Biology subjects

Prakash, G.

Publications and source records attributed to Prakash, G..

11 recordsLinked to original sources

bioRxiv2026

Hypoxia Induced Modulation of Cellular and in vivo uptake of DNA nanocage implications in therapeutics

DNA tetrahedra (DNA Td) are promising nanocarriers for drug delivery, but how hypoxia affects their cellular internalisation remains poorly understood. We synthesised and characterised Cy5-labelled DNA Td and established chemical hypoxia; in HeLa, MDA-MB-231, and MCF-7 cells. Hypoxia was confirmed by HIF-1; nuclear translocation. Confocal microscopy revealed significantly reduced DNA Td uptake under hypoxia, whereas transferrin and cholera toxin B uptake increased, indicating cargo-selective regulation. Temperature-arrest experiments confirmed reduced energy-dependent internalisation. Pharmacological profiling showed a shift from clathrin-mediated and galectin/glycan-dependent pathways toward lipid raft/cholesterol-dependent uptake. Hypoxia increased plasma membrane electronegativity, suggesting a biophysical barrier to DNA Td uptake. Importantly, DOTMA complexation restored uptake to normoxic levels, identifying electrostatic repulsion as a key determinant. In zebrafish larvae, hypoxia significantly enhanced whole-larva DNA Td accumulation. These findings highlight surface charge engineering as a strategy for improving DNA nanostructure delivery under hypoxic conditions.

bioinformatics↗

bioRxiv2026

Mitochondrial RNA processing promotes translation by resolving structured precursor RNAs

Mammalian mitochondrial mRNAs (mt-mRNAs) are excised from polycistronic precursors primarily through cleavage of flanking tRNAs. A subset of junctions lacks intervening tRNAs and is processed by a non-canonical mechanism involving members of the FASTK family of RNA-binding proteins; however, the consequences of non-canonical processing for downstream gene expression remain poorly understood. Long-read RNA sequencing of FASTKD4-deficient cells revealed selective processing defects at the ATP8/6-CO3 and ND5-CYB junctions. Although processing of both junctions was impaired, mitoribosome profiling showed that only CYB translational efficiency declined, explaining reduced CYB protein despite unchanged CYB mRNA abundance. Mitoribosomes preferentially initiated on processed CYB transcripts despite the accumulation of unprocessed precursors, but engaged both processed and unprocessed CO3 transcripts. Chemical probing of RNA structure revealed that the unprocessed ND5-CYB junction contains a predicted RNA secondary structure adjacent to the CYB start codon, while the ATP8/6-CO3 junction is unstructured. Our results show that FASTKD4-dependent processing of the ND5-CYB precursor is required for efficient CYB translation and support a model in which non-canonical processing promotes translation of select transcripts by removing precursor RNA structures that hinder mitoribosome engagement.

molecular biology↗

bioRxiv2026

Hamelia patens-Derived Red-Emitting Carbon Quantum Dots: Surface-State Luminescence, Antioxidant Potency, and In Vitro Bioimaging

Red-emitting carbon quantum dots (HP-CQDs) were synthesised for the first time from aqueous leaf extracts of Hamelia patens through single-step, reagent-free microwave-assisted carbonisation (750 W). The resulting nanoparticles displayed a narrow hydrodynamic size distribution centred at 3.9 nm, consistent with atomic force microscopy measurements showing a maximum height of 2.81 nm. Under 400 nm excitation, the CQDs exhibited a characteristic red emission maximum at 675 nm, representing a rare example of long-wavelength-emitting green CQDs derived from plant biomass. UV-Vis absorption bands at 224 and 256 nm were assigned to {pi}-{pi}* transitions of aromatic carbon domains and n-{pi}* transitions associated with carbonyl-containing surface groups, respectively. X-ray photoelectron spectroscopy (XPS) indicated a carbon-rich composition (C: 67.24%, O: 31.25%, N: 1.52%) with prominent C-O (42.67%) and C-C/C=C (42.64%) contributions. ATR-FTIR further confirmed the retention of hydroxyl, ether, and aliphatic functionalities following carbonisation. The excitation-wavelength-independent emission peak position implicates discrete surface molecular states rather than a heterogeneous distribution of emitters. HP-CQDs exhibit potent DPPH radical scavenging activity (ICOO = 141.8 {micro}g mLO{superscript 1}), comparable to ascorbic acid (ICOO = 114.8 {micro}g mLO{superscript 1}), and maintain >95% cell viability in both HeLa and RPE-1 cells up to 250 {micro}g mLO{superscript 1}. Confocal microscopy demonstrates concentration-dependent cytoplasmic accumulation and selective perinuclear localization at 300 {micro}g mLO{superscript 1}. In vivo biodistribution in zebrafish larvae confirms systemic uptake with statistically significant fluorescence enhancement at 500 {micro}g mLO{superscript 1} (p < 0.01), establishing HP-CQDs as biocompatible red-fluorescent probes with dual imaging-antioxidant functionality. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/724069v2_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@17be668org.highwire.dtl.DTLVardef@153d874org.highwire.dtl.DTLVardef@1a7e6edorg.highwire.dtl.DTLVardef@29f13a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Curcumin-Magnesium complex loaded DNA hydrogels: concentration dependent swelling kinetics and selective cytotoxicity via Oxidative Stress induced apoptosis

