Biology subjects

Kay, E.

Publications and source records attributed to Kay, E..

12 recordsLinked to original sources

bioRxiv2026

Development and evaluation of a dual target glycoconjugate vaccine against Shigella sonnei

BackgroundShigellosis morbidity and mortality, combined with the increase in multidrug-resistant infections make Shigella vaccine development a global imperative. Glycoconjugate vaccines that couple immunogenic O-antigen to protein derived from Shigella may provide broader protection across Shigella species and serogroups. Such an approach also circumvents immunotolerance arising from repeated use of the same carrier. Here we use bioconjugation, exploiting an oligosaccharyltransferase (OST) enzyme to couple O-antigen and carrier protein in vivo, to generate a "double-hit" Shigella glycoconjugate vaccine. MethodGlycoconjugates were synthesised in E. coli SDB1 cells expressing S. sonnei O-antigen, the OST PglS, and one of two Shigella carrier proteins. Recombinant glycoconjugate was purified using anion exchange chromatography and then used to immunise mice. Antibody responses were measured and compared by ELISA. ResultsWhen co-produced in E. coli, PglS was able to transfer the cloned S. sonnei O-antigen onto three carrier proteins, modified to accept glycans from the PglS transferase enzymes- the standard bioconjugate carrier ExoA and two immunogenic Shigella-specific outer membrane proteins, EmrK and MdtA. Production of MdtA or ExoA glycoconjugates for immunisation studies utilised successive rounds of anion exchange chromatography, to remove unglycosylated material and obtain highly purified glycoconjugate proteins for us in vaccination. Analysis of murine sera following immunisation revealed an IgG response was raised against both carrier protein and the S. sonnei O-antigen for each glycoconjugate. ConclusionA novel, conserved Shigella protein can be utilised as an effective carrier for the generation of a "double-hit", immunogenic Shigella glycoconjugate vaccine that elicits IgG responses to both carrier protein and S. sonnei O-antigen.

microbiology↗

bioRxiv2024

Host-specific adaptation of Legionella pneumophila to single and multiple hosts

Legionella pneumophila is an endosymbiotic bacterial species able to infect and reproduce in various protist and human hosts. Upon entry in human lungs, they may infect lung macrophages, causing Legionnaires disease (LD), an atypical pneumonia, using similar mechanisms as in their protozoan hosts, despite the two hosts being separated by a billion years of evolution. In this study, we used experimental evolution to identify genes conferring host-specificity to L. pneumophila. To this end, we passaged L. pneumophila in two different hosts - Acanthamoeba castellanii and the human macrophage-like cells U937 - separately and by switching between the hosts twice a week for a year. In total, we identified 1518 mutations present in at least 5% of the population at the time of sampling. Half of these were localized in five groups of repeated sequences, likely to be recombination hotspots. Forty-nine mutations were fixed in the 18 populations at the end of the experiment, representing four different groups. The first two groups involve adaptation to the specific selection conditions, including (i) two specific mutations in the 30S ribosomal protein S12 (RpsL) that conferred resistance to streptomycin, to which the bacteria were unexpectedly exposed to during serial passage and (ii) two mutations, one in the 30S ribosomal protein S4 (RpsD), and one in the chaperonin GroES, both of which are likely to be fitness-restoring compensatory mutations to the original RpsL mutations. Two more interesting groups of mutations included (iii) mutations in 4 different strain-specific genes involved in LPS synthesis, found only in the lineages passaged with A. castellanii and (iv) mutations in the gene coding for LerC, a key regulator of protein effector expression, which was independently mutated in 6 lineages grown in presence of the U937 cells. We propose that the mutations degrading the function of the regulator LerC improve the fitness of L. pneumophila in human-derived cells, and that modifications in the LPS are beneficial for growth in A. castellanii. This study is a first step in further investigating determinants of host specificity in L. pneumophila.

evolutionary biology↗

bioRxiv2024

Wolbachia introgression in Ae. aegypti is accompanied by variable loss - a multi-country assessment

