Biology subjects

Kenny, S.

Publications and source records attributed to Kenny, S..

10 recordsLinked to original sources

bioRxiv2026

De novo design of selective kinase modulators

Protein kinases are critical regulators of cellular signaling, but precise modulation of their activity remains challenging due to their high structural conservation. Here, we present de novo designed genetically encoded miniproteins capable of activating or inhibiting focal adhesion kinase (FAK) by directly targeting the kinase domain itself. Among 96 binders designed to stabilize distinct conformational states of FAK, 33 modulated kinase activity. Biochemical characterization of the four most potent modulators revealed that two designs inhibit FAK with low-nanomolar IC50 values while the remaining two potentiated FAK activity by more than two-fold. When expressed in cells, the modulators preserved the same inhibitory and activating effects observed in vitro, establishing that designed conformational binders can directly tune FAK signaling in living cells. Taking advantage of the high similarity between kinases, we redesigned the FAK inhibitors to inhibit Src kinase. Our approach establishes a versatile platform for selective and genetically encoded kinase control as a way to rewire cell signaling and as a starting point for the discovery of novel modulatory sites of kinases.

biochemistry↗

bioRxiv2026

Reprogramming CAR T-Cells with designed bioPROTACs

Gene editing has been used to enhance CAR T-cell function by disrupting negative regulators but has limitations. Here we show that de novo-designed generated targeted degraders (bioPROTACs) provide an alternative approach. Expression of bioPROTACs in CAR T-cells targeting DNMT3A, a key regulator of T-cell exhaustion, phenocopied gene knockout. Our reversible, non-gene editing approach provides a tunable strategy to reprogram T-cell fate which should be broadly applicable for next-generation cell therapies.

immunology↗

bioRxiv2024

DIYNAFLUOR: An Affordable DIY Plug-and-Play Nucleic Acid Fluorometer for eDNA Quantification in Resource Limited Settings

Nucleic Acid (NA) fluorometry is widely employed for quantifying environmental DNA (eDNA) samples and their downstream DNA sequencing libraries, owing to its sensitivity, accuracy, and speed. However, the high cost of NA fluorometers presents a barrier to eDNA sequencing in resource limited settings (RLSs). For instance, at [~]$1.5-3.3k USD, current NA fluorometers present a greater capital cost than ONTs $1k USD portable MinION third-generation Nanopore sequencing platform. The collapse of international scientific device and consumable supply chains during the COVID-19 pandemic also highlighted the need for distributed manufacturing of molecular research tools to mitigate the impact of increased pricing to RLSs. To address these challenges, we have developed the "DIYNAFLUOR" (DIY Nucleic Acid FLUORometer), a portable, open-source, <$40 USD NA fluorometer, designed using readily available off-the-shelf components, simple 3D-printed parts, and plug-and-play, solder-free assembly. The DIYNAFLUOR was primarily designed to be compatible with the popular DNA-centric Qubit High Sensitivity (HS) and Broad Range (BR) assay kits. Notably, the DIYNAFLUOR demonstrated an in-assay Limit of Detection with the Qubit HS kit of 0.0028 ng/L, and an average absolute bias of 0.018 ng/L across a 0-10 ng/L working range using a 2-point linear calibration methodology. Device verification was performed by comparative measurements with a Qubit 4 fluorometer in a busy biotechnology laboratory, and build instructions were validated through assembly and qualification of three DIYNAFLUOR devices by researchers outside the primary design team. We also describe a custom "extreme"low-cost assay, <13{cents} USD per-measurement, that uses SYBR Safe dye to quantify DNA across a working range of 0-0.5 ng/L. This assay reports a lower sensitivity and accuracy than commercial kits but may be of use to RLSs in times of extreme resource constraints or as a teaching tool for STEM educators. To demonstrate its practical application for field-based eDNA analysis in RLSs, the DIYNAFLUOR was used to perform all quality control measurements throughout the preparation of a 16S and 18S metabarcoding library generated from eDNA extracted from Australian lake water, leading to the successful identification of Australian fauna via Nanopore sequencing. Finally, configurations of the DIYNAFLUOR for RNA and Protein quantification are briefly described. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/626200v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@28244dorg.highwire.dtl.DTLVardef@16749acorg.highwire.dtl.DTLVardef@15c31aorg.highwire.dtl.DTLVardef@bc03e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Limits of subjective and objective vection for ultra-high frame rate visual displays

