Biology subjects

Do, M.

Publications and source records attributed to Do, M..

11 recordsLinked to original sources

bioRxiv2024

Mechanical evolution of metastatic cancer cells in three-dimensional microenvironment

Cellular biomechanics plays critical roles in cancer metastasis and tumor progression. Existing studies on cancer cell biomechanics are mostly conducted in flat 2D conditions, where cells behavior can differ considerably from those in 3D physiological environments. Despite great advances in developing 3D in vitro models, probing cellular elasticity in 3D conditions remains a major challenge for existing technologies. In this work, we utilize optical Brillouin microscopy to longitudinally acquire mechanical images of growing cancerous spheroids over the period of eight days. The dense mechanical mapping from Brillouin microscopy enables us to extract spatially resolved and temporally evolving mechanical features that were previously inaccessible. Using an established machine learning algorithm, we demonstrate that incorporating these extracted mechanical features significantly improves the classification accuracy of cancer cells, from 74% to 95%. Building on this finding, we have developed a deep learning pipeline capable of accurately differentiating cancerous spheroids from normal ones solely using Brillouin images, suggesting the mechanical features of cancer cells could potentially serve as a new biomarker in cancer classification and detection.

biophysics↗

bioRxiv2021

PERK Activation by SB202190 Attenuates Amyloidogenesis via TFEB-induced Autophagy-Lysosomal Pathway

The protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK), a key ER stress sensor of the unfolded protein response (UPR), can confer beneficial effects by facilitating the removal of cytosolic aggregates through the autophagy-lysosome pathway (ALP). In neurodegenerative diseases, the ALP ameliorates the accumulation of intracellular protein aggregates in the brain. Transcription factor-EB (TFEB), a master regulator of the ALP, positively regulates key genes involved in the cellular degradative pathway. However, in neurons, the role of PERK activation in mitigating amyloidogenesis by ALP remains unclear. In this study, we found that SB202190 selectively activates PERK independently of its inhibition of p38 mitogen-activated protein kinase, but not inositol-requiring transmembrane kinase/endoribonuclease-1 (IRE1) or activating transcription factor 6 (ATF6), in human neuroblastoma cells. PERK activation by SB202190 was dependent on mitochondrial ROS production and promoted Ca2+-calcineurin activation. The activation of the PERK-Ca2+-calcineurin axis by SB202190 positively affects TFEB activity to increase ALP in neuroblastoma cells. Collectively, our study reveals a novel physiological mechanism underlying ALP activation, dependent on PERK activation, for ameliorating amyloidogenesis in neurodegenerative diseases.

neuroscience↗

bioRxiv2021

A single- and paired-pulse TMS-EEG investigation of the N100 and long interval cortical inhibition in autism spectrum disorder

BackgroundThere is evidence to suggest a disruption of gamma-aminobutyric acid (GABA) in autism spectrum disorder (ASD), but findings are mixed. Concurrent electroencephalography and transcranial magnetic stimulation (TMS-EEG) provides a novel method by which to probe GABA-mediated cortical inhibition. MethodsWith a particular focus on GABAB-ergic mechanisms, we investigated the N100 peak of the TMS evoked potential (TEP), as well as long interval cortical inhibition (LICIEEG) in adults with ASD (n = 23; 12 female) without intellectual disability, and a neurotypical comparison group (n =22; 12 female) matched for age, sex, and IQ. Seventy-five single-(spTMS) and 75 paired-(ppTMS; 100 ms inter-stimulus-interval) pulses were applied to the right primary motor cortex (M1), right temporoparietal junction (TPJ), and right dorsolateral prefrontal cortex (DLPFC) while EEG was recorded from 20 scalp electrodes. Additionally, electromyography (EMG) was used to investigate corticospinal inhibition following ppTMS to M1 (LICIEMG). ResultsThere were no group differences in the N100 amplitude or latency following spTMS. LICI outcomes following ppTMS, as measured by either EEG or EMG, also did not differ between groups. These findings were further supported by Bayesian analyses, which provided weak-moderate support for the null hypothesis. LimitationsData presented here reflect adults without intellectual disability, and the generalisability of these results is therefore limited. ConclusionsThe findings of this study argue against GABAB-ergic impairment in adults with ASD without intellectual disability, at least at the cortical regions examined. Further research investigating these mechanisms in ASD at various ages, with varying degrees of symptomatology, and at different brain sites is an important factor in understating the role of GABA in the neuropathophysiology of ASD.