The wMel and wAlbB strains of the bacterial endosymbiont Wolbachia are being introgressed into Aedes aegypti populations as a biocontrol method to reduce the transmission of medically important arboviruses. Successful introgression of Wolbachia relies on both persistence of Wolbachia throughout the host life cycle and a high fidelity of maternal transmission of Wolbachia between generations. wMel has been introgressed into field populations in 14 countries to date. Monitoring of field sites has shown that wMel prevalence can fluctuate substantially over time, prompting concerns this could lead to reduced efficacy of the biocontrol method. To explore the fidelity of wMel persistence and transmission, we developed molecular methods to measure the prevalence of Ae. aegypti negative for Wolbachia infection but carrying the "founder" mitochondrial haplotype of the single female first transinfected. As all released wMel-infected mosquitoes and any subsequent offspring will carry this founder mitochondrial haplotype, any mosquitoes with this mitochondrial haplotype and without wMel indicate that wMel was lost from this lineage at some point. We observed loss of wMel ranging from 0 to 20.4% measured at various time intervals after wMel-infected mosquito releases in five different countries. Despite some field sites showing Wolbachia loss, overall Wolbachia prevalence was sustained during the time periods studied. We then employed laboratory studies to explore factors that could contribute to the loss of wMel. Surprisingly, near-perfect maternal transmission was measured across laboratory conditions of early blood feeding, starvation, and salinity. Collectively, these findings underscore that although wMel transmission can be imperfect it does not necessarily undermine population-level establishment, providing encouragement that the intervention will be robust in most dengue-endemic environments.

ecology↗

bioRxiv2024

Metabolic adaptations of micrometastases alter EV production to generate invasive microenvironments

Altered cellular metabolism has been associated with acquisition of invasive phenotypes during metastasis. To study this, we combined a genetically engineered mouse model of mammary carcinoma with syngeneic transplantation and primary tumour resection to generate isogenic cells from primary tumours and their corresponding lung micrometastases. Metabolic analyses indicated that micrometastatic cells increase proline production at the expense of glutathione synthesis leading to a reduction in total glutathione levels. Micrometastatic cells also have altered sphingomyelin metabolism leading to increased intracellular levels of specific ceramides. The combination of these two metabolic adaptations alters small extracellular vesicle (sEV) production to drive generation of an invasive microenvironment. Indeed, micrometastatic cells shut-down Rab27-dependent production of sEVs and, instead, switch-on neutral sphingomyelinase-2 (nSM2)-dependent sEV release. sEVs released in a nSM2-dependent manner from micrometastatic cells, in turn, influence the ability of fibroblasts to deposit extracellular matrix which promotes cancer cell invasiveness. These data provide evidence that metabolic rewiring drives invasive processes in metastasis by influencing sEV release. SummaryBreast cancer cells isolated from lung micrometastases have altered metabolism which influences extracellular vesicle production to generate invasive microenvironments.

cancer biology↗

bioRxiv2023

Dual control of host actin polymerization by a Legionella effector pair

Host actin cytoskeleton is often targeted by pathogenic bacteria through the secretion of effectors. Legionella pneumophila virulence relies on the injection of the largest known arsenal of bacterial proteins, over 300 Dot/Icm Type 4 Secretion System effectors, into the host cytosol. Here we define the functional interactions between VipA and LegK2, two effectors with antagonistic activities towards actin polymerization that have been proposed to interfere with the endosomal pathway. We confirmed the prominent role of LegK2 effector in Legionella infection, as the deletion of legK2 results in defects in the inhibition of actin polymerization at the Legionella Containing Vacuole, as well as in endosomal escape of bacteria and subsequent intracellular replication. More importantly, we observed the restoration of the{Delta} legK2 mutant defects, upon deletion of vipA gene, making LegK2/VipA the first example of effector-effector suppression pair that targets the actin cytoskeleton and whose functional interaction impacts L. pneumophila virulence. We demonstrated that LegK2 and VipA do not modulate each others activity in a metaeffector relationship. Instead, the antagonistic activities of the LegK2/VipA effector pair would target different substrates, Arp2/3 for LegK2 and G-actin for VipA, to temporally control actin polymerization at the LCV and interfere with phagosome maturation and endosome recycling, thus contributing to the intracellular life cycle of the bacterium. Strikingly, the functional interaction between LegK2 and VipA is consolidated by an evolutionary history that has refined the best effector repertoire for the benefit of L. pneumophila virulence.

