N-alkylamides from Litsea cubeba (Lour.) Pers. with potential anti-inflammatory activity.

Six amides, including a new N-alkylamide (1), four known N-alkylamides (2-5) and one nicotinamide (6) were isolated from Litsea cubeba (Lour.) Pers., which is a pioneer herb traditionally utilized in medicine. Their structures were elucidated on the basis of 1D and 2D NMR experiments and by comparison of their spectroscopic and physical data with the literature values. Cubebamide (1) is a new cinnamoyltyraminealkylamide and possessed obvious anti-inflammatory activity against NO production with IC50 values of 18.45 µM. Further in-depth pharmacophore-based virtual screening and molecular docking were carried out to reveal the binding mode of the active compound inside the 5-LOX enzyme. The results indicate that L. cubeba, and the isolated amides might be useful in the development of lead compounds for the prevention of inflammatory diseases.

Single-nucleus transcriptional profiling uncovers the reprogrammed metabolism of astrocytes in Alzheimer's disease.

Astrocytes play an important role in the pathogenesis of Alzheimer's disease (AD). It is widely involved in energy metabolism in the brain by providing nutritional and metabolic support to neurons; however, the alteration in the metabolism of astrocytes in AD remains unknown. Through integrative analysis of single-nucleus sequencing datasets, we revealed metabolic changes in various cell types in the prefrontal cortex of patients with AD. We found the depletion of some important metabolites (acetyl-coenzyme A, aspartate, pyruvate, 2-oxoglutarate, glutamine, and others), as well as the inhibition of some metabolic fluxes (glycolysis and tricarbocylic acid cycle, glutamate metabolism) in astrocytes of AD. The abnormality of glutamate metabolism in astrocytes is unique and important. Downregulation of GLUL (GS) and GLUD1 (GDH) may be the cause of glutamate alterations in astrocytes in AD. These results provide a basis for understanding the characteristic changes in astrocytes in AD and provide ideas for the study of AD pathogenesis.

Cysteine carboxyethylation generates neoantigens to induce HLA-restricted autoimmunity.

Autoimmune diseases such as ankylosing spondylitis (AS) can be driven by emerging neoantigens that disrupt immune tolerance. Here, we developed a workflow to profile posttranslational modifications involved in neoantigen formation. Using mass spectrometry, we identified a panel of cysteine residues differentially modified by carboxyethylation that required 3-hydroxypropionic acid to generate neoantigens in patients with AS. The lysosomal degradation of integrin αIIb [ITGA2B (CD41)] carboxyethylated at Cys96 (ITGA2B-ceC96) generated carboxyethylated peptides that were presented by HLA-DRB1*04 to stimulate CD4+ T cell responses and induce autoantibody production. Immunization of HLA-DR4 transgenic mice with the ITGA2B-ceC96 peptide promoted colitis and vertebral bone erosion. Thus, metabolite-induced cysteine carboxyethylation can give rise to pathogenic neoantigens that lead to autoreactive CD4+ T cell responses and autoantibody production in autoimmune diseases.

GGC repeat expansion in NOTCH2NLC induces dysfunction in ribosome biogenesis and translation.

GGC repeat expansion in the 5' untranslated region (UTR) of NOTCH2NLC is associated with a broad spectrum of neurological disorders, especially neuronal intranuclear inclusion disease (NIID). Studies have found that GGC repeat expansion in NOTCH2NLC induces the formation of polyglycine (polyG)-containing protein, which is involved in the formation of neuronal intranuclear inclusions. However, the mechanism of neurotoxicity induced by NOTCH2NLC GGC repeats is unclear. Here, we used NIID patient-specific induced pluripotent stem cell (iPSC)-derived 3D cerebral organoids (3DCOs) and cellular models to investigate the pathophysiological mechanisms of NOTCH2NLC GGC repeat expansion. IPSC-derived 3DCOs and cellular models showed the deposition of polyG-containing intranuclear inclusions. The NOTCH2NLC GGC repeats could induce the upregulation of autophagic flux, enhance integrated stress response and activate EIF2α phosphorylation. Bulk RNA sequencing for iPSC-derived neurons and single-cell RNA sequencing (scRNA-seq) for iPSC-derived 3DCOs revealed that NOTCH2NLC GGC repeats may be associated with dysfunctions in ribosome biogenesis and translation. Moreover, NOTCH2NLC GGC repeats could induce the NPM1 nucleoplasm translocation, increase nucleolar stress, impair ribosome biogenesis and induce ribosomal RNA sequestration, suggesting dysfunction of membraneless organelles in the NIID cellular model. Dysfunctions in ribosome biogenesis and phosphorylated EIF2α and the resulting increase in the formation of G3BP1-positive stress granules may together lead to whole-cell translational inhibition, which may eventually cause cell death. Interestingly, scRNA-seq revealed that NOTCH2NLC GGC repeats may be associated with a significantly decreased proportion of immature neurons while 3DCOs were developing. Together, our results underscore the value of patient-specific iPSC-derived 3DCOs in investigating the mechanisms of polyG diseases, especially those caused by repeats in human-specific genes.

