Enhanced production of isobutyl and isoamyl acetate using Yarrowia lipolytica.

Short-chain esters, particularly isobutyl acetate and isoamyl acetate, hold significant industrial value due to their wide-ranging applications in flavors, fragrances, solvents, and biofuels. In this study, we demonstrated the biosynthesis of acetate esters using Yarrowia lipolytica as a host by feeding alcohols to the yeast culture. Initially, we screened for optimal alcohol acyltransferases for ester biosynthesis in Y. lipolytica. Strains of Y. lipolytica expressing atf1 from Saccharomyces cerevisiae, produced 251 or 613 mg/L of isobutyl acetate or of isoamyl acetate, respectively. We found that introducing additional copies of ATF1 enhanced ester production. Furthermore, by increasing the supply of acetyl-CoA and refining the culture conditions, we achieved high production of isoamyl acetate, reaching titers of 3404 mg/L. We expanded our study to include the synthesis of a range of acetate esters, facilitated by enriching the culture medium with various alcohols. This study underscores the versatility and potential of Y. lipolytica in the industrial production of acetate esters.

Repair of a right coronary artery rupture with perforated right ventricle following spontaneous pseudoaneurysm: a case report.

BACKGROUND: Following the rupture of a coronary artery, a patient's condition usually deteriorates rapidly due to cardiac tamponade. A pseudoaneurysm due to a coronary artery rupture is rare; however, when a spontaneous coronary artery pseudoaneurysm occurs without tamponade, it creates a fistula in the right ventricle, often requiring surgical repair. CASE PRESENTATION: This report describes the case of a 68-year-old man who presented with chest discomfort after a 12-day course of antibiotic treatment for bacteremia. Following coronary angiography, echocardiography, and enhanced computed tomography, he was diagnosed with a right coronary artery pseudoaneurysm accompanied with perforation of the right ventricle. Severe adhesions were observed during emergency surgery surrounding the entire heart. The patient presented with risk factors for coronary artery disease, including hypertension and smoking history. His coronary artery was severely calcified due to end-stage renal failure requiring dialysis; thus, a covered stent could not fit inside the arterial lumen. Consequently, coronary artery bypass grafting to the right coronary artery and right ventricle repair were performed. Unfortunately, the patient died postoperatively due to sepsis from intestinal translocation. This rare development was hypothesized to be an incidental result of the combination of severe post-inflammatory adhesions, extensive coronary artery calcification, and rupture of the calcification crevices. CONCLUSIONS: In the case of a severe post-inflammatory response, shock without cardiac tamponade may require further scrutiny by assuming the possibility of inward rupture. For patients in poor condition, two-stage surgical treatment might be considered after stabilization with a covered stent.

Construction of a screening system for lipid-derived radical inhibitors and validation of hit compounds to target retinal and cerebrovascular diseases.

Recent studies have highlighted the indispensable role of oxidized lipids in inflammatory responses, cell death, and disease pathogenesis. Consequently, inhibitors targeting oxidized lipids, particularly lipid-derived radicals critical in lipid peroxidation, which are known as radical-trapping antioxidants (RTAs), have been actively pursued. We focused our investigation on nitroxide compounds that have rapid second-order reaction rate constants for reaction with lipid-derived radicals. A novel screening system was developed by employing competitive reactions between library compounds and a newly developed profluorescence nitroxide probe with lipid-derived radicals to identify RTA compounds. A PubMed search of the top hit compounds revealed their wide application as repositioned drugs. Notably, the inhibitory efficacy of methyldopa, selected from these compounds, against retinal damage and bilateral common carotid artery stenosis was confirmed in animal models. These findings underscore the efficacy of our screening system and suggest that it is an effective approach for the discovery of RTA compounds.

Mitophagy mediated by BNIP3 and NIX protects against ferroptosis by downregulating mitochondrial reactive oxygen species.