Large-field optic flow generates the illusory percept of self-motion, termed vection. Smoother visual motion displays generate a more compelling subjective sense of vection and objective postural responses, as well as a greater sense of immersiveness for the user observing the visual display. Research suggests that the function linking frame rate and vection asymptotes at 60 frames per second (FPS), but previous studies have used only moderate frame rates that do not approach the limits of human perception. Here, we measure vection using subjective and objective (mean frequency and path length of postural centre-of-pressure (COP) excursions) responses following the presentation of high-contrast optic flow stimuli at slow and fast speeds and low and ultra-high frame rates. We achieve this using a novel rendering method implemented with a projector capable of sub-millisecond temporal resolution in order to simulate refresh rates ranging from very low (15 FPS) to ultra-high frame rates (480 FPS). The results suggest that subjective vection was experienced most strongly at 60 FPS. Below and above 60 FPS, subjective vection is generally weaker, shorter, and starts later, although this pattern varied slightly according to the speed of stimuli. For objective measures, while the frequency of postural sway was unaffected by frame rate, COP path length was greatest for 480 FPS stimuli. Together, our results support diminishing returns for vection above 60 FPS and provide insight into the use of high frame rate for enhancing the user experience in visual displays.

neuroscience↗

bioRxiv2020

Bio-upcycling of polyethylene terephthalate

Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, we present the sequential conversion of polyethylene terephthalate (PET) into two types of bioplastics: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films were hydrolyzed by a thermostable polyester hydrolase yielding 100% terephthalate and ethylene glycol. A terephthalate-degrading Pseudomonas was evolved to also metabolize ethylene glycol and subsequently produced PHA. The strain was further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which were used as monomers for the chemo-catalytic synthesis of bio-PU. In short, we present a novel value-chain for PET upcycling, adding technological flexibility to the global challenge of end-of-life management of plastics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/993592v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1433e0dorg.highwire.dtl.DTLVardef@19efc71org.highwire.dtl.DTLVardef@cf97f7org.highwire.dtl.DTLVardef@101f123_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Murine models of renal ischaemia reperfusion injury: An opportunity for refinement using non-invasive monitoring methods

BackgroundRenal Ischaemia Reperfusion Injury (R-IRI) can cause Acute Kidney Injury (AKI) and Chronic Kidney Disease (CKD), resulting in significant morbidity and mortality. To understand the underlying mechanisms, reproducible small-animal models of AKI and CKD are needed. We describe how innovative technologies for measuring kidney function non-invasively in small rodents allow successful refinement of the R-IRI models, and offer the unique opportunity to monitor longitudinally in individual animals the transition from AKI to CKD. MethodsMale BALB/c mice underwent bilateral renal pedicle clamping (AKI) or unilateral renal pedicle clamping with delayed contralateral nephrectomy (CKD) under isoflurane anaesthetic. Transdermal GFR monitoring and multi-spectral optoacoustic tomography in combination with statistical analysis were used to identify and standardise variables within these models. ResultsPre-clamping anaesthetic time was one of the most important predictors of AKI severity after R-IRI. Standardising pre-clamping time resulted in a more predictably severe AKI model. In the CKD model, initial improvement in renal function was followed by significant progressive reduction in function between weeks 2 and 4. Performing contralateral nephrectomy on day 14 enabled the development of CKD in a survivable way. ConclusionsNon-invasive monitoring of global and individual renal function after R-IRI is feasible, reproducible and correlates well with classical markers of injury. This facilitates refinement of kidney injury models and enables the degree of injury seen in pre-clinical models to be translated to those seen in the clinical setting. Thus, future therapies can be tested in a clinically relevant, non-invasive manner. What is already knownThe severity of Renal Ischaemia Reperfusion injury (R-IRI) varies between animal strain, gender and age. Experimental variables including temperature and clamping time are usually tightly controlled but significant variability still exists. Classically, small rodent experiments depend on endpoint evaluation of serum and histological features of disease. However, new technologies including transdermal glomerular filtration rate (GFR) monitoring and Multispectral Optoacoustic Tomography (MSOT) may enable renal function to be accurately monitored longitudinally, enabling better refinement of these models. What this study addsThis study shows that transdermal GFR measurements have reliably enabled refinement of the R-IRI model by standardisation of the duration of isoflurane prior to commencing surgery. Individual kidney function can be assessed in-vivo after unilateral R-IRI using MSOT imaging. The excretion tmax of IRDye-800 reliably represents the relative function of the injured kidney, permitting longitudinal in-vivo assessment of differential kidney function. What impact this may have on practiceThis study demonstrates the utility of two minimally-invasive in-vivo methods of monitoring kidney function which have advantages over classical methods and potentially enable fewer animals to be used in future studies. The study demonstrates refinement of bilateral and unilateral R-IRI models which will also enable a reduction in the number of animals needed for experimentation.

physiology↗

bioRxiv2019

Narrative and gaming experience interact to affect presence and cybersickness in virtual reality

