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Pelled, G.

Publications and source records attributed to Pelled, G..

17 recordsLinked to original sources

Magneto-Photonic Gene Circuit for Minimally Invasive Control of Gene Expression in Mammalian Cells

Precise control of gene expression is one of the fundamental goals of synthetic biology. Whether the objective is to modify endogenous cellular function or induce the expression of molecules for diagnostic and therapeutic purposes, gene regulation remains a key aspect of biological systems. Over time, advances in protein engineering and molecular biology have led to the creation of gene circuits capable of inducing the expression of specific proteins in response to external stimulus such as light. These optogenetic, or light-activated circuits hold significant potential for gene therapy as a tool for regulating the expression of therapeutic genes within cells. However, the applications of optogenetic systems can be limited by the lack of efficient ways for light delivery inside cells or tissue. Our approach to address this challenge is to harness the power of bioluminescence to produce light directly inside cells using a luminescent enzyme. Combined with a photosensitive transcription factor, we report the development of a fully genetically encoded optogenetic circuit for control of gene expression. Furthermore, we utilized a magneto sensitive protein to engineer a split protein version of this luminescent enzyme, where its reconstitution is driven by a 50mT magnetic stimulus. Thus, resulting in a first-of-its-kind gene circuit activated by a combination of light and magnetic stimulus. We expect this work to advance the implementation of light-controlled systems without the need of external light sources, as well as serve as a basis for the development of future magneto-sensitive tools.

synthetic biology↗

Using Manganese-Enhanced MRI to visualize Magnetogenetic-based Neuromodulation

PurposeInvestigation of the electromagnetic perceptive gene (EPG) protein and garnering evidence to suggest its use as a magnetogenetic tool for neuromodulation. Activation of EPG by electromagnetic field increases intracellular calcium levels, thus, we aimed to determine whether EPG influences the analogous manganese ion. This work yields potential for manganese-enhanced MRI (MEMRI) to be used to monitor EPG acting as a neuromodulator. MethodsHEK293FT cells expressing EPG were exposed to a MnCl2 solution and stimulated with a static or electromagnet. Excess MnCl2 was washed off the cells, followed by their collection and lysis. T1 map measurements of the lysates were obtained to gauge the presence of intracellular Mn2+. Several controls were employed to critically evaluate whether EPG can influence Mn2+ dynamics. ResultsLysate of cells expressing EPG showed significantly lower T1 values compared to cells without EPG that were exposed to the same MnCl2 solution and magnetic stimulus. ConclusionMagnetic activation of EPG increases the uptake of Mn2+ into the cell. By influencing ions pertinent to neuronal function, we demonstrate the potential of MEMRI to monitor EPG neuromodulatory activity.

biophysics↗

Evidence of Gait Impairment After mTBI in an Adolescent Pig Model

This study investigates the effects of mild traumatic brain injury (mTBI) on gait in Yucatan minipigs, a relevant animal model for assessing balance impairments after TBI. Retro-reflective markers were placed on different anatomical locations on the pig, and eight motion capture cameras were set up along the sides of the pig walking path to capture natural gait data. Pigs were tested both before and after induced head injury, allowing for direct comparison of gait alterations. Our findings reveal significant impairments, including reduced shoulder flexibility, altered head movement, and compensatory stabilization strategies, such as increased vertical nodding. These observations suggest that, like human mTBI patients, pigs exhibit balance deficits and modified movement patterns. The results demonstrate the utility of the adolescent pig model in studying mTBI-related balance disturbances, providing valuable insights into its neurological and biomechanical consequences. This research lays the groundwork for future studies focused on developing diagnostic and therapeutic strategies for mTBI-induced gait impairments.