Mitophagy plays an important role in the maintenance of mitochondrial homeostasis and can be categorized into two types: ubiquitin-mediated and receptor-mediated pathways. During receptor-mediated mitophagy, mitophagy receptors facilitate mitophagy by tethering the isolation membrane to mitochondria. Although at least five outer mitochondrial membrane proteins have been identified as mitophagy receptors, their individual contribution and interrelationship remain unclear. Here, we show that HeLa cells lacking BNIP3 and NIX, two of the five receptors, exhibit a complete loss of mitophagy in various conditions. Conversely, cells deficient in the other three receptors show normal mitophagy. Using BNIP3/NIX double knockout (DKO) cells as a model, we reveal that mitophagy deficiency elevates mitochondrial reactive oxygen species (mtROS), which leads to activation of the Nrf2 antioxidant pathway. Notably, BNIP3/NIX DKO cells are highly sensitive to ferroptosis when Nrf2-driven antioxidant enzymes are compromised. Moreover, the sensitivity of BNIP3/NIX DKO cells is fully rescued upon the introduction of wild-type BNIP3 and NIX, but not the mutant forms incapable of facilitating mitophagy. Consequently, our results demonstrate that BNIP3 and NIX-mediated mitophagy plays a role in regulating mtROS levels and protects cells from ferroptosis.

Ethoxyquin, a Lipid Peroxidation Inhibitor, Has Protective Effects against White Matter Lesions in a Mouse Model of Chronic Cerebral Hypoperfusion.

White matter lesions induced by chronic cerebral hypoperfusion can cause vascular dementia; however, no appropriate treatments are currently available for these diseases. In this study, we investigated lipid peroxidation, which has recently been pointed out to be associated with cerebrovascular disease and vascular dementia, as a therapeutic target for chronic cerebral hypoperfusion. We used ethoxyquin, a lipid-soluble antioxidant, in a neuronal cell line and mouse model of the disease. The cytoprotective effect of ethoxyquin on glutamate-stimulated HT-22 cells, a mouse hippocampal cell line, was comparable to that of a ferroptosis inhibitor. In addition, the administration of ethoxyquin to bilateral common carotid artery stenosis model mice suppressed white matter lesions, blood-brain barrier disruption, and glial cell activation. Taken together, we propose that the inhibition of lipid peroxidation may be a useful therapeutic approach for chronic cerebrovascular disease and the resulting white matter lesions.

MRP1-Dependent Extracellular Release of Glutathione Induces Cardiomyocyte Ferroptosis After Ischemia-Reperfusion.

BACKGROUND: The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential for maintaining cellular functions. However, the relationship between disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. We investigated the mechanisms underlying the disruption of the glutathione-mediated reduction system related to ferroptosis during IR and developed intervention strategies to suppress ferroptosis. METHODS: In vivo fluctuations of both intra- and extracellular metabolite levels during IR were explored via microdialysis and tissue metabolome analysis. Oxidized phosphatidylcholines were assessed using liquid chromatography high-resolution mass spectrometry. The areas at risk following IR were assessed using triphenyl-tetrazolium chloride/Evans blue stain. RESULTS: Metabolomic analysis combined with microdialysis revealed a significant release of glutathione from the ischemic region into extracellular spaces during ischemia and after reperfusion. The release of glutathione into extracellular spaces and a concomitant decrease in intracellular glutathione concentrations were also observed during anoxia-reperfusion in an in vitro cardiomyocyte model. This extracellular glutathione release was prevented by chemical inhibition or genetic suppression of glutathione transporters, mainly MRP1 (multidrug resistance protein 1). Treatment with MRP1 inhibitor reduced the intracellular reactive oxygen species levels and lipid peroxidation, thereby inhibiting cell death. Subsequent in vivo evaluation of endogenously oxidized phospholipids following IR demonstrated the involvement of ferroptosis, as levels of multiple oxidized phosphatidylcholines were significantly elevated in the ischemic region 12 hours after reperfusion. Inhibition of the MRP1 transporter also alleviated intracellular glutathione depletion in vivo and significantly reduced the generation of oxidized phosphatidylcholines. Administration of MRP1 inhibitors significantly attenuated infarct size after IR injury. CONCLUSIONS: Glutathione was released continuously during IR, primarily in an MRP1-dependent manner, and induced ferroptosis. Suppression of glutathione release attenuated ferroptosis and reduced myocardial infarct size following IR.

