HMOX1 regulates ferroptosis via mic14 and its impact on chemotherapy resistance in small-cell lung cancer.

This study aimed to investigate the role and molecular mechanism of heme oxygenase-1 (HMOX1) in chemotherapy resistance in small-cell lung cancer (SCLC). Employed bioinformatics, qPCR, and Western Blot to assess HMOX1 levels in SCLC versus normal tissues and its prognostic relevance. CCK-8, flow cytometry, and thiobarbituric acid assays determined HMOX1's impact on SCLC chemosensitivity, ferroptosis markers, lipid peroxidation, and mic14's role in chemoresistance. In the GSE40275 and GSE60052 cohorts, HMOX1 expression was downregulated in SCLC tissues compared to normal tissues. Higher HMOX1 expression was associated with improved prognosis in the Sun Yat-sen University Cancer Hospital cohort and GSE60052 cohort. The RNA and protein levels of HMOX1 were reduced in drug-resistant SCLC cell lines compared to chemosensitive cell lines. Upregulation of HMOX1 increased chemosensitivity and reduced drug resistance in SCLC, while downregulation of HMOX1 decreased chemosensitivity and increased drug resistance. Upregulation of HMOX1 elevated the expression of ferroptosis-related proteins ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while decreasing the expression of GPX4 and xCT. Conversely, downregulation of HMOX1 decreased the expression of ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while increasing the expression of GPX4 and xCT. Upregulation of HMOX1 promoted cellular lipid peroxidation, whereas downregulation of HMOX1 inhibited cellular lipid peroxidation. Upregulation of HMOX1 reduced the RNA level of mic14, while downregulation of HMOX1 increased the RNA level of mic14. mic14 exhibited inhibitory effects on cellular lipid peroxidation in SCLC cells and contributed to reduced chemosensitivity and increased drug resistance in chemoresistant SCLC cell lines. HMOX1 plays a role in ferroptosis by regulating mic14 expression, thereby reversing chemoresistance in SCLC.

Protein Coronas Derived from Mucus Act as Both Spear and Shield to Regulate Transferrin Functionalized Nanoparticle Transcellular Transport in Enterocytes.

The epithelial mucosa is a key biological barrier faced by gastrointestinal, intraoral, intranasal, ocular, and vaginal drug delivery. Ligand-modified nanoparticles demonstrate excellent ability on this process, but their efficacy is diminished by the formation of protein coronas (PCs) when they interact with biological matrices. PCs are broadly implicated in affecting the fate of NPs in vivo and in vitro, yet few studies have investigated PCs formed during interactions of NPs with the epithelial mucosa, especially mucus. In this study, we constructed transferrin modified NPs (Tf-NPs) as a model and explored the mechanisms and effects that epithelial mucosa had on PCs formation and the subsequent impact on the transcellular transport of Tf-NPs. In mucus-secreting cells, Tf-NPs adsorbed more proteins from the mucus layers, which masked, displaced, and dampened the active targeting effects of Tf-NPs, thereby weakening endocytosis and transcellular transport efficiencies. In mucus-free cells, Tf-NPs adsorbed more proteins during intracellular trafficking, which enhanced transcytosis related functions. Inspired by soft coronas and artificial biomimetic membranes, we used mucin as an "active PC" to precoat Tf-NPs (M@Tf-NPs), which limited the negative impacts of "passive PCs" formed during interface with the epithelial mucosa and improved favorable routes of endocytosis. M@Tf-NPs adsorbed more proteins associated with endoplasmic reticulum-Golgi functions, prompting enhanced intracellular transport and exocytosis. In summary, mucus shielded against the absorption of Tf-NPs, but also could be employed as a spear to break through the epithelial mucosa barrier. These findings offer a theoretical foundation and design platform to enhance the efficiency of oral-administered nanomedicines.

Iron Deficiency in Chronic Pediatric Heart Failure: Overall Assessment and Outcomes in Dilated Cardiomyopathy.

