Biology subjects

Doshi, A.

Publications and source records attributed to Doshi, A..

10 recordsLinked to original sources

bioRxiv2025

Lipid droplets promote the aberrant liquid-liquid phase separation of alpha-synuclein leading to impaired energy homeostasis

Alpha-synuclein (Syn) inclusions, termed Lewy bodies, are the characteristic neuropathological feature of Parkinsons disease. Growing evidence points towards a role of aberrant liquid-liquid phase separation in the dysregulation of Syn and sequence of events that lead to the formation of Lewy bodies. However, the triggers leading to aberrant phase separation are unknown, as is the relevance of this phenomenon to the neurodegeneration process. In this study, we showed that Syn spontaneously phase separates into condensates in the presence of lipid droplets. These lipid droplet-rich condensates represent a toxic species of Syn that prevents the turnover of the entrapped lipid droplets; they are also toxic to neighbouring mitochondria which are depolarized and undergo increased mitophagy. These findings underscore the increasing importance of lipid droplets in the pathogenesis of neurodegenerative diseases, and Parkinsons disease in particular. The lipid droplets are significantly enriched within the neuromelanin in midbrain dopaminergic neurons in the substantia nigra and could therefore uniquely facilitate the early Syn-associated neurodegeneration of this region in PD. Our findings reveal a novel pathway implicated in the dysregulation of Syn that connects aberrant liquid-liquid phase separation, lipid droplets and mitochondrial toxicity.

neuroscience↗

bioRxiv2025

Multiscale Spatial Mapping of Microbial Communities for Biotherapeutic Development

Live biotherapeutic products (LBPs) are emerging as powerful tools to modulate the microbiome using well-defined microbial communities. Yet, designing, manufacturing, and delivering LBPs remains challenging, in part due to a lack of technologies capable of analyzing LBPs as complete, spatially organized consortia. Conventional sequencing-based methods lack sensitivity and specificity and do not provide critical spatial information. To address this, we present high-phylogenetic-resolution spatial mapping platform (HiPR-Map), a state-of-the-art spectral imaging technology that enables precise enumeration and spatial localization of microbial cells at species-level within complex communities. Through these advantages, HiPR-Map provides unique insights for LBP discovery and development. Applying HiPR-Map to an LBP designed to complement immune checkpoint therapy, we profiled over 1.8 million microbial cells engrafted in the murine gut. Our analysis revealed distinctive microbial spatial organization, underscoring the power of imaging-based microbiome profiling to optimize LBP design and characterization. This work highlights the transformative potential of spatial microbiome analysis for next-generation LBP development.

microbiology↗

bioRxiv2025

Neuropixels Opto: Combining high-resolution electrophysiology and optogenetics

High-resolution extracellular electrophysiology is the gold standard for recording spikes from distributed neural populations, and is especially powerful when combined with optogenetics for manipulation of specific cell types with high temporal resolution. We integrated these approaches into prototype Neuropixels Opto probes, which combine electronic and photonic circuits. These devices pack 960 electrical recording sites and two sets of 14 light emitters onto a 70 m wide, 1 cm long shank, allowing spatially addressable optogenetic stimulation with blue and red light. In mouse cortex, Neuropixels Opto probes delivered high-quality recordings together with spatially addressable optogenetics, differentially activating or silencing neurons at distinct cortical depths. In mouse striatum and other deep structures, Neuropixels Opto probes delivered efficient optotagging, facilitating the identification of two cell types in parallel. Neuropixels Opto probes represent an unprecedented tool for recording, identifying, and manipulating neuronal populations.

neuroscience↗

bioRxiv2024

Degradation of IKAROS prevents epigenetic progression of T cell exhaustion in a novel antigen-specific assay

In cancer, chronic antigen stimulation drives effector T cells to exhaustion, limiting the efficacy of T cell therapies. Recent studies have demonstrated that epigenetic rewiring governs the transition of T cells from effector to exhausted states and makes a subset of exhausted T cells non-responsive to PD1 checkpoint blockade. Here, we describe an antigen-specific assay for T cell exhaustion that generates T cells that are phenotypically and transcriptionally similar to those found in human tumors. We performed a screen of human epigenetic regulators, identifying and validating IKAROS as a driver of T cell exhaustion. We found that the IKAROS degrader iberdomide prevents exhaustion by blocking chromatin remodeling at T cell effector enhancers and preserving binding of AP-1, NF-{kappa}B, and NFAT. Thus, our study uncovered a role for IKAROS as a driver of T cell exhaustion through epigenetic modulation, providing a rationale for the potential use of iberdomide in solid tumors to prevent T cell exhaustion. HighlightsO_LINovel in vitro assay generates antigen-specific exhausted T cells from human T cells C_LIO_LIIKAROS (IKZF1) is a key driver of T cell exhaustion C_LIO_LIIberdomide (IKZF1/3 degrader) prevents the progression of exhaustion C_LIO_LITF footprinting reveals IKAROS silences effector genes by inhibiting AP-1, NF-{kappa}B and NFAT binding C_LI

genomics↗

bioRxiv2022

SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) samples reveal extensive cellular heterogeneity. Using single-cell RNA sequencing, we uncover multiple tumor cell populations distinguished by their differentiation state and associated with different stages of tumor progression in a mouse model of PDA. We identify Spdef as a factor required for tumorigenesis in pancreatic cancer cells of epithelial and mucinous nature. By comparative analysis of cell differentiation states in mice and humans, we find that the Spdef program is highly expressed by human PDAs of the classical subtype. Mouse and human PDA cells expressing elevated levels of Spdef are dependent upon this transcription factor for tumor progression in vivo. The tumor-promoting function of Spdef is recapitulated by two Spdef target genes that regulate protein folding and endoplasmic reticulum activity, Agr2 and Ern2/Ire1{beta}. These findings offer insights into the factors controlling differentiation states in PDA and identify new vulnerabilities in the most common subtype of pancreatic cancer.