Curcumin is a naturally occurring polyphenol that demonstrates considerable anti-cancer activity, however the aqueous insolubility, rapid metabolism and relatively low bioavailability are limiting to its clinical application. As such, a curcumin-magnesium (Cur-Mg) coordination complex was synthesized and subsequently encapsulated within DNA hydrogels (Cur-Mg-Hgel). The Cur-Mg complex was fully characterized using UV-Vis spectroscopy, FTIR and X-ray diffraction (XRD). UV-Vis, FTIR and XRD all support the formation of a coordination complex and suggest a decreased level of crystallinity compared to free curcumin. DNA hydrogels were formed and characterized using atomic force microscopy, rheology and swelling kinetic studies. In vitro cytotoxicity studies utilizing an MTT assay demonstrate dose dependent inhibition of HeLa cell proliferation and a slightly better retention of RPE-1 viability at low concentrations (suggesting some difference in sensitivity) though significant cell death is seen at higher concentrations and both cells. Intracellular production of ROS was measured using the DCFH-DA assay and is seen to increase when HeLa cells are treated with Cur-Mg-Hgel in comparison to un-treated controls. Annexin V/PI staining demonstrates primarily late or early apoptotic activity with minimal necrosis following treatment with Cur-Mg-Hgel. The evidence presented strongly supports the notion that Cur-Mg-Hgel is a ROS-modulating, pro-apoptotic Hydrogel suitable for cancer treatment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/724072v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@d23f8corg.highwire.dtl.DTLVardef@14a30f6org.highwire.dtl.DTLVardef@1d485e9org.highwire.dtl.DTLVardef@1a3adf7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Membrane sialylation orchestrates cellular gateways: A spatiotemporal analysis of cellular transport using DNA nanocages via membrane charge modulation

Negatively charged DNA nanostructures, such as tetrahedral nanocages, are internalized by cells despite the electrostatic repulsion from the anionic cell membrane, and, paradoxically, cancer cells, which carry intrinsically higher negative charge due to overexpression of sialic acids on their cell surface, show markedly higher uptake than normal cells. This contradiction exposes a fundamental gap in our understanding of how these anionic nanostructures overcome this repulsion. Using chemical modulation of cell-surface sialylation in RPE1 cells to create three groups with altered sialylation levels, together with inhibitor-based dissection of endocytic pathways, we demonstrate that an increase in cell surface sialylation governs the uptake of DNA tetrahedra not through electrostatics but by structurally remodeling the cell membrane via rearrangement of the GM1 lipid raft microdomain, recruiting caveolae-mediated endocytosis as an additional pathway alongside clathrin-mediated endocytosis, thereby increasing the intake of the nanostructure. These findings reframe tumor hyper-sialylation as a determinant of the uptake of anionic nanostructures, such as DNA tetrahedra, and as a targetable parameter for rational optimization of DNA-based nanotherapeutics against cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/722926v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@10eede7org.highwire.dtl.DTLVardef@124dd56org.highwire.dtl.DTLVardef@13f5355org.highwire.dtl.DTLVardef@780ecf_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

A family of lethal exotoxins defined by cell entry via the Attractin receptor

Although bacterial genomes encode numerous potential toxins, it is unclear how evolution drives the specificity of these important virulence factors. Using an insect CRISPR screen, we identified the transmembrane protein Attractin (ATRN) as the receptor for Nigritoxin (Ntx), a Vibrio toxin that causes seasonal shrimp pandemics. We found that Ntxs effector "warhead" inhibits translation via a previously uncharacterized mechanism. Moreover, we show that two related toxins require ATRN for entry but possess unrelated effector domains. One has a Rho-GTPase AMPylation function and the other an actin-targeting/proteolysis function. Our findings reveal the mechanism of Ntx entry and toxicity and show that the ATRN-targeting domain can deliver disparate effector domains, strongly indicating that this class of exotoxins can evolve as modular proteins using a common entry domain.

microbiology↗

bioRxiv2024

Methionine cycle in a pair of serotonergic neurons regulates diet-dependent behavior and longevity through a neuron-gut signaling

