Biology subjects

Howard, J.

Publications and source records attributed to Howard, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Artefact-free EEG during temporal interference stimulation reveals phase-specific modulation of the human visual cortex

Background: Temporal interference (TI) stimulation applies kHz electric fields whose envelope is modulated at a difference frequency ({Delta}f), allowing non-invasive deep-brain targeting. Measuring the neural response during stimulation with EEG is essential for establishing mechanisms and for closed-loop control. However, non-linearities in the recording chain mix the kHz carriers, producing an artefact at exactly {Delta}f. Objectives: To establish a method for recording EEG during TI without the {Delta}f artefact, and to test whether TI modulates neural activity at {Delta}f. Methods: A front-end analogue low-pass filter (7th order; 90 Hz cut-off; [≥]130 dB attenuation at 1 kHz) was bench-tested. In 22 healthy participants, residual artefact was assessed per recording with a two-stage detection pipeline in stimulation-only blocks. TI targeted the right primary visual cortex at 10 or 15.5 Hz, in-phase or anti-phase with multi-frequency photic stimulation (4.5, 10, 15.5 Hz), during a choice reaction time task. Results: The filter suppressed the {Delta}f artefact below the noise floor on a resistive load. In vivo, residual artefact was confirmed in two participants and could not be excluded in two more; all four were excluded from EEG analyses. Steady-state visual evoked potential (SSVEP) amplitude at 11, 14.5 and 15.5 Hz predicted faster responses. In-phase 15.5 Hz TI increased SSVEP amplitude at 14.5 and 15.5 Hz and shortened response times relative to both sham and anti-phase TI, which did not differ from sham. Conclusions: With front-end filtering and per-recording verification, EEG can measure TI neuromodulation during stimulation and at {Delta}f, revealing phase-specific modulation of visual entrainment linked to behaviour.

neuroscience↗

bioRxiv2026

Rapid volumetric reconstruction and tracking for Fourier light-field microscopy enables real-time calcium imaging in freely behaving Hydra.

Fourier light field microscopy (FLFM) enables high-speed volumetric imaging by encoding multiple angular perspectives of a three-dimensional sample onto a single image. For this reason, FLFM is well-suited to sparse and rapidly evolving biological systems. To aid in the adoption of FLFM, we present OpenFLR, an open-source software framework for real-time volumetric reconstruction, three-dimensional particle tracking, and calcium image processing using FLFM. OpenFLR reconstruction is distributed as four interchangeable interfaces: a Python library, a command-line script, an interactive web application, and an ImageJ/micromanager plugin, so that the pipeline is accessible to both developers and bench biologists. Building on established Richardson-Lucy deconvolution, we use a hybrid experimental-computational PSF calibration strategy and a triangulation approach to tracking to extract particle positions in 3D directly from raw light field frames, bypassing reconstruction. We validate the complete pipeline on GCaMP6s recordings of freely behaving Hydra vulgaris, tracking sparse populations of neurons as they undergo large three-dimensional displacements.

biophysics↗

bioRxiv2026

Sticker number modulates pyrenoid condensate assembly to support algal fitness

The valency of intrinsically disordered proteins underpins liquid-liquid phase separation (LLPS), yet how this parameter shapes condensate function and cellular fitness remains poorly understood. Here we exploit the algal pyrenoid-a minimal, two component LLPS system-to directly link condensate properties to physiological performance. Pyrenoid assembly is driven by a disordered, multivalent Linker protein that binds Rubisco at symmetry-related surface sites, with the number of binding motifs ("stickers") varying across species. Using Chlamydomonas reinhardtii, we systematically tuned sticker number from two to nine and examined effects on Rubisco condensation, pyrenoid architecture and CO2 fixation. Three stickers were sufficient for condensation in vitro, but at least four were required for pyrenoid assembly in vivo. Cryo-electron tomography and single-molecule tracking revealed that increasing sticker number enhances Rubisco packing and mobility, while time-resolved imaging and competition assays demonstrated that sticker number governs the kinetics of pyrenoid formation and determines cellular fitness under fluctuating carbon conditions. Our findings establish sticker number as an evolutionary tuning parameter that balances condensate formation, dynamics, and function, providing a quantitative framework for linking the molecular grammar of phase separation to biological fitness.

