Targeting metabolic sensing switch GPR84 on macrophages for cancer immunotherapy.

INTRODUCTION: As one of the major components of the tumor microenvironment, tumor-associated macrophages (TAMs) possess profound inhibitory activity against T cells and facilitate tumor escape from immune checkpoint blockade therapy. Converting this pro-tumorigenic toward the anti-tumorigenic phenotype thus is an important strategy for enhancing adaptive immunity against cancer. However, a plethora of mechanisms have been described for pro-tumorigenic differentiation in cancer, metabolic switches to program the anti-tumorigenic property of TAMs are elusive. MATERIALS AND METHODS: From an unbiased analysis of single-cell transcriptome data from multiple tumor models, we discovered that anti-tumorigenic TAMs uniquely express elevated levels of a specific fatty acid receptor, G-protein-coupled receptor 84 (GPR84). Genetic ablation of GPR84 in mice leads to impaired pro-inflammatory polarization of macrophages, while enhancing their anti-inflammatory phenotype. By contrast, GPR84 activation by its agonist, 6-n-octylaminouracil (6-OAU), potentiates pro-inflammatory phenotype via the enhanced STAT1 pathway. Moreover, 6-OAU treatment significantly retards tumor growth and increases the anti-tumor efficacy of anti-PD-1 therapy. CONCLUSION: Overall, we report a previously unappreciated fatty acid receptor, GPR84, that serves as an important metabolic sensing switch for orchestrating anti-tumorigenic macrophage polarization. Pharmacological agonists of GPR84 hold promise to reshape and reverse the immunosuppressive TME, and thereby restore responsiveness of cancer to overcome resistance to immune checkpoint blockade.

A self-activating electron transfer antibacterial strategy: Co3O4/TiO2 P-N heterojunctions combined with photothermal therapy.

Implant-associated infections are significant impediments to successful surgical outcomes, often resulting from persistent bacterial contamination. It has been hypothesized that bacteria can transfer electrons to semiconductors with comparable potential to the biological redox potential (BRP). Building on this concept, we developed an antibiotic-free bactericidal system, Co3O4/TiO2-Ti, capable of achieving real-time and sustainable bactericidal effects. Our study demonstrated that Co3O4/TiO2-Ti, possessing an appropriately set valence band, initiated charge transfer, reactive oxygen species (ROS) production, and membrane damage in adherent Staphylococcus aureus (S. aureus). Notably, in vivo experiments illustrated the remarkable antibacterial activity of Co3O4/TiO2-Ti, while promoting soft-tissue reconstruction and demonstrating excellent cytocompatibility. Transcriptomic analysis further revealed a down-regulation of aerobic respiration-associated genes and an up-regulation of ROS-associated genes in S. aureus in the presence of Co3O4/TiO2-Ti compared to Ti. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA) identified alterations in respiratory metabolism, oxidative phosphorylation, and the synthesis of amino acid in S. aureus cultured on Co3O4/TiO2-Ti. Furthermore, when combined with near-infrared (NIR) irradiation and photothermal therapy (PTT), Co3O4/TiO2-Ti eliminated 95.71% of floating and adherent S. aureus in vitro. The findings suggest that this antibiotic-free strategy holds substantial promise in enhancing implant sterilization capabilities, thereby contributing to the prevention and treatment of bacterial infections through bandgap engineering of implants and NIR irradiation.

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in the development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health, and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from patients with PAH. Endothelial haploinsufficiency of RAB7 caused spontaneous pulmonary hypertension (PH) in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA-Seq, and RAB7-silenced ECs showed impaired angiogenesis and expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, suggesting inhibition of autophagy at the predegradation level. Furthermore, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in rats with chronic hypoxia/SU5416. In conclusion, we demonstrate for the first time to our knowledge the fundamental impairment of EC function by loss of RAB7, causing PH, and show RAB7 activation to be a potential therapeutic strategy in a preclinical model of PH.

A Biomimetic Nanogenerator to Enhance Bone Regeneration by Restoring Electric Microenvironments.

Piezoelectric materials have received increasing attention in bone regeneration due to their prominent role in bioelectricity in bone homeostasis. This study aimed to develop bioactive barium titanate-chitosan-graphene oxide piezoelectric nanoparticles (BCG-NPs) to improve biocompatibility and stimulate bone repair. Butterfly loops, hysteresis loops, and in vitro microcurrent studies on BCG-NPs confirmed their good piezoelectric properties. BCG-NPs exhibited enhanced alkaline phosphatase activity, mineralized nodule formation, and expression of osteogenic-associated proteins and genes in human umbilical cord Wharton's jelly-derived mesenchymal stem cells by creating microelectric environments in response to noninvasive ultrasound stimulation. Further, BCG-NPs upregulated intracellular calcium ions via electrical stimulation. They acted synergistically with piezo-type mechanosensitive ion channel component 1 and calcium-permeable cation channel transient receptor potential vanilloid 4 to activate osteogenic differentiation. In conclusion, ultrasound-assisted BCG-NPs created a microelectric environment that putatively promoted bone repair in a noninvasive manner.

