Biology subjects

Hubbard, J.

Publications and source records attributed to Hubbard, J..

8 recordsLinked to original sources

bioRxiv2025

Small siphophage binding to an open state of the LptDE outer membrane lipopolysaccharide translocon

Bacteriophages are bacterial viruses that provide alternatives to small-molecule drugs to combat infections by antibiotic-resistant bacteria. To infect a bacterial host, a phage needs to bind to the bacterial surface via receptor binding proteins (RBPs), which are critical for determining host specificity. For functionally important receptors, the RBP-receptor interaction could be exploited via phage steering, where emerging bacterial resistance due to receptor modification could make bacteria less fit or virulent. Despite this, relatively little is known about RBP-receptor interactions. Here we build on the recent discovery of coliphages that have the outer membrane (OM) lipopolysaccharide translocon LptDE as their terminal receptor and show via cryogenic electron microscopy (cryoEM) that, surprisingly, the RBP of the small siphophage Oekolampad binds to a hitherto unobserved, open state of LptDE. The open lateral gate of LptD is occupied by a {beta}-strand peptide originating from the degraded N-terminal jellyroll domain of LptD, suggesting the possibility of LptD inhibition via peptidomimetics. A structure of LptDE in complex with the superinfection exclusion (SE) protein Rtp45 of the Oekolampad-related phage Rtp shows a mechanism of SE where Rtp45-induced conformational changes in LptD resulting from steric clashes preclude RBP binding. Finally, analysis of spontaneous Oekolampad-resistant E. coli mutants identifies mutations in LptD that abolish the LptDE-RBP interaction in vitro. SDS-EDTA sensitivity assays of the mutants show no major defects in LptDE function, suggesting that phage steering via LptDE might be challenging. SignificanceThe outer membrane (OM) of Gram-negative bacteria is a protective barrier generated by lipopolysaccharide (LPS), a complex glycolipid that makes up the outer leaflet of the OM. Following its synthesis and transport from the inner membrane, LPS is inserted into the OM by LptDE, an OM protein complex essential for most Gram-negative bacteria. LPS insertion requires a hitherto unobserved, open state of LptDE. Here we report the unexpected finding that the receptor binding protein (RBP) of a bacteriophage binds to an open state of LptDE, allowing isolation and visualisation via cryogenic electron microscopy. The lateral gate of the LptDE-RBP complex is occupied by a {beta}-strand peptide, suggesting that the LptDE of pathogenic bacteria could be inhibited by peptidomimetics.

microbiology↗

bioRxiv2025

Anionic polymers stabilize Cas9 ribonucleoprotein nanoparticles to improve direct protein delivery and genome editing efficiencies in plant protoplasts

The application of CRISPR-based genome editing tools in plants is often challenged by low editing efficiencies, requiring most plant editing workflows to proceed through the delivery of pre-formed ribonucleoprotein (RNP) complexes to protoplasts. Here, we report increases in protoplast-based RNP delivery and genome editing efficiencies through the addition of anionic polymers to standard protoplast transfection protocols. We test addition of various polymers and peptides for their ability to increase genome editing efficiencies in both Nicotiana benthamiana and Arabidopsis thaliana protoplasts, by adding these components to standard PEG-based protoplast transfection protocols: i) non-covalent addition of charged polymers, ii) non-covalent addition of amphiphilic peptide A5K, and iii) tyrosinase-mediated covalent conjugation of various relevant peptide motifs directly to the RNP. Incorporation of the amphiphilic peptide A5K or covalent attachment of peptides to the RNP had no positive effect on editing efficiencies. However, we found that addition of anionic polymer polyglutamic acid to standard PEG transfection protocols significantly improved editing efficiencies in both Nicotiana benthamiana and Arabidopsis thaliana protoplasts relative to RNPs alone without negatively impacting protoplast viability. Our results suggest anionic polymers stabilize the RNP and increase the colloidal stability of the protoplast transfection workflow. This simple and straightforward method of stabilizing Cas9 RNPs can be easily adopted by others working on direct protein delivery to plant protoplasts to increase genome editing efficiencies. Key messageThe addition of anionic polymer polyglutamic acid to standard protoplast PEG transfection workflows enhance CRISPR-Cas9-mediated gene editing in plant protoplasts. Our results suggest the mechanism of increased transfection efficiency is due to colloidal stabilization of RNPs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/635105v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1101f0org.highwire.dtl.DTLVardef@964402org.highwire.dtl.DTLVardef@14afe61org.highwire.dtl.DTLVardef@82a982_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2024

