Biology subjects

Alam, S.

Publications and source records attributed to Alam, S..

At least 19 recordsLinked to original sources

bioRxiv2026

GEO-BERT: A Retrieval augmented LLM Framework for Intelligent Dataset Retrieval from the Gene Expression Omnibus

The Gene Expression Omnibus (GEO) is a commonly used data repository of high throughput gene expression and other functional genomics data. This platform is globally used by researchers for submitting experimental datasets, accessing these datasets for hypothesis validation, reusing them in data-driven research, generating ideas for new research directions. Despite global usage, searching and identifying relevant and usable datasets in GEO remains challenging with an underlying keyword based search system needing manual intervention for the retrieval of desired datasets. GEO's inconsistent metadata and varying data annotation practices across submissions, variable data quality are contributing to its challenge for building structured query tools. With the recent advancement of Large Language Models (LLMs), it is timely to revisit the GEO datasets and its query system. Here, we present an AI chat-bot, GEO-BERT, leveraging an underlying retrieval augmented generator framework on the GEO metadata with a novel hierarchical hybrid field aware retrieval system to mine the datasets. GEO-BERT is locally installable, available for programmers and project developers (https://github.com/mrumi/GEOBERTtest) and readily usable by regular users on subset of test data. We demonstrated that GEO-BERT enhances the semantic search performance not currently available by any of the leading existing AI-powered chatbots, while improving the performance of GEO's traditional search performance for project development. Given the global usage of GEO and the latest advancement of the large language models, GEO-BERT fills the gap for context-aware extraction of GEO datasets.

bioinformatics↗

bioRxiv2026

A meta-interaction basis for cell-cell communication in tissues

Tissue function depends on signals exchanged between cells and the responses they elicit. Yet whether diverse cell-cell interactions in situ form recurrent sender-receiver programs remains unclear. We present SpiderNet, an interpretable representation-learning framework that discovers such directed programs as a compact basis of cell-cell meta-interactions (MIs) from spatial transcriptomics. SpiderNet jointly learns which sender regulators, ligand-receptor pairs, and receiver targets define each MI and where each program is active across neighboring cell pairs. The resulting representation traces multicellular relays and links communication to cell states, perturbation responses, and phenotypes. SpiderNet recovers ground-truth MIs and their molecular components in simulations and, in real tissues, shows stronger direction-specific agreement with independently curated regulatory programs in senders and receivers than alternative methods. Across more than 5.8 million spatially profiled cells, SpiderNet resolves an SPP1-THBS relay linking monocytes, fibroblasts, and tumor cells within an immune-suppressive ovarian cancer niche, predicts T-cell responses to held-out melanoma-cell perturbations, and identifies a T-cell-associated brain-aging program and age-predictive signals that transfer across regions and platforms. It reveals a recurrent pan-cancer COLLAGEN-linked fibroblast-tumor program whose projected abundance in independent cohorts is associated with poorer survival and non-response to immunotherapy. SpiderNet thus establishes MIs as a reusable organizational layer between molecular interactions and tissue phenotypes, providing a framework to resolve, compare, trace, and perturb multicellular regulation in situ.

bioinformatics↗

bioRxiv2026

Integron-Mediated Convergence of Carbapenemase and Disinfectant Resistance in Acinetobacter spp. from Critical Care Units

Acinetobacter spp. represents critical opportunistic pathogens driving severe bloodstream infections (BSIs) in intensive care unit (ICU) and neonatal intensive care unit (NICU) settings. The convergence of carbapenem resistance and emerging biocide tolerance, often mediated by mobile genetic elements, has intensified concerns regarding co-selection and persistence in clinical environments. A total of 90 molecularly confirmed Acinetobacter isolates (ICU = 44; NICU = 46) from bloodstream infections were analyzed. Antimicrobial susceptibility was determined using the Kirby-Bauer disk diffusion method in accordance with CLSI M100 (2024) guidelines and extended-spectrum {beta}-lactamase production was assessed by combined disc diffusion. Polymerase chain reaction (PCR) was employed to detect carbapenemase genes (blaVIM, blaNDM, blaIMP, blaOXA-23, blaOXA-58), biocide resistance determinants (qacE, qac{Delta}E1), and the class 1 integron-integrase gene (intI1). Multidrug-resistant (MDR) and extensively drug-resistant (XDR) phenotypes were identified in 71.1% (64/90) and 22.2% (20/90) of isolates, respectively. High resistance (>71%) was observed against meropenem and cephalosporins, whereas colistin (58.8%, 53/90) and amikacin (47.8%, 43/90) showed moderate susceptibility. The most prevalent genotypes were qac{Delta}E1 (76.6%, 69/90) and blaVIM (56.6%, 51/90). Statistical and network analyses revealed significant correlations between biocide and carbapenemase genes, identifying IntI1 as a primary driver of co-resistance. The findings indicate a significant co-occurrence of carbapenemase genes, biocide resistance determinants, and class 1 integrons among Acinobacter spp. isolates. These associations suggest that mobile genetic elements may contribute to the dissemination of resistance traits.

