Biology subjects

Shukla, C.

Publications and source records attributed to Shukla, C..

9 recordsLinked to original sources

bioRxiv2024

G-quadruplex Structures in Dysregulated Long Non-Coding RNA of Ovarian Cancer and their Binding Interactions with Human Serum Albumin

Ovarian carcinoma (OC) is a major cause of cancer-related mortality among women worldwide, with especially poor outcomes in resource-constrained regions. Long non-coding RNAs (lncRNAs) have emerged as critical players in OC progression, influencing drug resistance, metastasis, and other cellular processes. This study examines the potential of dysregulated lncRNAs in OC to form G-quadruplex (G4) structures. Using in silico predictions and experimental validation, we identified five lncRNAs ERLNC1, DLX6-AS1, LINC01127, FMNL1-DT, and LINP1 as candidates with a high propensity towards G4 formation. A combination of circular dichroism (CD) spectroscopy, Thioflavin T (ThT) fluorescence assay, Dot Blot assay, and reverse transcriptase stop assay, confirmed the ability of the lncRNAs to fold into stable G4 structures. Competitive DNA binding assays enabled the identification of essential G-tracts within each G4 motif highlighting the subtle differences in structure and stability of the different quadruplexes. Human serum albumin (HSA), a major circulatory protein, was found to interact with these G4-forming lncRNAs, albeit with different affinities and structural implications on the G4 motifs. This study provides the first detailed characterization of G4 structures in OC-dysregulated lncRNAs and elucidates their interactions with HSA. The G4 structures identified in these lncRNAs and their interaction with HSA can be of potential value for early OC diagnosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/624450v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@75dc3eorg.highwire.dtl.DTLVardef@1ce7b18org.highwire.dtl.DTLVardef@f371e4org.highwire.dtl.DTLVardef@ba5212_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

G-quadruplex formation in long non-coding RNAs dysregulated in colorectal cancer

Non-coding RNAs (ncRNAs) in human cells do not lead to protein synthesis and constitute a substantial portion of the transcriptome. Human long non-coding RNAs (lncRNAs) orchestrate critical cellular functions influencing development, differentiation, and metabolism. Dysregulation of lncRNAs has been correlated with several pathological conditions such as neurodegenerative and autoimmune disorders, diabetes, and cancer. Recent reports have suggested the involvement of G4s in lncRNAs to regulate colorectal cancer (CRC) carcinogenesis. In this study, we investigate the occurrence and distribution of G4s in the LINC01589, MELTF-AS1, and UXT-AS1 lncRNAs, which have been reported to be dysregulated in CRC. Using a combination of in silico tools and in vitro biophysical techniques, we show that these lncRNAs form stable, parallel, and intramolecular G4s. Furthermore, we establish the formation of G4s within these lncRNAs in CRC using cell-based assays, including RNA G4-Immuno-FISH and G4RP-RT-qPCR. This is the first systematic study of G4s in lncRNAs dysregulated in CRC, and our findings highlight the diagnostic and therapeutic potential of G4s in CRC.

biochemistry↗

bioRxiv2024

Prosapip1 in the dorsal hippocampus mediates synaptic protein composition, long-term potentiation, and spatial memory

Prosapip1 is a brain-specific protein localized to the postsynaptic density, where it promotes dendritic spine maturation in primary hippocampal neurons. However, nothing is known about the role of Prosapip1 in vivo. To examine this, we utilized the Cre-loxP system to develop a Prosapip1 neuronal knockout mouse. We found that Prosapip1 controls the synaptic localization of its binding partner SPAR, along with PSD-95 and the GluN2B subunit of the NMDA receptor (NMDAR) in the dorsal hippocampus (dHP). We next sought to identify the potential contribution of Prosapip1 to the activity and function of the NMDAR and found that Prosapip1 plays an important role in NMDAR-mediated transmission and long-term potentiation (LTP) in the CA1 region of the dHP. As LTP is the cellular hallmark of learning and memory, we examined the consequences of neuronal knockout of Prosapip1 on dHP-dependent memory. We found that global or dHP-specific neuronal knockout of Prosapip1 caused a deficit in learning and memory whereas developmental, locomotor, and anxiety phenotypes were normal. Taken together, Prosapip1 in the dHP promotes the proper localization of synaptic proteins which, in turn, facilitates LTP driving recognition, social, and spatial learning and memory.

neuroscience↗

bioRxiv2024

A Mechanically Resilient Soft Hydrogel Improves Drug Delivery for Treating Post-Traumatic Osteoarthritis in Physically Active Joints

