Biology subjects

Cole, D.

Publications and source records attributed to Cole, D..

12 recordsLinked to original sources

bioRxiv2026

A 3D printed mini-gel electrophoresis system for rapid and inexpensive DNA nanoswitch biosensing

Gel electrophoresis has been a cornerstone laboratory technique for decades, yet it is often viewed as cumbersome, costly, and has remained confined to laboratory settings. Recent advances in DNA nanotechnology have repurposed electrophoresis as a primary readout for some biosensing applications such as DNA nanoswitches, where a conformational change in a DNA structure indicates the presence of a target molecule. Conventional gel electrophoresis setups not ideal for such targeted applications, with moderate equipment cost, excessive reagent use, and time-consuming processes. Here, we adopt a reductionist, application-driven approach to redesign gel electrophoresis specifically for DNA nanoswitch-based detection. We present a fully 3D-printable mini gel electrophoresis system that incorporates conductive plastic electrodes, demonstrating performance comparable to conventional systems using platinum electrodes. By optimizing the inter-electrode distance and running parameters, our system resolves the on/off states of DNA nanoswitches in as little as one minute. We further show that the device operates reliably at low voltages, including when powered by a USB power bank, and even enables instrument-free nanoswitch readout using an LED with a cell-phone camera. Our design substantially reduces the cost, voltage requirements, material usage, operational complexity, and experiment time. These improvements make gel-based biosensing more practical outside traditional laboratory environments, paving the way for broader adoption of gel electrophoresis in point-of-care and resource-limited settings.

biochemistry↗

bioRxiv2025

Stable Somatotopy in Chronic Low Back Pain: Is Cortical Map Reorganization a Myth?

Chronic low back pain (CLBP) has been linked to maladaptive cortical plasticity in sensorimotor regions, which may contribute to pain persistence through less distinct ("smudged") somatotopic maps of afferent input from the back. However, empirical support for such functional reorganization processes, especially in the primary somatosensory cortex (S1), is limited. To delineate precise sensory representations of the back and test for altered somatotopic processing in CLBP, we utilized an MR-compatible pneumatic vibration device and applied frequency-specific vibrotactile stimuli (20 Hz and 80 Hz) at nine thoracolumbar paraspinal sites in 45 patients with CLBP and 41 healthy controls. Representational similarity analysis (RSA) was employed via a whole-brain searchlight method, and the neural representational patterns were contrasted against theoretical models including a segmental (based on anatomical proximity), simple (upper vs. lower back division) and a random model. In addition, a machine learning classifier was trained to predict afferent input from the upper vs lower back in healthy controls and tested in CLBP patients. Both groups displayed well-organized segmental representations spanning somatosensory, motor, and posterior parietal cortices. Posterior parietal regions exhibited the best model fits, followed by S1. No evidence for differences in representational patterns between groups was found. In CLBP patients, these patterns did not show associations with pain duration, severity, fear of movement or self-reported back perception. The classifier, trained on healthy controls, accurately predicted upper vs lower back afferent input in CLBP patients. These findings indicate preserved cortical maps of the back in CLBP, challenging the hypothesis of sensory cortical reorganization in this condition. Significance StatementChronic low back pain (CLBP) is often linked to maladaptive reorganization of somatotopic maps in sensorimotor cortices, providing the mechanistic rationale for therapies like sensory discrimination training to "normalize" these maps and alleviate pain. Using frequency-specific vibrotactile stimulation of the back and multivariate fMRI analysis, we found well-organized, segmental cortical representations in both CLBP patients and healthy controls, with no evidence of group differences. Representational patterns showed no associations with clinical measures such as pain duration or intensity. These findings challenge the cortical reorganization hypothesis in CLBP and suggest sensory training benefits may involve alternative mechanisms.

neuroscience↗

bioRxiv2024

Tuning the stability of DNA tetrahedra with base-stacking interactions

DNA nanotechnology relies on programmable anchoring of regions of single-stranded DNA through base pair hybridization to create nanoscale objects such as polyhedra, tubes, sheets, and other desired shapes. Recent work from our lab measured the energetics of base-stacking interactions and suggested that terminal stacking interactions between two adjacent strands could be an additional design parameter for DNA nanotechnology. Here, we explore that idea by creating DNA tetrahedra held together with sticky ends that contain identical base pairing interactions but different terminal stacking interactions. Testing all 16 possible combinations, we found that the melting temperature of DNA tetrahedra varied by up to 10 {degrees}C from altering a single base stack in the design. These results can inform stacking design to control DNA tetrahedra stability in a substantial and predictable way. To that end, we show that a 4 bp sticky end with weak terminal stacking does not form stable tetrahedra, while strengthening the stacks confers high stability with a 46.8 {+/-} 1.2 {degrees}C melting temperature, comparable to a 6 bp sticky end with weak stacking (49.7 {+/-} 2.9 {degrees}C). The results likely apply to other types of DNA nanostructures and suggest that terminal stacking interactions play an integral role in formation and stability of DNA nanostructures.