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from PAH patients. Endothelial haploinsufficiency of RAB7 caused spontaneous PH in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA sequencing and RAB7 silenced ECs showed impaired angiogenesis, expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, which suggests inhibition of autophagy at the pre-degradation level. Further, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in chronic hypoxia/SU5416 rats. In conclusion, we demonstrate here for the first time the fundamental impairment of EC function by loss of RAB7 that leads to PH and show RAB7 activation as a potential therapeutic strategy in a preclinical model of PH.

The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling.

Mitophagy is an intracellular mechanism to maintain mitochondrial health by removing dysfunctional mitochondria. The E3 ligase Parkin ubiquitinates the membrane proteins on targeted mitochondria to initiate mitophagy, whereas USP30 antagonizes Parkin-dependent mitophagy by removing ubiquitin from Parkin substrates. The AKT/mTOR signaling is a master regulator of cell proliferation, differentiation, apoptosis, and autophagy. Although mounting evidence suggests that perturbations in the AKT/mTOR signaling pathway may contribute to mitophagy regulation, the specific mechanisms between Parkin/USP30 and AKT/mTOR signaling have not been elucidated. In this study, we employ a set of genetic reagents to investigate the role of Parkin and USP30 in regulating the AKT/mTOR signaling during mitophagy. We demonstrated that, in the setting of mitochondrial stress, the AKT/mTOR signaling is regulated, at least in part, by the activity of Parkin and USP30. Parkin inhibits AKT/mTOR signaling following an in vitro mitochondrial stress, thereby promoting apoptosis. However, USP30 overexpression antagonizes the activity of Parkin to sustain AKT/mTOR activity and inhibit apoptosis. These findings provide new insights into Parkin and USP30's role in apoptosis and suggest that inhibiting USP30 might provide a specific strategy to synergize with AKT/mTOR inhibitors in cancer treatment.

Molecular signature of cardiac remodeling associated with Polymerase Gamma mutation.

AIMS: Metabolic function/dysfunction is central to aging biology. This is well illustrated by the Polymerase Gamma (POLG) mutant mouse where a key residue of the mitochondrial DNA polymerase is mutated (D257A), causing loss of mitochondrial DNA stability and dramatically accelerated aging processes. Given known cardiac phenotypes in the POLG mutant, we sought to characterize the course of cardiac dysfunction in the POLG mutant to guide future intervention studies. MATERIALS AND METHODS: Cardiac echocardiography and terminal hemodynamic analyses were used to define the course of dysfunction in the right and left cardiac ventricles in the POLG mutant. We also conducted RNA-seq analysis on cardiac right ventricles to identify mechanisms engaged by severe metabolic dysfunction and compared this analysis to several publically available datasets. KEY FINDINGS: Interesting sex differences were noted as female POLG mutants died earlier than male POLG mutants and LV chamber diameters were impacted earlier in females than males. Moreover, male mutants showed LV wall thinning while female mutant LV walls were thicker. Both males and females displayed significant RV hypertrophy. POLG mutants displayed a gene expression pattern associated with inflammation, fibrosis, and heart failure. Finally, comparative omics analyses of publically available data provide additional mechanistic and therapeutic insights. SIGNIFICANCE: Aging-associated cardiac dysfunction is a growing clinical problem. This work uncovers sex-specific cardiac responses to severe metabolic dysfunction that are reminiscent of patterns seen in human heart failure and provides insights to the molecular mechanisms engaged downstream of severe metabolic dysfunction that warrant further investigation.

Assessment of mitophagy in human iPSC-derived cardiomyocytes.