Triacetic acid lactone production using 2-pyrone synthase expressing Yarrowia lipolytica via targeted gene deletion.

An environmentally sustainable world can be realized by using microorganisms to produce value-added materials from renewable biomass. Triacetic acid lactone (TAL) is a high-value-added compound that is used as a precursor of various organic compounds such as food additives and pharmaceuticals. In this study, we used metabolic engineering to produce TAL from glucose using an oleaginous yeast Yarrowia lipolytica. We first introduced TAL-producing gene 2-pyrone synthase into Y. lipolytica, which enabled TAL production. Next, we increased TAL production by engineering acetyl-CoA and malonyl-CoA biosynthesis pathways by redirecting carbon flux to glycolysis. Finally, we optimized the carbon and nitrogen ratios in the medium, culminating in the production of 4078 mg/L TAL. The strategy presented in this study had the potential to improve the titer and yield of polyketide biosynthesis.

Structural Analysis of Intracellular Lipid Radicals by LC/MS/MS Using a BODIPY-Based Profluorescent Nitroxide Probe.

Free radical-mediated lipid peroxidation (LPO) induces the formation of numerous lipid radicals, which contribute to the development of several oxidative diseases. To understand the mechanism of LPO in biological systems and the significance of these radicals, identifying the structures of individual lipid radicals is imperative. In this study, we developed an analytical method based on liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and a profluorescent nitroxide probe, N-(1-oxyl-2,2,6-trimethyl-6-pentylpiperidin-4-yl)-3-(5,5-difluoro-1,3-dimethyl-3H,5H-5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-7-yl)propanamide (BDP-Pen), for the detailed structural analysis of lipid radicals. The MS/MS spectra of BDP-Pen-lipid radical adducts showed product ions and thus allow the prediction of the lipid radical structures and individual detection of isomeric adducts. Using the developed technology, we separately detected the isomers of arachidonic acid (AA)-derived radicals generated in AA-treated HT1080 cells. This analytical system is a powerful tool for elucidating the mechanism of LPO in biological systems.

Involvement of Mast-Cell-Tryptase- and Protease-Activated Receptor 2-Mediated Signaling and Urothelial Barrier Dysfunction with Reduced Uroplakin II Expression in Bladder Hyperactivity Induced by Chronic Bladder Ischemia in the Rat.

We aimed to investigate the relationship between mast cell (MC) infiltration into the bladder with urothelial barrier dysfunction and bladder hyperactivity in a chronic bladder ischemia (CBI) rat model. We compared CBI rats (CBI group; n = 10) with normal rats (control group; n = 10). We measured the expression of mast cell tryptase (MCT) and protease-activated receptor 2 (PAR2), which are correlated with C fiber activation via MCT, and Uroplakins (UP Ia, Ib, II and III), which are critical to urothelial barrier function, via Western blotting. The effects of FSLLRY-NH2, a PAR2 antagonist, administered intravenously, on the bladder function of CBI rats were evaluated with a cystometrogram. In the CBI group, the MC number in the bladder was significantly greater (p = 0.03), and the expression of MCT (p = 0.02) and PAR2 (p = 0.02) was significantly increased compared to that of the control group. The 10 µg/kg FSLLRY-NH2 injection significantly increased the micturition interval of CBI rats (p = 0.03). The percentage of UP-II-positive cells on the urothelium with immunohistochemical staining was significantly lower in the CBI group than in the control group (p < 0.01). Chronic ischemia induces urothelial barrier dysfunction via impairing UP II, consequently inducing MC infiltration into the bladder wall and increased PAR2 expression. PAR2 activation by MCT may contribute to bladder hyperactivity.

Detection and structural analysis of pyrimidine-derived radicals generated on DNA using a profluorescent nitroxide probe.

The oxidative damage of DNA is associated with aging and the development of various diseases. Although nucleoside-derived radicals play an important role in DNA oxidation, their analysis methods are limited. Herein, we propose a fluorometric detection and structural analysis of radicals on the surface of oxidatively damaged DNA using a profluorescent nitroxide probe combined with liquid chromatography-fluorometry and high-resolution tandem mass spectrometry.