OBJECTIVE: To evaluate the frequency of iron status assessment in pediatric heart failure and the prevalence and adverse effects of absolute iron deficiency in dilated cardiomyopathy-induced heart failure. STUDY DESIGN: We retrospectively reviewed records of children with chronic heart failure at our center between 2010 and 2020. In children with dilated cardiomyopathy, we analyzed baseline cardiac function, hemoglobin level, and subsequent risk of composite adverse events (CAE), including death, heart transplant, ventricular assist device (VAD) placement, and transplant registry listing. Absolute iron deficiency and iron sufficiency were defined as transferrin saturations <20% and ≥30%, respectively; and indeterminant iron status as 20%-29%. RESULTS: Of 799 patients with chronic heart failure, 471 (59%) had no iron-related laboratory measurements. Of 68 children with dilated cardiomyopathy, baseline transferrin saturation, and quantitative left ventricular ejection fraction (LVEF), 33 (49%) and 14 (21%) were iron deficient and sufficient, respectively, and 21 (31%) indeterminant. LVEF was reduced to 23.6 ± 12.1% from 32.9 ± 16.8% in iron deficiency and sufficiency, respectively (P = .04), without a significant difference in hemoglobin. After stratification by New York Heart Association classification, in advanced class IV, hemoglobin was reduced to 10.9 ± 1.3 g/dL vs 12.7 ± 2.0 g/dL in iron deficiency and sufficiency, respectively (P = .01), without a significant difference in LVEF. CONCLUSIONS: In this single-center study, iron deficiency was not monitored in most children with chronic heart failure. In pediatric dilated cardiomyopathy-induced heart failure, absolute iron deficiency was prevalent and associated with clinically consequential and possibly correctable decreases in cardiac function and hemoglobin concentration.

Glutathione S-transferase zeta 1 alters the HMGB1/GPX4 axis to drive ferroptosis in bladder cancer cells.

OBJECTIVE: The ability of glutathione S-transferase zeta 1 (GSTZ1) to modulate homeostasis of cellular redox and induce ferroptosis was explored in bladder cancer cells, and the involvement of the high mobility group protein 1/glutathione peroxidase 4 (HMGB1/GPX4) in these effects was studied. METHODS: BIU-87 cells stably overexpressing GSTZ1 were transfected with appropriate plasmids to deplete HMGB1 or overexpress GPX4, then treated with deferoxamine and ferrostatin-1. Antiproliferative effects were assessed by quantifying levels of ferroptosis markers, such as iron, glutathione (GSH), malondialdehyde (MDA), reactive oxygen species (ROS), GPX4, transferrin, and ferritin. RESULTS: GSTZ1 was significantly downregulated in bladder cancer cells. GSTZ1 overexpression downregulated GPX4 and GSH, while greatly increasing levels of iron, MDA, ROS, and transferrin. GSTZ1 overexpression also decreased proliferation of BIU-87 cells and activated HMGB1/GPX4 signaling. The effects of GSTZ1 on ferroptosis and proliferation were antagonized by HMGB1 knockdown or GPX4 overexpression. CONCLUSION: GSTZ1 induces ferroptotic cell death and alters cellular redox homeostasis in bladder cancer cells, and these effects involve activation of the HMGB1/GPX4 axis.

A Novel Triple Combination To Combat Serious Infections with Carbapenem-Resistant Acinetobacter baumannii in a Mouse Pneumonia Model.