Defective mitophagy contributes to normal aging and various neurodegenerative and cardiovascular diseases. The newly developed methodologies to visualize and quantify mitophagy allow for additional progress in defining the pathophysiological significance of mitophagy in various model organisms. However, current knowledge regarding mitophagy relevant to human physiology is still limited. Model organisms such as mice might not be optimal models to recapitulate all the key aspects of human disease phenotypes. The development of the human-induced pluripotent stem cells (hiPSCs) may provide an exquisite approach to bridge the gap between animal mitophagy models and human physiology. To explore this premise, we take advantage of the pH-dependent fluorescent mitophagy reporter, mt-Keima, to assess mitophagy in hiPSCs and hiPSC-derived cardiomyocytes (hiPSC-CMs). We demonstrate that mt-Keima expression does not affect mitochondrial function or cardiomyocytes contractility. Comparison of hiPSCs and hiPSC-CMs during different stages of differentiation revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima hiPSC-CMs to analyze how mitophagy is altered under certain pathological conditions including treating the hiPSC-CMs with doxorubicin, a chemotherapeutic drug well known to cause life-threatening cardiotoxicity, and hypoxia that stimulates ischemia injury. We have further developed a chemical screening to identify compounds that modulate mitophagy in hiPSC-CMs. The ability to assess mitophagy in hiPSC-CMs suggests that the mt-Keima hiPSCs should be a valuable resource in determining the role mitophagy plays in human physiology and hiPSC-based disease models. The mt-Keima hiPSCs could prove a tremendous asset in the search for pharmacological interventions that promote mitophagy as a therapeutic target.Abbreviations: AAVS1: adeno-associated virus integration site 1; AKT/protein kinase B: AKT serine/threonine kinase; CAG promoter: cytomegalovirus early enhancer, chicken ACTB/ß-actin promoter; CIS: cisplatin; CRISPR: clustered regularly interspaced short palindromic repeats; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide p-trifluoromethoxyphenylhydrazone; hiPSC: human induced pluripotent stem cell; hiPSC-CMs: human induced pluripotent stem cell-derived cardiomyocytes; ISO: isoproterenol; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RT: room temperature; SB: SBI-0206965; ULK1: unc-51 like autophagy activating kinase 1.

Significance of mitochondrial activity in neurogenesis and neurodegenerative diseases.

Mitochondria play a multidimensional role in the function and the vitality of the neurological system. From the generation of neural stem cells to the maintenance of neurons and their ultimate demise, mitochondria play a critical role in regulating our neural pathways' homeostasis, a task that is critical to our cognitive health and neurological well-being. Mitochondria provide energy via oxidative phosphorylation for the neurotransmission and generation of an action potential along the neuron's axon. This paper will first review and examine the molecular subtleties of the mitochondria's role in neurogenesis and neuron vitality, as well as outlining the impact of defective mitochondria in neural aging. The authors will then summarize neurodegenerative diseases related to either neurogenesis or homeostatic dysfunction. Because of the significant detriment neurodegenerative diseases have on the quality of life, it is essential to understand their etiology and ongoing molecular mechanics. The mitochondrial role in neurogenesis and neuron vitality is essential. Dissecting and understanding this organelle's role in the genesis and homeostasis of neurons should assist in finding pharmaceutical targets for neurodegenerative diseases.

Hyper-Synergistic Antifungal Activity of Rapamycin and Peptide-Like Compounds against Candida albicans Orthogonally via Tor1 Kinase.

Candida albicans is a life-threatening, opportunistic fungal pathogen with a high mortality rate, especially within the immunocompromised populations. Multidrug resistance combined with limited antifungal drugs even worsens the situation. Given the facts that the current drug discovery strategies fail to deliver sufficient antifungals for the emerging multidrug resistance, we urgently need to develop novel approaches. By systematically investigating what caused the different antifungal activity of rapamycin in RPMI 1640 and YPD, we discovered that peptide-like compounds can generate a hyper-synergistic antifungal effect with rapamycin on both azole-resistant and sensitive clinical C. albicans isolates. The minimum inhibitory concentration (MIC) of rapamycin reaches as low as 2.14 nM (2-9 µg/mL), distinguishing this drug combination as a hyper-synergism by having a fractional inhibitory concentration (FIC) index ≤ 0.05 from the traditional defined synergism with an FIC index < 0.5. Further studies reveal that this hyper-synergism orthogonally targets the protein Tor1 and affects the TOR signaling pathway in C. albicans, very likely without crosstalk to the stress response, Ras/cAMP/PKA, or calcineurin signaling pathways. These results lead to a novel strategy of controlling drug resistant C. albicans infection in the immunocompromised populations. Instead of prophylactically administering other antifungals with undesirable side-effects for extended durations, we now only need to coadminister some nontoxic peptide additives. The novel antifungal strategy approached in this study not only provides a new therapeutic method to control fungal infections in rapamycin-taking immunocompromised patients but also mitigates the immunosuppressive side-effects of rapamycin, repurposing rapamycin as an antifungal agent with wide applications.

Pharmacological inhibition of USP30 activates tissue-specific mitophagy.