cell biology↗

bioRxiv2025

A correlative quantitative phase contrast and fluorescence super-resolution microscope for imaging molecules in their cellular context

Fluorescence microscopy has been widely used to reveal the spatial distribution of specifically labeled molecules, but it is blind to cellular context. Quantitative phase contrast microscopy (QPC) provides such complementary information. Here we have developed a platform that combines the QPC technique of correlative orientation-independent differential interference contrast (OI-DIC) microscopy with single-molecule super-resolution fluorescence microscopy. We demonstrate a detection sensitivity of 0.05 nm optical path difference, sufficient to detect single microtubules, and show its capability of 3D super-resolution fluorescence imaging in the cellular context. Additionally, we report deep-learning enabled digital staining, identifying nuclei, mitochondria and lipid droplets from OI-DIC data and demonstrate the potential of this approach for long-term live-cell imaging of organelles of interest without the need for fluorescence. OI-DIC can be easily integrated into most fluorescence microscopes and is readily adoptable by microscopy labs. ONE-SENTENCE TEASERA highly sensitive technique to visualize sub-cellular structures, dynamics, and molecules in their cellular context.

cell biology↗

bioRxiv2025

Computational analysis of therapeutic potential for simplified Piper. spp- derived medicinal mixtures in anxiety, sleep, pain and seizure.

Phytomedicines have played a vital role in traditional medical systems globally, particularly in providing culturally relevant and accessible healthcare solutions. Piper methysticum, known as Kava, is a traditional Pacific Island phytomedicine with clinically validated anxiolytic properties, primarily attributed to its Kavalactones. However, the biogeographically restricted distribution of Piper methysticum and the ecological and cultural concerns surrounding its widespread adoption highlight the need to explore alternative sources within the Piper genus. This study investigates whether other species within the Piper genus, used phytomedically in non-Pacific contexts, exhibit similar therapeutic efficacy for anxiety, stress, and related disorders including Post-Traumatic Stress Disorder (PTSD). We employed a computational approach utilizing a novel data platform of non-Western phytomedical pharmacopeias to analyze the secondary metabolomes of various Piper species. Network analysis and multidimensional data projections were used to compare the chemical composition and therapeutic indications of these species with those of Piper methysticum. Our findings suggest that while Kavalactones are predominantly unique to Piper methysticum, other Piper species also contain bioactive compounds associated with anxiolytic and stress-relieving effects. These results provide insight into the potential for culturally and biogeographically contextualized approaches to PTSD treatment, beyond the exclusive use of Kava, and lay the groundwork for future research into alternative phytomedicinal therapies within the Piper genus.

pharmacology and toxicology↗

bioRxiv2025

A pathway to next-generation mast cell stabilizers identified through the novel Phytomedical Analytics for Research Optimization at Scale data platform.

Mast cell stabilizers (MCS) have the potential to address unmet therapeutic need in allergy and inflammation management. MCS chronically suppress all arms of the pro-inflammatory mast cell response to stimulation (e.g., histamine, protease, lipid mediators, cytokines, chemokines). They may therefore outperform approaches such as H1, H2 and H4 inhibitors (antihistamines) which block only the acute histamine release by mast cells and leave the rest of the functional response untouched. Despite their potential, current MCS (e.g., cromolyn sodium, Tranilast, nedocromil) in clinical use are hindered by poor bioavailability, frequent dosing, long lags to onset of relief, and enigmatic mechanisms of action. MCS have their origins in phytomedicine: cromolyn sodium is the longest standing drug in the class and is a derivatized form of Khellin from Ammi visnaga, used as an anti-inflammatory. Other phytopharmacopeias may offer candidate next generation MCS (ngMCS) and in this study we hypothesized that a coupled pharmacoanalytic and in vitro pharmacology approach could be used to identify, prioritize and derisk additional candidate MCS from phytomedical sources for later pre-clinical and clinical evaluation. Here, we report a novel data analytics workflow starting with a newly developed phytopharmacopeia data platform with >3.5B linkage pathways (country [->] medical system [->] formulation [->] indication [->] ingredient organism [->] chemical component [->] other parameters), covering 22 M sq. miles of biogeography and historical and contemporary timeframes. Additional data layers include druggability indices, target and pathway analyses. The current study validates a subset of candidate phytomedical ngMCS using in silico workflow and in vitro pharmacology, and develops a new harmonic mean-based MCS score for further streamlining of the candidate prioritization process. This proof-of-concept study may have particular relevance for complex presentations such as Mast Cell Activation Syndrome (MCAS) where ngMCS may outperform current management approaches.

