Mitochondrial dysfunction and type I interferon signaling induce anxiodepressive-like behaviors in mice with neuropathic pain.

Clinical evidence indicates that major depression is a common comorbidity of chronic pain, including neuropathic pain; however, the cellular basis for chronic pain-mediated major depression remains unclear. Mitochondrial dysfunction induces neuroinflammation and has been implicated in various neurological diseases, including depression. Nevertheless, the relationship between mitochondrial dysfunction and anxiodepressive-like behaviors in the neuropathic pain state remains unclear. The current study examined whether hippocampal mitochondrial dysfunction and downstream neuroinflammation are involved in anxiodepressive-like behaviors in mice with neuropathic pain, which was induced by partial sciatic nerve ligation (PSNL). At 8 weeks after surgery, there was decreased levels of mitochondrial damage-associated molecular patterns, such as cytochrome c and mitochondrial transcription factor A, and increased level of cytosolic mitochondrial DNA in the contralateral hippocampus, suggesting the development of mitochondrial dysfunction. Type I interferon (IFN) mRNA expression in the hippocampus was also increased at 8 weeks after PSNL surgery. The restoration of mitochondrial function by curcumin blocked the increased cytosolic mitochondrial DNA and type I IFN expression in PSNL mice and improved anxiodepressive-like behaviors. Blockade of type I IFN signaling by anti-IFN alpha/beta receptor 1 antibody also improved anxiodepressive-like behaviors in PSNL mice. Together, these findings suggest that neuropathic pain induces hippocampal mitochondrial dysfunction followed by neuroinflammation, which may contribute to anxiodepressive-behaviors in the neuropathic pain state. Improving mitochondrial dysfunction and inhibiting type I IFN signaling in the hippocampus might be a novel approach to reducing comorbidities associated with neuropathic pain, such as depression and anxiety.

Orotic acid protects pancreatic ß cell by p53 inactivation in diabetic mouse model.

Impairment of pancreatic ß cells is a principal driver of the development of diabetes. Restoring normal insulin release from the ß cells depends on the ATP produced by the intracellular mitochondria. In maintaining mitochondrial function, the tumor suppressor p53 has emerged as a novel regulator of metabolic homeostasis and participates in adaptations to nutritional changes. In this study, we used orotic acid, an intermediate in the pathway for de novo synthesis of the pyrimidine nucleotide, to reduce genotoxicity. Administration of orotic acid reduced p53 activation of MIN6 ß cells and subsequently reduced ß cell death in the db/db mouse. Orotic acid intake helped to maintain the islet size, number of ß cells, and protected insulin secretion in the db/db mouse. In conclusion, orotic acid treatment maintained ß cell function and reduced cell death, and may therefore, be a future therapeutic strategy for the prevention and treatment of diabetes.

ACLY Nuclear Translocation in Human Macrophages Drives Proinflammatory Gene Expression by NF-κB Acetylation.

Macrophage stimulation by pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS) or lipoteichoic acid (LTA) drives a proinflammatory phenotype and induces a metabolic reprogramming to sustain the cell's function. Nevertheless, the relationship between metabolic shifts and gene expression remains poorly explored. In this context, the metabolic enzyme ATP citrate lyase (ACLY), the producer of citrate-derived acetyl-coenzyme A (CoA), plays a critical role in supporting a proinflammatory response. Through immunocytochemistry and cytosol-nucleus fractionation, we found a short-term ACLY nuclear translocation. Protein immunoprecipitation unveiled the role of nuclear ACLY in NF-κB acetylation and in turn its full activation in human PBMC-derived macrophages. Notably, sepsis in the early hyperinflammatory phase triggers ACLY-mediated NF-κB acetylation. The ACLY/NF-κB axis increases the expression levels of proinflammatory genes, including SLC25A1-which encodes the mitochondrial citrate carrier-and ACLY, thus promoting the existence of a proinflammatory loop involving SLC25A1 and ACLY genes.

Abnormal Calcium Handling in Atrial Fibrillation Is Linked to Changes in Cyclic AMP Dependent Signaling.