microbiology↗

bioRxiv2026

Ribosome biogenesis bottlenecks reveal vulnerabilities in cancer

Cell growth requires elevated protein synthesis, which depends on the production of ribosomes. Ribosome biogenesis is a complex, multi-step pathway in which newly transcribed precursor ribosomal RNA (rRNA) undergoes coordinated processing and assembly in the nucleolus to produce the small and large ribosomal subunits (SSU and LSU).1-3 Oncogene activation stimulates rRNA transcription and processing, giving rise to enlarged nucleoli that produce thousands of ribosomes every minute.4,5 However, efficient ribosome production requires tight coordination across numerous maturation steps, and it remains unclear if elevated rDNA transcription is proportionally converted into mature ribosomes, or whether imperfect coordination constrains the output yield. Here, we quantify pre-rRNA transcription (input) and compare it with newly-assembled cytoplasmic ribosomes (output), revealing that oncogene activation reduces the efficiency of ribosome production. Using a quantitative pulse-chase sequencing approach with mathematical modeling to resolve rRNA maturation kinetics, we found that oncogene activation creates late-stage processing bottlenecks, characterized by delayed precursor maturation and increased degradation. Perturbation of late-stage ribosome biogenesis factors preferentially impaired oncogene-driven cell growth, and limited tumor growth in mouse models, suggesting that these bottlenecks represent selective vulnerabilities in cancer, created by imbalanced biosynthetic flux. Together, these findings reveal that oncogene-driven ribosome production is imperfectly coordinated across maturation steps, and suggest that capacity limits in multi-step assembly pathways may be therapeutically exploitable in cancer and other diseases.

cell biology↗

bioRxiv2026

Successful gene editing in tetraploid alfalfa using the open-source, AI-derived OpenCRISPR-1

While CRISPR/Cas-based gene editing technologies have the potential to greatly advance crop breeding endeavours, intellectual property-related challenges can hinder the ability to move such varieties to the market. Recently, an open-access Cas enzyme derived from large language models (OpenCRISPR-1) was developed and shown to function effectively in human cells. In this study, we demonstrate the successful use of this nuclease in a polyploid plant species (Medicago sativa), with mono- or bi-allelic editing observed in 30% of genotypes bearing OpenCRISPR-1. These findings indicate that OpenCRISPR-1 holds promise to expand the use of gene editing technology in the breeding of polyploid crops.

plant biology↗

bioRxiv2026

Fmr1 mutation reshapes gut microbiome structure, diversity, intestinal barrier integrity, and function in a sex and genotype-dependent manner

Fragile X Syndrome (FXS) is the leading monogenic cause of autism spectrum disorder (ASD), caused by mutation in the Fmr1 gene. In addition to cognitive and behavioral challenges, FXS patients often experience altered gut microbiome-induced gastrointestinal (GI) problems. Evidence suggests that an altered gut microbiome can disrupt mucosal barrier and promote inflammation, leading to impaired intestinal barrier integrity and function. However, the mechanisms by which the gut microbiome alters the barrier integrity and contributes to GI pathology in FXS remain poorly understood. To address this gap, we tested the hypothesis that Fmr1 mutation reshapes the gut microbiome composition, altering transcriptional markers of intestinal barrier regulation and gut barrier physiology in mice. To test our hypothesis, we used an Fmr1 knockout (KO) mouse model with wild-type (WT) littermates as controls. First, we performed 16S ribosomal RNA sequencing to characterize gut microbial community structure and diversity across genotypes and sexes. Among the alpha-diversity metrics, only Chao1 showed significant differences across female genotypes in both fecal and cecal contents. Additionally, qRT-PCR analysis of ileal samples revealed reduced barrier and mucosal defense gene expression in female KO and Het mice compared with WT females. Next, we utilized a Ussing chamber assay to test gut epithelial permeability and function. Our physiological data showed that female Het mice had increased transepithelial resistance compared to WT females, indicating a tighter epithelial barrier. Overall, FXS is associated with modest genotype and sex-specific microbiome variation, impaired gut barrier integrity, and altered epithelial barrier function in female mice.

