Biology subjects

Mahapatra, S.

Publications and source records attributed to Mahapatra, S..

33 records · Page 2Linked to original sources

bioRxiv2024

Post-GWAS functional analyses of CNTNAP5 suggests its role in glaucomatous neurodegeneration

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.

genetics↗

bioRxiv2024

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer

Mammalian switch/sucrose non-fermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, a first-in-class, orally bioavailable proteolysis targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 (BRD4) and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise. Significance StatementThe mSWI/SNF complex is a promising therapeutic target for enhancer-driven cancers. PROTACs, which enable the targeting of "undruggable" proteins, often face the challenge of achieving oral bioavailability. Here, we present AU-24118, a first-in-class, orally bioavailable mSWI/SNF ATPase dual degrader with remarkable efficacy in in vitro and in vivo models. Additionally, our study describes two distinct mechanisms of resistance to PROTAC degraders, providing crucial insights into potential challenges facing their clinical application. These findings are critical for advancing PROTAC-based therapies to clinical settings as targeted therapies for cancers.

cancer biology↗

bioRxiv2024

Unleashing precision and freedom of optical manipulation: Software-aided real-time precision opto-control of intracellular molecular activities and cell functions

The traditional method in biological science to regulate cell functions often employs chemical interventions, which commonly lack precision in space and time. While optical manipulation offers superior spatial precision, existing technologies are constrained by limitations in flexibility, accuracy, and response time. Here, we present an adaptable and interactive optical manipulation platform that integrates laser scanning, chemical sensing, synchronized multi-laser control, adaptable target selection, flexible decision-making, and real-time monitoring of sample responses. This software-assisted real-time precision opto-control (S-RPOC) platform facilitates automatic target selection driven by optical signals while permitting user-defined manual delineation. It allows the treatment of mobile or stationary targets with varying laser dosages and wavelengths simultaneously at diffraction-limited spatial precision and optimal accuracy. Significantly, S-RPOC showcases versatile capabilities including adaptive photobleaching, comprehensive quantification of protein dynamics, selective organelle perturbation, control of cell division, and manipulation of individual cell behaviors within a population. With its unprecedented spatiotemporal precision and adaptable decision-making, S-RPOC holds the potential for extensive applications in biological science.

cell biology↗

bioRxiv2024

Targeting the mSWI/SNF Complex in POU2F-POU2AF Transcription Factor-Driven Malignancies

The POU2F3-POU2AF2/3 (OCA-T1/2) transcription factor complex is the master regulator of the tuft cell lineage and tuft cell-like small cell lung cancer (SCLC). Here, we found that the POU2F3 molecular subtype of SCLC (SCLC-P) exhibits an exquisite dependence on the activity of the mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complex. SCLC-P cell lines were sensitive to nanomolar levels of a mSWI/SNF ATPase proteolysis targeting chimera (PROTAC) degrader when compared to other molecular subtypes of SCLC. POU2F3 and its cofactors were found to interact with components of the mSWI/SNF complex. The POU2F3 transcription factor complex was evicted from chromatin upon mSWI/SNF ATPase degradation, leading to attenuation of downstream oncogenic signaling in SCLC-P cells. A novel, orally bioavailable mSWI/SNF ATPase PROTAC degrader, AU-24118, demonstrated preferential efficacy in the SCLC-P relative to the SCLC-A subtype and significantly decreased tumor growth in preclinical models. AU-24118 did not alter normal tuft cell numbers in lung or colon, nor did it exhibit toxicity in mice. B cell malignancies which displayed a dependency on the POU2F1/2 cofactor, POU2AF1 (OCA-B), were also remarkably sensitive to mSWI/SNF ATPase degradation. Mechanistically, mSWI/SNF ATPase degrader treatment in multiple myeloma cells compacted chromatin, dislodged POU2AF1 and IRF4, and decreased IRF4 signaling. In a POU2AF1-dependent, disseminated murine model of multiple myeloma, AU-24118 enhanced survival compared to pomalidomide, an approved treatment for multiple myeloma. Taken together, our studies suggest that POU2F-POU2AF-driven malignancies have an intrinsic dependence on the mSWI/SNF complex, representing a therapeutic vulnerability.