Heterodimers with a cucurbitane-type triterpenoid skeleton from the branches of Elaeocarpus dubius.

Four undescribed and two known cucurbitane-type triterpenoids, including two heterodimers, elaeocarpudubins A and B, were isolated from the branches of Elaeocarpus dubius (Elaeocarpaceae). The chemical structures of these undescribed isolates were determined by analyses of 1D and 2D NMR and MS data, electronic circular dichroism (ECD) calculations, and chemical transformation. Biogenetically, elaeocarpudubins A and B might be derived from cucurbitacin F through Michael addition with vitamin C and (-)-catechin, respectively. These six isolates were evaluated for their cytotoxic activities against human leukemia HL-60, human lung adenocarcinoma A549, human hepatoma SMMC-7721, human breast cancer MCF-7, human colon cancer SW480, and paclitaxel-resistant A549 (A549/Taxol) cell lines, for their antioxidant properties using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and for their differentiation effects on nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells. Cucurbitacins F (IC50 of 4.98-38.11 µM) and D (IC50 of 0.03-4.40 µM) showed growth-inhibitory activities against these six cancer cell lines. Elaeocarpudubin B (IC50 of 61.04 µM) and elaeocarpudoside B (IC50 of 6.93 µM) showed antioxidant activities. Elaeocarpudubin B and elaeocarpudoside B also showed neurite outgrowth-promoting activities in PC12 cells at a concentration of 10 µM.

Association Between the Fatigue and Sleep Quality of Kidney Transplant Recipients: The Mediating Role of Rumination.

ABSTRACT: Sleep disorders persist in renal transplant patients. Previous studies have showed that fatigue and rumination are an important determinant of sleep quality. However, very few studies have explored the mediating role of rumination in the relationship between fatigue and sleep quality in kidney transplant recipients. A descriptive cross-sectional research design was implemented, and 192 kidney transplant patients completed the short questionnaire about their recent experiences of fatigue, rumination, and sleep quality. The prevalence of sleep disorders among kidney transplant recipients was 19.3%. With rumination as a partial mediator, fatigue indirectly affected the patients' sleep quality. This indirect effect was 0.10 (95% confidence interval, 0.154-0.419). Our results indicate that the incidence of sleep disorders after renal transplantation was high, and the more tired kidney transplant recipients become, the more likely they are to ruminate, which leads to a decline in sleep quality.

Differential Post-Translational Modifications of Proteins in Bladder Ischemia.

Clinical and basic research suggests that bladder ischemia may be an independent variable in the development of lower urinary tract symptoms (LUTS). We have reported that ischemic changes in the bladder involve differential expression and post-translational modifications (PTMs) of the protein's functional domains. In the present study, we performed in-depth analysis of a previously reported proteomic dataset to further characterize proteins PTMs in bladder ischemia. Our proteomic analysis of proteins in bladder ischemia detected differential formation of non-coded amino acids (ncAAs) that might have resulted from PTMs. In-depth analysis revealed that three groups of proteins in the bladder proteome, including contractile proteins and their associated proteins, stress response proteins, and cell signaling-related proteins, are conspicuously impacted by ischemia. Differential PTMs of proteins by ischemia seemed to affect important signaling pathways in the bladder and provoke critical changes in the post-translational structural integrity of the stress response, contractile, and cell signaling-related proteins. Our data suggest that differential PTMs of proteins may play a role in the development of cellular stress, sensitization of smooth muscle cells to contractile stimuli, and deferential cell signaling in bladder ischemia. These observations may provide the foundation for future research to validate and define clinical translation of the modified biomarkers for precise diagnosis of bladder dysfunction and the development of new therapeutic targets against LUTS.

Chromosome-level genome assembly of bunching onion illuminates genome evolution and flavor formation in Allium crops.

