Biology subjects

Bose, D.

Publications and source records attributed to Bose, D..

17 recordsLinked to original sources

bioRxiv2026

Evolutionary tuning of complexin-synaptotagmin fusion clamp governs regulated neurotransmitter release

Synaptic vesicles must be held in a fusion-ready but restrained state until calcium influx triggers release. Synaptotagmin-1 (Syt1) and Complexin (CPX) both contribute to this fusion clamp, yet removing either protein produces opposite effects in different species. Whether this reflects genuine divergence in their molecular roles, or species-specific tuning of a conserved mechanism, has remained unresolved. Here we address this by comparing mammalian and C. elegans orthologs in a reconstituted single-vesicle assay that isolates clamping from upstream docking and priming. We find that clamping in both species requires the combined action of CPX and Syt1, with neither sufficient on its own. CPX clamps only with its cognate SNARE complex, a specificity conferred entirely by its accessory helix. The Syt1 orthologs differ markedly in intrinsic clamping strength, which maps to C2B mediated higher-order oligomerization and sets the functional dependence on CPX in each system. These results support a dual-regulator model of exocytosis in which Syt1 and CPX act sequentially and cooperatively through discrete molecular states to control vesicle clamping and calcium-triggered release. This framework reconciles long-standing functional discrepancies across species and reveals how evolutionary divergence modulates conserved mechanisms governing synaptic fidelity.

neuroscience↗

bioRxiv2026

Kinetochore Proteins CENPB and LIC2 are critical for Episome Persistence and Replication of Kaposis's Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes lifelong latency by maintaining its genome as circular episomes tethered to host chromatin and faithfully segregated during mitosis. This process is primarily mediated by the latency-associated nuclear antigen (LANA), which interacts with host chromosome-associated factors to ensure viral genome persistence. Several kinetochore proteins, including CENP-F, Bub1, BubR1, Aurora B, Survivin, and NuMA, have been implicated in this process, yet the broader contribution of kinetochore components remains poorly defined. We performed a systematic shRNA-based screen of the 37 host genes encoding kinetochore-associated proteins to evaluate their role in KSHV episome maintenance. We validated involvement of previously reported factors and identified Centromere Protein B (CENP-B) and Light Intermediate Chain 2 of the dynein complex (LIC2) as novel kinetochore-associated proteins critical for KSHV episome persistence. Loss of these proteins significantly reduced episome stability, highlighting previously unrecognized host-virus interactions at the chromosome segregation machinery. These findings identify critical host factors that expand the repertoire of kinetochore proteins co-opted by KSHV for episome maintenance and provide new mechanistic insights into how viral episomes exploit host chromosome segregation pathways for long-term persistence. Our work underscores kinetochore proteins as a broader class of host dependency factors in gamma-herpesvirus biology and suggests potential therapeutic targets for KSHV-associated malignancies.

microbiology↗

bioRxiv2026

IFN-driven lipid synthesis shutdown in CD4⁺ T cells during acute SIV infection and persistent OXPHOS with ART initiation

Cellular metabolism regulates HIV/SIV replication and reservoir establishment, yet how infection and antiretroviral therapy initiation (ARTi) shape CD4 T cell metabolism in vivo remains poorly defined. Using the SIVmac239 macaque model, we integrated single-cell metabolic profiling (MIST), transcriptomics, lipidomics and genome-scale metabolic modeling to characterize their metabolic remodeling. At peak viremia, CD4 T cells exhibited shutdown of de novo fatty-acid (FA) synthesis, reflected by acetyl-CoA carboxylase-1 (ACC1) downregulation, inhibition of lipid-anabolic reactions, and depletion of membrane phospholipids. This state was driven by type I interferon (IFN-I) responses, and IFN-I suppressed ACC1 in vitro. Pharmacologic inhibition of FA synthesis enhanced T cell activation and exerted direct antiviral effects. Following ARTi, most metabolic pathways in CD4+ T cells were suppressed, whereas oxidative phosphorylation (OXPHOS) remained elevated and its levels in effector memory cells correlated with cell-associated-vDNA These findings identify IFN-driven FA synthesis suppression as a novel effector mechanism during acute viral infection, and persistent OXPHOS in effector CD4 T cells as a metabolic correlate of early reservoir establishment.

immunology↗

bioRxiv2026

Intranasal Delivery of HIV/SIV Antigens with NE/AS01B Adjuvants Enhances Cellular Immunity and Reduces Viral Loads in SHIV-Challenged Macaques

