Biology subjects

Gupta, S.

Publications and source records attributed to Gupta, S..

At least 181 records · Page 10Linked to original sources

bioRxiv2024

The need for high-resolution gut microbiome characterization to design efficient strategies for sustainable aquaculture production

Microbiome-directed dietary interventions such as microbiota-directed fibers (MDFs) have a proven track record in eliciting responses in beneficial gut microbes and are increasingly being promoted as an effective strategy to improve animal production systems. Here we used initial metataxonomic data on fish gut microbiomes as well as a wealth of a priori mammalian microbiome knowledge on -MOS and {beta}-mannan-derived MDFs to study effects of such feed supplements in Atlantic salmon (Salmo salar) and their hitherto poorly characterized gut microbiomes. Our multi-omic analysis revealed that the investigated MDFs (two -mannans and an acetylated {beta}-galactoglucomannan), at a dose of 0.2%, had negligible effects on both host gene expression, and gut microbiome structure and function under studied conditions. While a subsequent trial using a higher (4%) dietary inclusion of {beta}-mannan significantly shifted the gut microbiome composition, there were still no biologically relevant effects on salmon metabolism and physiology. Only a single Burkholderia-Caballeronia-Paraburkholderia (BCP) population demonstrated consistent and significant abundance shifts across both feeding trials, although with no evidence of {beta}-mannan utilization capabilities or changes in gene transcripts for producing metabolites beneficial to the host. In light of these findings, we revisited our omics data to predict and outline novel and potentially beneficial endogenous lactic acid bacteria that should be targeted with future, conceivably more suitable, MDF strategies for salmon. IMPORTANCEThis study focuses on the potential of MDFs to improve aquaculture production. Despite preliminary 16S rRNA amplicon data suggested that populations in the salmon gut microbiome could utilize structurally complex mannans, our findings indicates that endogenous microbes could not metabolize it, nor the host responds to its dietary inclusion, at least not under the trial conditions investigated in this study. We highlight that high-resolution and host-specific microbiome characterization can greatly improve trial design and selection of candidate MDFs for future nutritional interventions. Understanding the intricate interplay between host and its gut microbiome is paramount in studies seeking to leverage endogenous microbial communities to benefit the host. While each new condition, whether it is a disease onset or a nutritional stressor, has the potential to profoundly reshape the microbial diversity, composition and outputs, the functional microbiome information gained under healthy conditions represent a pivotal step towards designing more effective trials involving microbiome-reprogramming feed additives. Overall, we envisage that these results will lead to improved focus on coupling fundamental microbiome characterization to the design of next-generation feeds for salmon aquaculture.

microbiology↗

bioRxiv2024

Cellulose fermentation by the gut microbiota is likely not essential for the nutrition of millipedes

Millipedes are thought to depend on their gut microbiome for processing plant-litter-cellulose through fermentation, similar to many other arthropods. However, this hypothesis lacks sufficient evidence. To investigate this, we disrupted the gut microbiota of juvenile Epibolus pulchripes (tropical, CH4-emitting) and Glomeris connexa (temperate, non-CH4-emitting) using chemical inhibitors and isotopic labelling. Feeding the millipedes sterile or antibiotics-treated litter notably reduced faecal production and microbial load without major impacts on survival or weight. Bacterial diversity remained similar, with Bacteroidota dominant in E. pulchripes and Pseudomonadota in G. connexa. Sodium-2-bromoethanesulfonate treatment halted CH4 emissions and reduced the faecal mcrA copies in E. pulchripes after 14 days, but emissions resumed after returning to normal feeding. Methanogens in the order Methanobacteriales and Methanomasscilliicoccales associated with protists were detected using Catalysed Reporter Deposition Fluorescence In situ Hybridization (CARD-FISH) on day 21, despite suppressed CH4-emission. Employing 13C-labeled leaf litter and RNA-SIP revealed a slow and gradual prokaryote labelling, indicating a significant density shift only by day 21. In addition to labelling of taxa from orders well-recognized for their role in (ligno)cellulose fermentation (e.g., Bacteroidales, Burkholderiales, and Enterobacterales), others, such as members of Desulfovibrionales were also labelled. Surprisingly, labelling of the fungal biomass was somewhat quicker. Our findings suggest that fermentation by the gut microbiota is likely not essential for the millipedes nutrition. ImportanceMillipedes (Diplopoda) constitute the third most significant group of detritivores after termites and earthworms, yet they have been comparatively understudied. Traditionally, it was believed that millipedes gain energy from fermenting cellulose using their gut microbiota, similar to wood-feeding termites, but this belief lacks evidence. This study used two model millipede species, Epibolus pulchripes (large, tropical, and methane emitter) and Glomeris connexa (small, temperate, and non-methane emitter) to test this belief. We used chemical manipulation experiments, stable isotope labelling, and DNA sequencing to comprehend the microbiotas role in the millipedes nutrition. The findings suggest that cellulose fermentation by the gut microbiota may not be essential for millipede nutrition; instead, bacteriovory and fungivory might be the dominant feeding strategies of millipedes.