Both, the decreased L-type Ca2+ current (ICa,L) density and increased spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), have been associated with atrial fibrillation (AF). In this study, we tested the hypothesis that remodeling of 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is linked to these compartment-specific changes (up- or down-regulation) in Ca2+-handling. Perforated patch-clamp experiments were performed in atrial myocytes from 53 patients with AF and 104 patients in sinus rhythm (Ctl). A significantly higher frequency of transient inward currents (ITI) activated by spontaneous Ca2+ release was confirmed in myocytes from AF patients. Next, inhibition of PKA by H-89 promoted a stronger effect on the ITI frequency in these myocytes compared to myocytes from Ctl patients (7.6-fold vs. 2.5-fold reduction), while the ß-agonist isoproterenol (ISO) caused a greater increase in Ctl patients (5.5-fold vs. 2.1-fold). ICa,L density was larger in myocytes from Ctl patients at baseline (p < 0.05). However, the effect of ISO on ICa,L density was only slightly stronger in AF than in Ctl myocytes (3.6-fold vs. 2.7-fold). Interestingly, a significant reduction of ICa,L and Ca2+ sparks was observed upon Ca2+/Calmodulin-dependent protein kinase II inhibition by KN-93, but this inhibition had no effect on ITI. Fluorescence resonance energy transfer (FRET) experiments showed that although AF promoted cytosolic desensitization to ß-adrenergic stimulation, ISO increased cAMP to similar levels in both groups of patients in the L-type Ca2+ channel and ryanodine receptor compartments. Basal cAMP signaling also showed compartment-specific regulation by phosphodiesterases in atrial myocytes from 44 Ctl and 43 AF patients. Our results suggest that AF is associated with opposite changes in compartmentalized PKA/cAMP-dependent regulation of ICa,L (down-regulation) and ITI (up-regulation).

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells.

Although the causes of hepatotoxicity among alcohol-abusing HIV patients are multifactorial, alcohol remains the least explored "second hit" for HIV-related hepatotoxicity. Here, we investigated whether metabolically derived acetaldehyde impairs lysosomes to enhance HIV-induced hepatotoxicity. We exposed Cytochrome P450 2E1 (CYP2E1)-expressing Huh 7.5 (also known as RLW) cells to an acetaldehyde-generating system (AGS) for 24 h. We then infected (or not) the cells with HIV-1ADA then exposed them again to AGS for another 48 h. Lysosome damage was assessed by galectin 3/LAMP1 co-localization and cathepsin leakage. Expression of lysosome biogenesis-transcription factor, TFEB, was measured by its protein levels and by in situ immunofluorescence. Exposure of cells to both AGS + HIV caused the greatest amount of lysosome leakage and its impaired lysosomal biogenesis, leading to intrinsic apoptosis. Furthermore, the movement of TFEB from cytosol to the nucleus via microtubules was impaired by AGS exposure. The latter impairment appeared to occur by acetylation of α-tubulin. Moreover, ZKSCAN3, a repressor of lysosome gene activation by TFEB, was amplified by AGS. Both these changes contributed to AGS-elicited disruption of lysosome biogenesis. Our findings indicate that metabolically generated acetaldehyde damages lysosomes and likely prevents their repair and restoration, thereby exacerbating HIV-induced hepatotoxicity.

Calcineurin Activation by Prion Protein Induces Neurotoxicity via Mitochondrial Reactive Oxygen Species.

Prion diseases are caused by PrPsc accumulation in the brain, which triggers dysfunctional mitochondrial injury and reactive oxygen species (ROS) generation in neurons. Recent studies on prion diseases suggest that endoplasmic reticulum (ER) stress induced by misfolding proteins such as misfolded prion protein results in activation of calcineurin. Calcineurin is a calcium-related protein phosphatase of type 2B that exists in copious quantities in the brain and acts as a critical nodal component in the control of cellular functions. To investigate the relationship between calcineurin and intracellular ROS, we assessed the alteration of CaN and ROS induced by prion peptide (PrP) 106-126. Human prion peptide increased mitochondrial ROS by activating calcineurin, and the inhibition of calcineurin activity protected mitochondrial function and neuronal apoptosis in neuronal cells. These results suggest that calcineurin plays a pivotal role in neuronal apoptosis by mediating mitochondrial injury and ROS in prion diseases.

Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90ß.

Hsp90ß is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90ß for proper folding and/or activity. Regulation of Hsp90ß is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90ß, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90ß is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90ß activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90ß globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90ß and its secretion, through changes in the conformation of the chaperone.

Palmitic acid induces insulin resistance by a mechanism associated with energy metabolism and calcium entry in neuronal cells.