neuroscience↗

bioRxiv2020

Assessing cerebellar-cortical connectivity using concurrent TMS-EEG: A Feasibility Study

BackgroundCombined single-pulse transcranial magnetic stimulation (TMS) and electroencephalography (EEG) has been used to probe the features of local networks in the cerebral cortex. Here we investigate whether we can use this approach to explore long-range connections between the cerebellum and cerebral cortex. ObjectiveTo assess the feasibility of using cerebellar TMS-EEG for the exploration of cerebellar-cerebral network dynamics. MethodsTen healthy adults received single-pulse suprathreshold TMS to the cerebellum and an occipital/parietal control site with double-cone and figure-of-eight coils while cerebral activity was recorded. A multisensory electrical control condition was used to simulate the sensation of the double-cone coil at the cerebellar site. Two cleaning pipelines were compared, and the spatiotemporal relationships of the EEG output between conditions were examined at sensor and source levels. ResultsCerebellar stimulation with the double-cone coil resulted in large artefacts in the EEG trace. The addition of SOUND filtering to the cleaning pipeline improved the signal such that further analyses could be undertaken. The cortical potentials evoked by the active TMS conditions showed strong relationships with the responses to the multisensory control condition after ~50 ms. A distinct parietal component at ~42 ms was found following cerebellar double-cone stimulation. ConclusionsCerebellar double-cone stimulation produces large artefacts in the EEG. Cerebellar-specific responses could not be reliably differentiated from sensory evoked potentials after ~50 ms. While evoked potentials differed across conditions at early latencies, it is unclear as to whether these represented TMS-related network activation of the cerebellarthalamocortical tract, or whether components were dominated by sensory contamination and/or coil-driven artefacts. Further work will be required to clarify the specific contribution of cerebellar-cortical connectivity to the observed early latency signals.

neuroscience↗

bioRxiv2020

Selective activation of FZD7 promotes mesendodermal differentiation of human pluripotent stem cells.

WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here we describe an engineered protein, called F7L6, comprised of antibody-derived single chain variable fragments that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/{beta}-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.

developmental biology↗

bioRxiv2020

IRF8 Governs Tumor-Associated Macrophage Control of T Cell Exhaustion

Tumor progression is associated with overstimulation of cytotoxic T lymphocytes (CTLs), resulting in a dysfunctional state of exhaustion. How T cell exhaustion is elicited in the tumor remains poorly understood. Here we show that tumor-associated macrophages (TAMs) present cancer cell antigen and induce CTL exhaustion through a gene expression program dependent on the transcription factor interferon regulatory factor-8 (IRF8). In a transgenic model of murine breast cancer, CTL priming was supported by IRF8-dependent dendritic cells; yet, CTL exhaustion required TAM expression of IRF8, and its ablation suppressed tumor growth. An analysis of the highly immune-infiltrated human renal cell carcinoma tumors revealed abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. The IRF8 signature co-segregated with T cell exhaustion markers and was negatively associated with long-term patient survival. Thus, CTL exhaustion is promoted by TAMs via IRF8, and this crosstalk may be disrupted in TAM-targeted therapies.

immunology↗

bioRxiv2020

Glycolysis Fuels Phosphoinositide 3-Kinase Signaling to Bolster T Cell Immunity

Infection triggers clonal expansion and effector differentiation of microbial antigen-specific T cells in association with metabolic reprograming. Here, we show that the glycolytic enzyme lactate dehydrogenase A (LDHA) is induced in CD8+ T effector cells via phosphoinositide 3-kinase (PI3K)-dependent mechanisms. In turn, ablation of LDHA inhibits PI3K-dependent phosphorylation of Akt and its transcription factor target Foxo1, causing defective antimicrobial immunity. LDHA deficiency cripples cellular redox control and diminishes glycolytic adenosine triphosphate (ATP) production in effector T cells, resulting in attenuated PI3K signaling. Thus, nutrient metabolism and growth factor signaling are highly integrated processes with glycolytic ATP serving as a rheostat to gauge PI3K/Akt/Foxo1 signaling in T cell immunity control. Such a bioenergetics mechanism of signaling regulation implies a root cause for the century-old phenomenon of Warburg effect, and could guide development of novel therapeutics for infectious diseases and cancer. One Sentence SummaryA PI3K and LDHA circuit enables T cell immunity