microbiology↗

bioRxiv2022

A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation

Cancer cells outgrowing in distant organs of metastasis rewire their metabolism to fuel on the available nutrients. While this is often considered an adaptive pressure limiting metastasis formation, some nutrients available at the metastatic site naturally or through changes in organ physiology may inherently promote metastatic growth. We find that the lung, a frequent site of metastasis, is a lipid-rich environment. Moreover, we observe that pathological conditions such as pre-metastatic niche formation and obesity further increase the availability of the fatty acid palmitate in the lung. We find that targeting palmitate processing inhibits spheroid growth in vitro and metastasis formation in lean and obese mice. Mechanistically, we discover that breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a (CPT1a)-dependent manner. Lysine acetyltransferase 2a (KAT2a), whose expression is promoted by palmitate availability, relies on the available acetyl-CoA to acetylate the NF-{kappa}B subunit p65. This favors nuclear location of p65 and activates a pro-metastatic transcriptional program. Accordingly, deletion of KAT2a phenocopies CPT1a silencing in vitro as well as in vivo and patients with breast cancer show co-expression of both proteins in metastases across palmitate-rich metastatic sites. In conclusion, we find that palmitate-rich environments foster metastasis growth by increasing p65 acetylation resulting in elevated NF-{kappa}B signaling.

cancer biology↗

bioRxiv2021

Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Antimicrobial resistance (AMR) is threatening the lives of millions worldwide. Antibiotics which once saved countless lives, are now failing, ushering in vaccines development as a current global imperative. Conjugate vaccines produced either by chemical synthesis or biologically in Escherichia coli cells, have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccines assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel method that overcomes the limitations of the current conjugate vaccine design method(s). We demonstrate at least 2-fold increase in glycoconjugate yield via MAGIC when compared to conventional bioconjugate method(s). Furthermore, the modularity of the MAGIC platform also allowed us to perform glycoengineering in genetically intractable bacterial species other than E. coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against bacteria, especially AMR pathogens, and could be applied for biopreparedness.

microbiology↗

bioRxiv2021

Extraction and high-throughput sequencing of oak heartwood DNA: assessing the feasibility of genome-wide DNA methylation profiling

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus spp.) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

plant biology↗

bioRxiv2021

BRAIN-BASED AUTHENTICATION: TOWARDS A SCALABLE, COMMERCIAL GRADE SOLUTION USING NONINVASIVE BRAIN SIGNALS.

Here we report on a field test where we asked if it is feasible to deliver a scalable, commercial-grade solution for brain-based authentication given currently available head wearables. In this study, forty-nine (49) participants completed multiple sessions in their natural home environment over a single week. Participants used an off-the-shelf brain signal measuring headband to record their own brain activity while completing various tasks. Recording sessions were self-operated by the participants and unsupervised by any expert or technician to simulate real world use cases, while also contrasting common research approaches to this topic that rely on data from controlled laboratory conditions. Although brain signals have a non-stationary, complex nature, when participants watched rapidly presented images, our authentication system was able to successfully construct a unique and robust "brain ID" for each participant. Based on this brain ID, we developed a simplified brain-based authentication method that captures distinguishable information with reliable, commercial-grade performance from participants at their own homes. We conclude that noninvasively measured brain signals are ideal for use in biometric authentication systems, especially in environments where head wearables such as headphones or AR/VR devices are used as these devices offer a natural form factor for capturing participant brain ID continuously.

neuroscience↗

bioRxiv2021

DIFFERENCES IN THE EFFECTS ON HUMAN FOCUS OF MUSIC PLAYLISTS AND PERSONALIZED SOUNDSCAPES, AS MEASURED BY BRAIN SIGNALS.