Palmitic acid (PA) is a saturated fatty acid whose high consumption has been largely associated with the development of different metabolic alterations, such as insulin resistance, metabolic syndrome, and type 2 diabetes. Particularly in the brain, insulin signaling disruption has been linked to cognitive decline and is considered a risk factor for Alzheimer's disease. Cumulative evidence has demonstrated the participation of PA in the molecular cascade underlying cellular insulin resistance in peripheral tissues, but its role in the development of neuronal insulin resistance and the mechanisms involved are not fully understood. It has generally been accepted that the brain does not utilize fatty acids as a primary energy source, but recent evidence shows that neurons possess the machinery for fatty acid ß-oxidation. However, it is still unclear under what conditions neurons use fatty acids as energy substrates and the implications of their oxidative metabolism in modifying insulin-stimulated effects. In the present work, we have found that neurons differentiated from human neuroblastoma MSN exposed to high but nontoxic concentrations of PA generate ATP through mitochondrial metabolism, which is associated with an increase in the cytosolic Ca2+ and diminished insulin signaling in neurons. These findings reveal a novel mechanism by which saturated fatty acids produce Ca2+ entry and insulin resistance that may play a causal role in increasing neuronal vulnerability associated with metabolic diseases.

Hydrogen Sulfide Is a Regulator of Hemoglobin Oxygen-Carrying Capacity via Controlling 2,3-BPG Production in Erythrocytes.

Hydrogen sulfide (H2S) is naturally synthesized in a wide range of mammalian tissues. Whether H2S is involved in the regulation of erythrocyte functions remains unknown. Using mice with a genetic deficiency in a H2S natural synthesis enzyme cystathionine-γ-lyase (CSE) and high-throughput metabolomic profiling, we found that levels of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), an erythroid-specific metabolite negatively regulating hemoglobin- (Hb-) oxygen (O2) binding affinity, were increased in CSE knockout (Cse -/-) mice under normoxia. Consistently, the 50% oxygen saturation (P50) value was increased in erythrocytes of Cse -/- mice. These effects were reversed by treatment with H2S donor GYY4137. In the models of cultured mouse and human erythrocytes, we found that H2S directly acts on erythrocytes to decrease 2,3-BPG production, thereby enhancing Hb-O2 binding affinity. Mouse genetic studies showed that H2S produced by peripheral tissues has a tonic inhibitory effect on 2,3-BPG production and consequently maintains Hb-O2 binding affinity in erythrocytes. We further revealed that H2S promotes Hb release from the membrane to the cytosol and consequently enhances bisphosphoglycerate mutase (BPGM) anchoring to the membrane. These processes might be associated with S-sulfhydration of Hb. Moreover, hypoxia decreased the circulatory H2S level and increased the erythrocyte 2,3-BPG content in mice, which could be reversed by GYY4137 treatment. Altogether, our study revealed a novel signaling pathway that regulates oxygen-carrying capacity in erythrocytes and highlights a previously unrecognized role of H2S in erythrocyte 2,3-BPG production.

Cadmium disrupts mitochondrial distribution and activates excessive mitochondrial fission by elevating cytosolic calcium independent of MCU-mediated mitochondrial calcium uptake in its neurotoxicity.

Cadmium (Cd) is a ubiquitous environmental and occupational pollutant that is considered as a high-risk factor for neurodegenerative diseases. However, the mechanism underlying Cd-induced neurotoxicity has not been fully elucidated. Abnormal mitochondrial distribution and excessive mitochondrial fission are increasingly implicated in various neurological pathologies. Herein, by exposing primary cortical neurons to Cd (10 and 100 µM) for various times (0, 6, 12, and 24 h), we observed that the rapid motility of the mitochondria in neurons progressively slowed. Many more mitochondria were transported and distributed to the somas of Cd-treated neurons. Coupled with abnormal mitochondrial distribution, Cd exposure triggered excessive mitochondrial fragmentation, followed by mitochondrial membrane potential loss and neuronal damage. However, BAPTA-AM, a chelator of cytosolic calcium ([Ca2+]c), significantly attenuated Cd-induced abnormal mitochondrial distribution and excessive mitochondrial fission, which protected against Cd-induced mitochondrial damage and neuronal toxicity. In contrast to the increase in [Ca2+]c, Cd exposure had no effect on the level of mitochondrial calcium ([Ca2+]m). Inhibiting [Ca2+]m uptake, either by ruthenium 360 (Ru360) or by knock-out of mitochondrial calcium uniporter (MCU), failed to alleviate Cd-induced mitochondrial damage and neuronal toxicity. Additionally, in MCU knock-out neurons, BAPTA-AM effectively prevented Cd-induced abnormal mitochondrial distribution and excessive mitochondrial fission. Taken together, Cd exposure disrupts mitochondrial distribution and activates excessive mitochondrial fission by elevating [Ca2+]c independent of MCU-mediated mitochondrial calcium uptake, thereby leading to neurotoxicity. Chelating overloaded [Ca2+]c is a promising strategy to prevent the neurotoxicity of Cd.