The primary route of HIV transmission is across mucosal tissues; therefore, developing a protective mucosal vaccine is a top priority. In a pilot study, using a macaque model, we delivered HIV gp140 envelope glycoprotein and SIVmac239 P55 Gag and Nef antigens using heterologous prime/boost via the intranasal route with a soybean oil-based nanoemulsion (NE) adjuvant and through the intramuscular route with the AS01B adjuvant system to generate enhanced cell-mediated immunity. We used a NE adjuvant to promote gut-homing cell-mediated immunity and the AS01B system to enhance humoral immune responses. Following intrarectal challenge with SHIV 4MTF.tHy, vaccinated macaques acquired the virus but experienced lower viral loads in plasma (P=0.003) and CSF (P=0.001), and potent polyfunctional gag-specific (CD107a+, IFN{gamma}, TNF+) responses across diverse lymph nodes. Significant antibody-dependent complement deposition (ADCD) and antibody-dependent cellular phagocytosis (ADCP) responses were induced, and gut-microbiome crosstalk could be modulated and showing reduced SHIV-dysbiosis. Notably, vaccination preserved mucosal all-trans retinoic acid levels (atRA) (p<0.05). However, no significant differences were observed for antibody responses between vaccinated and unvaccinated macaques. In summary, the induced gut-homing properties by the NE adjuvant are effective at generating cell-mediated immunity and reducing viral set points and warrant further investigations as a mucosal adjuvant in HIV vaccine design. ImportanceThree major non-mucosal vaccine trials (RV144, HVTN702, and 706) failed to reduce HIV infection rates. Therefore, new approaches in developing a mucosal vaccine remain an effective strategy to attempt to control HIV infection. Coherent vaccine approaches against HIV were focused on immune correlates related to viral loads, persistent reservoirs, and antibody responses. As a proof-of-principle, we developed a vaccine regimen consisting of AS01B and an adjuvanted oil-in-water NE cleaved HIV clade C gp140 protein and non-cleaved Gag, and nef particles administered through intranasal, subcutaneous, and intramuscular routes, followed by intrarectal challenge with clade C SHIV. This vaccine elicited strong ADCD and ADCP responses, modulated immune-microbiome crosstalk, and reduced susceptibility to SHIV-infection-associated dysbiosis. Additionally, it preserved mucosal all trans retinoic acid (atRA) levels, suggesting a potential role for this approach in HIV vaccine development.

microbiology↗

bioRxiv2025

Mapping EGFR1 sorting domains in endosomes with a calibrated 3D expansion microscopy toolkit

Endosomes are nanoscale intracellular compartments that sort and recycle cell surface receptors such as epidermal growth factor receptor 1 (EGFR1). Nanometre-scale interactions and co-clustering of signalling proteins, cargo, and the membrane are critical to this process. Direct visualisation of these interactions has been hindered by the limited 3D resolution achievable with conventional and super-resolution microscopies. Here, we present the adaptation of expansion microscopy (ExM) to visualise, and quantify nanoclusters of endosomal proteins of human retinal pigment epithelial (RPE-1) cells. A 3D distortion analysis was developed leveraging the Farneback optical flow principle for detecting anisotropies in the hydrogel expansion. Analysis of pre- and post- ExM image volumes for 3D anisotropies revealed under-expansion of cytoplasmic regions within ExM hydrogels, often leading to over-estimation of size and distance measurements of small compartments such as endosomes. A self-assembling protein nanocage that reports the true local and nanoscale expansion factor was genetically introduced into the cells to calibrate ExM images of cytoplasmic regions containing endosomes. To stimulate and visualise the internalisation and sorting of EGFR1 in mammalian cells, a pulse-chase protocol was carried out with fluorescently-tagged EGF. The cells were subsequently fixed at 15- and 30- minute time points and subjected to 10-fold ExM and multiplexed 3D Airyscan microscopy to map cargo and EGFR1 vs other endosomal proteins. A volume tracing pipeline was developed to visualise the changes in the labelled EGF and EGFR1 densities at the limiting membrane of the endosomes. With multiplexed 3D ExM image volumes, we observed the enrichment of both EGF and EGFR1 in the endosomal interior and the accumulation of endosomal protein Rab5a near the limiting membrane during this early maturation of the endosomes. Taken together, the multiplexed 3D ExM toolkit offers a quantitative framework for visualising and measuring the intrinsic biology of small sub-cellular organelles like endosomes at true molecular-scale resolution.

biophysics↗

bioRxiv2024

G-quadruplexes in the RSV genome: potential anti-viral targets.