Conflicts between the DNA replication and repair machineries promote cell death in Gram-positive bacteria

Cellular proliferation relies on successful coordination and completion of genome replication and segregation. To help achieve this, many bacteria utilise regulatory pathways that ensure DNA replication initiation only occurs once per cell cycle. When dysregulated, loss of DNA replication control can have severe consequences. In Escherichia coli it has been established that hyper-initiation of DNA synthesis leads to pleiotropic genome instability and cell death. Therefore, targeting DNA replication initiation proteins to promote hyper-initiation may be an approach to generate novel antimicrobials. However, the pathways and potential consequences of replication hyper-initiation in Gram-positive species remain enigmatic. To address this question, we devised genetic systems to artificially induce hyper-initiation in the model organism Bacillus subtilis and the pathogen Staphylococcus aureus. In both species, hyper-initiation elicited cellular degeneration culminating in growth inhibition by cell death. During this process in B. subtilis, temporal analyses revealed the early onset of the DNA damage response, followed by membrane depolarisation and cell lysis. This phenotype could be supressed by removing pathways that repair damaged DNA, suggesting that cell death is a consequence of conflicts between DNA replication and repair. In S. aureus, cells quickly accumulated striking morphological changes associated with rapid loss of chromosomal DNA and death via a lysis-independent pathway. Moreover, inducing hyper-initiation in S. aureus was observed to decrease bacterial survival during infection of murine macrophages. Taken together, the data suggest that stimulating initiation of bacterial DNA synthesis could be an alternative approach to inhibiting microbial growth, particularly in combination with compounds that inhibit or poison DNA repair, akin to cancer therapies.

microbiology↗

bioRxiv2023

Interactive Bioacoustic Playback as a Tool for Detecting and Exploring Nonhuman Intelligence: Conversing with an Alaskan Humpback Whale

Here we report on a rare and opportunistic acoustic "conversation" with an adult female humpback whale, known as Twain, in Southeast Alaska. Post hoc acoustic and statistical analyses of a 20-minute acoustic exchange between the broadcast of a recorded contact call, known as a whup/throp, with call responses by Twain revealed an intentional human-whale acoustic (and behavioral) interaction. Our results show that Twain participated both physically and acoustically in three phases of interaction (Phase 1: Engagement, Phase 2: Agitation, Phase 3: Disengagement), independently determined by blind observers reporting on surface behavior and respiratory activity of the interacting whale. A close examination of both changes to the latency between Twains calls and the temporal matching to the latency of the exemplar across phases indicated that Twain was actively engaged in the exchange during Phase 1 (Engagement), less so during Phase 2 (Agitation), and disengaged during Phase 3 (Disengagement). These results, while preliminary, point to several key considerations for effective playback design, namely the importance of salient, dynamic and adaptive playbacks, that should be utilized in experimentation with whales and other intelligent nonhuman species.

animal behavior and cognition↗

bioRxiv2020

Melanopsin-dependent direct photic effects are equal to clock-driven effects in shaping the nychthemeral sleep-wake cycle

Nychthemeral sleep-wake cycles (SWc) are known to be generated by the circadian clock in suprachiasmatic nuclei (SCN), entrained to the light-dark cycle. Light also exerts direct acute effects on sleep and waking. However, under longer photic exposure such as the 24-hour day, the precise significance of sustained direct light effects (SDLE) and circuitry involved have been neither clarified nor quantified, as disentangling them from circadian influence is difficult. Recording sleep in mice lacking a circadian pacemaker and/or melanopsin-based phototransduction, we uncovered, contrary to prevailing assumptions, that circadian-driven input shapes only half of SWc, with SDLE being equally important. SDLE were primarily mediated (>80%) through melanopsin, of which half were relayed through SCN, independent of clock function. These findings were used for a model that predicted SWc under simulated jet-lag, and revealed SDLE as a crucial mechanism influencing behavior, and should be considered for circadian/sleep disorder management and societal lighting optimization.

neuroscience↗

bioRxiv2019

Reassessing the validity of slow-wave dynamics as a proxy for NREM sleep homeostasis