microbiology↗

bioRxiv2024

PTF-Vac: Ab-initio discovery of plant transcription factors binding sites using deep co-learning encoders-decoders

Discovery of transcription factors (TFs) binding sites (TFBS) and their motifs in plants pose significant challenges due to high cross-species variability. The interaction between TFs and their binding sites is highly specific and context dependent. Most of the existing TFBS finding tools are not accurate enough to discover these binding sites in plants. They fail to capture the cross-species variability, interdependence between TF structure and its TFBS, and context specificity of binding. Since they are coupled to predefined TF specific model/matrix, they are highly vulnerable towards the volume and quality of data provided to build the motifs. All these software make a presumption that the user input would be specific to any particular TF which renders them of very limited use for practical applications like genomic annotations of newly sequenced species. Here, we report an explainable Deep Encoders-Decoders generative system, PTF-V[a]c, founded on a universal model of deep co-learning on variability in binding sites and TF structure, PTFSpot, making it completely free from the bottlenecks mentioned above. It has successfully decoupled the process of TFBS discovery from the prior step of motif finding and requirement of TF specific motif models. Due to the universal model for TF:DNA interactions as its guide, it can discover the binding motifs in total independence from data volume, species and TF specific models. In a comprehensive benchmarking study across a huge volume of experimental data, it has outperformed most advanced motif finding deep learning (DL) algorithms. With this all, PTF-V[a]c brings a completely new chapter in ab-initio TFBS discovery through generative AI. Short SummaryThe discovery of transcription factor binding sites (TFBS) in plants is challenging due to high variability across species and context-specific interactions. Traditional tools rely on predefined models and often fail in their cross-species applications. PTF-V[a]c has implemented generative a universal deep-learning model that decouples TFBS discovery from predefined motifs, enabling accurate, species-independent TFBS/motif identification while outperforming existing methods by huge leads.

bioinformatics↗

bioRxiv2024

The integration of network biology and pharmacophore modeling suggests repurposing Clindamycin as an inhibitor of pyroptosis via Caspase-1 blockage in tumor-associated macrophages