immunology↗

bioRxiv2025

Neurons exploit stochastic growth to rapidly and economically build dense radially oriented dendritic arbors

Dendrites grow by stochastic branching, elongation, and retraction. A key question is whether such a mechanism is sufficient to form highly branched dendritic morphologies. Alternatively, are signals from other cells or is the topological hierarchy of the growing network necessary for dendrite geometry? To answer these questions, we developed a mean-field model in which branch dynamics is isotropic and homogenous (i.e., no extrinsic instruction) and depends only on the average lengths and densities of branches. Branching is modeled as density-dependent nucleation so there are no tree structures and no network topology. Despite its simplicity, the model predicted several key morphological properties of class IV Drosophila sensory dendrites, including the exponential distribution of branch lengths, the parabolic scaling between dendrite number and length densities, the tight spacing of the dendritic meshwork (which required minimal total branch length), and the radial orientation of branches. Stochastic growth also accelerated the overall expansion rate of the arbor. Therefore, stochastic dynamics is an economical and rapid space-filling mechanism for building dendritic arbors without external guidance or hierarchical branching mechanisms. Our model provides a general theoretical framework for understanding how macroscopic branching patterns emerge from microscopic dynamics.

developmental biology↗

bioRxiv2025

PI(3)P signaling regulates endosomal flux underlying developmental synaptic remodeling via Rab4

Rab4 GTPase, essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) of Drosophila larvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed, transient contraction of synapses in the ventral neuropil region at a specific larval stage. This coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruit a PX-domain-containing motor protein, Klp98A, accelerating synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via insulin signaling and directed axonal transport of endosomes. Author summaryHealthy brain function relies on the growth and remodeling of synaptic connections between nerve cells. This process involves carefully orchestrated movements of proteins and membranes inside the neurites. For instance, endosome movement in the nerve cell extensions helps guide neuronal growth, recycle parts of the synapse, and alter signal transmissions between neurons. Rab GTPases, a group of small G-proteins, are crucial for these processes. Here, we aimed to understand what triggers these processes and how the supply chain within nerve cell extensions is connected to changes at the synapse. In fruit fly larvae, we observed that the movement of Rab4 vesicles in the nerve cell extensions is inversely related to gross synapse content. A further examination revealed that onset of insulin signaling accelerates the Rab4 vesicles towards synapse through a lipid kinase called Vps34, which makes a signaling lipid called phospatidylinnositol-3-phosphate. Higher levels this lipid molecule on Rab4 vesicles attract a motor protein, Klp98A, speeding up their movement during the synaptic contraction phase. Our findings reveal a new mechanism that regulates Rab4 vesicle transport and its impact on synapse stability. The study further suggests potential targets for controlling age-related dementia likely to be caused by excessive insulin signaling in neurons.

neuroscience↗

bioRxiv2024

A Discard-and-Restart MD algorithm for the sampling of realistic protein transition states and enhance structure-based drug discovery

We introduce a Discard-and-Restart molecular dynamics (MD) algorithm tailored for the sampling of realistic protein transition states. It aids computational structure-based drug discovery by reducing the simulation times to compute transition pathways by up to 2000x. The algorithm iteratively performs short MD simulations and measures their proximity to a target state via a collective variable (CV) loss, which can be defined in a flexible fashion, locally or globally. Using the loss, if the trajectory proceeds toward the target, the MD simulation continues. Otherwise, it is discarded and a new MD simulation is restarted, with new initial velocities randomly drawn from a Boltzmann distribution. The discard-and-restart algorithm demonstrates efficacy and atomistic accuracy in capturing the folding pathways in several contexts: (1) fast-folding small protein domains; (2) the folding intermediate of the prion protein PrP; and (3) the spontaneous partial unfolding of -Tubulin, a crucial event for microtubule severing. During each iteration of the algorithm, we are able to perform AI-based analysis of the transitory conformations to find binding pockets, which could potentially represent druggable sites. Overall, our algorithm enables systematic and computationally efficient exploration of conformational landscapes, enhancing the design of ligands targeting dynamic protein states.