microbiology↗

bioRxiv2026

Loss of Fmr1 reorganizes the multi-elemental composition of neural and somatic tissues in Fragile X mice

Fragile X Syndrome (FXS) results from a genetic mutation which silences the expression of Fragile X Messenger Ribonucleoprotein (FMRP). FMRP serves various roles regulating cellular protein synthesis including mRNAs that code for proteins regulating ion flux. However, there are few studies measuring the elemental balance between FXS genotypes and tissues. Here, we measured the multivariate balance of 10 elements in tissues of wild-type and Fmr1-knockout mice to compare elemental composition of brain and somatic tissues within and across genotypes. Using a Bayesian mixed model approach, we found that the main differences between groups were between tissues, with significant effects of genotype and interaction of tissue on genotype. Wild-type feces were significantly higher in magnesium and sodium than knockout. Fur was significantly higher in potassium in wild-type, which was supported by the interaction effect of genotype with tissue. These results align with previous work showing FXS pathologies alter electrolytic and metal ion regulation, neuronal excitability, and gastrointestinal function. Future work should additionally test how elemental differences relate to function at the cellular level, as well as patterns of individual intake, digestion, assimilation, and/or excretion.

neuroscience↗

bioRxiv2025

A neuron type-specific microexon in Ank3/ankyrin-G modulates calcium activity and neuronal excitability

Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but included in cortical GABAergic neurons and cerebellar neurons, which is dictated by multiple neuronal splicing factors. In E35a-deletion mice we generated, interneurons show increased excitability and somatic Ca2+ activity, without disruption in AIS. Biochemical analyses suggest that E35a inclusion facilitates AnkG interaction with a protein complex involving inositol trisphosphate receptors (InsP3Rs) important for intracellular Ca2+ signaling. Alternative splicing therefore allows AnkG to modulate neuron type-specific excitability in addition to its ubiquitous pan-neuronal role in organizing the AIS.

neuroscience↗

bioRxiv2025

A modular lentiviral system for multiplexed gene perturbation and functional analysis reveals interdependence of hormone receptors in breast cancer growth in vivo

Precise and flexible control of gene expression is essential for dissecting gene function in complex biological systems. Although recent developments in genetic engineering and CRISPR/Cas9 technology have expanded tools for gene activation, suppression and editing, their application in physiologically relevant models remains challenging, time consuming, and expensive. Here, we present a modular, doxycycline-inducible vector system that integrates gene overexpression, shRNA-mediated knockdown, and CRISPR/Cas9-mediated regulation within a single, lentivirus-compatible system. The modular design allows rapid exchange of selection markers, epitope tags, and reporters via Gateway cloning, providing broad adaptability across experimental settings. In addition to standard fluorescent and luminescent reporters, the system includes advanced sensors, such as Fucci cell cycle reporters, to enable monitoring of cellular processes. By combining fluorescence barcoding with combinatorial genetic perturbations, the platform supports multiplexed analysis of gene function and genetic interactions through phenotypic characterization by fluorescence imaging or flow cytometry. We demonstrate its utility in vivo with breast cancer intraductal xenografts, which suggest that ER+ breast cancer cells (MCF7) rely on androgen (AR), estrogen (ER) and progesterone receptors (PR) for in vivo growth. This versatile gene perturbation system provides tight temporal control, streamlined implementation, and high-content phenotyping capacity facilitating efficient in vitro and in vivo studies while reducing the use of animals in in vivo validation experiments. It thus expands the experimental repertoire for dynamic, multigene interrogation in complex systems.

molecular biology↗

bioRxiv2025

NUDT5 regulates the global efficacy of nucleoside analog drugs by coordinating purine synthesis and PRPP allocation