bioengineering↗

bioRxiv2024

Deepening biomedical research training: Community-Building Wellness Workshops for Post-Baccalaureate Research Education Program (PREP) Trainees

ProblemAll trainees, especially those from historically minoritized backgrounds, experience stresses that may reduce their continuation in science, technology, engineering, math, and medicine (STEMM) careers. The Johns Hopkins University School of Medicine is one of [~]45 institutions with a National Institutes of Health funded Postbaccalaureate Research Education Program (PREP) that provides mentoring and a year of fulltime research to prepare students from historically excluded groups for graduate school. Having experienced the conflation of stresses during the COVID-19 pandemic and related shutdown, we realized our program lacked a component that explicitly helped PREP Scholars recognize and cope with non-academic stresses (financial, familial, social, mental) that might threaten their confidence and success as scientists and future in STEMM. InterventionWe developed an early-intervention program to help Scholars develop life-long skills to become successful and resilient scientists. We developed a year-long series comprised of 9 workshops focused on community, introspection, financial fitness, emotional intelligence, mental health, and soft-skills. We recruited and compensated a cohort of PhD students and postdoctoral fellows to serve as Peer Mentors, to provide a community and the safest space for Scholars to discuss personal concerns. Peer Mentors were responsible for developing and facilitating these Community-Building Wellness Workshops (CBWW). ContextCBWW were created and exectued as part of the larger PREP program. Workshops included a PowerPoint presentation by Peer Mentors that featured several case studies that prompted discussion and provided time for small-group discussions between Scholars and Peer Mentors. We also included pre- and post-work for each workshop. These touch-points helped Scholars cultivate the habit of introspection. ImpactThe CBWW exceeded our goals. Both Peer Mentors and Scholars experienced strong mutual support, and Scholars developed life-long skills. Notably, several Scholars who had been experiencing financial, mental or mentor-related stress immediately brought this to the attention of program leadership, allowing early and successful intervention. At the completion of CBWW, PREP Scholars reported implementing many workshop skills into practice, were reshaping their criteria for choosing future mentors, and evaluating career decisions. Strikingly, Peer Mentors found they also benefitted from the program as well, suggesting a potential larger scope for the role of CBWW in academia. Lessons LearnedPeer Mentors were essential in creating a safe supportive environment that facilitated discussions, self-reflection, and self-care. Providing fair compensation to Peer Mentors for their professional mentoring and teaching contributions was essential and contributed meaningfully to the positive energy and impact of this program.

scientific communication and education↗

bioRxiv2023

Myosin-5 varies its steps along the irregular F-actin track

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering (iSCAT) microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably-spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

biophysics↗

bioRxiv2020

Allosteric activation of T cell antigen receptor signalling by quaternary structure relaxation

The mechanism of T cell antigen receptor (TCR-CD3) signalling remains elusive. Here, we identified mutations in the transmembrane region of TCR{beta} or CD3{zeta} that augmented pMHC-induced signalling, not explicable by enhanced ligand binding, lateral diffusion, clustering or co-receptor function. Using a novel biochemical assay and molecular dynamics simulation, we demonstrated that the gain-of-function mutations loosened interaction between TCR{beta} and CD3{zeta}. We found that, similar to the activating mutations, pMHC binding reduced TCR{beta} cohesion with CD3{zeta}. This event occurred prior to CD3{zeta} phosphorylation and at 0{degrees}C. Moreover, we demonstrated that soluble monovalent pMHC alone induced signalling and reduced TCR{beta} cohesion with CD3{zeta} in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCR{beta} to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to licence CD3{zeta} phosphorylation and initiate signal propagation.

immunology↗

bioRxiv2020

The fiber knob protein of human adenovirus type 49 mediates highly efficient and promiscuous infection of cancer cell lines using a novel cell entry mechanism

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of pre-existing immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases where they induce robust immunity in pre-clinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids to HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, whilst potential interaction with CAR is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, oesophageal and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumour types. ImportanceAdenoviruses are powerful tools experimentally and clinically. To maximise efficacy, the development of serotypes with low pre-existing levels of immunity in the population is desirable. Consequently, attention has focussed on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pan-tropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

microbiology↗

bioRxiv2019

Sensitivity to gains during risky decision-making differentiates chronic cocaine users from stimulant-naive controls