Prolonged unfolded protein reaction is involved in the induction of chronic myeloid leukemia cell death upon oprozomib treatment.

To select the most efficient chemical to induce apoptosis in leukemia cells, a multidrug screen was applied on bone marrow mononuclear cells from chronic myeloid leukemia (CML) patients. Oprozomib (Cpd 21) was chosen for the subsequent experiments. The isobaric tags for relative and absolute quantitation (iTRAQ) was then performed to identify the responsible pathway relative to apoptosis and the results showed that endoplasmic reticulum (ER) chaperones were upregulated. Apoptosis was attributed to a joint effect of calcium leakage andPERK and IRE1α phosphorylation. The PERK branch was responsible for the first wave of cell death that occurred within 24 hours. The later wave of apoptosis was mediated by IRE1α, which transmit apoptotic signals through the ASK-JNK-BIM axis. Release of Ca2+ from ER into cytosol resulted in activation of calpain, which, in turn, cleaved caspase-12. Our data also explained the selective killing effects of oprozomib on CML cells, which relied on proteasome activity. The present study demonstrated that prolonged inhibition of proteasome to trigger unfolded protein response could be an alternative strategy for treating CML in light of tyrosine kinase inhibitors resistance.

Glutathione depletion induces oxidative injury and apoptosis via TRPM2 channel activation in renal collecting duct cells.

Oxidative stress (OS)-induced glutathione (GSH) depletion plays an essential role in several kidney diseases such as chronic kidney disease and nephrotoxicity. The OS-dependent activation of TRPM2 cation channel in several neurons and cells were modulated by the concentration of intracellular GSH. However, the effects of GSH alteration on TRPM2 activation, OS, and apoptosis in the cortical collecting duct (mpkCCDc14) cells still remain elusive. We investigated the effects of GSH supplementation on OS-induced TRPM2 activation, mitochondrial oxidative stress, and apoptosis in the human embryonic kidney 293 (HEK293) and mpkCCDc14 cells treated with buthionine-sulfoximine (BSO), a GSH synthase inhibitor. The HEK293 and mpkCCDc14 cells were divided into five groups as control, GSH (10 mM for 2 h), BSO (0.5 mM for 6 h), BSO + GSH, and BSO + TRPM2 channel blockers. Apoptosis, cell death, mitochondrial OS, caspase -3, caspase -9, cytosolic free Zn2+, and Ca2+ concentrations were increased in the BSO group of the TRPM2 expressing mpkCCDc14 cells, although they were diminished by the treatments of GSH, PARP-1 inhibitors (PJ34 and DPQ), and TRPM2 blockers (ACA and 2-APB). The BSO-induced decreases in the levels of cell viability and cytosolic GSH were increased by the treatments of GSH, ACA, and 2-APB. However, the effects of BSO and GSH were not observed in the non-TRPM2 expressing HEK293 cells. Current results show that maintaining GSH homeostasis is not only important for quenching OS in the cortical collecting duct cells but equally critical to modulate TRPM2 activation. Thus, suppressing apoptosis and mitochondrial OS responses elicited by oxidant action of GSH depletion.

Minoxidil Cannot Be Used To Target Lysyl Hydroxylases during Postnatal Mouse Lung Development: A Cautionary Note.