Cancer cells require nucleotides to meet proliferation demands, which has been effectively exploited by nucleoside analog (NA) drugs. Despite their success, we still do not completely understand factors dictating their anticancer effect. Here, we report that a NUDT5 scaffolding function indirectly regulates the global efficacy of anti-cancer NA drugs. Mechanistically, PROTAC- or RNAi-mediated NUDT5 depletion, not inhibition, desensitizes cells to 6-thioguanine (6TG) and other NA drugs proportionally with NUDT5 abundance. NUDT5-depleted cells appear locked into de novo purine biosynthesis (DNPB), thus impairing salvage of nucleotide precursors and NA drug activation. Specifically, NUDT5 interacts with phosphoribosyl amidotransferase (PPAT), the rate-limiting DNPB enzyme, to putatively regulate its activity, DNPB flux, and resultant phosphoribosyl pyrophosphate (PRPP) allocation. Collectively, these results suggest that NUDT5 controls NA drug efficacy by coordinating nucleotide synthesis and PRPP utilization, making it a potential biomarker of clinical efficacy and target for finetuning antimetabolite therapies. Statement of significanceNUDT5 non-enzymatically regulates the global efficacy of anti-cancer nucleoside analog drugs by interacting with PPAT to control DNPB and allocation of PRPP, thereby establishing it as a biomarker of clinical significance.

biochemistry↗

bioRxiv2025

HEIMDALL: A Modular Framework for Tokenization in Single-Cell Foundation Models

Foundation models for single-cell RNA-sequencing (scRNA-seq) data are emerging as powerful tools for single-cell analysis, yet their performance depends critically on how cells are tokenized into model inputs. Single-cell data lack a canonical tokenization scheme, and many design choices in current single-cell foundation models (scFMs) remain heuristic, entangled, and difficult to evaluate. Here, we introduce HO_SCPLOWEIMDALLC_SCPLOW, a unified framework for dissecting and redesigning tokenizers in scFMs. By decomposing existing tokenization strategies into individual design choices, HO_SCPLOWEIMDALLC_SCPLOW enables attribution of the components that underlie robust generalization, allowing more principled design of improved tokenizers. Combining HO_SCPLOWEIMDALLC_SCPLOW with a minimal transformer backbone, we find that tokenizer design is instrumental for generalization in challenging distribution-shift settings such as cross-tissue, cross-species, and cross-gene-panel cell type classification, as well as reverse perturbation prediction. We show that, while tokenizer choice has little effect in scenarios with matched train and test data, it becomes imperative under distribution shift. Rather than identifying a single globally optimal tokenizer, HO_SCPLOWEIMDALLC_SCPLOW reveals that robust transfer depends on a small number of tokenization design axes - especially gene identity, expression encoding, and ordering - that expose different biological priors to the model. In this sense, universal transferability in scFMs still depends on a non-universal tokenizer interface. Together, these findings establish tokenization as a critical design axis in scFMs and provide design principles and reusable infrastructure for more robust scFMs.

bioinformatics↗

bioRxiv2025

Reversible therapeutic resistance in EGFR mutant lung cancer caused by RB1 loss induced lineage plasticity

Treatment with EGFR tyrosine kinase inhibitors (EGFR TKI) is standard of care for patients with lung cancer initiated by activating mutations in the EGFR gene. While EGFR TKI treatment is effective, virtually all patients progress on therapy. Loss of function mutations in the RB1 tumor suppressor gene are associated with poor EGFR TKI outcomes, but underlying mechanisms remain unclear. Analysis of genetically engineered experimental models and human clinical specimens described here demonstrates RB1 loss has minimal impact on initial cell cycle responses to EGFR TKI but markedly accelerates acquired resistance over time. RB1 loss promotes transcriptional plasticity that enables rare cells to spontaneously assume reversible, resistance-conferring lineage states. These findings highlight the importance of non-genetic adaptations in driving acquired EGFR TKI resistance, advance our understanding of how RB1 impacts cancer, and identify opportunities to improve EGFR TKI treatment outcomes by targeting reversible epigenetic resistance mechanisms.

cancer biology↗

bioRxiv2025

A cell-based degrader assessment platform facilitates discovery of functional NUDT5 PROTACs