Sleep-wake driven changes in NREM sleep (NREMS) EEG delta ({delta}: [~]0.75-4.5Hz) power are widely used as proxy for a sleep homeostatic process. We noted frequency increases in {delta}-waves in sleep-deprived (SD) mice, prompting us to re-evaluate how slow-wave characteristics relate to prior sleep-wake history. We discovered two types of {delta}-waves; one responding to SD with high initial power and fast, discontinuous decay ({delta}2: [~]2.5-3.5Hz) and another unrelated to time-spent-awake with slow, linear decays ({delta}1: [~]0.75-1.75Hz). Human experiments confirmed this {delta}-band heterogeneity. Similar to SD, silencing of centromedial thalamus neurons boosted {delta}2-waves, specifically. {delta}2-dynamics paralleled that of temperature, muscle tone, heart-rate, and neuronal UP/DOWN state lengths, all reverting to characteristic NREMS levels within the first recovery hour. Thus, prolonged waking seems to necessitate a physiological recalibration before typical NREMS can be reinstated. These short-lasting {delta}2-dynamics challenge accepted models of sleep regulation and function based on the merged {delta}-band as sleep-need proxy.

neuroscience↗

bioRxiv2019

Simple and complex interactions between sleep-wake driven and circadian processes shape daily genome regulatory dynamics in the mouse

The timing and duration of sleep results from the interaction between a sleep-wake driven, or homeostatic, process (S) and a circadian process (C), and involves changes in gene expression and genomic regulation. Unraveling the respective contributions of S and C, and their interaction, to transcriptional and epigenomic regulatory dynamics requires sampling over time under unperturbed conditions and conditions of perturbed sleep. Here, we profiled mRNA expression and chromatin accessibility in the cerebral cortex of mice over a three-day period, including a 6-hour sleep deprivation (SD) on day two. Mathematical modeling established that a large proportion of rhythmic genes are actually governed by Process S with varying degrees of interaction with Process C, sometimes working in opposition. Remarkably, SD causes long-term effects on gene expression dynamics, outlasting phenotypic recovery, most strikingly illustrated by a dampening of the oscillation of most core clock genes, including Bmal1, suggesting that enforced wakefulness directly impacts the molecular clock machinery. Chromatin accessibility proved highly plastic and dynamically affected by SD. Distal regions, rather than promoters, display dynamics corresponding to gene transcription, implying that changes in mRNA expression result from constantly accessible promoters under the influence of distal enhancers or repressors. Srf was predicted as a transcriptional regulator driving immediate response, suggesting that Srf activity mirrors the build-up and release of sleep pressure. Our results demonstrate that a single, short SD has long-term aftereffects at the genomic regulatory level. Such effects might accumulate with repeated sleep restrictions, thereby contributing to their adverse health effects.\n\nSignificance statementWhen and how long we sleep is determined by the time-of-day and how long we have been awake, which are tracked molecularly by a circadian and a sleep-wake driven process, respectively. We measured the long-term consequences of a short-term sleep deprivation (SD) on gene expression and regulation in the mouse brain, and used mathematical models to determine the relative contributions of the circadian and sleep-wake driven processes. We find that many genes, including most of the genes that constitute the molecular circadian clock, are perturbed by SD long after the mice ceased showing behavioral signs of sleep loss. Our results have implications for human health, given the high prevalence of insufficient and poor quality sleep in our contemporary society.

genomics↗

bioRxiv2017

EEG Decoding Reveals the Temporal Dynamics and Functional Relevance of Goal-Relevant Representations

Models of action control assume that abstract task-set settings regulate lower-level stimulus/response representations. Yet, we know little about the functional and dynamic properties of task-set representations in humans. Using a cued task-switching paradigm, we show that information about task sets and lower-level stimulus/response aspects can be extracted through decoding analyses from the scalp electrophysiological signal (EEG) on the single-trial level and with high temporal resolution. Task-sets are active throughout the entire processing cascade and trial-to-trial variations in task-set strength emerges as a remarkably strong predictor of variability in performance, both within and between individuals. Also, taskset strength is related to stimulus representation strength at an early period and to the strength of response representations at a later period, consistent with the notion that task-sets coordinate successive, lower-level representations in a concurrent manner. These results demonstrate a powerful approach towards uncovering stages of information processing and their relative importance for performance.

neuroscience↗