The lysyl hydroxylases (procollagen-lysine 5-dioxygenases) PLOD1, PLOD2, and PLOD3 have been proposed as pathogenic mediators of stunted lung development in bronchopulmonary dysplasia (BPD), a common complication of preterm birth. In affected infants, pulmonary oxygen toxicity stunts lung development. Mice lacking Plod1 exhibit 15% mortality, and mice lacking Plod2 or Plod3 exhibit embryonic lethality. Therefore, to address any pathogenic role of lysyl hydroxylases in stunted lung development associated with BPD, minoxidil was administered to newborn mice in an oxygen toxicity-based BPD animal model. Minoxidil, which has attracted much interest in the management of systemic hypertension and androgenetic alopecia, can also be used to reduce lysyl hydroxylase activity in cultured cells. An in vivo pilot dosing study established 50 mg⋅kg-1⋅day-1 as the maximum possible minoxidil dose for intraperitoneal administration in newborn mouse pups. When administered at 50 mg⋅kg-1⋅day-1 to newborn mouse pups, minoxidil was detected in the lungs but did not impact lysine hydroxylation, collagen crosslinking, or lysyl hydroxylase expression in the lungs. Consistent with no impact on mouse lung extracellular matrix structures, minoxidil administration did not alter the course of normal or stunted lung development in newborn mice. At doses of up to 50 mg⋅kg⋅day-1, pharmacologically active concentrations of minoxidil were not achieved in neonatal mouse lung tissue; thus, minoxidil cannot be used to attenuate lysyl hydroxylase expression or activity during mouse lung development. These data also highlight the need for new and specific lysyl hydroxylase inhibitors. SIGNIFICANCE STATEMENT: Extracellular matrix crosslinking is mediated by lysyl hydroxylases, which generate hydroxylated lysyl residues in procollagen peptides. Deregulated collagen crosslinking is a pathogenic component of a spectrum of diseases, and thus, there is interest in validating lysyl hydroxylases as pathogenic mediators of disease and potential "druggable" targets. Minoxidil, administered at the maximum possible dose, did not inhibit lysyl hydroxylation in newborn mouse lungs, suggesting that minoxidil was unlikely to be of use in studies that pharmacologically target lysyl hydroxylation in vivo.

Direct and quantitative analysis of altered metabolic flux distributions and cellular ATP production pathway in fumarate hydratase-diminished cells.

Fumarate hydratase (FH) is an enzyme in the tricarboxylic acid (TCA) cycle, biallelic loss-of-function mutations of which are associated with hereditary leiomyomatosis and renal cell cancer. However, how FH defect modulates intracellular metabolic fluxes in human cells has remained unclear. This study aimed to reveal metabolic flux alterations induced by reduced FH activity. We applied 13C metabolic flux analysis (13C-MFA) to an established cell line with diminished FH activity (FHdim) and parental HEK293 cells. FHdim cells showed reduced pyruvate import flux into mitochondria and subsequent TCA cycle fluxes. Interestingly, the diminished FH activity decreased FH flux only by about 20%, suggesting a very low need for FH to maintain the oxidative TCA cycle. Cellular ATP production from the TCA cycle was dominantly suppressed compared with that from glycolysis in FHdim cells. Consistently, FHdim cells exhibited higher glucose dependence for ATP production and higher resistance to an ATP synthase inhibitor. In summary, using FHdim cells we demonstrated that FH defect led to suppressed pyruvate import into mitochondria, followed by downregulated TCA cycle activity and altered ATP production pathway balance from the TCA cycle to glycolysis. We confirmed that 13C-MFA can provide direct and quantitative information on metabolic alterations induced by FH defect.

Design, synthesis, and evaluation of pyrrolidine based CXCR4 antagonists with in vivo anti-tumor metastatic activity.

The chemokine receptor CXCR4 has been proposed as a drug target based on its important functions in HIV infection, inflammation/autoimmune diseases and cancer metastasis. Herein we report the design, synthesis and evaluation of novel CXCR4 antagonists based on a pyrrolidine scaffold. The structural exploration/optimization identified numerous potent CXCR4 antagonists, represented by compound 46, which displayed potent binding affinity to CXCR4 receptor (IC50 = 79 nM competitively displacing fluorescent 12G5 antibody) and inhibited CXCL12 induced cytosolic calcium flux (IC50 = 0.25 nM). Moreover, in a transwell invasion assay, compound 46 significantly mitigated CXCL12/CXCR4 mediated cell migration. Compound 46 exhibited good physicochemical properties (MW 367, logD7.4 1.12, pKa 8.2) and excellent in vitro safety profiles (e.g., hERG patch clamp IC50 > 30 µM and minimal CYP isozyme inhibition). Importantly, 46 displayed much improved metabolic stability in human and rat liver microsomes. Lastly, 46 demonstrated marked efficacy in a cancer metastasis model in mice. These results strongly support 46 as a prototypical lead for the development of promising CXCR4 antagonists as clinical candidates.