Intra-articular delivery of disease-modifying osteoarthritis drugs (DMOADs) is likely to be most effective in early post-traumatic osteoarthritis (PTOA) when symptoms are minimal and patients are physically active. DMOAD delivery systems therefore must withstand repeated mechanical loading without affecting the drug release kinetics. Although soft materials are preferred for DMOAD delivery, mechanical loading can compromise their structural integrity and disrupt drug release. Here, we report a mechanically resilient soft hydrogel that rapidly self-heals under conditions resembling human running while maintaining sustained release of the cathepsin-K inhibitor L-006235 used as a proof-of-concept DMOAD. Notably, this hydrogel outperformed a previously reported hydrogel designed for intra-articular drug delivery, used as a control in our study, which neither recovered nor maintained drug release under mechanical loading. Upon injection into mouse knee joints, the hydrogel showed consistent release kinetics of the encapsulated agent in both treadmill-running and non-running mice. In a mouse model of aggressive PTOA exacerbated by treadmill running, L-006235 hydrogel markedly reduced cartilage degeneration. To our knowledge, this is the first hydrogel proven to withstand human running conditions and enable sustained DMOAD delivery in physically active joints, and the first study demonstrating reduced disease progression in a severe PTOA model under rigorous physical activity, highlighting the hydrogels potential for PTOA treatment in active patients.

bioengineering↗

bioRxiv2024

CanLncG4: A database curated for the assessment of G4s in the lncRNAs dysregulated in various human cancers

Long non-coding RNAs (lncRNAs) comprise a substantive part of the human genome and have emerged as crucial participants of cellular processes and disease pathogenesis. Dysregulated expression of lncRNAs in cancer contributes to various hallmarks of the disease, presenting novel opportunities for diagnosis and therapy. G-quadruplexes (G4s) within lncRNAs have gained attention, though their systematic evaluation in cancer biology is yet to be performed. In this work, we have formulated CanLncG4, a comprehensive database integrating experimentally validated associations between lncRNAs and cancer, and detailed predictions of their G4-forming potential. CanLncG4 categorizes predicted G4 motifs into anticipated G4 types and offers insights into the subcellular localization of the corresponding lncRNAs. It provides information on lncRNA-RNA and lncRNA-protein interactions, together with the RNA G4-binding capabilities of these proteins. To ensure the accuracy and validity of the data sourced from various databases, a meticulous examination of the output data was conducted to identify any discrepancies, including incorrect, missing, or duplicate entries. Additionally, scientific literature mining was performed to cross-validate the gathered information. Data from G4-prediction tools was generated using multiple parameter combinations to determine the parameters that yield more relevant and accurate predictions of the G4-forming potential. We validate our in silico G4-prediction pipeline through in vitro experiments, affirming the presence of G4s within specific cancer-dysregulated lncRNAs, thereby illustrating the predictive capability of CanLncG4. CanLncG4 represents a valuable resource for investigating G4-mediated lncRNA functions in diverse human cancers. It is expected to provide distinctive leads about G4-mediated lncRNA-protein interactions. CanLncG4 comprehensively documents 17,666 entries, establishing correlations between 6,408 human lncRNAs encompassing their transcript variants, and 15 distinct types of human cancers. The database is freely available at https://canlncg4.com/, offering researchers a valuable tool for exploring lncRNA and G4 biology towards cancer diagnosis and therapeutics.

bioinformatics↗

bioRxiv2023

The small G-protein Rac1 in the dorsomedial striatum promotes alcohol-dependent structural plasticity and goal-directed learning in mice

The small G-protein Rac1 promotes the formation of filamentous actin (F-Actin). Actin is a major component of dendritic spines, and we previously found that alcohol alters actin composition and dendritic spine structure in the nucleus accumbens (NAc) and the dorsomedial striatum (DMS). To examine if Rac1 contributes to these alcohol-mediated adaptations, we measured the level of GTP-bound active Rac1 in the striatum of mice following 7 weeks of intermittent access to 20% alcohol. We found that chronic alcohol intake activates Rac1 in the DMS of male mice. In contrast, Rac1 is not activated by alcohol in the NAc and DLS of male mice, or in the DMS of female mice. Similarly, closely related small G-proteins are not activated by alcohol in the DMS, and Rac1 activity is not increased in the DMS by moderate alcohol or natural reward. To determine the consequences of alcohol-dependent Rac1 activation in the DMS of male mice, we inhibited endogenous Rac1 by infecting the DMS of mice with an AAV expressing a dominant negative form of the small G-protein (Rac1-DN). We found that overexpression of AAV-Rac1-DN in the DMS inhibits alcohol-mediated Rac1 signaling and attenuates alcohol-mediated F-actin polymerization, which corresponded with a decrease in dendritic arborization and spine maturation. Finally, we provide evidence to suggest that Rac1 in the DMS plays a role in alcohol-associated goal-directed learning. Together, our data suggest that Rac1 in the DMS plays an important role in alcohol-dependent structural plasticity and aberrant learning. Significance StatementAddiction, including alcohol use disorder, is characterized by molecular and cellular adaptations that promote maladaptive behaviors. We found that Rac1 was activated by alcohol in the dorsomedial striatum (DMS) of male mice. We show that alcohol-mediated Rac1 signaling is responsible for alterations in actin dynamics and neuronal morphology. We also present data to suggest that Rac1 is important for alcohol-associated learning processes. These results suggest that Rac1 in the DMS is an important contributor to adaptations that promote alcohol use disorder.