The Allium genus is cultivated globally as vegetables, condiments, or medicinal plants and is characterized by large genomes and strong pungency. However, the genome evolution and genomic basis underlying their unique flavor formation remain poorly understood. Herein, we report an 11.27-Gb chromosome-scale genome assembly for bunching onion (A. fistulosum). The uneven bursts of long-terminal repeats contribute to diversity in genome constituents, and dispersed duplication events largely account for gene expansion in Allium genomes. The extensive duplication and differentiation of alliinase and lachrymatory factor synthase manifest as important evolutionary events during flavor formation in Allium crops. Furthermore, differential selective preference for flavor-related genes likely lead to the variations in isoalliin content in bunching onions. Moreover, we reveal that China is the origin and domestication center for bunching onions. Our findings provide insights into Allium genome evolution, flavor formation and domestication history and enable future genome-assisted breeding of important traits in these crops.

Exosome biogenesis: machinery, regulation, and therapeutic implications in cancer.

Exosomes are well-known key mediators of intercellular communication and contribute to various physiological and pathological processes. Their biogenesis involves four key steps, including cargo sorting, MVB formation and maturation, transport of MVBs, and MVB fusion with the plasma membrane. Each process is modulated through the competition or coordination of multiple mechanisms, whereby diverse repertoires of molecular cargos are sorted into distinct subpopulations of exosomes, resulting in the high heterogeneity of exosomes. Intriguingly, cancer cells exploit various strategies, such as aberrant gene expression, posttranslational modifications, and altered signaling pathways, to regulate the biogenesis, composition, and eventually functions of exosomes to promote cancer progression. Therefore, exosome biogenesis-targeted therapy is being actively explored. In this review, we systematically summarize recent progress in understanding the machinery of exosome biogenesis and how it is regulated in the context of cancer. In particular, we highlight pharmacological targeting of exosome biogenesis as a promising cancer therapeutic strategy.

Formation of Double Stranded RNA Provokes Smooth Muscle Contractions and Structural Modifications in Bladder Ischemia.

Purpose: Growing evidence suggests that ischemia provokes detrusor overactivity and degenerative responses in the bladder. Underlying mechanisms appear to involve modification of smooth muscle contractile rudiments by hypoxia, redox, cellular stress and cell survival signaling. Downstream pathways of cellular stress and stress response molecules eliciting bladder dysfunction in ischemia remain largely elusive. Our goal was to define the role of double stranded RNA (dsRNA), a stress response molecule provoked by redox, in ischemia mediated bladder dysfunction. Methods: A rat model of pelvic ischemia along with a cell culture hypoxia model were used to investigate the expression levels, functional consequences, structural aspects, and regulatory mechanisms of dsRNA in the bladder. Gene and protein expression were examined by reverse transcription polymerase chain reaction (RT-PCR), dot blot, and Western blotting, respectively. Tissue structure and function were assessed using histological staining and organ bath. Regulatory mechanisms were analyzed in cultured bladder smooth muscle cells. Results: The data presented here provide the first evidence of the formation of dsRNA in the overactive bladder. dsRNA is a cellular stress response molecule that sensitizes smooth muscle and regulates inflammatory and degenerative rejoinders. Our data suggest that the production of dsRNA in the bladder is provoked by ischemia. Formation of dsRNA appears to augment bladder smooth muscle contractions and provoke fibrotic and apoptotic responses. Downstream actions of dsRNA in the bladder may involve upregulation of dsRNA-activated protein kinase R (PKR) and caspase-3, the executioner of apoptosis. Conclusion: Activation of dsRNA/PKR pathway may play a role in sensitization of bladder smooth muscle cells to contractile stimuli, whereas dsRNA and caspase-3 crosstalk appear to modulate cellular stress and instigate degenerative responses in bladder ischemia. These observations suggest the role of dsRNA in bladder dysfunction and may open new perspectives to overcome overactive smooth muscle contractions and structural damage in the bladder.

Robust Corrupted Data Recovery and Clustering via Generalized Transformed Tensor Low-Rank Representation.