bioengineering↗

Volumetric and Diffusion Tensor Imaging biomarkers indicating long lasting post-concussion abnormalities in a youth pig model of mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) caused by sports-related incidents in children and youth can lead to prolonged cognitive impairments, underscoring the importance of improved diagnosis and comprehension of its enduring impacts on neuropathology. A pig model was chosen for its similarities to the human brain in terms of gyrencephalic structure, size, and regional proportions, and a closed-head mTBI was induced in adolescent pigs. In this study, 12 (n=4 male and n=8 female) 16-weeks old Yucatan pigs were tested; n=6 received mTBI and n=6 received a Sham procedure. This study utilized T1-weighted imaging to assess volumetric alterations in different regions of the brain and diffusion tensor imaging (DTI) to examine microstructural damage in white matter. The pigs were imaged at one and three months post-injury. Our volumetric analysis of key white and gray matter regions showed significant longitudinal changes in pigs with mTBI compared to sham controls. The observed volume increases may be attributed to swelling, neuroinflammation, or hyperactivity. Fractional anisotropy (FA) values derived from DTI images demonstrated an increase in corpus callosum from 1 month to 3 months only in mTBI pigs. Additionally, comparisons of the left and right internal capsules revealed a decrease in FA in the right internal capsule for mTBI pigs, likely due to the impact being slightly localized to the right side of the brain, which may indicate demyelination. Thus, the injury has disrupted the maturation of white and gray matter of the developing brain. This signifies the need for longitudinal investigations after mTBI to comprehensively assess its long-term effects and contribute to the clinical management of concussion in youth.

bioengineering↗

Single unit electrophysiology recordings and computational modeling can predict octopus arm movement

The octopus simplified nervous system holds the potential to reveal principles of motor circuits and improve brain-machine interface devices through computational modeling with machine learning and statistical analysis. Here, an array of carbon electrodes providing single-unit electrophysiology recordings were implanted into the octopus anterior nerve cord. The number of spikes and arm movements in response to stimulation at different locations along the arm were recorded. We observed that the number of spikes occurring within the first 100ms after stimulation were predictive of the resultant movement response. Computational models showed that temporal electrophysiological features could be used to predict whether an arm movement occurred with 88.64% confidence, and if it was a lateral arm movement or a grasping motion with 75.45% confidence. Both supervised and unsupervised methods were applied to gain streaming measurements of octopus arm movements and how their motor circuitry produces rich movement types in real time. Deep learning models and unsupervised dimension reduction identified a consistent set of features that could be used to distinguish different types of arm movements. These models generated predictions for how to evoke a particular, complex movement in an orchestrated sequence for an individual motor circuit.

bioengineering↗

Playtime for Cephalopods: Understanding the Significance of Play Behavior inOctopus bimaculoides

Play is considered to be an essential part of development that supports learning, memory, and the development of flexible behavioral strategies. It may also serve as an informative factor in assessing an animals welfare state and in improving care and husbandry practices. An increasing number of non-mammalian species have been discovered to engage in play behavior, including several cephalopod species. Here, we characterized play behavior in wild-caught, laboratory-housed California Two-Spot Octopuses, Octopus bimaculoides, a species with growing relevance as a model in biomedical research, with the goal of establishing a behavioral repertoire and encouraging further research into the behavior and welfare of this species.

animal behavior and cognition↗

Magnetogenetic stimulation inside MRI induces spontaneous and evoked changes in neural circuits activity in rats

The ability to modulate specific neural circuits and simultaneously visualize and measure brain activity with MRI would greatly impact understanding brain function in health and disease. The combination of neurostimulation methods and MRI in animal models have already shown promise in elucidating fundamental mechanisms associated with brain activity. We developed an innovative magnetogenetics neurostimulation technology that can trigger neural activity through magnetic fields. Similar to other genetic-based neuromodulation methods, magnetogenetics offers cell-, area- and temporal- specific control of neural activity. However, the magnetogenetics protein (Electromagnetic Preceptive Gene (EPG)) are activated by non-invasive magnetic fields, providing a unique way to target neural circuits by the MRI gradients while simultaneously measure their effect on brain activity. EPG was expressed in rats visual cortex and the amplitude of low-frequency fluctuation (fALFF), resting-state functional connectivity (FC), and sensory activation was measured using a 7T MRI. The results demonstrate that EPG-expressing rats had significantly higher signal fluctuations in the visual areas and stronger FC in sensory areas consistent with known anatomical visuosensory and visuomotor connections. This new technology complements the existing neurostimulation toolbox and provides a mean to study brain function in a minimally-invasive way which was not possible previously.