AIM: Mitophagy is the regulated process that targets damaged or dysfunctional mitochondria for lysosomal-mediated removal. This process is an essential element of mitochondrial quality control, and dysregulation of mitophagy may contribute to a host of diseases, most notably neurodegenerative conditions such as Parkinson's disease. Mitochondria targeted for mitophagic destruction are molecularly marked by the ubiquitination of several outer mitochondrial membrane (OMM) proteins. This ubiquitination is positively regulated, in part, by the mitochondrial-targeted kinase PINK1 and the E3 ubiquitin ligase Parkin. In contrast, the reverse phenomenon, deubiquitination, removes ubiquitin from Parkin substrates embedded in the OMM proteins, antagonizing mitophagy. Recent evidence suggests that the mitochondrial deubiquitinase USP30 negatively regulates Parkin-mediated mitophagy, providing opportunities to identify USP30 inhibitors and test for their effects in augmenting mitophagy. Here we will characterize a USP30 inhibitor and demonstrate how the pharmacological inhibition of USP30 can augment stress-induced mitophagic flux. METHODS: We have conducted mitophagy and mitochondrial analyses in cultured cells. We have determined the plasma pharmacokinetics of the USP30 inhibitor in mice and conducted analyses using the mt-Keima mice to measure in vivo mitophagy directly. RESULTS: The compound has minimal mitochondrial toxicity in cultured cells and is tolerated well in mice. Interestingly, we demonstrated tissue-specific induction of mitophagy following USP30 pharmacological inhibition. In particular, pharmacological inhibition of USP30 induces a significant increase in cardiac mitophagy without detriment to cardiac function. CONCLUSION: Our data support the evidence that USP30 inhibition may serve as a specific strategy to selectively increase mitophagic flux, allowing for the development of novel therapeutic approaches.

Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p.

Clinical use of antimicrobials faces great challenges from the emergence of multidrug-resistant pathogens. The overexpression of drug efflux pumps is one of the major contributors to multidrug resistance (MDR). Reversing the function of drug efflux pumps is a promising approach to overcome MDR. In the life-threatening fungal pathogen Candida albicans, the major facilitator superfamily (MFS) transporter Mdr1p can excrete many structurally unrelated antifungals, leading to MDR. Here we report a counterintuitive case of reversing MDR in C. albicans by using a natural product berberine to hijack the overexpressed Mdr1p for its own importation. Moreover, we illustrate that the imported berberine accumulates in mitochondria and compromises the mitochondrial function by impairing mitochondrial membrane potential and mitochondrial Complex I. This results in the selective elimination of Mdr1p overexpressed C. albicans cells. Furthermore, we show that berberine treatment can prolong the mean survival time of mice with blood-borne dissemination of Mdr1p overexpressed multidrug-resistant candidiasis. This study provides a potential direction of novel anti-MDR drug discovery by screening for multidrug efflux pump converters.

A Healthy Heart and a Healthy Brain: Looking at Mitophagy.

Mitochondrial dysfunction is a hallmark of aging and is a major contributor to neurodegenerative diseases and various cardiovascular disorders. Mitophagy, a specialized autophagic pathway to remove damaged mitochondria, provides a critical mechanism to maintain mitochondrial quality. This function has been implicated in a tissue's ability to appropriately respond to metabolic and to bioenergetic stress, as well as to recover from mitochondrial damage. A global decline in mitophagic flux has been postulated to be linked to pathological alterations that occur in the heart and the brain as well as a general age-dependent decline in organ function. Cellular observation suggests multiple mechanistically distinct pathways converge upon and activate mitophagy. Over the past decade, additional molecular components within mitophagy have been discovered, including several disease-associated genes that are functionally implicated in mitophagy. However, the pathophysiological role of mitophagy, and how it is regulated within normal physiology or various disease states, is less well established. Here, we will review the evidence that a decline in mitophagy contributes to impaired mitochondrial homeostasis and may be particularly detrimental to postmitotic neurons and cardiomyocytes. We will discuss mitophagy's pathological significance in both neurodegenerative diseases and cardiovascular disorders. Additionally, signaling pathways regulating mitophagy are reviewed, with emphasis placed on how these pathways might contribute to disease progression. Understanding mitophagy's role in the mechanisms of disease pathogenesis should allow for the development of more efficient strategies to battle pathological conditions associated with mitochondrial dysfunction.

Mitophagy in cardiovascular homeostasis.