The Rous sarcoma virus (RSV) is an onco-retrovirus that infects avian species such as the chicken (Gallus gallus). RSV is the first oncovirus to be described and the oncogenic activity of this virus is related to the expression of a tyrosine kinase that induces carcinogenic transformation. Interestingly, we have noted that the RSV genome contains various potential G4 forming sequences. Among these, two sequences located in the GAG and POL genes, respectively, show high G4 forming potential. Additionally, the SRC oncogene also harbours a putative G4 forming sequence. In this study, we have characterised the G4 formation and topology in these three loci in the RSV-DNA. We have found that these sequences form dynamic G4 structures in physiological conditions and such dynamicity may be associated with their cellular functions. Further, we have also established that these G4s are recognized by G4 interacting small-molecule ligands and the G4-stabilizing protein nucleolin. Binding of these ligands induces structural shifts in the G4 leading to change in structure and stability. Thus, the RSV-DNA G4s may be further studied as targets to control its infection and oncogenic effects.

biophysics↗

bioRxiv2024

A minimal presynaptic protein machinery mediating synchronous and asynchronous exocytosis and short-term plasticity.

Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic Ca2+ influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically-relevant conditions to delineate the minimal protein machinery sufficient to account for different modes of Ca2+-triggered vesicle fusion and short-term facilitation. We find that Synaptotagmin-1, Synaptotagmin-7, and Complexin, synergistically restrain SNARE complex assembly, thus preserving vesicles in a stably docked state at rest. Upon Ca2+ activation, Synaptotagmin-1 induces rapid vesicle fusion, while Synaptotagmin-7 mediates delayed fusion. Competitive binding of Synaptotagmin-1 and Synaptotagmin-7 to the same SNAREs, coupled with differential rates of Ca2+-triggered fusion clamp reversal, govern the kinetics of vesicular fusion. Under conditions mimicking sustained neuronal activity, the Synaptotagmin-7 fusion clamp is destabilized by the elevated basal Ca2+ concentration, thereby enhancing the synchronous component of fusion. These findings provide a direct demonstration that a small set of proteins is sufficient to account for how nerve terminals adapt and regulate the Ca2+-evoked neurotransmitter exocytosis process to support their specialized functions in the nervous system.

neuroscience↗

bioRxiv2023

TGF-β blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of the anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirmed the latency reversal properties of in vivo TGF-{beta} blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on ART were treated with four 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption (ATI). 7 out of 8 macaques rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with 64Cu-DOTA-F(ab)2-p7D3-probe. A decrease in cell-associated (CA-)SIV DNA was detected in lymph nodes, gut and PBMC, while intact pro-virus in PBMC decreased by 3-fold. No systemic increase in inflammatory cytokines was observed. High-dimensions cytometry, bulk, and single-cell (sc)RNAseq revealed a shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrated that galunisertib, a clinical stage TGF-{beta} inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity. One-sentence summaryTGF-{beta} blockade drives an effector phenotype in immune cells leading to SIV latency reversal and enhanced immune responses in vivo.

immunology↗

bioRxiv2023

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in atrophic AMD

Introductory paragraphAge-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.

cell biology↗

bioRxiv2022

Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo

Elevated levels of TGF-{beta}, a potent immunosuppressive factor, are present in HIV-1 infected individuals even after years of antiretroviral therapy (ART). TGF-{beta} plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent one of the main cellular reservoirs after long term ART and the low inducibility of the latent provirus constitutes one of the major obstacles to "kick and kill" cure strategies. We hypothesized that releasing cells from TGF-{beta}-driven signaling would promote latency reversal. To test our hypothesis, we compared ex vivo models of HIV-1 latency reactivation with and without TGF-{beta} and a TGF-{beta} type 1 receptor (TGFBR1) inhibitor, galunisertib. We also tested the effect of galunisertib in SIV infected, ART treated macaques by monitoring SIV envelope (env) protein expression via PET/CT using the Cu64-anti gp120 Fab (7D3) probe, along with plasma and tissue viral loads (VL). Exogenous TGF-1{beta} reduced HIV-1 reactivation in U1 and ACH2 latency models. Galunisertib increased HIV-1 latency reversal both in ex vivo models and in PBMC from HIV-1 infected, cART treated aviremic donors. In vivo, oral galunisertib promoted increased SIV env protein total standardized uptake values (SUVtot) in PET/CT images of tissues (gut and lymph nodes) of 5 out of 7 aviremic, long-term ART-treated, SIV-infected, macaques. This increase correlated with an increase in SIV RNA in gut tissue. Two out of 7 animals also exhibited increases in plasma viral load. Higher anti-SIV T cell responses and anti-SIV env antibody titers were detected after galunisertib treatment in most animals. In summary, our data suggest that blocking TGF-{beta} signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.