Inhibition of Human Adenovirus Replication by the Importin α/ß1 Nuclear Import Inhibitor Ivermectin.

Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -ß1 (Imp-ß1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-ß1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/ß1-mediated nuclear import.IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/ß1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.

Small Molecule Enhancers of Endosome-to-Cytosol Import Augment Anti-tumor Immunity.

Cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumor immune responses. Yet, key steps involved in trafficking of antigens taken up by DCs remain incompletely understood. Here, we screen 700 US Food and Drug Administration (FDA)-approved drugs and identify 37 enhancers of antigen import from endolysosomes into the cytosol. To reveal their mechanism of action, we generate proteomic organellar maps of control and drug-treated DCs (focusing on two compounds, prazosin and tamoxifen). By combining organellar mapping, quantitative proteomics, and microscopy, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release. Enhancing antigen import facilitates cross-presentation of soluble and cell-associated antigens. Systemic administration of prazosin leads to reduced growth of MC38 tumors and to a synergistic effect with checkpoint immunotherapy in a melanoma model. Thus, inefficient antigen import into the cytosol limits antigen cross-presentation, restraining the potency of anti-tumor immune responses and efficacy of checkpoint blockers.

Epothilone B prevents lipopolysaccharide-induced inflammatory osteolysis through suppressing osteoclastogenesis via STAT3 signaling pathway.

Inflammatory osteolysis is a common osteolytic specificity that occurs during infectious orthopaedic surgery and is characterized by an imbalance in bone homeostasis due to excessive osteoclast bone resorption activity. Epothilone B (Epo B) induced α-tubulin polymerization and enhanced microtubule stability, which also played an essential role in anti-inflammatory effect on the regulation of many diseases. However, its effects on skeletal system have rarely been investigated. Our study demonstrated that Epo B inhibited osteoclastogenesis in vitro and prevented inflammatory osteolysis in vivo. Further analysis showed that Epo B also markedly induced mature osteoclasts apoptosis during osteoclastogenesis. Mechanistically, Epo B directly suppressed osteoclastogenesis by the inhibitory regulation of the phosphorylation and activation of PI3K/Akt/STAT3 signaling directly, and the suppressive regulation of the CD9/gp130/STAT3 signaling pathway indirectly. The negative regulatory effect on STAT3 signaling further restrained the translocation of NF-κB p65 and NFATc1 from the cytosol to the nuclei during RANKL stimulation. Additionally, the expression of osteoclast specific genes was also significantly attenuated during osteoclast fusion and differentiation. Taken together, these findings illustrated that Epo B protected against LPS-induced bone destruction through inhibiting osteoclastogenesis via regulating the STAT3 dependent signaling pathway.

Dual-Reporting Transcriptionally Linked Genetically Encoded Fluorescent Indicators Resolve the Spatiotemporal Coordination of Cytosolic Abscisic Acid and Second Messenger Dynamics in Arabidopsis.

Deciphering signal transduction processes is crucial for understanding how plants sense and respond to environmental changes. Various chemical compounds function as central messengers within deeply intertwined signaling networks. How such compounds act in concert remains to be elucidated. We have developed dual-reporting transcriptionally linked genetically encoded fluorescent indicators (2-in-1-GEFIs) for multiparametric in vivo analyses of the phytohormone abscisic acid (ABA), Ca2+, protons (H+), chloride (anions), the glutathione redox potential, and H2O2 Simultaneous analyses of two signaling compounds in Arabidopsis (Arabidopsis thaliana) roots revealed that ABA treatment and uptake did not trigger rapid cytosolic Ca2+ or H+ dynamics. Glutamate, ATP, Arabidopsis PLANT ELICITOR PEPTIDE, and glutathione disulfide (GSSG) treatments induced rapid spatiotemporally overlapping cytosolic Ca2+, H+, and anion dynamics, but except for GSSG, only weakly affected the cytosolic redox state. Overall, 2-in-1-GEFIs enable complementary, high-resolution in vivo analyses of signaling compound dynamics and facilitate an advanced understanding of the spatiotemporal coordination of signal transduction processes in Arabidopsis.