[Developments of Profluorescent Nitroxide Probes for Highly Sensitive and Selective Detection of Biological Redox Molecules].

Disruption of the redox balance in vivo is closely involved in the development of various diseases associated with oxidative stress. Therefore, methods for the in vivo analysis of antioxidants and free radicals are essential to elucidate the pathogenic mechanisms of such diseases. Although profluorescent nitroxide probes can be used to evaluate redox molecules with high sensitivity, these probes have low selectivity. Recently, we developed two profluorescent nitroxide probes, 15-((9-(ethylimino)-10-methyl-9Hbenzo[a]phenoxazin-5-yl)amino)-3,11-dioxa-7-azadispiro-hexadecan-7-yloxyl (Nile-DiPy) and 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen), which had high sensitivity and selectivity toward ascorbic acid and lipid-derived radicals, respectively. These probes can react sensitively and selectively to each target molecule and can be used in animal experiments. In this paper, we review the design strategies and application of these profluorescent nitroxide probes.

Structural library and visualization of endogenously oxidized phosphatidylcholines using mass spectrometry-based techniques.

Although oxidized phosphatidylcholines (oxPCs) play critical roles in numerous pathological events, the type and production sites of endogenous oxPCs remain unknown because of the lack of structural information and dedicated analytical methods. Herein, a library of 465 oxPCs is constructed using high-resolution mass spectrometry-based non-targeted analytical methods and employed to detect 70 oxPCs in mice with acetaminophen-induced acute liver failure. We show that doubly oxygenated polyunsaturated fatty acid (PUFA)-PCs (PC PUFA;O2), containing epoxy and hydroxide groups, are generated in the early phase of liver injury. Hybridization with in-vivo 18O labeling and matrix-assisted laser desorption/ionization-tandem MS imaging reveals that PC PUFA;O2 are accumulated in cytochrome P450 2E1-expressing and glutathione-depleted hepatocytes, which are the major sites of liver injury. The developed library and visualization methodology should facilitate the characterization of specific lipid peroxidation events and enhance our understanding of their physiological and pathological significance in lipid peroxidation-related diseases.

Usefulness of the mayo adhesive probability score as a predictive factor for renal function deterioration after partial nephrectomy: a retrospective case-control study.

PURPOSE: Whether the Mayo adhesive probability score, an index of the perinephric fat environment, could be a predictive factor for renal function deterioration after partial nephrectomy was investigated. METHODS: A retrospective case-control study of 78 patients who underwent laparoscopic partial nephrectomy was performed. An estimated glomerular filtration rate preservation rate at ≤ 90% at 3 months after surgery was defined as postoperative renal function deterioration. These patients were divided into two groups (non-deterioration and deterioration groups). Patient factors including Mayo adhesive probability scores (both tumor and unaffected sides) and surgical factors were evaluated to identify the predictors for postoperative renal function deterioration. The statistical analysis used univariate and multivariate logistic regression analyses. RESULTS: Thirty-seven (47.4%) patients had postoperative renal function deterioration after partial nephrectomy. Univariate analysis identified Mayo adhesive probability score on the unaffected side (p = 0.02), and warm ischemia time (p < 0.01) as predictors of postoperative renal function deterioration. On multivariate analyses, Mayo adhesive probability score on the unaffected side (odds ratio: 1.38 [1.05-1.79], p = 0.02) and warm ischemia time (odds ratio: 1.04 [1.01-1.07], p < 0.01) were significantly associated with postoperative renal function deterioration as same as univariate analysis. On receive operating characteristic curve analysis, Mayo adhesive probability score on the unaffected side (cutoff value 1.5; p = 0.02) and warm ischemia time (cutoff value 26.5 min; p = 0.01) were significant predictors of renal function deterioration 3 month after surgery. CONCLUSION: The Mayo adhesive probability score on the unaffected side and warm ischemia time are useful predictors for renal function deterioration after partial nephrectomy. TRIAL REGISTRATION NUMBER: 2019-249, January 21st, 2019, retrospectively registered.