immunology↗

bioRxiv2021

Prior exposure to microcystin alters host gut resistome and is associated with dysregulated immune homeostasis in translatable mouse models

The increased propensity of harmful algal blooms (HABs) and exposure from HABs-cyanotoxin causes human toxicity. It has been associated with the progression of several diseases that encompass the liver, kidneys, and immune system. Recently, a strong association of cyano-HAB toxicity with the altered host gut microbiome has been shown. We tested the hypothesis that prior exposure to cyanotoxin microcystin may alter the microbiome and induce microbiome-host-resistome crosstalk. Using both wild-type and humanized mice, we show that the mice exposed to microcystin had an altered microbiome signature that harbored antimicrobial resistance genes. Host resistome phenotypes such as mefA, msrD, mel, ant6, and tet40 increased in diversity and relative abundance following microcystin exposure. Interestingly, the increased abundance of these genes was traced to resistance to common antibiotics such as tetracycline, macrolides, glycopeptide, and aminoglycosides, crucial for modern-day treatment for several diseases. Increased abundance of these genes was positively associated with increased expression of PD1, a T-cell homeostasis marker, and pleiotropic inflammatory cytokine IL-6 with a concomitant negative association with immunosurveillance markers IL7 and TLR2. Microcystin exposure also caused decreased TLR2, TLR4, and REG3G expressions, increased immunosenescence, and higher systemic levels of IL-6 in both wild-type and humanized mice. In conclusion, the results show a first-ever characterization of the host resistome of microcystin exposure and its connection to host immune status and antibiotic resistance. The results may be crucial for understanding the ability of exposed subjects to fight future bacterial infections and the progression of the debilitating disease in hospital settings.

microbiology↗

bioRxiv2021

Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins

Hemoglobin oxidation due to oxidative stress and disease conditions leads to generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the RBC membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2, 2-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-{beta}-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.

biochemistry↗

bioRxiv2020

Spectrin Interactome under normal and HbE-disease conditions

Spectrin, the major component of the erythrocyte membrane skeleton is a key player in red cell biology. It has a significant role in signalling pathways and as such knowledge of spectrin interactors becomes crucial. Here we report the cytosolic interactome of human erythroid spectrin (ProteomeXchange id: PXD021525). This is to the best of our knowledge the first report of the interactome of human erythroid spectrin. We have further investigated the spectrin interactome under HbE disease conditions. Our findings indicate that there is no difference in the identity of the proteins interacting with spectrin between normal and disease conditions. However relative abundance of the interacting partners is seen to change. Very interestingly the interacting chaperone proteins, heme-containing proteins and redox active proteins are seen to be up-regulated in HbE disease state. This is consistent with our previous observation that presence of oxidation prone hemoglobin variants leads to an increase of redox regulators and chaperones in the red cell proteome. Spectrin can also interact with horse radish peroxidase and oxidatively crosslink hemoglobin, which has possible implications in oxidative stress management. Since a large fraction of spectrin interacting proteins are chaperones and redox active proteins, it is possible that spectrin may have a broader role in redox regulation, especially in cases where there are unstable hemoglobin variants present.

biochemistry↗

bioRxiv2019

Locally acting transcription factors are required for p53-dependent cis-regulatory element activity