BackgroundUveal melanoma (UM) is a highly malignant intraocular tumor with a poor prognosis and response to therapy, including immune checkpoint inhibitors (ICIs), after the onset of liver metastasis. The metastatic microenvironment contains high levels of tumor-associated macrophages (TAMs) that correlate positively with a worse patient prognosis. We hypothesized that one could increase the efficacy of ICIs in UM metastases by immunomodulating UM-associated macrophages. MethodsTo identify potential targets for the immunomodulation, we created a network-based representation of the biology of TAMs and employed (bulk and single-cell) differential gene expression analysis to obtain a regulatory core of UM macrophages-associated genes. We utilized selected targets for pharmacophore-based virtual screening against a library of FDA-approved chemical compounds, followed by refined flexible docking analysis. Finally, we ranked the interactions and selected one novel drug-target combination for in vitro validation. ResultsBased on the generated TAM-specific interaction network (3863 nodes, 9073 edges), we derived a UM macrophages-associated regulatory core (74 nodes, 286 edges). From the regulatory core genes, we selected eight potential targets for pharmacophore-based virtual screening (YBX1, GSTP1, NLRP3, ISG15, MYC, PTGS2, NFKB1, CASP1). Of 266 drug-target interactions screened, we identified the interaction between the antibiotic Clindamycin and Caspase-1 as a priority for experimental validation. Our in vitro validation experiments showed that Clindamycin specifically interferes with activated Caspase-1 and inhibits the secretion of IL-1{beta}, IL-18, and lactate dehydrogenase (LDH) in macrophages after stimulation. Our results suggest that repurposed Clindamycin could reduce pyroptosis in TAMs, a pro-inflammatory form of programmed immune cell death favouring tumor progression. ConclusionWe were able to predict a novel Clindamycin-Caspase-1 interaction that effectively blocks Caspase-1-mediated inflammasome activity and pyroptosis in vitro in macrophages. This interaction is a promising clinical immunomodulator of the tumor microenvironment for improving ICI responsivenss. This work demonstrates the power of combining network-based transcriptomic analysis with pharmacophore-guided screening for de novo drug-target repurposing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/576201v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@310a9aorg.highwire.dtl.DTLVardef@1af0dcaorg.highwire.dtl.DTLVardef@1b272aborg.highwire.dtl.DTLVardef@86572b_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2024

Intranasally Administered EVs from hiPSC-derived NSCs Alter the Transcriptomic Profile of Activated Microglia and Conserve Brain Function in an Alzheimer's Model

Antiinflammatory extracellular vesicles (EVs) derived from human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) hold promise as a disease-modifying biologic for Alzheimers disease (AD). This study directly addressed this issue by examining the effects of intranasal administrations of hiPSC-NSC-EVs to 3-month-old 5xFAD mice. The EVs were internalized by all microglia, which led to reduced expression of multiple genes associated with disease-associated microglia, inflammasome, and interferon-1 signaling. Furthermore, the effects of hiPSC-NSC-EVs persisted for two months post-treatment in the hippocampus, evident from reduced microglial clusters, inflammasome complexes, and expression of proteins and/or genes linked to the activation of inflammasomes, p38/mitogen-activated protein kinase, and interferon-1 signaling. The amyloid-beta (A{beta}) plaques, A{beta}-42, and phosphorylated-tau concentrations were also diminished, leading to better cognitive and mood function in 5xFAD mice. Thus, early intervention with hiPSC-NSC-EVs in AD may help maintain better brain function by restraining the progression of adverse neuroinflammatory signaling cascades.

neuroscience↗

bioRxiv2024

ATP Prevents Aggregation of Protein via Modulating its Monomeric Conformation

Proteins occurring in significantly high concentrations in cellular environments (over 100 mg/mL) and functioning in crowded cytoplasm, often face the prodigious challenges of aggregation which are the pathological hallmark of aging and are critically responsible for a wide spectrum of rising human diseases. Here we combine a joint-venture of complementary wet-lab experiment and molecular simulation to discern the potential ability of adenosine triphosphate (ATP) as solubilizer of protein aggregates. We show that ATP prevents both condensation of aggregation-prone intrinsically disordered protein A{beta}40 and promotes dissolution of pre-formed aggregates. Computer simulation links ATPs solubilizing role to its ability to modulate proteins structural plasticity by unwinding protein conformation. We show that ATP is positioned as a superior biological solubilizer of protein aggregates over traditional chemical hydrotropes, potentially holding promises in therapeutic interventions in protein-aggregation related diseases. Going beyond its conventional activity as energy currency, the amphiphilic nature of ATP enables its protein-specific interaction that would enhance ATPs efficiency in cellular processes.

biochemistry↗

bioRxiv2024

Comparing multifunctional viral and eukaryotic proteins for generating scission necks in membranes

