Biology subjects

Raghavan, P.

Publications and source records attributed to Raghavan, P..

9 recordsLinked to original sources

bioRxiv2024

Open-source cell culture automation system with integrated cell counting for passaging microplate cultures

Tissue culture in 96-well microplates is conventionally a tedious, highly manual process sensitive to individual technique and experimenter error. Here, we describe the Automated Cell Culture Splitter (ACCS), a system for passaging plates of adherent or suspension cells, for routine culture maintenance or specialized applications such as seeding plates for microscopy. The system is built around the Opentrons OT-2 liquid handling robot and incorporates a novel on-deck imaging-based cell counter which allows it to compensate for density disparities across a source plate and control the number of cells seeded on a per-well basis. We find this solution can cut hands-on time by 61% and the results compare favorably to our existing manual cell culture processes in terms of both seeding density precision and bio-logical outcomes, achieving a control of seeding density with a well-to-well coefficient of variation (CV) under 11%. The system is designed to be adaptable and an accessible entry point into automation for high-throughput cell culture; to that end, all of the source code and hardware designs are released under open source licenses.

bioengineering↗

bioRxiv2024

Dissecting autonomous enzyme variability in single cells

Metabolic enzymes perform life-sustaining functions in various cellular compartments. Anecdotally, metabolic activity is observed to vary between genetically identical cells, which impacts drug resistance, differentiation, and immune cell activation. However, no large-scale resource systematically reporting metabolic cellular heterogeneity exists. Here, we leverage imaging-based single-cell spatial proteomics to reveal the extent of non-genetic variability of the human enzymatic proteome, as a proxy for metabolic states. Nearly two fifths of enzymes exhibit cell-to-cell variable expression, and half localize to multiple cellular compartments. Metabolic heterogeneity arises largely autonomously of cell cycling, and individual cells reestablish these myriad metabolic phenotypes over several cell divisions. Multiplexed imaging revealed that metabolic states are continuous and that the correlation between metabolic pathways is metabolic state dependent. These results establish cell-to-cell enzymatic heterogeneity as an organizing principle of cell biology that may rewire our understanding of drug resistance, treatment design, and other aspects of medicine.

cell biology↗

bioRxiv2023

Improvisation and Live Accompaniment Increase Motor Response and Reward During a Music Playing Task

Music provides an abstract reward that can enhance learning and motivation in humans. While music is often combined with exercise to improve performance and to upregulate mood, the relationship between music-induced reward and motor output is poorly understood. Here, we study music reward and motor output at the same time by capitalizing on music playing. Specifically, we investigate the effects of music improvisation and live accompaniment on motor, autonomic, and affective responses. Thirty adults performed a rhythm tapping task while (i) improvising or maintaining the beat and (ii) with live or recorded accompaniment. Motor response was characterized by acceleration of hand movements (accelerometry), wrist flexor and extensor muscle activation (electromyography), and the number of beats played. Autonomic arousal was measured by tonic response of electrodermal activity (EDA) and heart rate (HR). Affective responses were measured by a 12-item Likert scale. The combination of improvisation and live accompaniment, as compared to all other conditions, significantly increased acceleration of hand movements and muscle activation, as well as participant reports of enjoyment during music-playing. Improvisation, regardless of type of accompaniment, increased the number of beats played and autonomic arousal (including tonic EDA responses and several measures of HR), as well as participant reports of challenge. Importantly, increased motor response was associated with increased enjoyment during music improvisation only and not while participants were maintaining the beat. The increased motor responses achieved with improvisation and live accompaniment have important implications for enhancing dose of movement during music-based interventions for stroke rehabilitation. Significance StatementMusic provides a rewarding stimulus and improves motor performance and learning. However, the relationship between music reward and motor output is poorly understood. Here, we show that music improvisation with live accompaniment increased acceleration and muscle activation during movement, and that this increase in motor response was associated with increased enjoyment only when improvising. These findings are important for developing music interventions that target improved motor performance and learning in exercise and physical rehabilitation.

physiology↗

bioRxiv2022

InfoMuNet: Information-theory-based Functional Muscle Network Tracks Sensorimotor Integration Post-stroke

Sensory information is critical for motor coordination. However, understanding sensorimotor integration is complicated, especially in individuals with nervous system impairment. This research presents a novel functional biomarker, based on a nonlinear network graph of muscle connectivity, called InfoMuNet, to quantify the role of sensory information in motor performance. Thirty-two individuals with post-stroke hemiparesis performed a grasp-and-lift task while muscle activities were measured using eight surface electromyography (sEMG) sensors. Subjects performed the task with their affected hand before and after exposure to the sensory stimulation elicited by performing the task with the less-affected hand. For the first time, this work shows that InfoMuNet robustly quantifies functional muscle connectivity improvements in the affected hand after exposure of the less-affected side to sensory information. >90% of the subjects conformed with the improvement resulting from this sensory exposure. InfoMuNet also shows high sensitivity to tactile, kinesthetic, and visual input alterations at the subject level, highlighting the potential use in precision rehabilitation interventions.