neuroscience↗

bioRxiv2021

G4-Selective Ligand Induced Autophagy

G-quadruplex (G4) structures have emerged as singular therapeutic targets for cancer and neurodegeneration. Autophagy is a housekeeping cellular homeostatic mechanism and deregulation of autophagy is common in cancer and in neurodegenerative diseases. In this study, we identified the presence of 46 putative G4 sequences in the MTOR gene by use of QGRS mapper tool. We sought to connect these putative G4 sequences to a functional context by leveraging G4-targeting ligands. A G4-selective dimeric carbocyanine dye Bis-4,3 and the porphyrin TMPyP4 were used to affect the replication, transcription and translation of the MTOR gene. The ligand-induced induction of autophagic pathway via MTOR gene regulation was monitored upon treatment of HeLa and SHSY-5Y cells with G4-targeting ligands. The use of Bis-4,3 was compared with the known G4-stabilizing activity of TMPyP4. Our results show that treatment with G4-selective ligands downregulates mTOR activity and leads to the induction of excessive autophagy. This is first report on effect of G4-selective ligands on MTOR regulation and mTOR expression. mTOR being the key negative regulator of autophagy, the current work suggests potential of G4 stabilizing ligands towards induction of autophagy through the downregulation of mTOR.

biochemistry↗

bioRxiv2019

Basal forebrain parvalbumin neurons mediate arousals from sleep induced by hypercarbia or auditory stimuli

Brief arousals from sleep in patients with sleep apnea and other disorders prevent restful sleep, and contribute to cognitive, metabolic and physiologic dysfunction. Little is currently known about which neural systems mediate these brief arousals, hindering the development of treatments. The basal forebrain (BF) receives inputs from many nuclei of the ascending arousal system. These inputs include the brainstem parabrachial neurons which promote arousal in response to elevated blood carbon dioxide levels, as seen in sleep apnea. Optical inhibition of the terminals of parabrachial neurons in the BF impairs cortical arousals to hypercarbia, but which cell types within the BF mediate cortical arousals in response to hypercarbia or other sensory stimuli is unknown. Here using optogenetic techniques in mice, we show that BF parvalbumin (PV) neurons fulfill several criteria for a system mediating brief arousals from sleep. Optical stimulation of BF PV neurons during the light period, when mice normally sleep, caused rapid transitions to wakefulness and increased wake bout durations. Unlike many other ascending arousal systems, arousals induced by stimulation of BF PV neurons were brief, resulting in only a small (13.6%) increase in the total amount of wakefulness. Bilateral optical inhibition of BF PV neurons increased the latency to arousal produced by hypercarbia or auditory stimuli. Thus, BF PV neurons are an important component of the brain circuitry which generates brief arousals from sleep in response to internal and external sensory stimuli.

neuroscience↗

bioRxiv2018

A practical guide to methods controlling false discoveries in computational biology

BackgroundIn high-throughput studies, hundreds to millions of hypotheses are typically tested. Statistical methods that control the false discovery rate (FDR) have emerged as popular and powerful tools for error rate control. While classic FDR methods use only p-values as input, more modern FDR methods have been shown to increase power by incorporating complementary information as \"informative covariates\" to prioritize, weight, and group hypotheses. However, there is currently no consensus on how the modern methods compare to one another. We investigated the accuracy, applicability, and ease of use of two classic and six modern FDR-controlling methods by performing a systematic benchmark comparison using simulation studies as well as six case studies in computational biology\n\nResultsMethods that incorporate informative covariates were modestly more powerful than classic approaches, and did not underperform classic approaches, even when the covariate was completely uninformative. The majority of methods were successful at controlling the FDR, with the exception of two modern methods under certain settings. Furthermore, we found the improvement of the modern FDR methods over the classic methods increased with the informativeness of the covariate, total number of hypothesis tests, and proportion of truly non-null hypotheses.\n\nConclusionsModern FDR methods that use an informative covariate provide advantages over classic FDR-controlling procedures, with the relative gain dependent on the application and informativeness of available covariates. We present our findings as a practical guide and provide recommendations to aid researchers in their choice of methods to correct for false discoveries.

bioinformatics↗