The goal of this study was to investigate the effect of sounds on human focus and to identify the properties that contribute most to increasing and decreasing focus in people within their natural, everyday environment. Participants (N=62, 18-65y) performed various tasks on a tablet computer while listening to either no background sounds (silence), popular music playlists designed to increase focus (pre-recorded songs in a particular sequence), or engineered soundscapes that were personalized to individual listeners (digital audio composed in real-time based on input parameters such as heart rate, time of day, location, etc.). Sounds were delivered to participants through headphones while simultaneously their brain signals were recorded by a portable electroencephalography headband. Participants completed four one-hour long sessions at home during which different sound content played continuously. Using brain decoding technology, we obtained individual participant focus levels over time and used this data to analyze the effects of various properties of sound. We found that while participants were working, personalized soundscapes increased their focus significantly above silence (p=0.008), while music playlists did not have a significant effect. For the young adult demographic (18-36y), all sound content tested was significantly better than silence at producing focus (p=0.001-0.009). Personalized soundscapes increased focus the most relative to silence, but playlists of pre-recorded songs also increased focus significantly during specific time intervals. Ultimately we found that it is possible to accurately predict human focus levels that will be experienced in response to sounds a priori based on the sounds physical properties. We then applied this finding to compare between music genres and revealed that classical music, engineered soundscapes, and natural sounds were the best genres for increasing focus, while pop and hip-hop were the worst. These insights can enable human and artificial intelligence composers to produce increases or decreases in listener focus with high temporal (millisecond) precision. Future research will include real-time adaptation of sound libraries for other functional objectives beyond affecting focus, such as affecting listener enjoyment, stress, and memory.

neuroscience↗

bioRxiv2021

Ultraviolet light-induced collagen degradation inhibits melanoma invasion

Ultraviolet radiation (UVR) increases the incidence of cutaneous melanoma1-4. The ageing, sun-exposed dermis accumulates UVR damage5, and older patients develop more melanomas at UVR-exposed sites4,6,7. As fibroblasts are functionally heterogeneous and play key roles in the stromal contribution to cancer8,9, we asked whether UVR modifies dermal fibroblast function. Here we confirmed the expression of collagen-cleaving matrix metalloprotein-1 (MMP1) by UVR-damaged fibroblasts was persistently upregulated to reduce local levels of collagen 1 (COL1A1), and found dermal COL1A1 degradation by MMP1 decreased melanoma invasion. Conversely, we show inhibiting extracellular matrix degradation and MMP1 expression restored melanoma invasion to UVR damaged dermis. We confirmed in vitro findings in a cohort of primary cutaneous melanomas of aged humans, showing more cancer cells invade as single cells at the invasive front of melanomas expressing and depositing more collagen. We found collagen and single melanoma cell invasion are robust predictors of poor melanoma-specific survival. These data indicate melanomas arising over UVR-damaged, collagen-poor skin of the elderly are less invasive, and this reduced invasion improves survival. Consequently, although UVR increases tumour incidence, it delays primary melanoma invasion by degrading collagen. However, we show melanoma-associated fibroblasts can restore invasion in low-collagen primary tumours by increasing collagen synthesis. Finally, we demonstrate high COL1A1 gene expression is a biomarker of poor outcome across a broad range of primary cancers.

cancer biology↗

bioRxiv2019

Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that are distinct from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more deleterious, a larger proportion has successfully adapted for the new activity, exhibiting different functional profiles: (i) both strong and weak trade-off in original vs promiscuous activity are observed; (ii) trade-off is more severe (10- to 20-fold increased kcat/KM in arylesterase with [~]100-fold decreases in the original phosphotriesterase activity) and (iii) improvements show up in kcat rather than KM, suggesting novel adaptive solution. These distinct features make TRIAD an alternative to widely used point mutagenesis, providing access to functional innovations and traversing unexplored fitness landscape regions.

biochemistry↗