cancer biology↗

bioRxiv2024

Quantification of cellular phototoxicity of organelle stains by the dynamics of microtubule polymerization

Being able to quantify the phototoxicity of dyes and drugs in live cells allows biologists to better understand cell responses to exogenous stimuli during imaging. This capability further helps to design fluorescent labels with lower phototoxicity and drugs with better efficacy. Conventional ways to evaluate cellular phototoxicity rely on late-stage measurements of individual or different populations of cells. Here, we developed a quantitative method using intracellular microtubule polymerization as a rapid and sensitive marker to quantify early-stage phototoxicity. Implementing this method, we assessed the photosensitization induced by organelle dyes illuminated with different excitation wavelengths. Notably, fluorescent markers targeting mitochondria, nuclei, and endoplasmic reticulum exhibited diverse levels of phototoxicity. Furthermore, leveraging a real-time precision opto-control technology allowed us to evaluate the synergistic effect of light and dyes on specific organelles. Studies in hypoxia revealed enhanced phototoxicity of Mito-Tracker Red CMXRos that is not correlated with the generation of reactive oxygen species but a different deleterious pathway in low oxygen conditions. TeaserMicrotubule dynamics in live cells allow quantification of cellular phototoxicity of fluorescent dyes in various conditions

cell biology↗

bioRxiv2023

Physiological roles of endocytosis and presynaptic scaffold in vesicle replenishment at fast and slow central synapses

After exocytosis, release sites are cleared of vesicular residues to replenish with transmitter-filled vesicles. Endocytic and scaffold proteins are thought to underlie this site-clearance mechanism. However, the physiological significance of this mechanism at diverse mammalian central synapses remains unknown. Here, we tested this in a physiologically optimized condition using action potential evoked EPSCs at fast calyx synapse and relatively slow hippocampal CA1 synapse, in post-hearing mice brain slices at 37{degrees}C and in 1.3 mM [Ca2+]. Pharmacological block of endocytosis enhanced synaptic depression at the calyx synapse, whereas it attenuated synaptic facilitation at the hippocampal synapse. Block of scaffold protein activity likewise enhanced synaptic depression at the calyx but had no effect at the hippocampal synapse. At the fast calyx synapse, block of endocytosis or scaffold protein activity significantly enhanced synaptic depression as early as 10 ms after the stimulation onset. Unlike previous reports, neither endocytic blockers nor scaffold protein inhibitors prolonged the recovery from short-term depression. We conclude that the release-site clearance by endocytosis can be a universal phenomenon supporting vesicle replenishment at both fast and slow synapses, whereas the presynaptic scaffold mechanism likely plays a specialized role in vesicle replenishment predominantly at fast synapses.

neuroscience↗

bioRxiv2023

Spatiotemporal precise optical manipulation of intracellular molecular activities

Controlling chemical processes in live cells is a challenging task. The spatial heterogeneity of biochemical reactions in cells is often overlooked by conventional means of incubating cells with desired chemicals. A comprehensive understanding of spatially diverse biochemical processes requires precise control over molecular activities at the subcellular level. Herein, we develop a closed-loop optoelectronic control system that allows the manipulation of biomolecular activities in live cells at high spatiotemporal precision. Chemical-selective fluorescence signals are utilized to command lasers that trigger specific chemical reactions or control the activation of photoswitchable inhibitors at desired targets. We demonstrate the capability to selectively produce reactive oxygen species (ROS) solely at targeted organelles using blue light. Notably, the induction of ROS in the endoplasmic reticulum leads to a more pronounced disruption of tubulin polymerization and a reduction in green fluorescent protein signals, in comparison to that in lipid droplets. Moreover, when combined with a photoswitchable inhibitor, we selectively inhibit tubulin polymerization within subcellular compartments. This technology enables spatiotemporal control over chemical processes and drug activities, exclusively at desired targets, while minimizing undesired effects on non-targeted locations.