Research has established a link between presence and cybersickness in virtual environments, but there is significant disagreement regarding the directionality of the relationship (positive or negative) between the two factors, and if the relationship is modulated by other top-down influences. Several studies have revealed a negative association between the factors, highlighting the prospect that manipulating one factor might affect the other. Here we examined if a top-down factor (narrative context) enhances presence, and whether this effect is associated with a decrease in cybersickness. We analyzed the association between responses to questionnaire measures of cybersickness and presence, as well as the degree to which their relationship was affected by the administration of an enriched or minimal verbal narrative context. The results of the first experiment, conducted in a controlled laboratory environment, revealed that enriched narrative was associated with increased presence, but that the reductive effect of narrative on cybersickness depended on video gaming experience. We also observed the expected negative association between presence and cybersickness, but only in the enriched narrative group. In a second experiment, conducted with a diverse sample at a public museum, we confirmed our previous finding that presence and cybersickness are negatively correlated, specifically when participants experienced an enriched narrative. We also confirmed the interaction between narrative and gaming experience with respect to cybersickness. These results highlight the complexity of the presence-cybersickness relationship, and confirm that both factors can be modulated in a beneficial manner for virtual reality users by means of top-down interventions.

neuroscience↗

bioRxiv2019

Super-resolution microscopy unveils FIP200-scaffolded, cup-shaped organization of mammalian autophagic initiation machinery

Autophagy is an essential physiological process by which eukaryotic cells degrade and recycle cellular materials. Although the biochemical hierarchies of the mammalian autophagy pathway have been identified, questions remain regarding the sequence, subcellular location, and structural requirements of autophagosome formation. Here, we characterize the structural organization of key components of the mammalian autophagic initiation machinery at [~]20 nm spatial resolution via three-color, three-dimensional super-resolution fluorescence microscopy. We thus show that upon cell starvation, FIP200, a large structural protein of the ULK1 complex with no direct yeast homolog, scaffolds the formation of cup-like structures located at SEC12-enriched remodeled ER-exit sites prior to LC3 lipidation. This cup scaffold, then, provides a structural asymmetry to enforce the directional recruitment of downstream components, including the Atg12-Atg5-Atg16 complex, WIPI2, and LC3, to the cup inside. Moreover, we provide evidence that the early autophagic machinery is recruited in its entirety to these cup structures prior to LC3 lipidation, and gradually disperses and dissociates on the outer face of the phagophore membrane during elongation. We thus shed new light on the physical process of mammalian autophagic initiation and development at the nanometer-scale.

cell biology↗

bioRxiv2018

Asymmetrically Positioned Flagellar Control Units Regulate Human Sperm Rotation

The ability of sperm to fertilize an egg is controlled by ion channels, one of which is the pH-dependent calcium channel of sperm CatSper. For CatSper to be fully activated, the cytoplasmic pH must be alkaline, which is accomplished by either proton transporters, or a faster mechanism, such as the voltage-gated proton channel Hv1. To ensure effective regulation, these channels and regulatory proteins must be tightly compartmentalized. Here, we characterize human sperm nanodomains that are comprised of Hv1, CatSper and regulatory protein ABHD2. Super-resolution microscopy revealed that Hv1 forms asymmetrically positioned bilaterally distributed longitudinal lines that span the entire length of the sperm tail. Such a distribution provides a direct structural basis for the selective activation of CatSper, and subsequent flagellar rotation along the long axis that, together with hyperactivated motility, enhances sperm fertility. Indeed, Hv1 inhibition leads to a decrease in sperm rotation. Thus, sperm ion channels are organized in distinct regulatory nanodomains that control hyperactivated motility and rotation.

cell biology↗

bioRxiv2017

Remodeling of ER-exit sites initiates a membrane supply pathway forautophagosome biogenesis

Autophagosomes are double-membrane vesicles generated during autophagy. Biogenesis of the autophagosome requires membrane acquisition from intracellular compartments, the mechanisms of which are unclear. We previously found that a relocation of COPII machinery to the ER-Golgi intermediate compartment (ERGIC) generates ERGIC-derived COPII vesicles which serve as a membrane precursor for the lipidation of LC3, a key membrane component of the autophagosome. Here we employed super-resolution microscopy to show that starvation induces the enlargement of ER-exit sites (ERES) positive for the COPII activator, SEC12, and the remodeled ERES patches along the ERGIC. A SEC12 binding protein, CTAGE5, is required for the enlargement of ERES, SEC12 relocation to the ERGIC, and modulates autophagosome biogenesis. Moreover, FIP200, a subunit of the ULK protein kinase complex, facilitates the starvation-induced enlargement of ERES independent of the other subunits of this complex and associates via its C-terminal domain with SEC12. Our data indicate a pathway wherein FIP200 and CTAGE5 facilitate starvation-induced remodeling of the ERES, a prerequisite for the production of COPII vesicles budded from the ERGIC that contribute to autophagosome formation.

cell biology↗