biophysics↗

bioRxiv2024

Human TRMT1 and TRMT1L paralogs ensure the proper modification state, stability, and function of tRNAs

The tRNA methyltransferase 1 (TRMT1) enzyme catalyzes m2,2G modification in tRNAs. Intriguingly, vertebrates encode an additional tRNA methyltransferase 1-like (TRMT1L) paralog. Here, we use a comprehensive tRNA sequencing approach to decipher targets of human TRMT1 and TRMT1L. We find that TRMT1 methylates all known tRNAs containing guanosine at position 26 while TRMT1L represents the elusive enzyme catalyzing m2,2G at position 27 in tyrosine tRNAs. Surprisingly, TRMT1L is also necessary for maintaining acp3U modifications in a subset of tRNAs through a process that can be uncoupled from methyltransferase activity. We also demonstrate that tyrosine and serine tRNAs are dependent upon m2,2G modifications for their stability and function in translation. Notably, human patient cells with disease-associated TRMT1 variants exhibit reduced levels of tyrosine and serine tRNAs. These findings uncover unexpected roles for TRMT1 paralogs, decipher functions for m2,2G modifications, and pinpoint tRNAs dysregulated in human disorders caused by tRNA modification deficiency.

molecular biology↗

bioRxiv2024

Bacterial stress granule protects mRNA through ribonucleases exclusion

Membraneless droplets formed through liquid-liquid phase separation of ribonucleoprotein particles contribute to mRNA storage in eukaryotic cells. How such aggresomes contribute to mRNA dynamics under stress, and their functional role, is less understood in bacteria. Here we used multiple approaches including live-cell imaging, polymer physics modelling and transcriptomics to show that prolonged stress leading to ATP depletion in Escherichia coli results in increased aggresome formation, compaction, and selective mRNA enrichment within these aggresomes. Longer transcripts accumulate more in aggresomes than in the cytosol. Mass spectrometry and mutagenesis studies showed that mRNA ribonucleases are excluded from aggresomes due to electrostatic repulsion arising from their negative surface charges. Experiments with fluorescent reporters and disruption of aggresome formation showed that mRNA storage within aggresomes promoted rapid translation reactivation and associated with reduced lag phases during growth after stress removal. Our findings suggest that mRNA storage within aggresomes confers an advantage for bacterial survival and recovery from stress.

biophysics↗

bioRxiv2024

Insulin signaling accelerates the anterograde movement of Rab4 vesicles in axons through Klp98a/KIF16B recruitment via Vps34-PI3Kinase

Rab4 GTPase organizes endosomal sorting essential for maintaining the balance between recycling and degradative pathways. Rab4 localizes to many cargos whose transport in neurons is critical for regulating neurotransmission and neuronal health. Furthermore, elevated Rab4 levels in the CNS are associated with synaptic atrophy and neurodegeneration in Drosophila and humans, respectively. However, how the transport of Rab4-associated vesicles is regulated in neurons remains unknown. Using in vivo time-lapse imaging of Drosophila larvae, we show that activation of insulin signaling via Dilp2 and dInR increases the anterograde velocity, run length, and flux of Rab4 vesicles in the axons. Molecularly, we show that activation of neuronal insulin signaling further activates Vps34, elevates the levels of PI(3)P on Rab4-associated vesicles, recruits Klp98A (a PI(3)P-binding kinesin-3 motor) and activates their anterograde transport. Together, these observations delineate the role of insulin signaling in regulating axonal transport and synaptic homeostasis. HighlightsO_LIDilp2-mediated insulin signaling activates anterograde transport of Rab4 vesicles C_LIO_LIVps34 regulates anterograde velocity, run length and flux of Rab4 vesicles C_LIO_LILocal PI(3)P signaling on Rab4 vesicles regulates their motility in the axons C_LIO_LIPI(3)P production upon acute insulin stimulation recruits Klp98A on Rab4 vesicles C_LI

cell biology↗

bioRxiv2024

Loop-mediated Isothermal Amplification (LAMP) assay for reliable detection of Xanthomonas axonopodis pv. vasculorum