Targeted protein degradation (TPD) via PROTACs and molecular glues holds significant therapeutic promise but demands detailed mechanistic evaluation in live cells to fully understand compound behavior and optimize efficacy. Here, we present an integrated, cell-first platform that combines a modular degradation assay with E3 ligase target engagement readouts for comprehensive assessment of TPD molecules in cells and use it to evaluate PROTACs towards NUDT5. To mimic endogenous degradation conditions and TPD amenability, we established a fusion protein expression system consisting of a lysine-free FKBP12 F36V PROTAC handle (FKBPVK0) and used a HiBiT/akaLuc dual luciferase reporter to accurately measure degradation dynamics. This set-up identified a VHL-dependent NUDT5 PROTAC, DDD2, that induced robust NUDT5 degradation, despite impaired NUDT5 binding in vitro and in cellulo, but no CRBN-dependent degraders. NUDT5 lysine availability mapping with DDD2 and FKBP12 F36V-directed PROTACs suggested that the CRL4CRBN complex is more sensitive to target lysine accessibility than CRL2VHL, which may have implications for E3 ligase choice and therapeutic resistance. CeTEAM drug biosensors were also established towards CRBN and VHL to quantitatively monitor degrader engagement in living cells and confirmed that the tested CRBN-directed NUDT5 PROTACs poorly engaged the E3. All together, this platform provides a versatile and scalable framework for TPD molecule discovery in a cellular context.

biochemistry↗

bioRxiv2025

POMT-dependent O-mannosylation regulates integrin β1 maturation via N-glycan processing

Withdrawal StatementThe authors have withdrawn this manuscript because following a careful internal review conducted in the course of manuscript revision, the authors identified that parts of the presented data could not be reliably traced back to the corresponding original datasets in their current form. While the general biological interpretation is not necessarily invalidated by this finding, the integrity of the present data representation no longer meets the standard required for publication. For this reason, the authors have decided to withdraw this preprint and will refrain from pursuing publication until the underlying data situation has been fully clarified and, if possible, reconstructed. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The authors regret any inconvenience caused to the scientific community.

cell biology↗

bioRxiv2025

Uridine cytidine kinases dictate the therapeutic response of molnupiravir via its bioactivation

Molnupiravir is a nucleoside analogue antiviral drug with activities against a broad spectrum of RNA viruses, including its clinical indication SARS-CoV-2. Whilst its antiviral mechanism-of-action is well defined, host pharmacogenetic factors that regulate its therapeutic responses - efficacy as well as selectivity - have not been mechanistically deciphered and characterized. Here we identified that uridine cytidine kinase (UCK), the first and rate-limiting kinase of the pyrimidine salvage pathway, could effectively phosphorylate N4-hydroxycytidine (NHC), the active compound of molnupiravir, and thereby dictate its anti-SARS-CoV-2 efficacy, as well as its selectivity index. Using target engagement and enzyme kinetic assays, we demonstrate that recombinant UCK isoform 1 (UCK1) and 2 (UCK2) effectively bind and phosphorylate NHC, with UCK2 displaying 9-fold higher catalytic efficiency. Accordingly, in SARS-CoV-2-infected cells, downregulation of UCK2 via siRNA hampered the intracellular accumulation of the triphosphate antiviral metabolite of NHC, resulting in 10-fold reduction of antiviral efficacy. Furthermore, we could recapitulate this using a pan-UCK small molecule inhibitor. Critically, both UCK downregulation and inhibition significantly reduced the selectivity index of molnupiravir/NHC. Altogether, this work underscores the pivotal roles of UCK enzymes in upholding molnupiravir efficacy and therapeutic window, and furthermore, highlights their potential as pharmacologically tractable targets for tailoring the therapeutic response to this antiviral agent.

microbiology↗

bioRxiv2025

POPARI: Modeling multisample variation in spatial transcriptomics

Integrating spatially-resolved transcriptomics (SRT) across biological samples is essential for understanding dynamic changes in tissue architecture and cell-cell interactions in situ. While tools exist for multisample single-cell RNA-seq, methods tailored to multisample SRT remain limited. Here, we introduce PO_SCPLOWOPARIC_SCPLOW, a probabilistic graphical model for factor-based decomposition of multisample SRT that captures condition-specific changes in spatial organization. PO_SCPLOWOPARIC_SCPLOW jointly learns spatial metagenes - linear gene expression programs - and their spatial affinities across samples. Its key innovations include a differential prior to regularize spatial accordance and spatial downsampling to enable multiresolution, hierarchical analysis. Simulations show PO_SCPLOWOPARIC_SCPLOW outperforms existing methods on multisample and multi-resolution spatial metrics. Applications to real datasets uncover spatial metagene dynamics, spatial accordance, and cell identities. In mouse brain (STARmap PLUS), PO_SCPLOWOPARIC_SCPLOW identifies spatial metagenes linked to AD; in thymus (Slide-TCR-seq), it captures increasing colocalization of V(D)J recombination and T cell proliferation; and in ovarian cancer (CosMx), it reveals sample-specific malignant-immune interactions. Overall, PO_SCPLOWOPARIC_SCPLOW provides a general, interpretable framework for analyzing variation in multisample SRT.

bioinformatics↗

bioRxiv2025

Identification of new interactors of eIF3f by endogenous proximity-dependent biotin labelling in human muscle cells.