Deterministic formation of membrane scission necks by protein machinery with multiplexed functions is critical in biology. A microbial example is the M2 viroporin, a proton pump from the influenza A virus which is multiplexed with membrane remodeling activity to induce budding and scission in the host membrane during viral maturation. In comparison, the dynamin family constitutes a class of eukaryotic proteins implicated in mitochondrial fission, as well as various budding and endocytosis pathways. In the case of Dnm1, the mitochondrial fission protein in yeast, the membrane remodeling activity is multiplexed with mechanoenzyme activity to create fission necks. It is not clear why these functions are combined in these scission processes, which occur in drastically different compositions and solution conditions. In general, direct experimental access to changing neck sizes induced by individual proteins or peptide fragments is challenging due to the nanoscale dimensions and influence of thermal fluctuations. Here, we use a mechanical model to estimate the size of scission necks by leveraging Small-Angle X-ray Scattering (SAXS) structural data of protein-lipid systems under different conditions. The influence of interfacial tension, lipid composition, and membrane budding morphology on the size of the induced scission necks is systematically investigated using our data and molecular dynamic simulations. We find that the M2 budding protein from the influenza A virus has robust pH-dependent membrane activity that induces nanoscopic necks within the range of spontaneous hemi-fission for a broad range of lipid compositions. In contrast, the sizes of scission necks generated by mitochondrial fission proteins strongly depend on lipid composition, which suggests a role for mechanical constriction.

biophysics↗

bioRxiv2023

Distractors induce space-specific neural biases in visual working memory

Information held in working memory (WM) is remarkably resilient to distraction. Yet, perceptual distractors that share mnemonic features can impact WM profoundly; the neural basis of this phenomenon remains unclear. With multivariate decoding of human electroencephalography recordings, we investigate how delay-period perceptual distractors bias WM. Participants memorized the orientations of cued and uncued grating memoranda that appeared in opposite hemifields. A grating distractor, flashed during the delay period, produces space-specific biases: memorized features are attracted towards or repelled away from the distractors orientation depending, respectively, on when the distractor appeared in the same hemifield as the memorandum, or opposite to it. Neural prioritization in WM by cueing, and stronger memorandum maintenance mitigate this bias, whereas stronger distractor encoding enhances it. Lastly, a ring-attractor model with cross-hemifield inhibition mechanistically explains the origins of these spatially-antagonistic biases. Our results reveal how lateralized sensory buffers critically enable perceptual distractors to bias visual WM. Lay SummaryWorking memory (WM) - the ability to momentarily store important items - is remarkably robust to distraction. Yet, when a salient object with features resembling the memorized item ("perceptual distractor") appears in the environment, it can alter WM appreciably. What neural mechanisms render WM resilient to distraction, and how can perceptual distractors affect it so profoundly? We address this question by presenting a salient distractor, at unpredictable times, during the WM delay-period. Surprisingly, the distractors bias on WM depends on its proximity to the memorandums original location. With state-of-the-art neural decoding and computational modeling, we identify mechanisms that mediate these space-specific distractor effects. The findings advance our understanding of WMs resilience to distraction and may inform cognitive therapies for treating WM deficits.

neuroscience↗

bioRxiv2023

The Salmon Microbial Genome Atlas enables novel insights into bacteria-host interactions via functional mapping

The essential role of the gut microbiota for host health and nutrition is well established for many terrestrial animals, while its importance for fish and particularly Atlantic salmon is unclear. Here, we present the Salmon Microbial Genome Atlas (SMGA) originating from wild and farmed fish both in freshwater and seawater, and consisting of 211 high-quality bacterial genomes, recovered by cultivation (n=131) and gut metagenomics (n=80). Bacterial genomes were taxonomically assigned into 14 different orders, including 28 distinctive genera and 31 potentially novel species. Benchmarking the SMGA, we functionally characterized key populations in the salmon gut that were detected in vivo. This included the ability to degrade diet-derived fibers and release vitamins and other exo-metabolites with known beneficial effects, which were validated by in vitro cultivation and untargeted metabolomics. Together, the SMGA enables high resolution functional insight into salmon gut microbiota with relevance for salmon nutrition and health.

microbiology↗

bioRxiv2023

Deep Co-learning on transcription factors and their binding sites attains impeccable universality in plants