Xanthomonas axonopodis pv. vasculorum (Xav), the causative agent of sugarcane gumming disease, represents a significant threat to global sugarcane production due to its systemic and destructive nature. Despite the economic implications, a field-deployable, Xav-specific diagnostic tool has not been developed. This resulted in a loop-mediated isothermal amplification (LAMP) assay targeting the pelL gene, unique to Xav strains, as a rapid and precise diagnostic assay. The selection of the pelL gene was informed by comprehensive in silico analyses of Xav genomes and related Xanthomonas species and other close relatives. Validation against the NCBI GenBank database and internally sequenced genomes confirmed the genes exclusivity to Xav. Subsequent primers for both endpoint PCR and LAMP assays were designed using the pelL gene region. The LAMP assay underwent extensive testing against inclusivity and exclusivity panels. Use of exclusivity panel, comprising 81 strains from related species, other bacterial genera, and host genomes, demonstrated the assays specificity with no false positives. The assay exhibited a detection limit of 1 pg, and its effectiveness was unimpeded by crude host lysate (sugarcane). Further validation through multi-device and multi-operator testing underscored the assays 100% reproducibility and robustness. Application to infected plant samples resulted in the detection of all infected specimens without any false positives or negatives. This novel LAMP assay is accurate and reliable tool for Xav detection, with promising applications in routine diagnostics, biosecurity measures, microbial forensics, and epidemiological research.

plant biology↗

bioRxiv2024

Correlating fluorescence microscopy, optical and magnetic tweezers to study single chiral biopolymers, tested on DNA plectoneme formation dynamics

Biopolymer topology is critical for determining interactions inside cell environments, exemplified by DNA where its response to mechanical perturbation is as important as biochemical properties to its cellular roles. The dynamic structures of chiral biopolymers exhibit complex dependence with extension and torsion, however the physical mechanisms underpinning the emergence of structural motifs upon physiological twisting and stretching are poorly understood due to technological limitations in correlating force, torque and spatial localization information. We present COMBI-Tweez (Combined Optical and Magnetic BIomolecule TWEEZers), a transformative tool that overcomes these challenges by integrating optical trapping, time-resolved electromagnetic tweezers, and fluorescence microscopy, demonstrated on single DNA molecules, that can controllably form and visualise higher order structural motifs including plectonemes. This technology combined with cutting-edge MD simulations provides quantitative insight into complex dynamic structures relevant to DNA cellular processes and can be adapted to study a range of filamentous biopolymers.

biophysics↗

bioRxiv2023

Topology recapitulates ontogeny of dendritic arbors

Branching of dendrites and axons allows neurons to make synaptic contacts with large numbers of other neurons, facilitating the high connectivity of the nervous system. Neurons have geometric properties, such as the lengths and diameters of their branches, that change systematically throughout the arbor in ways that are thought to minimize construction costs and to optimize the transmission of electrical signals and the intracellular transport of materials. In this work, we investigated whether neuronal arbors also have topological properties that reflect the growth and/or functional properties of their dendritic arbors. In our efforts to uncover possible topological rules, we discovered a function that depends only on the topology of bifurcating trees such as dendritic arbors: the tip-support distribution, which is the average number of branches that support n dendrite tips. We found that for many, but not all, neurons from a wide range of invertebrate and vertebrate species, the tip-support distribution follows a power law with slopes ranging from -1.4 and -1.8 on a log-log plot. The slope is invariant under iterative trimming of terminal branches and under random ablation of internal branches. We found that power laws with similar slopes emerge from a variety of iterative growth processes including the Galton-Watson (GW) process, where the power-law behavior occurs after the percolation threshold. Through simulation, we show the slope of the power-law increases with the branching probability of a GW process, which corresponds to a more regular tree. Furthermore, the inclusion of postsynaptic spines and other terminal processes on branches causes a characteristic deviation of the tip-support distribution from a power law. Therefore, the tip-support function is a topological property that reflects the underlying branching morphogenesis of dendritic trees.