cell biology↗

bioRxiv2022

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. Here, we demonstrate that ATP modulates the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase-separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP dose independently disaggregates preformed NM fibrils. Furthermore, high concentrations of ATP delimited the number of seeds by generating compact, ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase. Additionally, (low) pathological ATP concentrations restricted autocatalytic amplification by forming structurally distinct seeding-inefficient amyloids. Our results provide mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions.

biochemistry↗

bioRxiv2022

MiR-1253 Potentiates Cisplatin Response in Pediatric Group 3 Medulloblastoma by Regulating Ferroptosis

Medulloblastomas (MB) are the most common malignant pediatric brain tumor and a leading cause of childhood mortality. Aggressive tumors belonging to group 3 (G3MB) are distinguished by a marked reduction in programmed cell death by ferroptosis. These aggressive tumors also enrich iron transport and glutathione metabolism. A highly enriched pathway in these tumors is iron-sulfur (Fe-S) cluster binding corresponding to significant upregulation of ABCB7, a poor prognostic feature linked with accelerated mortality. This study elucidated whether repressing ABCB7 activates ferroptosis to mitigate G3MB aggressiveness and whether this pathway is pharmacologically targetable. In silico and in vitro analyses confirmed upregulation of ABCB7 and GPX4, the central regulator of ferroptosis, in G3MB cell lines and tumors. Repressing ABCB7 (miR-1253OE, siABCB7, sh-ABCB7) induced iron overload, elicited oxidative stress, and triggered lipid peroxidation, leading to an abrogation of medullosphere formation and cell death by ferroptosis. Intriguingly, fractionation studies revealed that ABCB7 repression abrogated GPX4 expression, most likely by GSH depletion. Repressing ABCB7 induced mitochondrial dysfunction and reduced oxidative phosphorylation. Cisplatin, a chemotherapeutic mainstay of G3MB, induces cell death by DNA crosslinking. In ABCB7 repressed cell lines, the IC50 of cisplatin was halved, resulting in augmented oxidative stress and lipid peroxidation, culminating in a higher index of ferroptosis. Artesunate, an anti-malarial drug capable of triggering ferroptosis, was shown to synergize with cisplatin, reducing tumor burden and significantly prolonging survival. Taken together, the current study illustrates how targeting iron transport can augment ferroptosis in G3MBs. It further identifies an FDA-approved drug capable of recapitulating these effects and potentiating mainstay chemotherapy.

cancer biology↗

bioRxiv2021

MiR-212-3p functions as a tumor suppressor gene in group 3 medulloblastoma via targeting Nuclear Factor I/B (NFIB)

BackgroundMedulloblastoma (MB), the most frequent malignant pediatric brain tumor, is subdivided into four primary subgroups, wingless-type (WNT), sonic hedgehog (SHH), group 3, and group 4. Haploinsufficiency of chromosome 17p13.3 and c-Myc amplification distinguish high-risk group 3 tumors associated with rapid metastasis, recurrence and early mortality. We sought to identify the role of miR-212-3p, which resides on chromosome 17p13.3, in the pathophysiology of group 3 MB. MethodsWe first determined miR-212-3p expression in group 3 MB using several publicly-available datasets with confirmatory studies in vitro. We then identified epigenetic regulation by studying methylation and HDAC modifications along the promoter region. We used two systems for expression restoration, i.e. transient transfection or stable induction, to delineate miR-212-3ps tumor suppressive and biochemical properties via assays assessing cancer proliferation, migration, invasion, colony formation, along with cell cycle and apoptosis analyses. We then compared MB and miR target databases to isolate a putative target whose biochemical and oncogenic properties were similarly elucidated using either transient silencing of target expression or stable induction of miR-212-3p. ResultsRNA expression analyses revealed dramatically reduced miR-212-3p levels in group 3 tumors and cell lines mainly through epigenetic silencing via histone modifications. Restoring miR-212-3p expression reduced in vitro cancer cell proliferation, migration, colony formation, and wound healing. Elevated miR-212-3p levels shifted c-Myc phosphorylation (from serine-62 to threonine-58), triggering destabilization and degradation; concurrently, its pro-apoptotic binding partners, i.e., Bin-1 and P19ARF, were upregulated with subsequent elevated apoptotic signals. Using a combination of transcriptomic data and dual luciferase assay, we isolated an oncogenic target of miR-212-3p, i.e. NFIB, a nuclear transcription factor implicated in metastasis and recurrence in various cancers. Increased expression of NFIB was confirmed in group 3 tumors, with poor survival shown in high-expressing patients. Transient NFIB silencing in vitro reduced cancer cell proliferation, colony formation, migration, and invasion. Concurrently, in group 3 MB cells, reduced medullosphere formation along with decreased expression of stem cell markers (Nanog, Oct4, Sox2, CD133) were noted. ConclusionThese results substantiate the tumor-suppressive role of miR-212-3p in group 3 MB and provide a potential therapeutic oncogenic target implicated in metastasis and tumor recurrence.

