Americas and provides a framework for more accurately understanding outbreak dynamics.

epidemiology↗

bioRxiv2018

Utilizing Transcranial Direct Current Stimulation to Enhance Laparoscopic Technical Skills Training: A Randomized Controlled Trial

This study aimed to test the efficacy of transcranial direct current stimulation (tDCS) during laparoscopic skill training to determine if it has the capacity to accelerate technical skill acquisition. tDCS is a non-invasive brain stimulation technique that delivers constant, low electrical current resulting in changes to cortical excitability and prior work suggests it may enhance motor learning. We evaluate for the first time the potential of tDCS, coupled with motor skill training, to accelerate the development of laparoscopic technical skills. In this pre-registered, double-blinded and sham-controlled study, 60 healthy subjects were randomized into sham or active tDCS in either bilateral primary motor cortex (bM1) or supplementary motor area (SMA) electrode configurations. All subjects practiced the Fundamental of Laparoscopic Surgery Peg Transfer Task during a pre-test, six 20-minute training sessions, and a post-test. The primary outcome was change in laparoscopic skill performance over time, quantified by improvement in performance according to a seconds-per-object calculated score accounting for errors. Sixty participants were randomized equally into the three training cohorts (active bM1, active SMA, sham). The active groups had significantly greater improvement in performance from pre-test to post-test compared to the sham groups (108 vs 76 seconds, p = 0.018). Both bM1 and SMA active cohorts had significantly greater improvement in learning (p < 0.01), achieving the same skill level in 4 sessions compared to the 6 sessions required of the sham cohort. The SMA cohort had more variability in performance compared to the bM1 and control cohorts. Laparoscopic skill training with active, bM1 or SMA, tDCS exhibited significantly greater learning relative to training with sham tDCS. The potential for tDCS to enhance the training of surgical skills merits further investigation to determine if these preliminary results may be replicated.

clinical trials↗

bioRxiv2018

Utilisation of staphylococcal immune evasion protein Sbi as a novel vaccine adjuvant

Co-ligation of the B cell antigen receptor with complement receptor 2 on B-cells via a C3d-opsonised antigen complex significantly lowers the threshold required for B cell activation. Consequently, fusions of antigens with C3d polymers have shown great potential in vaccine design. However, these linear arrays of C3d multimers do not mimic the natural opsonisation of antigens with C3d. Here we investigate the potential of using the unique complement activating characteristics of Staphylococcal immune-evasion protein Sbi to develop a pro-vaccine approach that spontaneously coats antigens with C3 degradation products in a natural way. We show that Sbi rapidly triggers the alternative complement pathway through recruitment of complement regulators, forming a tripartite complex that acts as a competitive antagonist of factor H, resulting in enhanced complement consumption. These functional results are corroborated by the structure of this complement activating Sbi-III-IV:C3d:FHR-1 complex. Finally, we demonstrate that Sbi, fused with Mycobacterium tuberculosis antigen Ag85b, causes efficient opsonisation with C3 fragments, thereby enhancing the immune response significantly beyond that of Ag85b alone, providing proof of concept for our pro-vaccine approach.

biochemistry↗