BackgroundAlthough chronic cocaine use has been frequently associated with decision-making impairments that are supposed to contribute to the development and maintenance of cocaine addiction, it has remained unclear how risk-seeking behaviours observed in chronic cocaine users (CU) come about. Here we therefore test whether risky decision-making observed in CU is driven by alterations in individual sensitivity to the available information (gain, loss, and risk).\n\nMethodA sample of 96 participants (56 CU and 40 controls) performed the no-feedback (\"cold\") version of the Columbia Card Task. Structured psychiatric interviews and a comprehensive neuropsychological test battery were additionally conducted. Current and recent substance use was objectively assessed by toxicological urine and hair analysis.\n\nResultsCompared to controls, CU showed increased risk-seeking in unfavourable decision scenarios in which the risk was high and the returns were low, and a tendency for increased risk aversion in favourable decision scenarios. These differences arose from the fact that CU were less sensitive to gain, but similarly sensitive to loss and risk information in comparison to controls. Further analysis revealed that individual differences in sensitivity to loss and risk were related to cognitive performance and impulsivity.\n\nConclusionThe reduced sensitivity to gain information in people with CU may contribute to their propensity for making risky decisions. While these alterations in the sensitivity to gain might be directly related to cocaine use per se, the individual psychopathological profile of CU might moderate their sensitivity to risk and loss impulsivity.

neuroscience↗

bioRxiv2019

Estimating cause- and size- specific mortality hazard rates using mark-recapture-recovery data

O_LIEvidence-based management of natural populations under strong human influence frequently requires not only estimates of survival but also knowledge about how much mortality is due to anthropogenic versus natural causes. This is the case particularly when individuals vary in their vulnerability to different causes of mortality due to traits, life-history stages, or locations. C_LIO_LIHere, we estimated harvest and background (other cause) mortality of a landlocked migratory salmonid over half a century. In doing so, we quantified among-individual variation in vulnerability to cause-specific mortality resulting from differences in body size and spawning location relative to a hydropower dam. C_LIO_LIWe constructed a multistate mark-recapture model to estimate harvest and background mortality hazard rates as functions of a discrete state (spawning location) and an individual time-varying covariate (body size). We further accounted for among-year variation in mortality and migratory behavior and fit the model to a unique 50-year time-series of mark-recapture-recovery data on brown trout (Salmo trutta) in Norway. C_LIO_LIHarvest mortality was highest for intermediate-sized trout, and outweighed background mortality for most of the observed size range. Background mortality decreased with body size for trout spawning below the dam and increased for those spawning above. All vital rates varied substantially over time, but a trend was evident only in estimates of fishers reporting rate, which decreased from over 50% to less than 10% throughout the study period. C_LIO_LIWe highlight the importance of body size for cause-specific mortality and demonstrate how this can be estimated using a novel hazard rate parameterisation for mark-recapture models. Our approach allows estimating effects of individual traits and environment on cause-specific mortality without confounding, and provides an intuitive way to estimate temporal patterns within and correlation among different mortality sources. C_LI

ecology↗

bioRxiv2018

DNA Methylation Network Estimation with Sparse Latent Gaussian Graphical Model

Inferring molecular interaction networks from genomics data is important for advancing our understanding of biological processes. Whereas considerable research effort has been placed on inferring such networks from gene expression data, network estimation from DNA methylation data has received very little attention due to the substantially higher dimensionality and complications with result interpretation for non-genic regions. To combat these challenges, we propose here an approach based on sparse latent Gaussian graphical model (SLGGM). The core idea is to perform network estimation on q latent variables as opposed to d CpG sites, with q<<d. To impose a correspondence between the latent variables and genes, we use the distance between CpG sites and transcription starting sites of the genes to generate a prior on the CpG sites latent class membership. We evaluate this approach on synthetic data, and show on real data that the gene network estimated from DNA methylation data significantly explains gene expression patterns in unseen datasets.

genomics↗

bioRxiv2017

Complementary studies of lipid membrane dynamics using iSCAT and STED microscopy

Observation techniques with high spatial and temporal resolution, such as single-particle tracking (SPT) based on interferometric Scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye-tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work we have used lipid analogues tagged with a hybrid fluorescent tag - gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous Supported Lipid Bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 [≤] t [≤] 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye-tagged lipid analogues. These FCS measurements of hybrid fluorescent tag - gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially crosslinking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

biophysics↗

bioRxiv2017

Quantitative mass imaging of single molecules in solution

The cellular processes underpinning life are orchestrated by proteins and their interactions. Structural and dynamic heterogeneity, despite being key to protein and drug function, continues to pose a fundamental challenge to existing analytical and structural methodologies used to study these associations. Here, we use interferometric scattering microscopy to mass-image single biomolecules in solution with <2% mass error, up to 19-kDa resolution and 1-kDa precision. Thereby, we resolve oligomeric distributions at high dynamic range, detect small-molecule binding, and mass-image biomolecules composed not only of amino acids, but also heterogeneous species, such as lipo- and glycoproteins. These capabilities enable us to characterize the molecular mechanisms of processes as diverse as oligomeric selfassembly, glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry (iSCAMS) provides spatially resolved access to the dynamics of biomolecular interactions ranging from those involving small molecules to mesoscopic assemblies, one molecule at a time.

biophysics↗