Unlike animals, variability in transcription factors (TF) and their binding regions (TFBR) across the plants species is a major problem which most of the existing TFBR finding software fail to tackle, rendering them hardly of any use. This limitation has resulted into underdevelopment of plant regulatory research and rampant use of Arabidopsis like model species, generating misleading results. Here we report a revolutionary transformers based deep-learning approach, PTFSpot, which learns from TF structures and their binding regions co-variability to bring a universal TF-DNA interaction model to detect TFBR with complete freedom from TF and species specific models limitations. During a series of extensive benchmarking studies over multiple experimentally validated data, it not only outperformed the existing software by >30% lead, but also delivered consistently >90% accuracy even for those species and TF families which were never encountered during model building process. PTFSpot makes it possible now to accurately annotate TFBRs across any plant genome even in the total lack of any TF information, completely free from the bottlenecks of species and TF specific models.

bioinformatics↗

bioRxiv2023

Comprehensive evaluation of plant transcription factors binding sites discovery tools

Transcription factors (TFs) are regulatory proteins which bind to a specific DNA region known as the transcription factor binding regions (TFBRs) to regulate the rate of transcription process. The identification of TFBRs has been made possible by a number of experimental and computational techniques established during the past few years. The process of TFBR identification involves peak identification in the binding data, followed by the identification of motif characteristics. Using the same binding data attempts have been made to raise computational models to identify such binding regions which could save time and resources spent for binding experiments. These computational approaches depend a lot on what way they learn and how. These existing computational approaches are skewed heavily around human TFBRs discovery, while plants have drastically different genomic setup for regulation which these approaches have grossly ignored. Here, we provide a comprehensive study of the current state of the matters in plant specific TF discovery algorithms. While doing so, we encountered several software tools issues rendering the tools not usable to researches. We fixed them and have also provided the corrected scripts for such tools. We expect this study to serve as a guide for better understanding of software tools approaches for plant specific TFBRs discovery and the care to be taken while applying them, especially during cross-species applications. The corrected scripts of these software tools are made available at https://github.com/SCBB-LAB/Comparative-analysis-of-plant-TFBS-software.

bioinformatics↗

bioRxiv2023

Netrin1 patterns the dorsal spinal cord through modulation of Bmp signaling

We have identified an unexpected role for netrin1 as a suppressor of bone morphogenetic protein (Bmp) signaling in the developing dorsal spinal cord. Using a combination of gain- and loss-of-function approaches in chicken, embryonic stem cell (ESC), and mouse models, we have observed that manipulating the level of netrin1 specifically alters the patterning of the Bmp-dependent dorsal interneurons (dIs), dI1-dI3. Altered netrin1 levels also change Bmp signaling activity, as measured by bioinformatics, and monitoring phosophoSmad1/5/8 activation, the canonical intermediate of Bmp signaling, and Id levels, a known Bmp target. Together, these studies support the hypothesis that netrin1 acts from the intermediate spinal cord to regionally confine Bmp signaling to the dorsal spinal cord. Thus, netrin1 has reiterative activities shaping dorsal spinal circuits, first by regulating cell fate decisions and then acting as a guidance cue to direct axon extension.

developmental biology↗

bioRxiv2023

Circadian oscillation of perireceptor events influence olfactory sensitivity in diurnal and nocturnal mosquitoes

Olfaction and circadian rhythm gate different behaviors in mosquitoes that are important for their capacity to transmit disease. However, the mechanisms of odor detection, and the circadian-guided changes in olfactory sensitivity across different mosquito species, remain largely unexplored. To this end, we performed a circadian-dependent RNA-sequencing study of the peripheral olfactory- and brain tissues of female Anopheles culicifacies and Aedes aegypti mosquitoes. Data analysis revealed a significant upregulation of genes encoding: (a) odorant binding proteins (OBPs), required for transportation of odorant molecules towards the olfactory receptors, and (b) xenobiotic-metabolizing enzymes (XMEs) during the day time in Aedes aegypti and during the dusk-transition phase in Anopheles culicifacies. While XMEs primarily function in the elimination of toxic xenobiotics, concurrent elevation of XMEs and OBPs are hypothesized to act cumulatively to regulate perireceptor events and odorant sensitivity. Electroantennographic analysis with both Anopheles gambiae and Aedes aegypti against diverse behaviorally relevant odorants, combined with XMEs inhibitors and RNA interference, establish the proof-of-concept that XMEs function in perireceptor events during odorant detection and influence the odorant sensitivity in mosquitoes. Additionally, the RNA-sequencing and RNAi-mediated knockdown data revealed that daily temporal modulation of neuronal serine proteases may facilitate the consolidation of the brain function, and influence the odor detection process in both diurnal and nocturnal mosquitoes. These findings provide the impetus to further explore the species-specific rhythmic expression pattern of the neuro-olfactory encoded molecular factors, which could pave the way to develop and implement successful mosquito control methods. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/563057v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@181b845org.highwire.dtl.DTLVardef@168f22dorg.highwire.dtl.DTLVardef@f3994aorg.highwire.dtl.DTLVardef@d4fc06_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICircadian oscillation of perireceptor proteins possibly influences time-of-day dependent change olfactory sensitivity in diurnal and nocturnal mosquitoes C_LIO_LIDiurnal and nocturnal mosquitoes depict distinct dynamic change in perireceptor proteins C_LIO_LIInhibition of cytochrome P450 gene minimizes antennal response to different odorants C_LIO_LINeuronal serine protease may consolidate brain function and odor detection C_LI