cancer biology↗

bioRxiv2021

Sub-stoichiometric Hsp104 regulates the genesis and persistence of self-replicable amyloid seeds of a yeast prion protein

The prion-like self-perpetuating conformational conversion is involved in both transmissible neurodegenerative diseases and non-Mendelian inheritance traits. The transmissibility of amyloid-like aggregates is dependent on the stoichiometry of chaperones such as heat shock proteins. To provide the mechanistic underpinning of the generation and persistence of prefibrillar amyloid seeds that are critical for the prion-like propagation, we studied the effect of Hsp104 disaggregase on the assembly mechanism of a yeast prion determinant of Saccharomyces cerevisiae Sup35. At low sub-stoichiometric concentrations, Hsp104 exhibits a dual role and considerably accelerates the formation of seeding-competent prefibrillar amyloids by shortening the lag phase but also prolongs their persistence by introducing unusual kinetic halts and delaying their conversion into matured fibers. Hsp104-mediated amyloid species comprise a more ordered packing and display an enhanced autocatalytic self-templating ability compare to amyloids formed without Hsp104. Our findings underscore the key functional and pathological roles of sub-stoichiometric chaperones in prion-like propagation.

biophysics↗

bioRxiv2020

Toward Minimally Invasive Therapeutic Ultrasound: Ultrasound-guided Ablation in Neuro-oncology

IntroductionTo improve patient outcomes (eg, reducing blood loss and infection), practitioners have gravitated toward noninvasive and minimally invasive surgeries (MIS), which demand specialized toolkits. Focused ultrasound, for example, facilitates thermal ablation from a distance, thereby reducing injury to surrounding tissue. Focused ultrasound can often be performed noninvasively; however, it is more difficult to carry out in neuro-oncological tumors, as ultrasound is dramatically attenuated while propagating through the skull. This shortcoming has prompted exploration of MIS options for intracranial placement of focused ultrasound probes, such as within the BrainPath (NICO Corporation, Indianapolis, IN). Herein, we present the design, development, and in vitro testing of an image-guided, focused ultrasound prototype designed for use in MIS procedures. This probe can ablate neuro-oncological lesions despite its small size. Materials & MethodsPreliminary prototypes were iteratively designed, built, and tested. The final prototype consisted of three 8-mm-diameter therapeutic elements guided by an imaging probe. Probe functionality was validated on a series of tissue-mimicking phantoms. ResultsLesions were created in tissue-mimicking phantoms with average dimensions of 2.5x1.2x6.5mm and 3.4x3.25x9.36mm after 10- and 30-second sonification, respectively. 30s sonification with 118W power at 50% duty cycle generated a peak temperature of 68{degrees}C. Each ablation was visualized in real time by the built-in imaging probe. ConclusionWe developed and validated an ultrasound-guided focused ultrasound probe for use in MIS procedures. The dimensional constraints of the prototype were designed to reflect those of BrainPath trocars, which are MIS tools used to create atraumatic access to deep-seated brain pathologies. HIGHLIGHTSO_LIAn ultrasound-guided, focused ultrasound prototype was developed and validated C_LIO_LIThe therapeutic transducer (1.5MHz) consisted of three 8-mm circular elements C_LIO_LIElements were placed on a 9x32mm curved rectangular aperture: 45mm radius curvature C_LIO_LIFunctionality was examined on tissue-mimicking phantoms C_LIO_LI2.5x1.2x6.5mm and 3.4x3.25x9.36mm lesions were seen for 10 and 30s sonification C_LI