Mitochondria are essential organelles that generate energy to fuel myocardial contraction. Accumulating evidence also suggests that, in the heart, mitochondria may contribute to specific aspects of disease progression through the regulations of specific metabolic intermediates, as well as the transcriptional and epigenetic states of cells. If damaged, the mitochondria and their related pathways are hindered, which may result in or contribute to the development of a wide range of cardiovascular diseases. Therefore, the maintenance of cardiac mitochondrial function and integrity through specific mitochondrial quality control mechanisms is critical for cardiovascular health. Mitophagy is part of the overall mitochondrial quality control process, and acts as a specialized autophagic pathway that mediates the lysosomal clearance of damaged mitochondria. In response to cardiac stress and injury, the pathways associated with mitophagy are triggered resulting in the removal of damaged mitochondrial, thereby maintaining cardiac homeostasis. In addition, recent studies have demonstrated an essential role for mitophagy in both developmental and disease-related metabolic transitioning of cardiac mitochondria. Here, we discuss the physiological and the pathological roles of mitophagy in the heart, the underlying molecular mechanisms, as well as potential therapeutic strategies based on mitophagic modulation.

Detection of mitophagy in mammalian cells, mice, and yeast.

Selective elimination of superfluous or dysfunctional mitochondria is a fundamental process conserved among both uni- and multicellular eukaryotes, contributing to mitochondrial quality and quantity control. This process depends on autophagy, a cellular self-eating membrane trafficking system, and is thus called mitophagy. In this chapter, we describe methods to detect mitophagy in mammalian cells, mice, and yeast.

Zika Virus-Induced Neuronal Apoptosis via Increased Mitochondrial Fragmentation.

The 2015 to 2016 outbreak of Zika virus (ZIKV) infections in the Americas coincided with a dramatic increase in neurodevelopmental abnormalities, including fetal microcephaly, in newborns born to infected women. In this study, we observed mitochondrial fragmentation and disrupted mitochondrial membrane potential after 24 h of ZIKV infection in human neural stem cells and the SNB-19 glioblastoma cell line. The severity of these changes correlated with the amount of ZIKV proteins expressed in infected cells. ZIKV infection also decreased the levels of mitofusin 2, which modulates mitochondria fusion. Mitochondrial division inhibitor 1 (Mdivi-1), a small molecule inhibiting mitochondria fission, ameliorated mitochondria disruptions and reduced cell death in ZIKV-infected cells. Collectively, this study suggests that abnormal mitochondrial fragmentation contributes to ZIKV-induced neuronal cell death; rebalancing mitochondrial dynamics of fission-fusion could be a therapeutic strategy for drug development to treat ZIKV-mediated neuronal apoptosis.

Unique, Diverged, and Conserved Mitochondrial Functions Influencing Candida albicans Respiration.

Candida albicans is an opportunistic fungal pathogen of major clinical concern. The virulence of this pathogen is intimately intertwined with its metabolism. Mitochondria, which have a central metabolic role, have undergone many lineage-specific adaptations in association with their eukaryotic host. A screen for lineage-specific genes identified seven such genes specific to the CTG clade of fungi, of which C. albicans is a member. Each is required for respiratory growth and is integral to expression of complex I, III, or IV of the electron transport chain. Two genes, NUO3 and NUO4, encode supernumerary subunits of complex I, whereas NUE1 and NUE2 have nonstructural roles in expression of complex I. Similarly, the other three genes have nonstructural roles in expression of complex III (QCE1) or complex IV (COE1 and COE2). In addition to these novel additions, an alternative functional assignment was found for the mitochondrial protein encoded by MNE1MNE1 was required for complex I expression in C. albicans, whereas the distantly related Saccharomyces cerevisiae ortholog participates in expression of complex III. Phenotypic analysis of deletion mutants showed that fermentative metabolism is unable to support optimal growth rates or yields of C. albicans However, yeast-hypha morphogenesis, an important virulence attribute, did not require respiratory metabolism under hypoxic conditions. The inability to respire also resulted in hypersensitivity to the antifungal fluconazole and in attenuated virulence in a Galleria mellonella infection model. The results show that lineage-specific adaptations have occurred in C. albicans mitochondria and highlight the significance of respiratory metabolism in the pathobiology of C. albicansIMPORTANCECandida albicans is an opportunistic fungal pathogen of major clinical concern. The virulence of this pathogen is intimately intertwined with its metabolic behavior, and mitochondria have a central role in that metabolism. Mitochondria have undergone many evolutionary changes, which include lineage-specific adaptations in association with their eukaryotic host. Seven lineage-specific genes required for electron transport chain function were identified in the CTG clade of fungi, of which C. albicans is a member. Additionally, examination of several highly diverged orthologs encoding mitochondrial proteins demonstrated functional reassignment for one of these. Deficits imparted by deletion of these genes revealed the critical role of respiration in virulence attributes of the fungus and highlight important evolutionary adaptations in C. albicans metabolism.