Tensor analysis has received widespread attention in high-dimensional data learning. Unfortunately, the tensor data are often accompanied by arbitrary signal corruptions, including missing entries and sparse noise. How to recover the characteristics of the corrupted tensor data and make it compatible with the downstream clustering task remains a challenging problem. In this article, we study a generalized transformed tensor low-rank representation (TTLRR) model for simultaneously recovering and clustering the corrupted tensor data. The core idea is to find the latent low-rank tensor structure from the corrupted measurements using the transformed tensor singular value decomposition (SVD). Theoretically, we prove that TTLRR can recover the clean tensor data with a high probability guarantee under mild conditions. Furthermore, by using the transform adaptively learning from the data itself, the proposed TTLRR model can approximately represent and exploit the intrinsic subspace and seek out the cluster structure of the tensor data precisely. An effective algorithm is designed to solve the proposed model under the alternating direction method of multipliers (ADMMs) algorithm framework. The effectiveness and superiority of the proposed method against the compared methods are showcased over different tasks, including video/face data recovery and face/object/scene data clustering.

Single-cell analysis reveals transcriptomic reprogramming in aging primate entorhinal cortex and the relevance with Alzheimer's disease.

The entorhinal cortex is of great importance in cognition and memory, its dysfunction causes a variety of neurological diseases, particularly Alzheimer's disease (AD). Yet so far, research on entorhinal cortex is still limited. Here, we provided the first single-nucleus transcriptomic map of primate entorhinal cortex aging. Our result revealed that synapse signaling, neurogenesis, cellular homeostasis, and inflammation-related genes and pathways changed in a cell-type-specific manner with age. Moreover, among the 7 identified cell types, we highlighted the neuronal lineage that was most affected by aging. By integrating multiple datasets, we found entorhinal cortex aging was closely related to multiple neurodegenerative diseases, particularly for AD. The expression levels of APP and MAPT, which generate ß-amyloid (Aß) and neurofibrillary tangles, respectively, were increased in most aged entorhinal cortex cell types. In addition, we found that neuronal lineage in the aged entorhinal cortex is more prone to AD and identified a subpopulation of excitatory neurons that are most highly associated with AD. Altogether, this study provides a comprehensive cellular and molecular atlas of the primate entorhinal cortex at single-cell resolution and provides new insights into potential therapeutic targets against age-related neurodegenerative diseases.

5-Demethoxy-10'-ethoxyexotimarin F, a New Coumarin with MAO-B Inhibitory Potential from Murraya exotica L.

Rutaceae plants are known for being a rich source of coumarins. Preliminary molecular docking showed that there was no significant difference for coumarins in Clausena and Murraya, both of which had high scoring values and showed good potential inhibitory activity to the MAO-B enzyme. Overall, 32 coumarins were isolated from Murraya exotica L., including a new coumarin 5-demethoxy-10'-ethoxyexotimarin F (1). Their structures were elucidated on the basis of a comprehensive analysis of 1D and 2D NMR and HRMS spectroscopic data, and the absolute configurations were assigned via a comparison of the specific rotations and the ECD exciton coupling method. The potential of new coumarin (1) as a selective inhibitor of MAO-B was initially evaluated through molecular docking and pharmacophore studies. Compound (1) showed selectivity for the MAO-B isoenzyme and inhibitory activity in the sub-micromolar range with an IC50 value of 153.25 ± 1.58 nM (MAO-B selectivity index > 172).

Open Search-Based Proteomics Reveals Widespread Tryptophan Modifications Associated with Hypoxia in Lung Cancer.

The tryptophan residue has a large hydrophobic surface that plays a unique role in the folded protein conformation and functions. Tryptophan modifications are presumably to be readily detected in proteins due to the vulnerability of the indole structure to electrophilic attacks. In this study, we report a systematic identification of sequence variations at tryptophan, termed tryptophan variants, from the proteome of patients with nonsmall cell lung cancer (NSCLC). Using shotgun proteomics and a modified open search algorithm, 25 tryptophan variants on 2481 sites in over 858 proteins were identified. Among these, 6 tryptophan variants are previously identified, 15 are newly annotated, and 4 are still unknown, most of which are involved in the cascade of oxidation in the blood microparticle. Remarkably, Trp313 of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-oxidized whereas Trp16 and Trp38 of hemoglobin (HBB) were down-oxidized in NCSLC tissues. The results were further supported by an independent cohort of 103 lung adenocarcinoma samples, reflecting a negative feedback and potential detoxification mechanism against tumor glycolysis and hypoxia. Overall, the study reports a quick approach to explore tryptophan variants at the proteomic scale. Our findings highlight the predominant role of tryptophan oxidation in regulating the redox balance of cancer cells and its potential role as prognostic biomarker for patients with NSCLC.