molecular biology↗

bioRxiv2023

Parkinson's-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

Parkinsons (PD) is a multi-factorial disease that affects multiple brain systems and circuits. While defined by motor symptoms caused by degeneration of brainstem dopamine neurons, debilitating non-motor abnormalities in fronto-striatal based cognitive function are common, appear early and are initially independent of dopamine. Young adult mice expressing the PD-associated G2019S missense mutation in Lrrk2 also exhibit deficits in fronto-striatal-based cognitive tasks. In mice and humans, cognitive functions require dynamic adjustments in glutamatergic synapse strength through cell-surface trafficking of AMPA-type glutamate receptors (AMPARs), but it is unknown how LRRK2 mutation impacts dynamic features of AMPAR trafficking in striatal projection neurons (SPNs). Here, we used Lrrk2G2019S knockin mice to show that surface AMPAR subunit stoichiometry is altered biochemically and functionally in mutant SPNs to favor incorporation of GluA1 over GluA2. GluA1-containing AMPARs were resistant to internalization from the cell surface, leaving an excessive accumulation of GluA1 on the surface within and outside synapses. This negatively impacted trafficking dynamics that normally support synapse strengthening, as GluA1-containing AMPARs failed to increase at synapses in response to a potentiating stimulus and showed significantly reduced surface mobility. Surface GluA2-containing AMPARs were expressed at normal levels in synapses, indicating subunit-selective impairment. Abnormal surface accumulation of GluA1 was independent of PKA activity and was limited to D1R SPNs. Since LRRK2 mutation is thought to be part of a common PD pathogenic pathway, our data suggest that sustained, striatal cell-type specific changes in AMPAR composition and trafficking contribute to cognitive or other impairments associated with PD. SIGNIFICANCE STATEMENTMutations in LRRK2 are common genetic risks for PD. Lrrk2G2019S mice fail to exhibit long-term potentiation at corticostriatal synapses and show significant deficits in frontal-striatal based cognitive tasks. While LRRK2 has been implicated generally in protein trafficking, whether G2019S derails AMPAR trafficking at synapses on striatal neurons (SPNs) is unknown. We show that surface GluA1-AMPARs fail to internalize and instead accumulate excessively within and outside synapses. This effect is selective to D1R SPNs and negatively impacts synapse strengthening as GluA1-AMPARs fail to increase at the surface in response to potentiation and show limited surface mobility. Thus, LRRK2-G2019S narrows the effective range of plasticity mechanisms, supporting the idea that cognitive symptoms reflect an imbalance in AMPAR trafficking mechanisms within cell-type specific projections.

neuroscience↗

bioRxiv2023

Inhibition of Lactate Dehydrogenase A (LDH-A) by Diclofenac Sodium Induces Apoptosis in HeLa Cells by Activation of AMPK

