Biology subjects

Nepal, S.

Publications and source records attributed to Nepal, S..

7 recordsLinked to original sources

bioRxiv2026

Molecular Structure and DNA Binding Mode of Unsymmetric Cyanine Dyes RiboGreen and OliGreen

The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we de novo determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy and ultra-high resolution mass spectrometry. All three dyes were identified as unsymmetric cyanine dyes, where benzazolium and 4-quinolinium moieties are linked by a monomethine bridge. Complete 1H and 13C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and {pi}-{pi} stacking with accessible nucleobases of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes. HighlightsO_LIDetermination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreen C_LIO_LINMR spectroscopic characterization of all three dyes. C_LIO_LINMR and MD data indicate binding to be dominated by electrostatic and {pi}-{pi} stacking interactions C_LI

molecular biology↗

bioRxiv2026

Optimization of an automated system (ZEG) for rapid cellular extraction from live zebrafish

Collecting cells from zebrafish embryos for genotyping is critical to rapid research with these model organisms. The standard collection process is manual, labor-intensive, time-consuming, and requires a skilled person to perform it. To overcome this challenge, researchers are exploring the development of automated genotyping tools for live animals, which would significantly enhance the efficiency and accuracy of genetic screening in zebrafish and other species. The focus of this research was to optimize the Zebrafish Embryo Genotyper (ZEG), an automated system used for the rapid extraction of cellular material from zebrafish embryos. This system rapidly vibrates a roughened chip containing a zebrafish embryo to collect genetic material safely and efficiently. The aim was to improve the efficiency of DNA collection from the chips used with the ZEG by identifying the key factors that contribute to the process. First, the chips were modified to resolve issues associated with loss of sample volume from the chip wells due to evaporation during processing. Second, we experimented with three critical parameters - sample volume in the wells, the vibrational frequency of the system, and the operation time - on the quantity of DNA collected. The performance was evaluated by measuring embryo survival and quantifying the DNA collected. The sensitivity (previously 90%) of the DNA collection and embryo survival (previously 95%) of the were both found to be greater than 95% after optimization. The optimized design parameters (15 {micro}L solution volume, 2.4 V, and a 5-minute run with 5 s alternating on/off) provided a >50% increase in DNA collection compared to the previous designs and parameters. The proposed chip design and operation do not appear to cause any apparent adverse effects on the development or survival of the embryos.

bioengineering↗

bioRxiv2025

CD4+ T-cells sensitize quasi-mesenchymal breast tumors lacking CD73 to anti-CTLA4 immune checkpoint blockade therapy

Although immune checkpoint blockade therapy has generated dramatic responses in certain cancer types, breast tumors are largely unresponsive. Epithelial-mesenchymal plasticity leads to the assembly of an immunosuppressive tumor microenvironment and drives resistance of breast tumors to immunotherapies. Importantly, targeting CD73 completely sensitizes quasi-mesenchymal breast tumors to anti-CTLA4 immune checkpoint blockade therapy. However, the mechanism(s) of sensitization remained unknown. We demonstrate that targeting CD73 in quasi-mesenchymal breast tumors sensitizes them to anti-CTLA4 immune checkpoint blockade therapy in a CD4+ T-cell dependent manner. Moreover, epithelial-mesenchymal plasticity results in elevated expression of cancer cell-intrinsic CD73 in human triple negative breast cancers. Given the ability of quasi-mesenchymal cancer cells to metastasize and resist multiple therapies, these findings can instruct the formation of novel translational strategies for the treatment of human breast cancers. These findings also bring to the forefront the attractive possibility of utilizing the phenotypic plasticity of cancer cells along with CD73 and CD4+ T-cells as a predictive criterion for immunotherapy responsiveness. TeaserTargeting CD73 sensitizes breast cancer cells with mesenchymal traits to anti-CTLA4 therapy in a CD4+ T-cell dependent manner.

cancer biology↗

bioRxiv2024

Time spent in conversation over meals predicts default network function: Evidence from a passive mobile-sensing and fMRI study.