Regulation of protein synthesis is central to maintaining skeletal muscle integrity and its understanding is important for the treatment of muscular and neuromuscular pathologies. The eIF3f subunit of the translation initiation factor eIF3 has a key role, as it stands at the crossroad between protein-synthesis-associated hypertrophy and MAFbx/atrogin-1-dependent. To decipher the molecular mechanisms underpinning the role of eIF3f in regulating muscle mass, we established a cellular model that enables interrogation of eIF3f functionality via identification of proximal interactors. Using CRISPR-Cas9 molecular scissors, we generated single cell clones of immortalised human muscle cells expressing eIF3f fused to the BirA biotin ligase (eIF3f-BioID1 chimera) from the endogenous EIF3F locus. Biotinylated proteins, representing interactors of eIF3f in nanometer range distance, were identified by streptavidin pull-downs and mass spectrometry. In both proliferating and differentiated muscle cells, the eIF3f-BioID1 chimera co-sedimented with ribosomal complexes in polysome profiles and interacted mainly with components of the eIF3 complex, and with the eIF4E, eIF4G, and eIF5 initiation factors. Surprisingly, we identified several nucleus-localised interactors of eIF3f, and the immunofluorescence analyses revealed a previously unknown nuclear localization of eIF3f in both myoblasts and myotubes. We also identified novel cytoplasmic partners of eIF3f, responsible for the maintenance of skeletal muscle ultrastructure (sarcomeric/Z-disc (SYNPO2) bound proteins) and proteins of the lysosomal compartment (LAMP1). The established tagging system should be useful to further advance studies of eIF3f function in hypertrophic and atrophic conditions in skeletal muscle.

molecular biology↗

bioRxiv2025

Inhibition of adenylyl cyclase 1 (AC1) and exchange protein directly activated by cAMP (EPAC) restores ATP-sensitive potassium (KATP) channel activity after chronic opioid exposure

Prolonged exposure to Gi/o receptor agonists such as opioids can lead to a sensitization of adenylyl cyclases (ACs), resulting in heterologous sensitization or cyclic AMP (cAMP) overshoot. The molecular consequences of cAMP overshoot are not well understood, but this adaptive response is suggested to play a critical role in the development of opioid tolerance and withdrawal. We found that genetic reduction of AC1 and simultaneous upregulation of ATP-sensitive potassium (KATP) channel subunits, SUR1 or Kir6.2, significantly attenuated morphine tolerance and reduced naloxone-precipitated withdrawal. In vitro models utilized an EPAC2-GFP-cAMP biosensor to investigate sensitization of adenylyl cyclase in SH-SY5Y neuroblastoma cells and HEK{Delta}AC3/6 knockout cells. Acute application of DAMGO significantly decreased the cAMP signal from the EPAC2-GFP-cAMP biosensor, while chronic DAMGO administration resulted in enhanced cAMP production following AC stimulation. Inhibition of cAMP overshoot was observed with naloxone (NAL), pertussis toxin (PTX), and the neddylation inhibitor, MLN4924 (Pevonedistat), as well as co-expression of {beta}-adrenergic receptor kinase C-terminus ({beta}ARK-CT). After establishment of the AC1-EPAC sensitization in the in vitro models, we found that inhibition of AC1 or EPAC enhanced potassium channel activity after chronic morphine treatment, using a thallium-based assay in SH-SY5Y cells. Similar data were obtained in mouse dorsal root ganglia (DRG) after chronic morphine treatment. This study presents evidence for investigating further AC1 signaling as a target for opioid tolerance and withdrawal, by increasing EPAC activity and affecting potassium channels downstream of opioid receptors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/636278v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@185bb82org.highwire.dtl.DTLVardef@d97ea4org.highwire.dtl.DTLVardef@1097addorg.highwire.dtl.DTLVardef@1616fcf_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

neuroscience↗