The master tumor suppressor p53 controls transcription of a wide-ranging gene network involved in apoptosis, cell cycle arrest, DNA damage repair, and senescence. Recent studies revealed pervasive binding of p53 to cis-regulatory elements (CRE), which are non-coding segments of DNA that spatially and temporally control transcription through the combinatorial binding of local transcription factors (TFs). Although the role of p53 as a strong trans-activator of gene expression is well known, the co-regulatory factors and local sequences acting at p53-bound CREs are comparatively understudied. We designed and executed a massively parallel reporter assay (MPRA) to investigate the effect of transcription factor binding motifs and local sequence context on p53-bound CRE activity. Our data indicate that p53-bound CREs are both positively and negatively affected by alterations in local sequence context and changes to co-regulatory TF motifs. We identified a SP1/KLF family motif located in an intronic p53 CRE that is required for the endogenous expression of the p53-dependent gene CCNG1. We also identified ATF3 as a factor that co-regulates the expression of the p53-dependent gene GDF15 through binding with p53 in an upstream CRE. Loss of either p53 or ATF3 severely reduces CRE activity and alters endogenous GDF15 mRNA levels in the cell. Our data suggests that p53 has the flexibility to cooperate with a variety of transcription factors in order to regulate CRE activity. By utilizing different sets of co-factors across CREs, we hypothesize that p53 activity is guarded against loss of any one regulatory partner allowing for dynamic and redundant control of p53-mediated transcription.

molecular biology↗

bioRxiv2019

Interaction of NPC2 protein with Lysobisphosphatidic Acid is required for normal endolysosomal cholesterol trafficking

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates by the LE/LY phospholipid lysobisphosphatidic acid (LBPA) and in these studies sought to determine their functional relationship in normal LE/LY cholesterol egress. Here we demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Using its precursor phosphatidylglycerol (PG), we show that PG-induced LBPA enrichment results in clearance of accumulated cholesterol from NPC1-deficient cells but is ineffective in cells lacking functional NPC2. Together these studies reveal a heretofore unknown aspect of intracellular cholesterol trafficking, in which NPC2 and LBPA function together in an obligate step of sterol egress from the LE/LY compartment, which appears to be independent of NPC1.

biochemistry↗

bioRxiv2019

Localizing the chaperone activity of erythroid spectrin

Spectrin, the major protein of the RBC membrane skeleton has canonically been thought to only serve a structural function. We have described a novel chaperone-like property of spectrin and have shown that it is able to prevent the aggregation of other proteins such as alcohol dehydrogenase, insulin and free globin chains. We have tried to localize the molecular origin of chaperone-like activity in multi-domain spectrin by using recombinant spectrin fragments and investigating individual domains. We have characterized the recombinant domains using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Hydrophobic ligands Prodan (6-propionyl-2[dimethylamino]-naphthalene) and ANS (1-anilinonaphthalene-8-sulfonic acid) binding has been used to probe the hydrophobicity of the recombinant domains and it is seen that all domains have surface exposed hydrophobic patches; and in accordance with our previous hypothesis only the reconstituted self-association domain binds Prodan. Recombinant domains display comparable chaperone potential in preventing protein aggregation; and substrate selectivity of -over {beta}-globin is seen. Enzyme refolding studies show alternate pathways of chaperone action. Our current study points to the presence of hydrophobic patches on the surface of these domains as the source of the chaperone activity of spectrin, as notably seen in the self-association domain. There is no one domain largely responsible for the chaperone activity of spectrin; rather all domains appear to contribute equally, such that the chaperone activity of spectrin seems to be a linear sum of the individual activities of the domains.

biochemistry↗

bioRxiv2017

Aminoglycoside Antibiotics Perturb Physiologically Important MicroRNA Contributing To Drug Toxicity

miRNAs are key non-protein coding regulators of gene expression in various pathophysiological conditions. Targeting miRNA with small molecules offer an unconventional approach, where clinically active compounds with RNA binding activity can be tested for their ability to modulate miRNA levels and thus for drug repositioning. Aminoglycoside antibiotics are highly effective microbicidal RNA binding molecules that bind to prokaryotic rRNA secondary structures. Here, we report that specific subsets of miRNA can be modulated by aminoglycosides. However, ototoxicity (cochlear and vestibular) and nephrotoxicity of multiple origins resulting from prolonged use are a well-known disadvantage of aminoglycosides. Mature non-coding RNAs and their precursors can present off-target sites, by forming secondary structures that resemble ribosomal RNA, thus providing an additional molecular basis for the toxicity of aminoglycosides. Using in vitro, in cellulae and physiological responses, we provide evidence for the direct functional perturbation of the miR- 96 cluster leading to selective cell death in neuromasts- the zebrafish equivalent of cochlear hair cells, by Streptomycin, a prototype aminoglycoside antibiotic, thus contributing to the observed ototoxicity. Our observations, collectively underscore the importance of re- evaluating RNA binding drugs for their off-targeting effects in the context of miRNA and other functional non-coding RNA.

cell biology↗