neuroscience↗

Proposed three-phenylalanine motif involved in magnetoreception signaling of an Actinopterygii protein expressed in mammalian cells

Studies at the cellular and molecular level of magnetoreception - sensing and responding to magnetic fields - is a relatively new research area. As it appears that different mechanisms of magnetoreception in animals evolved from different origins, many questions about the mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C (PI-PLC) we show that EPG is associated to the membrane via glycosylphosphatidylinositol (GPI) anchor. EPGs function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues in order to function - stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the field. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/519643v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@19a4f54org.highwire.dtl.DTLVardef@1ce17b2org.highwire.dtl.DTLVardef@175b97borg.highwire.dtl.DTLVardef@1e45294_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefEPG is a magnetoreceptive GPI anchored protein. Critical to its function is a three-phenylalanine motif which allows EPG to sense and respond to EMF. When expressed in mammalian cell, an increase in intracellular calcium is observed using GCaMP6m. This work represents progress towards understanding magnetoreception for use in future technologies. HighlightsO_LIEPG is associated to the cell membrane via glycosylphosphatidylinositol anchoring C_LIO_LIIn mammalian cells, EPG increases intracellular calcium upon EMF stimulation C_LIO_LIHomologs of EPG from the uPAR/Ly6 family show different responses to EMF C_LIO_LIA three-phenylalanine motif in EPG is critical to its magnetoreceptive ability C_LI

molecular biology↗

A putative design for electromagnetic activation of split proteins for molecular and cellular manipulation

The ability to manipulate cellular function using an external stimulus is a powerful strategy for studying complex biological phenomena. One approach to modulate the function of the cellular environment is split proteins. In this method, a biologically active protein or an enzyme is fragmented so that it reassembles only upon a specific stimulus. While there are many tools available to induce these systems, nature has provided other mechanisms that can be utilized to expand the split protein toolbox. Here we show a novel method for reconstituting split proteins using magnetic stimulation. We have found that the Electromagnetic Perceptive Gene (EPG) changes conformation due to magnetic fields stimulation. By fusing split fragments of a certain protein to both termini of the EPG, the fragments can be reassembled into a functional protein under magnetic stimulation due to conformational change. We show this effect with three separate split proteins; NanoLuc, APEX2, and Herpes Simplex Virus Type-1 Thymidine Kinase. Our results show for the first time, that reconstitution of split proteins can be achieved only with magnetic fields. We anticipate that this study will be a starting point for future magnetically inducible split protein designs for cellular perturbation and manipulation. With this technology, we can help to expand the toolbox of the split protein platform and allow better elucidation of complex biological systems.

synthetic biology↗

Magnetogenetic closed-loop reduction of seizure activity in a rat model of epilepsy

On-demand neurostimulation has shown success in epilepsy patients with pharmacoresistant seizures. Seizures produce magnetic fields that can be recorded using magnetoencephalography. We developed a new closed-loop approach to control seizure activity based on magnetogenetics using the electromagnetic perceptive gene (EPG) that encodes a protein that responds to magnetic fields. The EPG transgene was expressed in inhibitory interneurons under hDlx promoter and kainic acid was used to induce acute seizures. In vivo electrophysiological signals were recorded. We found that hDlx EPG rats exhibited a significant delay in the onset of first seizure (1142.72 {+/-} 186.35s) compared to controls (644.03 {+/-} 15.06s) and significantly less seizures (4.11 {+/-} 1.03) compared to controls (8.33 {+/-} 1.58). These preliminary findings suggest that on-demand activation of EPG expressed in inhibitory interneurons suppress seizure activity, and magnetogenetics via EPG may be an effective strategy to alleviate seizure severity in a minimally invasive, closed-loop and cell-specific fashion.

neuroscience↗

Bioelectromagnetic platform for stimulation

Magnetogenetics is a new field that utilizes electromagnetic fields to remotely control cellular activity. In addition to the development of the biological genetic tools, this approach requires designing hardware with a specific set of demands for the electromagnets used to provide the desired stimulation for electrophysiology and imaging experiments. Here we present a universal stimulus delivery system comprised of four magnet designs compatible with electrophysiology, fluorescence and luminescence imaging, microscopy, and freely behaving animal experiments. The overall system includes a low-cost stimulation controller which enables rapid switching between active and sham stimulation trials as well as precise control of stimulation delivery.