Coronavirus disease (COVID-19) associated delayed-onset fulminant myocarditis in patient with a history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

A healthy 35-year-old man was admitted to a rural hospital with coronavirus disease (COVID-19). During 14 days of hospitalization, he had no symptoms and was not given supplemental oxygen. About 3 weeks after discharge, he was re-admitted to the same hospital with new-onset continuous fever and general weakness. At the time of his second admission, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RT-PCR was performed on a retro-nasal swab and the result was negative. Four days after admission, the patient was transferred to our intensive care unit (ICU) following deterioration of his respiratory and haemodynamic conditions, where he received mechanical ventilation, intra-aortic balloon pumping, and veno-arterial extracorporeal membrane oxygenation. A nasopharyngeal swab was obtained again at ICU admission, but RT-PCR was negative for SARS-CoV-2. All antibody titres measured against other viruses were low. Blood cultures were negative, and no bacteria were observed in sputum samples. However, SARS-CoV-2 RNA was detected by RT-PCR from sections obtained by myocardial biopsy. The patient's final diagnosis was delayed-onset SARS-CoV-2-induced fulminant myocarditis (FM). We strongly suggested that one of the proposed mechanisms of COVID-19-related myocardial injury will be the direct invasion of SARS-CoV-2 into cardiomyocytes even if delayed-onset. And this is the first case of delayed-onset FM in which diagnosis of active myocarditis was proven by pathological examination following endomyocardial biopsy and SARS-CoV-2 was detected in the myocardium by RT-PCR.

Hepatic resistance to cold ferroptosis in a mammalian hibernator Syrian hamster depends on effective storage of diet-derived α-tocopherol.

Mammalian hibernators endure severe and prolonged hypothermia that is lethal to non-hibernators, including humans and mice. The mechanisms responsible for the cold resistance remain poorly understood. Here, we found that hepatocytes from a mammalian hibernator, the Syrian hamster, exhibited remarkable resistance to prolonged cold culture, whereas murine hepatocytes underwent cold-induced cell death that fulfills the hallmarks of ferroptosis such as necrotic morphology, lipid peroxidation and prevention by an iron chelator. Unexpectedly, hepatocytes from Syrian hamsters exerted resistance to cold- and drug-induced ferroptosis in a diet-dependent manner, with the aid of their superior ability to retain dietary α-tocopherol (αT), a vitamin E analog, in the liver and blood compared with those of mice. The liver phospholipid composition is less susceptible to peroxidation in Syrian hamsters than in mice. Altogether, the cold resistance of the hibernator's liver is established by the ability to utilize αT effectively to prevent lipid peroxidation and ferroptosis.

Upregulation of glucocorticoid receptor-mediated glucose transporter 4 in enzalutamide-resistant prostate cancer.

Enzalutamide (Enz) is a second-generation androgen receptor (AR) antagonist for castration-resistant prostate cancer (CRPC) therapy, and it prolongs survival time in these patients. However, during Enz treatment, CRPC patients usually acquire resistance to Enz and often show cross-resistance to other AR signaling inhibitors. Although glucocorticoid receptor (GR) is involved in this resistance, the role of GR has not yet been clarified. Here, we report that chronic Enz treatment induced GR-mediated glucose transporter 4 (GLUT4) upregulation, and that upregulation was associated with resistance to Enz and other AR signaling inhibitors. Additionally, inhibition of GLUT4 suppressed cell proliferation in Enz-resistant prostate cancer cells, which recovered from Enz resistance and cross-resistance without changes in GR expression. Thus, a combination of Enz and a GLUT4 inhibitor could be useful in Enz-resistant CRPC patients.

Detection and structural analysis of lipid-derived radicals in vitro and in vivo.

Lipids can be oxidized by reactive oxygen species, resulting in lipid peroxidation and the formation of reactive metabolites such as lipid-derived electrophiles. These products have been reported to induce inflammation, angiogenesis, and ferroptosis. Lipid peroxidation can produce many different products, each of which performs a different function, and which can be challenging to detect in vivo. The initial products of lipid oxidation are lipid-derived radicals, which can cause extensive chain reactions leading to lipid peroxidation. Hence, the ability to detect lipid radicals may provide information about this important class of molecules and the mechanism by which they cause cellular and tissue damage in a wide range of oxidative conditions. In this review, we report recent scientific advances in the detection of lipid-derived radicals in vitro and in cultured cells. We also introduce the possibility of visualization and structural analysis of lipid-derived radicals generated not only in in cells but also in animal tissue samples from oxidative disease models, using fluorescence-based lipid radicals' detection probes. We anticipate that the various innovative techniques summarized in this paper will be applied and further developed to clarify the role of lipid peroxidation in the pathogenesis of oxidative stress-associated diseases.