bioengineering↗

bioRxiv2019

Persistent DNA Hyper-methylation Dampens Host Anti-Mycobacterial Immunity

Mycobacterium tuberculosis (Mtb) has co-evolved with humans for millennia and developed multiple mechanisms to evade host immunity. Restoring host immunity in order to shorten existing therapy and improve outcomes will require identifying the full complement by which host immunity is inhibited. Perturbing host DNA methylation is a mechanism induced by chronic infections such as HIV, HPV, LCMV and schistosomiasis to evade host immunity. Here, we evaluated the DNA methylation status of TB patients and their asymptomatic household contacts demonstrating that TB patients have DNA hyper-methylation of the IL-2-STAT5, TNF-NF-{varkappa}B and IFN-{gamma} signaling pathways. By MSRE-qPCR, multiple genes of the IL-12-IFN-{gamma} signaling pathway (IL12B, IL12RB2, TYK2, IFNGR1, JAK1 and JAK2) were hyper-methylated in TB patients. The DNA hyper-methylation of these pathways is associated with decreased immune responsiveness with decreased mitogen induced upregulation of IFN-{gamma}, TNF, IL-6 and IL-1{beta} production. The DNA hyper-methylation of the IL-12-IFN-{gamma} pathway was associated with decreased IFN-{gamma} induced gene expression and decreased IL-12 inducible up-regulation of IFN-{gamma}. This work demonstrates that immune cells from TB patients are characterized by DNA hyper-methylation of genes critical to mycobacterial immunity resulting in decreased mycobacteria-specific and non-specific immune responsiveness.

immunology↗

bioRxiv2018

Epidemiological Study of Autism Subgroups Using Autism Treatment Evaluation Checklist (ATEC) Score

Here we report the results of the subgroup analyses of an observational cohort of children whose parents completed the Autism Treatment Evaluation Checklist (ATEC) over the period of several years. A linear mixed effects model was used to evaluate longitudinal changes in ATEC scores within different patient subgroups. All groups decreased their mean ATEC score over time indicating improvement of symptoms, however there were significant differences between the groups. Younger children improved more than the older children. Children with milder ASD improved more than children with more severe ASD in the Communication subscale. There was no difference in improvement between females vs. males. One surprising finding was that children from developed English-speaking countries improved less than children from non-English-speaking countries.

epidemiology↗

bioRxiv2017

Linguistically deprived children: meta-analysis of published research underlines the importance of early syntactic language use for normal brain development

We analyzed all published reports of individuals not exposed to syntactic language until puberty: two feral children, who grew up without hearing any language, and eight deaf linguistic isolates, who grew up communicating to their families using homesign or kitchensign, a system of gestures which allows them to communicate simple commands but lacks much in the way of syntax. A common observation in these individuals is the lifelong difficulty understanding syntax and spatial prepositions, even after many years of rehabilitation. This debilitating condition stands in stark contrast to linguistic isolates performance on memory as well as semantic tests: they could easily remember hundreds of newly learned words and identify previously seen objects by name. The lack of syntactic language comprehension in linguistic isolates may stem from inability to understand words and/or grammar or inability to mentally synthesize known objects into novel configurations. We have previously shown that purposeful construction of novel mental images is the function of the lateral prefrontal cortex (LPFC) ability to dynamically control posterior cortex neurons 1. Here we have ranked all tests performed on linguistic isolates by their reliance on the LPFC control of the posterior cortex: a) the amount of posterior cortex territory that needs to be recruited by the LPFC and b) the number of disparate objects that have to be combined together by the LPFC in order to answer the test question. According to our analysis, linguistic isolates performed well in all tests that did not involve the LPFC control of the posterior cortex, showed decreasing scores in tests that involved greater recruitment of the posterior cortex by the LPFC, and failed in tests that involved greatest recruitment of posterior cortex necessary for mental synthesis of multiple objects. This pattern is consistent with inadequate frontoposterior connections in linguistic isolates. We discuss implications of these findings for the importance of early syntactic language exposure in formation of frontoposterior connections.

developmental biology↗