bioengineering↗

Multimodal characterization of Yucatan minipig behavior and physiology through maturation

Brain injuries that are induced by external forces are particularly challenging to model experimentally. In recent decades, the domestic pig has been gaining popularity as a highly relevant animal model to address the pathophysiological mechanisms and the biomechanics associated with head injuries. Understanding cognitive, motor, and sensory aspects of pig behavior throughout development is crucial for evaluating cognitive and motor deficits after injury. We have developed a comprehensive battery of tests to characterize the behavior and physiological function of the Yucatan minipig throughout maturation. Behavioral testing included assessments of learning and memory, executive functions, circadian rhythms, gait analysis, and level of motor activity. We applied traditional behavioral apparatus and analysis methods, as well as state-of-the-art sensor technologies to report on motion and activity, and artificial intelligent approaches to analyze behavior. We studied pigs from 16 weeks old through sexual maturity at 35 weeks old. The results show multidimensional characterization of minipig behavior, and how it develops and changes with age. This animal model may capitulate the biomechanical consideration and phenotype of head injuries in the developing brain and can drive forward the field of understanding pathophysiological mechanisms and developing new therapies to accelerate recovery in children who have suffered head trauma.

animal behavior and cognition↗

Multi-session delivery of synchronous rTMS and sensory stimulation induces long-term plasticity

BackgroundCombining training or sensory stimulation with non-invasive brain stimulation has shown to improve performance in healthy subjects and improve brain function in patients after brain injury. However, the plasticity mechanisms and the optimal parameters to induce long-term and sustainable enhanced performance remain unknown. ObjectiveThis work was designed to identify the protocols of which combining sensory stimulation with repetitive transcranial magnetic stimulation (rTMS) will facilitate the greatest changes in fMRI activation maps in the rats primary somatosensory cortex (S1). MethodsSeveral protocols of combining forepaw electrical stimulation with rTMS were tested, including a single stimulation session compared to multiple, daily stimulation sessions, as well as synchronous and asynchronous delivery of both modalities. High-resolution fMRI was used to determine how pairing sensory stimulation with rTMS induced short and long-term plasticity in the rat S1. ResultsAll groups that received a single session of rTMS showed short-term increases in S1 activity, but these increases did not last three days after the session. The group that received a stimulation protocol of 10 Hz forepaw stimulation that was delivered simultaneously with 10 Hz rTMS for five consecutive days demonstrated the greatest increases in the extent of the evoked fMRI responses compared to groups that received other stimulation protocols. ConclusionsOur results provide direct indication that pairing peripheral stimulation with rTMS induces long-term plasticity, and this phenomenon appears to follow a time-dependent plasticity mechanism. These results will be important to lead the design of new training and rehabilitation paradigms and training towards achieving maximal performance in healthy subjects. HighlightsO_LIA single rTMS session induced short-term changes but they were not sustainable C_LIO_LIMulti-session delivery of rTMS paired with sensory stimulation induced long-term plasticity C_LIO_LIrTMS paired with sensory stimulation induced plasticity via time-dependent mechanism C_LIO_LIDelivery of sensory stimulation only did not induce long-term plasticity C_LI

bioengineering↗

Functional Characterization of an Electromagnetic Perceptive Protein

Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and in-vivo. Here, we report for the first time the cellular location and orientation of the EPG protein. We utilized a differential labelling technique to determine that the EPG protein is a membrane anchored protein with an N-terminal extracellular domain. The kinetics and diffusion dynamics of the EPG protein in response to magnetic stimulation was also elucidated using single particle imaging and tracking. Pulse chase labelling and Total Internal Reflection Fluorescence (TIRF) imaging revealed an increase in EPG kinetics post magnetic activation at a single particle level. Trajectory analysis show notably different EPG protein kinetics before and after magnetic stimulation in both 2 (free vs bound particle) and 3 state (free vs intermediate vs bound particle) tracking models. This data provides additional information to support and understand the underlying biophysical mechanisms behind EPG activation by magnetic fields and provides evidence for the basis of magnetoreception in the EPG protein that will aid in future studies that seek to further understand this novel mechanism. This study is important for understanding magnetoreception as well as developing new technologies for magnetogenetics - the utilization of electromagnetic fields to remotely control cellular function. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/329946v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1ce4aborg.highwire.dtl.DTLVardef@e8bb89org.highwire.dtl.DTLVardef@1705113org.highwire.dtl.DTLVardef@dadf23_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Swimming direction of the Glass Catfish, Kryptopterus bicirrhis, is responsive to magnetic stimulation