Cancer cells exhibit a unique metabolic preference for choosing the glycolytic pathway over oxidative phosphorylation for maintaining the tumor microenvironment. Lactate dehydrogenase-A (LDH-A) is a key enzyme that facilitates glycolysis by converting pyruvate to lactate and has been shown to be upregulated in multiple cancers due to the hypoxic tumor microenvironment. Diclofenac (DCF), a non-steroidal anti-inflammatory drug, has been shown to exhibit anti-cancer effects by interfering with the glucose metabolism pathway. However, the specific targets remain unknown. Using in-silico, biochemical, and biophysical studies, we show that DCF binds to LDH-A adjacent to the substrate binding site and dose-dependently inhibits its activity in an allosteric manner in HeLa cells. Thus, DCF inhibits the hypoxic microenvironment and induces apoptosis-mediated cell death. DCF fails to induce cytotoxicity in LDH-A knocked-down HeLa cells, confirming that DCF renders its anti-mitotic effects via LDH-A inhibition. DCF-induced LDH-A inhibition alters pyruvate, lactate, NAD+, and ATP production in cells, and this could be a possible mechanism by which DCF inhibits glucose uptake in cancer cells. DCF-induced ATP deprivation leads to mitochondria-mediated oxidative stress, which results in DNA damage, lipid peroxidation, and apoptosis-mediated cell death. Reduction in intracellular ATP levels additionally activates AMPK, a sensor kinase, which further downregulates p-S6K, leading to apoptosis-mediated cell death. We find that in LDH-A knocked-down cells, intracellular ATP levels were depleted, resulting in the inhibition of p-S6K, implying the involvement of DCF-induced LDH-A inhibition in the activation of the AMPK/S6K signaling pathway.

cancer biology↗

bioRxiv2023

DNA Tetrahedral Nanocages as a Promising Nanocarrier for Dopamine Delivery in Neurological Disorders

Dopamine is a neurotransmitter in the central nervous system that is essential for many bodily and mental processes, and a lack of it can cause Parkinsons disease. DNA tetrahedral (TD) nanocages are promising in bio-nanotechnology, especially as a nanocarrier. TD is highly programmable, biocompatible, and capable of cell differentiation and proliferation. It also has tissue and blood-brain barrier permeability, making it a powerful tool that could overcome potential barriers in treating neurological disorders. In this study, we used DNA-TD as a carrier for Dopamine to cells and zebrafish embryos. We investigated the mechanism of complexation between TD and dopamine hydrochloride using gel electrophoresis, fluorescence and circular dichroism (CD) spectroscopy, atomic force microscopy (AFM), and molecular dynamic (MD) simulation tools. Further, we demonstrate these Dopamine-loaded DNA tetrahedral nanostructures cellular uptake and differentiation ability in SH-SY5Y neuroblastoma cells. Furthermore, we extended the study to zebrafish embryos as a model organism to examine survival and uptake. The research provides valuable insights into the complexation mechanism and cellular uptake of dopamine-loaded DNA tetrahedral nanostructures, paving the way for further advancements in nanomedicine for Parkinsons disease and other neurological disorders.

bioengineering↗

bioRxiv2023

Probing Molecular Diversity and Ultrastructure of Brain Cells with Fluorescent Aptamers

Detergent-free immunolabeling has been proven feasible for correlated light and electron microscopy, but its application is restricted by the availability of suitable affinity reagents. Here we introduce CAptVE, a method using slow off-rate modified aptamers for cell fluorescence labeling on ultrastructurally reconstructable electron micrographs. CAptVE provides labeling for a wide range of biomarkers, offering a pathway to integrate molecular analysis into recent approaches to delineate neural circuits via connectomics.

neuroscience↗

bioRxiv2023

Nature-Inspired Peptide of MtDef4 C-terminus Tail Enables Protein Delivery in Mammalian Cells

Cell-penetrating peptides hold great promise as versatile tools for the intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. In this study, we investigated the mammalian cell-penetrating properties of a 16-residue peptide derived from the C-terminus tail of the Medicago truncatula defensin protein, with the sequence GRCRHGFRRRCFCTTHC. We evaluated the ability of this peptide to penetrate multiple types of cells. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a sub-micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers the GFP into the cells. Despite its remarkable cellular penetration, the peptide has only a minor effect on cellular viability, making it a promising candidate for the development of a cell-penetrating peptide, with potential therapeutic applications.

cell biology↗