A radioiodinated nitroxide probe with improved stability against bioreduction for in vivo detection of lipid radicals.

It is well known that lipid carbon radicals (lipid radicals) are the origin of lipid peroxidation and are involved in various diseases such as cancer. Therefore, the in vivo detection of lipid radicals would be expected to lead to early diagnosis of these diseases. However, there are no methods for measuring lipid radicals in vivo. Nitroxides are known to be highly reactive with lipid radicals, but they tend to be reduced in vivo. Focusing on the excellent detection sensitivity of nuclear medical imaging, we have developed a radioiodinated nitroxide derivative with resistance to bioreduction for the in vivo detection of lipid radicals. The desired compound was obtained successfully and was highly stable against bioreduction while maintaining high reactivity toward lipid radicals. The I-125 labeling was efficacious with radiochemical yields of 84-87% and radiochemical purities of >99%. A cellular uptake assay showed that the radioiodinated compound was significantly taken up by cells under lipid radical-producing conditions compared to that in the absence of lipid radical production. A biodistribution study indicated that the radioiodinated compound accumulated more in organs where lipid peroxidation was promoted than the methoxyamine derivative, which lost reactivity to lipid radicals. These results indicated that the developed probe became trapped in cells or organs by reacting with lipid radicals. Thus, the radioiodinated nitroxide is a candidate probe for in vivo detection of lipid radicals.

Method for Structural Determination of Lipid-Derived Radicals.

Diversified oxidized-lipid molecules are responsible for inflammation and cell death, including ferroptosis. Lipid radicals are the source of these oxidized lipids, which are the initial key molecules in the lipid peroxidation chain reaction. However, owing to their extremely high reactivity and short half-life, an established detection technique is not available. Here, we propose a high-performance liquid chromatography fluorometry and high-resolution tandem mass spectrometry system combined with a fluorescent probe as a structural analysis method for lipid-derived radicals. We detected 132 lipid-derived radicals, including 111 new species, from five polyunsaturated fatty acids. In addition, a database was constructed for which the initial fatty acid could be determined using the radical structure. Further, 12 endogenous lipid-derived radicals were identified in carcinogen-induced liver cancer mouse models. Therefore, this method and its corresponding database will provide novel insights into mechanisms underlying the lipid peroxidation, including the associated inflammation and ferroptosis.

Reaction targets of antioxidants in azo-initiator or lipid hydroperoxide induced lipid peroxidation.

Lipid peroxidation (LPO) is reported to be involved in the pathogenesis of several oxidative diseases, and several therapeutic approaches using antioxidants have been proposed. LPO is thought to progress via a complicated series of multistep reactions suggesting that the activity of each antioxidant may be different, and depends on the reacting molecules. Hence, in this study, we evaluated the inhibitory mechanisms of several antioxidants toward arachidonic acid (AA) peroxidation induced by the azo initiator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) or a lipid hydroperoxide, hydroperoxyoctadecadienoic acid (HpODE)/hemin. Edaravone, ferrostatin-1, TEMPO and trolox effectively inhibited the production of malondialdehyde (MDA) and several oxidised AAs generated in the AAPH-induced LPO because of their scavenging ability toward lipid peroxyl radicals. In contrast, ebselen and ferrostatin-1 showed strong antioxidative activity in the HpODE/hemin-induced peroxidation. Under this condition, ebselen and ferrostatin-1 were thought to reduce HpODE and its derived alkoxyl radicals to the corresponding lipid alcohols. In conclusion, we found that each antioxidant had different antioxidative activities that prevented the progression of LPO. We expect that these findings will contribute to the design of novel therapeutic strategies using an appropriate antioxidant targeted to each step of the development of oxidative stress diseases.