neuroscience↗

bioRxiv2021

Polyelectrolyte Nanocomplex Formation Combined with Electrostatic Self-Assembly Enables the Co-Delivery of Synergistic Antimicrobials to Treat Bacterial Biofilms

New approaches are needed to treat bacterial biofilm infections, particularly those of Pseudomonas aeruginosa (PA), which have high rates of antimicrobial resistance and are commonly found in chronic wound and cystic fibrosis lung infections. Combination therapeutics that act synergistically can overcome resistance; however, the delivery of multiple therapeutics at relevant dosages remains a challenge. We therefore developed a new nanoscale drug carrier for antimicrobial co-delivery by combining approaches from polyelectrolyte nanocomplex (NC) formation and layer-by-layer electrostatic self-assembly. This strategy led to NC drug carriers loaded with tobramycin antibiotics and antimicrobial silver nanoparticles (AgTob-NCs). AgTob-NCs displayed synergistic enhancements in antimicrobial activity against both planktonic and biofilm PA cultures, with positively charged NCs leading to complete biofilm eradication. NCs were evaluated in mouse models of lung infection, leading to reduced bacterial burden and improved survival outcomes. This approach therefore shows promise for nanoscale therapeutic co-delivery to overcome antimicrobial resistant bacterial infections.

bioengineering↗

bioRxiv2021

WASP integrates substrate topology and cell polarity to guide neutrophil migration

To control their shape and movement, cells leverage nucleation promoting factors (NPFs) to regulate when and where they polymerize actin. Here we investigate the role of the immune-specific NPF WASP during neutrophil migration. Endogenously-tagged WASP localizes to substrate-induced plasma membrane deformations. Super-resolution imaging of live cells reveals that WASP preferentially enriches to the necks of these substrate-induced membrane invaginations, a distribution that could support substrate pinching. Unlike other curvature-sensitive proteins, WASP only enriches to membrane deformations at the cell front, where it controls Arp2/3 complex recruitment and actin polymerization. Despite relatively normal migration on flat substrates, WASP depletion causes defects in topology sensing and directed migration on textured substrates. WASP therefore both responds to and reinforces cell polarity during migration. Surprisingly, front-biased WASP puncta continue to form in the absence of Cdc42. We propose that WASP integrates substrate topology with cell polarity for 3D guidance by selectively polymerizing actin around substrate-induced membrane deformations at the leading edge. A misregulation of WASP-mediated contact guidance could provide insight into the immune disorder Wiskott-Aldrich syndrome.

cell biology↗

bioRxiv2021

OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization

Elucidating the wiring diagram of the human cell is a central goal of the post-genomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery, and uncovers that RNA-binding proteins form a specific sub-group defined by unique interaction and localization properties. Furthermore, we discover that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions. Paired with a fully interactive website opencell.czbiohub.org, we provide a resource for the quantitative cartography of human cellular organization.

cell biology↗

bioRxiv2020

Multi-Modal Biomarkers of Cerebral Edema in Low Resolution MRI

A central challenge of medical imaging studies is to extract biomarkers that characterize disease pathology or outcomes. Modern automated approaches have found tremendous success in high-resolution, high-quality magnetic resonance images (MRI). These methods, however, may not translate to low resolution images acquired on MRI scanners with lower magnetic field strength. In low-resource settings where low-field scanners are more common and there is a shortage of radiologists to manually interpret MRI scans, it is critical to develop automated methods that can augment or replace manual interpretation, while accommodating reduced image quality. We present a fully automated framework for translating radiological diagnostic criteria into image-based biomarkers, inspired by a project in which children with cerebral malaria were imaged using low-field 0.35 Tesla MRI. We integrate multi-atlas label fusion, which leverages high-resolution images from another sample as prior spatial information, with parametric Gaussian hidden Markov models based on image intensities, to create a robust method for determining ventricular cerebrospinal fluid volume. We also propose normalized image intensity and texture measurements to determine the loss of gray-to-white matter tissue differentiation and sulcal effacement. These integrated biomarkers have excellent classification performance for determining severe brain swelling due to cerebral malaria.

neuroscience↗

bioRxiv2019

Compromised function of an ESCRT complex promotes endolysosomal escape of tau seeds and propagation of tau aggregation

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimers Disease and frontotemporal dementia. However, we lack a systematic understanding of the cellular path-ways controlling prion-like propagation. To uncover such pathways, we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation. Our screens uncovered that knockdown of several components of the ESCRT machinery, including CHMP6, or CHMP2A in combination with CHMP2B (a gene linked to familial fronto-temporal dementia), promote propagation of tau aggregation. We found that knockdown of these genes caused damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation.

cell biology↗