cancer biology↗

bioRxiv2022

Engineered bacterial swarm patterns as spatial records of environmental inputs

A diverse array of bacteria species naturally self-organize into durable macroscale patterns on solid surfaces via swarming motility--a highly coordinated, rapid movement of bacteria powered by flagella1-5. Engineering swarming behaviors is an untapped opportunity to increase the scale and robustness of coordinated synthetic microbial systems. Here we engineer Proteus mirabilis, which natively forms centimeter-scale bullseye patterns on solid agar through swarming, to "write" external inputs into a visible spatial record. Specifically, we engineer tunable expression of swarming-related genes that accordingly modify pattern features, and develop quantitative approaches to decode input conditions. Next, we develop a two-input system that modulates two swarming-related genes simultaneously, and show the resulting patterns can be interpreted using a deep learning classification model. Lastly, we show a growing colony can record dynamic environmental changes, which can be decoded from endpoint images using a segmentation model. This work creates an approach for building a macroscale bacterial recorder and expands the framework for engineering emergent microbial behaviors.

synthetic biology↗

bioRxiv2022

A deep learning pipeline for segmentation of Proteus mirabilis colony patterns

The motility mechanisms of microorganisms are critical virulence factors, enabling their spread and survival during infection. Motility is frequently characterized by qualitative analysis of macroscopic colonies, yet the standard quantification method has mainly been limited to manual measurement. Recent studies have applied deep learning for classification and segmentation of specific microbial species in microscopic images, but less work has focused on macroscopic colony analysis. Here, we advance computational tools for analyzing colonies of Proteus mirabilis, a bacterium that produces a macroscopic bullseye-like pattern via periodic swarming, a process implicated in its virulence. We present a dual-task pipeline for segmenting (1) the macroscopic colony including faint outer swarm rings, and (2) internal ring boundaries, unique features of oscillatory swarming. Our convolutional neural network for patch-based colony segmentation and U-Net with a VGG-11 encoder for ring boundary segmentation achieved test Dice scores of 93.28% and 83.24%, respectively. The predicted masks at times improved on the ground truths from our automated annotation algorithms. We demonstrate how application of our pipeline to a typical swarming assay enables ease of colony analysis and precise measurements of more complex pattern features than those which have been historically quantified.

microbiology↗

bioRxiv2020

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

We previously described the design of triacetic acid lactone (TAL) biosensor "AraC-TAL1", based on the AraC regulatory protein. While useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity, and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a "decoy" ligand during negative selection, resulting in isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the IPRO computational framework was employed, followed by two rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

synthetic biology↗

bioRxiv2019

Novel cytokinetic ring components limit RhoA activity and contractility

Actomyosin cortical contractility drives many cell shape changes including cytokinetic furrowing. While positive regulation of contractility is well characterized, counterbalancing negative regulation and mechanical brakes are less well understood. The small GTPase RhoA is a central regulator, activating cortical actomyosin contractility during cytokinesis and other events. Here we report how two novel cytokinetic ring components, GCK-1 and CCM-3, participate in a negative feedback loop among RhoA and its cytoskeletal effectors to inhibit contractility. GCK-1 and CCM-3 are recruited by active RhoA and anillin to the cytokinetic ring, where they in turn limit RhoA activity and contractility. This is evidenced by increased RhoA activity, anillin and non-muscle myosin II in the cytokinetic ring, and faster cytokinetic furrowing, following depletion of GCK-1 or CCM-3. GCK-1 or CCM-3 depletion also reduced RGA-3 levels in pulses, and increased baseline RhoA activity and pulsed contractility during zygote polarization. Together, our findings suggest that GCK-1 and CCM-3 regulate cortical actomyosin contractility via negative feedback. SummaryNovel cytokinetic ring proteins, the Ste20 family kinase GCK-1 and its heterodimeric cofactor Cerebral Cavernous Malformations-3, close a negative feedback loop involving the RhoA GAP RGA-3/4, RhoA, and its cytoskeletal effector anillin to limit actomyosin contractility in cytokinesis and during polarization of the C. elegans zygote.

cell biology↗

bioRxiv2018

Two new plasmid post-segregational killing mechanisms for the implementation of synthetic gene networks in E. coli

Plasmids are the workhorse of both industrial biotechnology and synthetic biology, but ensuring they remain in bacterial cells is a challenge. Antibiotic selection, commonly used in the laboratory, cannot be used to stabilise plasmids in most real-world applications, and inserting dynamical gene networks into the genome is difficult. Plasmids have evolved several mechanisms for stability, one of which, post-segregational killing (PSK), ensures that plasmid-free cells do not grow or survive. Here we demonstrate the plasmid-stabilising capabilities of the axe/txe two component system and the microcin-V system in the probiotic bacteria Escherichia coli Nissle 1917 and show they can outperform the hok/sok system commonly used in biotechnological applications. Using plasmid stability assays, automated flow cytometry analysis, mathematical models and Bayesian statistics we quantified plasmid stability in vitro. Further, we used an in vivo mouse cancer model to demonstrate plasmid stability in a real-world therapeutic setting. These new PSK systems, plus the developed Bayesian methodology, will have wide applicability in clinical and industrial biotechnology.

synthetic biology↗