Tensor Completion via Complementary Global, Local, and Nonlocal Priors.

Completing missing entries in multidimensional visual data is a typical ill-posed problem that requires appropriate exploitation of prior information of the underlying data. Commonly used priors can be roughly categorized into three classes: global tensor low-rankness, local properties, and nonlocal self-similarity (NSS); most existing works utilize one or two of them to implement completion. Naturally, there arises an interesting question: can one concurrently make use of multiple priors in a unified way, such that they can collaborate with each other to achieve better performance? This work gives a positive answer by formulating a novel tensor completion framework which can simultaneously take advantage of the global-local-nonlocal priors. In the proposed framework, the tensor train (TT) rank is adopted to characterize the global correlation; meanwhile, two Plug-and-Play (PnP) denoisers, including a convolutional neural network (CNN) denoiser and the color block-matching and 3 D filtering (CBM3D) denoiser, are incorporated to preserve local details and exploit NSS, respectively. Then, we design a proximal alternating minimization algorithm to efficiently solve this model under the PnP framework. Under mild conditions, we establish the convergence guarantee of the proposed algorithm. Extensive experiments show that these priors organically benefit from each other to achieve state-of-the-art performance both quantitatively and qualitatively.

Protein modifications throughout the lung cancer proteome unravel the cancer-specific regulation of glycolysis.

Glycolytic reprogramming is a typical feature of cancer. However, the cancer-specific modulation of glycolytic enzymes requires systematic elucidation. Here, we report a range of dysregulated modifications in association with a family of enzymes specifically related to the glycolysis pathway by systematic identification of delta masses at the proteomic scale in human non-small-cell lung cancer. The most significant modification is the delta mass of 79.967 Da at serine 58 (Ser58) of triosephosphate isomerase (TPI), which is confirmed to be phosphorylation. Blocking TPI Ser58 phosphorylation dramatically inhibits glycolysis, cancer growth, and metastasis. The protein kinase PRKACA directly phosphorylates TPI Ser58, thereby enhancing TPI enzymatic activity and glycolysis. The upregulation of TPI Ser58 phosphorylation is detected in various human tumor specimens and correlates with poor survival. Therefore, our study identifies a number of cancer-specific protein modifications spanned on glycolytic enzymes and unravels the significance of TPI Ser58 phosphorylation in glycolysis and lung cancer development.

Adverse Effects Associated With Currently Commonly Used Antifungal Agents: A Network Meta-Analysis and Systematic Review.

Background: Invasive fungal infections (IFI) is an important contributing factor in morbidity and mortality of immunocompromised and critically ill patients. Although the therapeutic effects of these drugs on IFI have been well documented, the long-term use of antifungal agents has raised concerns about drug tolerability and treatment-related toxicity risks. Methods: We searched articles published before June 30, 2020 in four electronic databases: Web of Science, Cochrane Library, embase and PubMed. Results: 66 trials were determined to meet our inclusion criteria, providing data on 18,230 participants. We sorted out 23 AEs by system organ classes and six laboratory AEs, 13 of these were used to construct 13 network meta-analyses. Compared with LAmB, anidulafungin, caspofungin, micafungin, fluconazole, and posaconazole had a significantly low incidence of discontinuation of therapy due to AEs (OR = 0.24 (0.09,0.65), 0.24 (0.13,0.43), 0.32 (0.19,0.52), 0.38 (0.23,0.62) and 0.35 (0.17,0.69), respectively). Conclusion: We found that echinocandins are the most tolerated antifungal agents with high safety. The AEs of triazole drugs are mainly concentrated on the increase in liver enzymes, nervous system disorders, especially visual disorders, gastrointestinal disorders, and cardiac diseases. LAmB is the least tolerated and has the most abundant AEs.

Cellular Stress and Molecular Responses in Bladder Ischemia.

The concept of bladder ischemia as a contributing factor to detrusor overactivity and lower urinary tract symptoms (LUTS) is evolving. Bladder ischemia as a consequence of pelvic arterial atherosclerosis was first documented in experimental models and later in elderly patients with LUTS. It was shown that early-stage moderate ischemia produces detrusor overactivity, while prolonged severe ischemia provokes changes consistent with detrusor underactivity. Recent studies imply a central role of cellular energy sensors, cellular stress sensors, and stress response molecules in bladder responses to ischemia. The cellular energy sensor adenosine monophosphate-activated protein kinase was shown to play a role in detrusor overactivity and neurodegeneration in bladder ischemia. The cellular stress sensors apoptosis signal-regulating kinase 1 and caspase-3 along with heat shock proteins were characterized as important contributing factors to smooth muscle structural modifications and apoptotic responses in bladder ischemia. Downstream pathways seem to involve hypoxia-inducible factor, transforming growth factor beta, vascular endothelial growth factor, and nerve growth factor. Molecular responses to bladder ischemia were associated with differential protein expression, the accumulation of non-coded amino acids, and post-translational modifications of contractile proteins and stress response molecules. Further insight into cellular stress responses in bladder ischemia may provide novel diagnostic and therapeutic targets against LUTS.