Sharing meals with others, often referred to as commensality, fosters social connection and may have been essential to human brain evolution. Here, we investigate whether shared meals impact social brain processes through the conversations they afford. If so, then time spent in conversation over meals should affect the default network- a set of interconnected cortical regions reliably associated with the social exchange of ideas. To test this possibility, we combined passive-mobile sensing with neuroimaging. Undergraduate participants had an application on their smartphone for two months that unobtrusively captured their time spent in conversation, as well as their location on and around campus. In between these two months, participants completed a resting state scan. Time spent in conversations around meals (e.g., at cafeterias and restaurants) during the prior month predicted greater functional connectivity in the dorsomedial default network subsystem, specifically between the left inferior frontal gyrus (LIFG) portion and the rest of the subsystem regions. This relationship was preferential to: (1) time spent in conversation over the past (vs. future) month, (2) connectivity with the LIFG (vs. other dorsomedial subsystem nodes), (3) the dorsomedial subsystem (vs. other default network subsystems), (4) eateries (vs. other locations), and (5) time spent around conversation at eateries, rather than time at eateries more generally. Follow-up analyses further revealed results were driven by a ventral portion of the LIFG, with peaks in voxel-connectivity associated with language, social, memory, and affective processes, as determined by the meta-analytic platform, NeuroSynth. Time spent in conversation over meals may exercise social cognitive processes supported by the dorsomedial subsystem, a network key to social communication, understanding, and connection.

neuroscience↗

bioRxiv2021

Computer-Vision Stabilized Intravital Imaging Reveals Lung Capillary Neutrophil Dynamics Crucial for Lung Host-Defense Function

Polymorphonuclear neutrophils (PMN) are highly dynamic innate immune cells which are essential for lung host defense. However, in vivo intravital imaging in moving organs such as the lung remains challenging due to motion artifacts. Here we describe a novel intravital imaging method with high-throughput analytical capability based on a computer vision stabilization algorithm, Computer-vision-Assisted STabilized intravital imaging (CASTii). The sub-micron precision of this approach enables analysis of compartmentalized intravital PMN dynamics. We quantified in real-time a novel patrolling function of lung intracapillary circulating PMN. We also describe the dynamics of intracapillary PMN pooling (marginated PMN pool) using direct imaging of PMNs. The pool was formed by repeated catch-and-release kinetics involving PMN deformation inside microvessels during the passage of PMNs in vessels. We observed rapid PMN recruitment into the lung tissue compartments from pooled PMNs in response to alveolar chemoattract exposure. In contrast, endotoxemia-induced intracapillary sequestration of PMN impaired PMN transmigration into the alveolar space and defective phagocytosis of live bacteria. Intravital imaging of PMN dynamics with CASTii provides fundamental insights into host-defense functions of lung capillary PMN.

microbiology↗

bioRxiv2021

ARID1A-mutant and deficient bladder cancer is sensitive to EZH2 pharmacologic inhibition

Metastatic urothelial carcinoma of the bladder is generally incurable by current systemic therapy. Molecular characterization of bladder cancer (BLCa) has revealed multiple candidate driver genes for BLCa tumorigenesis. Epigenetic/chromatin modifiers have been shown to be frequently mutated in BLCa, with ARID1A mutations highly prevalent in nearly 20% of early and late stage tumors. EZH2 is a histone methyltransferase that acts as an oncogene. The data herein show that ARID1A deficient tumors, but not ARID1A wild-type tumors are sensitive to EZH2 inhibition. Specifically, EZH2 inhibitor-treated ARID1A deficient bladder cancer cells show significantly reduced cell viability, colony formation, and in vivo tumor growth relative to ARID1A-wild type bladder cancer cells. Thus, our study suggests that a specific subset of bladder cancer patients with ARID1A mutations can be therapeutically treated with pharmacologic inhibitors targeting EZH2.

cancer biology↗

bioRxiv2018

Effects of Temperatures and High Pressures on the Growth and Survivability of Methanogens and Stable Carbon Isotope Fractionation: Implications for Deep Subsurface Life on Mars

In order to examine the potential survivability of life in the Martian deep subsurface, we have investigated the effects of temperature (45{degrees}C, 55{degrees}C, and 65{degrees}C) and pressure (1 atm, 400 atm, 800 atm, and 1200 atm) on the growth, carbon isotopic data, and morphology of chemolithoautotrophic anaerobic methanogenic archaea, Methanothermobacter wolfeii. The growth and survivability of this methanogen were determined by measuring the methane concentration in headspace gas samples after the cells were returned to their conventional growth conditions. Interestingly, this methanogen survived at all the temperatures and pressures tested. M. wolfeii demonstrated the highest methane concentration following exposure to pressure of 800 atm and a temperature of 65{degrees}C. We found that the stable carbon isotopic fractionation of methane, {delta}13C(CH4), was slightly more enriched in 12C at 1 atm and 55{degrees}C than the carbon isotopic data obtained in other temperature and pressure conditions. A comparison of the images of the cells before and after the exposure to different temperatures and pressures did not show any obvious alteration in the morphology of M. wolfeii. The research reported here suggests that at least one methanogen, M. wolfeii, may be able to survive under hypothetical Martian subsurface conditions with respect to temperature and pressure.

microbiology↗