Several marine species have developed a magnetic perception that is essential for navigation and detection of prey and predators. One of these species is the transparent glass catfish that contains an ampullary organ dedicated to sense magnetic fields. Here we examine the behavior of the glass catfish in response to static magnetic fields which will provide valuable insight on function of this magnetic response. By utilizing state of the art animal tracking software and artificial intelligence approaches, we quantified the effects of magnetic fields on the swimming direction of glass catfish. The results demonstrate that glass catfish placed in a radial arm maze, consistently swim away from magnetic fields over 20 {micro}T and show adaptability to changing magnetic field direction and location.

animal behavior and cognition↗

Non-invasive neuromodulation using rTMS and the Electromagnetic-Preceptive Gene (EPG) facilitates plasticity after nerve injury

Peripheral nerve injury leads to altered cortical excitation-inhibition balance which is associated with sensory dysfunctions. We tested if non-invasive repetitive transcranial magnetic stimulation (rTMS) which has shown to induce neuronal excitability, and cell-specific magnetic activation via the Electromagnetic-perceptive gene (EPG) which is a novel gene that was identified and cloned from Kryptopterrus bicirrhis and demonstrated to evoke neural responses when magnetically stimulated, can restore cortical excitability. A battery of behavioral tests, fMRI and immunochemistry were performed in the weeks following limb denervation in rats. The results demonstrate that neuromodulation significantly improved long-term mobility, decreased anxiety and enhanced neuroplasticity. The study also identifies the acute post-injury phase as a critical time for intervention. Moreover, the results implicate EPG as an effective cell-specific neuromodulation approach. Together, these results reinforce the growing amount of evidence from human and animal studies that are establishing neuromodulation as an effective strategy to promote plasticity and rehabilitation.

neuroscience↗

High-resolution MEMRI characterizes laminar specific ascending and descending spinal cord pathways in rats

Manganese Enhanced MRI (MEMRI) utilizing different manganese chloride (MnCl2) delivery methods, has yielded valuable architectural, functional and connection information about the brain. MEMRI also has the potential in characterizing neural pathways in the spinal cord. The spinal cord grey matter is anatomically composed of nine distinct cellular laminae, where each of the laminae receives input from a specific type of neuronal population and process or serves as a relay region in a specific sensory or motor pathway. This type of laminar arrangement in the spinal cord is currently only visualized by histological methods. It is of significant interest to determine whether laminar specific enhancement by Mn2+ can be achieved in the spinal cord, as has been reported in the brain and olfactory pathway. Here we focus on using MEMRI to determine the specific laminae of the thoracic region of the spinal cord. We focus on MnCl2 changes in the ascending and descending tracts of the spinal cord. Major factors in applying this technique in the spinal cord are the ability to acquire high-resolution spinal cord images and to determine a noninvasive route of administration which will result in uptake by the central nervous system.\n\nWe have applied the MEMRI approach by intraperitoneal (i.p). delivery of MnCl2 and imaged lumbar and thoracic spinal cord levels in rats to determine whether T1 weighted MRI can detect spinal cord laminae 48 hours following MnCl2 administration. T1 weighted images of the lower lumbar level were obtained from MnCl2 injected and control rats. Here we demonstrate laminar specific signal enhancement in the spinal cord of rats administered with MnCl2 vs. controls in MRI of the cord with ultra-high, 69 m in-plane resolution. We also report reduced T1 values over time in MnCl2 groups across laminae I-IX. The regions with the largest T1 enhancements were observed to correspond to laminae that contain either high cell density or large motor neurons, making MEMRI an excellent tool for studying spinal cord architecture, physiology and function in different animal models.

neuroscience↗