biophysics↗

bioRxiv2022

Dynamic microtubules slow down during their shrinkage phase

Microtubules are dynamic polymers that undergo stochastic transitions between growing and shrinking phases. The structural and chemical properties of these phases remain poorly understood. The transition from growth to shrinkage, termed catastrophe, is not a first-order reaction but is rather a multi-step process whose frequency increases with the growth time: the microtubule ages as the older microtubule tip becomes more unstable. Aging shows that the growing phase is not a single state but comprises several substates of increasing instability. To investigate whether the shrinking phase is also multi-state, we characterized the kinetics of microtubule shrinkage following catastrophe using an in vitro reconstitution assay with purified tubulins. We found that the shrinkage speed is highly variable across microtubules and that the shrinkage speed of individual microtubules slows down over time by as much as several fold. The shrinkage slowdown was observed in both fluorescently labeled and unlabeled microtubules as well as in microtubules polymerized from tubulin purified from different species, suggesting that the shrinkage slowdown is a general property of microtubules. These results indicate that microtubule shrinkage, like catastrophe, is time-dependent and that the shrinking microtubule tip passes through a succession of states of increasing stability. We hypothesize that the shrinkage slowdown is due to destabilizing events that took place during growth which led to multi-step catastrophe. This suggests that the aging associated with growth is also manifest during shrinkage with the older, more unstable growing tip being associated with a faster depolymerizing shrinking tip. Statement of SignificanceThe dynamics of the microtubule cytoskeleton is crucial for several functions in eukaryotic cells. Microtubule dynamics is traditionally described by constant growth and shrinkage speeds with first order transitions between the growth and shrinkage phases. However, catastrophe, the transition from growth to shrinkage, increases with microtubule age and is not a first order process. In contrast to the common assumption that microtubules shrink with constant speed, here we show that shrinking microtubule tips undergo step-wise slowdown during depolymerization. Our results suggest that microtubule shrinkage, like catastrophe, is a multi-step process. This finding is important for understanding the molecular nature of microtubule dynamic instability and how microtubule shrinkage can be modulated by microtubule associated proteins.

biophysics↗

bioRxiv2022

Scaled-expansion of the membrane associated cytoskeleton requires conserved kinesin adaptors

A periodic lattice of actin rings and spectrin tetramers scaffolds the axonal membrane. How spectrin is delivered to this structure to scale its size to that of the growing axon is unknown. We found that endogenous spectrin, visualized with singe axon resolution in vivo, is delivered to hotspots in the lattice that support its expansion at rates set by axon stretch-growth. Unlike other cytoskeletal proteins, whose apparent slow movement consists of intermittent bouts of fast movements, spectrin moves slowly and processively. We identified a pair of coiled coil proteins that mediate this slow movement and the expansion of the lattice by linking spectrin to kinesin-1. Thus, processive slow transport and local lattice incorporation support scaled cytoskeletal expansion during axon stretch-growth. One-Sentence SummaryKinesin adaptors control spectrin transport and expansion of the membrane periodic skeleton.

cell biology↗

bioRxiv2022

The force required to remove tubulin from the microtubule lattice

Severing enzymes and molecular motors extract tubulin from the walls of microtubules by exerting mechanical force on subunits buried in the lattice. However, how much force is needed to remove tubulin from microtubules is unknown, as is the pathway by which subunits are removed. Using a site-specific functionalization method, we applied forces to the C-terminus of -tubulin with an optical tweezer and found that a force of [~]30 pN is required to extract tubulin from the microtubule wall. Consistent with this force, we show that several kinesins can also extract tubulin. Additionally, we discovered that partial unfolding is an intermediate step in tubulin removal. The unfolding and extraction forces are similar to those generated by AAA-unfoldases, suggesting that severing proteins such as spastin and katanin use an unfoldase mechanism. Our results reveal the response of tubulin to mechanical force and advance our understanding of severing enzymes and microtubule stability.

biophysics↗