Bcl-2-associated transcription factor 1 Ser290 phosphorylation mediates DNA damage response and regulates radiosensitivity in gastric cancer.

BACKGROUND: DNA damage response plays critical roles in tumor pathogenesis and radiotherapy resistance. Protein phosphorylation is a critical mechanism in regulation of DNA damage response; however, the key mediators for radiosensitivity in gastric cancer still needs further exploration. METHODS: A quick label-free phosphoproteomics using high-resolution mass spectrometry and an open search approach was applied to paired tumor and adjacent tissues from five patients with gastric cancer. The dysregulated phosphoproteins were identified and their associated-pathways analyzed using Gene Set Enrichment Analysis (GSEA). The mostly regulated phosphoproteins and their potential functions were validated by the specific antibodies against the phosphorylation sites. Specific protein phosphorylation was further analyzed by functional and clinical approaches. RESULTS: 832 gastric cancer-associated unique phosphorylated sites were identified, among which 25 were up- and 52 down-regulated. Markedly, the dysregulated phosphoproteins were primarily enriched in DNA-damage-response-associated pathways. Particularly, the phosphorylation of Bcl-2-associated transcription factor 1 (BCLAF1) at Ser290 was significantly upregulated in tumor. The upregulation of BCLAF1 Ser290 phosphorylation (pBCLAF1 (Ser290)) in tumor was confirmed by tissue microarray studies and further indicated in association with poor prognosis of gastric cancer patients. Eliminating the phosphorylation of BCLAF1 at Ser290 suppressed gastric cancer (GC) cell proliferation. Upregulation of pBCLAF1 (Ser290) was found in association with irradiation-induced γ-H2AX expression in the nucleus, leading to an increased DNA damage repair response, and a marked inhibition of irradiation-induced cancer cell apoptosis. CONCLUSIONS: The phosphorylation of BCLAF1 at Ser290 is involved in the regulation of DNA damage response, indicating an important target for the resistance of radiotherapy.

Impairment of AMPK-α2 augments detrusor contractions in bladder ischemia.

PURPOSE: Ischemia disrupts cellular energy homeostasis. Adenosine monophosphate-activated protein kinase alpha-2 (AMPK-α2) is a subunit of AMPK that senses cellular energy deprivation and signals metabolic stress. Our goal was to examine the expression levels and functional role of AMPK-α2 in bladder ischemia. MATERIALS AND METHODS: Iliac artery atherosclerosis and bladder ischemia were engendered in apolipoprotein E knockout rats by partial arterial endothelial denudation using a balloon catheter. After eight weeks, total and phosphorylated AMPK-α2 expression was analyzed by western blotting. Structural integrity of AMPK-α2 protein was assessed by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Functional role of AMPK-α2 was examined by treating animals with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D ribofuranoside (AICAR). Tissue contractility was measured in the organ bath and bladder nerve density was examined by immunostaining. RESULTS: Total AMPK-α2 expression increased in bladder ischemia, while phosphorylated AMPK-α2 was significantly downregulated. LC-MS/MS suggested post-translational modification of AMPK-α2 functional domains including phosphorylation sites, suggesting accumulation of catalytically inactive AMPK-α2 in bladder ischemia. Treatment of rats with AICAR diminished the force of overactive detrusor contractions and increased bladder capacity but did not have a significant effect on the frequency of bladder contractions. AICAR diminished contractile reactivity of ischemic tissues in the organ bath and prevented loss of nerve fibers in bladder ischemia. CONCLUSIONS: Ischemia induces post-translational modification of AMPK-α2 protein. Impairment of AMPK-α2 may contribute to overactive detrusor contractions and loss of nerve fibers in bladder ischemia. AMPK activators may have therapeutic potential against detrusor overactivity and neurodegeneration in bladder conditions involving ischemia.