immunology↗

bioRxiv2020

TGFβ Suppresses Type 2 Immunity to Cancer

The immune system employs two distinct defense strategies against infections: pathogen elimination typified by type 1 immunity, and pathogen containment exemplified by type 2 immunity in association with tissue repair. Akin to infectious diseases, cancer progresses with cancer cell acquisition of microorganism-like behavior propagating at the expense of the host. While immunological mechanisms of cancer cell elimination are well defined, whether immune-mediated cancer cell containment can be induced is poorly understood. Here we show that ablation of transforming growth factor-{beta} receptor II (TGF{beta}RII) in CD4+ T cells promotes tumor tissue healing and halts cancer progression. Notably, the restorative response is dependent on the T helper 2 cytokine IL-4 fortifying vasculature organization that spares only proximal layers of cancer cells from hypoxia, nutrient starvation and death. Thus, type 2 immunity represents an effective cancer defense mechanism, and TGF{beta} signaling in helper T cells may be targeted for novel cancer immunotherapy.

immunology↗

bioRxiv2020

Strong intracellular signal inactivation produces sharper and more robust signaling from cell membrane to nucleus

For a chemical signal to propagate across a cell, it must navigate a tortuous environment involving a variety of organelle barriers. In this work we study mathematical models for a basic chemical signal, the arrival times at the nuclear membrane of proteins that are activated at the cell membrane and diffuse throughout the cytosol. Organelle surfaces within human B cells are reconstructed from soft X-ray tomographic images, and modeled as reflecting barriers to the molecules diffusion. We show that signal inactivation sharpens signals, reducing variability in the arrival time at the nuclear membrane. Inactivation can also compensate for an observed slowdown in signal propagation induced by the presence of organelle barriers, leading to arrival times at the nuclear membrane that are comparable to models in which the cytosol is treated as an open, empty region. In the limit of strong signal inactivation this is achieved by filtering out molecules that traverse non-geodesic paths.

biophysics↗

bioRxiv2019

Antibiotic degradation by commensal microbes shields pathogens

The complex bacterial populations that constitute the gut microbiota can harbor antibiotic-resistance genes (ARGs), including those encoding for {beta}-lactamase enzymes (BLA), which degrade commonly prescribed antibiotics such as ampicillin. While it is known that ARGs can be transferred between bacterial species, with dramatic public health implications, whether expression of such genes by harmless commensal bacterial species shields antibiotic-sensitive pathogens in trans by destroying antibiotics in the intestinal lumen is unknown. To address this question, we colonized GF mice with a model intestinal commensal strain of E. coli that produces either functional or defective BLA. Mice were subsequently infected with Listeria monocytogenes or Clostridioides difficile followed by treatment with oral ampicillin. Production of functional BLA by commensal E. coli markedly reduced clearance of these pathogens and enhanced systemic dissemination during ampicillin treatment. Pathogen resistance was independent of ARG acquisition via horizontal gene transfer but instead relied on antibiotic degradation in the intestinal lumen by BLA. We conclude that commensal bacteria that have acquired ARGs can mediate shielding of pathogens from the bactericidal effects of antibiotics.

microbiology↗

bioRxiv2019

Divergence from conserved residues underlies intracellular retention of mammalian odorant receptors

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in non-olfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here, we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing "consensus" odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations. Significance StatementOdor detection in mammals depends on the largest family of G protein-coupled receptors, the odorant receptors, which represent [~]2% of our protein-coding genes. The vast majority of odorant receptors are trapped within the cell when expressed in non-olfactory cells. The underlying causes of why odorant receptors cannot be functionally expressed in non-olfactory cells have remained enigmatic for over 20 years. Our study points to divergence from a consensus sequence as a key factor in a receptors inability to function in non-olfactory cells, which in turn, helps explain odorant receptors exceptional functional diversity and rapid evolution. We also show the success of protein engineering strategies for promoting odorant receptor cell surface expression.

cell biology↗