The folate-methionine cycle (Met-C) is a central metabolic pathway that is regulated by vitamin B12 (B12), a micronutrient obtained exclusively from diet and microbiota. This metabolic hub supports amino acid, nucleotide and lipid biosynthesis apart from its central role of providing one carbon (-CH3) moiety for methylation reactions. While deficiency of B12 as well as polymorphism in enzymes of the Met-C has been clinically attributed to neurological and metabolic disorders, how this pathway cell non-autonomously regulates systemic physiological processes is less understood. Using a B12-sensitive mutant of Caenorhabditis elegans, we show that the neuronal Met-C responds to differential B12 content in diet to regulate p38-MAPK activation in intestinal cells, thereby modulating cytoprotective gene expression, stress tolerance and longevity. Mechanistically, B12-driven changes in the metabolic flux through the Met-C in the serotonergic ADF neurons of the mutant lead to the release of serotonin (5-hydroxytryptamine, 5-HT). 5-HT activates its receptor, MOD-1, in the post-synaptic interneurons that then secretes the neuropeptide FLR-2. FLR-2 binds to FSHR-1, its cognate receptor in the intestine, and induces the phase transition of the SARM domain protein TIR-1, thereby activating the p38-MAPK pathway. Importantly, this cascade influences the foraging behaviour of the mutant worms such that they prefer a B12-rich diet. Together, our study reveals a dynamic neuron-gut signaling axis that helps an organism modulate behaviour and life history traits based on the neuronal Met-C metabolic flux determined by B12 availability in its diet. Understandably, disruption of the optimum functioning of this axis may have debilitating effects on the health of an organism and the survival of the species.

cell biology↗

bioRxiv2023

Genome-wide screens for mitonuclear co-regulators uncover links between compartmentalized metabolism and mitochondrial gene expression

Mitochondrial oxidative phosphorylation (OXPHOS) complexes are assembled from proteins encoded by both nuclear and mitochondrial DNA. These dual-origin enzymes pose a complex gene regulatory challenge for cells, in which gene expression must be coordinated across organelles using distinct pools of ribosomes. How cells produce and maintain the accurate subunit stoichiometries for these OXPHOS complexes remains largely unknown. To identify genes involved in dual-origin protein complex synthesis, we performed FACS-based genome-wide screens analyzing mutant cells with unbalanced levels of mitochondrial- and nuclear-encoded subunits of cytochrome c oxidase (Complex IV). We identified novel genes involved in OXPHOS biogenesis, including two uncharacterized genes: PREPL and NME6. We found that PREPL specifically regulates Complex IV biogenesis by interacting with mitochondrial protein synthesis machinery, while NME6, an uncharacterized nucleoside diphosphate kinase (NDPK), controls OXPHOS complex biogenesis through multiple mechanisms reliant on its NDPK domain. First, NME6 maintains local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Second, through stabilizing interactions with RCC1L, NME6 modulates the activity of mitoribosome regulatory complexes, leading to disruptions in mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression. Finally, we present these screens as a resource, providing a catalog of genes involved in mitonuclear gene regulation and OXPHOS biogenesis.

molecular biology↗

bioRxiv2023

Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle

Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared to nuclear mRNAs, mt-mRNAs were produced 700-fold higher, degraded 5-fold faster, and accumulated to 170-fold higher levels. Quantitative modeling and depletion of mitochondrial factors, LRPPRC and FASTKD5, identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation.

cell biology↗

bioRxiv2022

MobsPy: A Meta-Species Language for Chemical Reaction Networks

Chemical reaction networks are widely used to model biochemical systems. However, when the complexity of these systems increases, the chemical reaction networks are prone to errors in the initial modeling and subsequent updates of the model. We present the Meta-species-oriented Biochemical Systems Language (MobsPy), a language designed to simplify the definition of chemical reaction networks in Python. MobsPy is built around the notion of meta-species, which are sets of species that can be multiplied to create higher-dimensional orthogonal characteristics spaces and inheritance of reactions. Reactions can modify these characteristics. For reactants, queries allow to select a subset from a meta-species and use them in a reaction. For products, queries specify the dimensions in which a modification occurs. We demonstrate the simplification capabilities of the MobsPy language at the hand of a running example and a circuit from literature. The MobsPy Python package includes functions to perform both deterministic and stochastic simulations, as well as easily configurable plotting. The MobsPy package is indexed in the Python Package Index and can thus be installed via pip.

bioinformatics↗

bioRxiv2020

MCART1 is required for mitochondrial NAD transport

The nicotinamide adenine dinucleotide (NAD+/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) cycle to ATP generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD+ transporter has been identified in yeast, how NAD enters mitochondria in higher eukaryotes is unknown. Here, we mine gene essentiality data from human cell lines to identify MCART1 (SLC25A51) as co-essential with ETC components. MCART1-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD+ and NADH. Isolated mitochondria from cells lacking or overexpressing MCART1 have greatly decreased or increased NAD uptake in vitro, respectively. Moreover, MCART1 and NDT1, a yeast mitochondrial NAD+ transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells.

cell biology↗