The ongoing crisis of antimicrobial resistance demands novel combinations between antimicrobials and nonantimicrobials to manage infections caused by highly resistant pathogens. This study aimed to evaluate the effect of combining sodium ascorbate and/or apo-transferrin with imipenem, forming double and triple combinations, against 20 multiple-carbapenemase-producing Acinetobacter baumannii strains using the checkerboard test, time-kill assay, and disc diffusion test. The results of the checkerboard assay revealed that all double combinations showed indifference, while only triple combination recorded a synergistic effect (fractional inhibitory concentration index [FICI] < 0.8) in 95% the test isolates. Moreover, the MIC of imipenem (MICimp) was strongly reduced (up to 128-fold reduction) after treatment with the triple combination against highly resistant isolates and reached the susceptible range. The time-kill assay revealed that the triple combination led to a 4-log10 reduction in the CFU at 8 h compared with the initial bacterial count, and no viable count was recorded at 10 h. The mouse pneumonia model showed restoration of lung function and structure, with mild to moderate residual inflammation and moderately congested vessels observed 8 h following administration of the triple rescue therapy. Additionally, normal lungs with normal patent alveoli were detected 72 h following treatment. Accordingly, sodium ascorbate and apo-transferrin are promising adjunct biological agents with the potential to restore the effectiveness of critically essential antibiotics like imipenem, commonly used for the treatment of A. baumannii infections. IMPORTANCE Combination therapy provides a perspective to threat multidrug-resistant (MDR) strains. The present study sheds light on a novel and effective triple combination against carbapenem-resistant A. baumannii. Our in vitro results showed that combining imipenem with apo-transferrin and sodium ascorbate yielded synergism in 95% of test isolates, and this was associated with a marked reduction in imipenem MIC, shifting it below the breakpoint. Furthermore, a bactericidal effect was recorded, with no viable count detected at 10 h. An in vivo murine model of pneumonia was induced to mimic human disease. The triple combination therapy restored lung function and structure, with mild to moderate residual inflammation and moderately congested vessels observed 8 h following the initiation of therapy. Therefore, our findings suggest novel insights about a promising new combination therapy against highly resistant carbapenemase-producing A. baumannii to restore the effectiveness of imipenem.

Iron Deficiency in Heart Failure and Effect of Dapagliflozin: Findings From DAPA-HF.

BACKGROUND: Iron deficiency is common in heart failure and associated with worse outcomes. We examined the prevalence and consequences of iron deficiency in the DAPA-HF trial (Dapagliflozin and Prevention of Adverse-Outcomes in Heart Failure) and the effect of dapagliflozin on markers of iron metabolism. We also analyzed the effect of dapagliflozin on outcomes, according to iron status at baseline. METHODS: Iron deficiency was defined as a ferritin level <100 ng/mL or a transferrin saturation <20% and a ferritin level 100 to 299 ng/mL. Additional biomarkers of iron metabolism, including soluble transferrin receptor, erythropoietin, and hepcidin were measured at baseline and 12 months after randomization. The primary outcome was a composite of worsening heart failure (hospitalization or urgent visit requiring intravenous therapy) or cardiovascular death. RESULTS: Of the 4744 patients randomized in DAPA-HF, 3009 had ferritin and transferrin saturation measurements available at baseline, and 1314 of these participants (43.7%) were iron deficient. The rate of the primary outcome was higher in patients with iron deficiency (16.6 per 100 person-years) compared with those without (10.4 per 100 person-years; P<0.0001). The effect of dapagliflozin on the primary outcome was consistent in iron-deficient compared with iron-replete patients (hazard ratio, 0.74 [95% CI, 0.58-0.92] versus 0.81 [95% CI, 0.63-1.03]; P-interaction=0.59). Similar findings were observed for cardiovascular death, heart failure hospitalization, and all-cause mortality. Transferrin saturation, ferritin, and hepcidin were reduced and total iron-binding capacity and soluble transferrin receptor increased with dapagliflozin compared with placebo. CONCLUSIONS: Iron deficiency was common in DAPA-HF and associated with worse outcomes. Dapagliflozin appeared to increase iron use but improved outcomes, irrespective of iron status at baseline. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03036124.

Differential effects of high-altitude exposure on markers of oxidative stress, antioxidant capacity, and iron profiles.

High-altitude (HA) exposure may stimulate significant physiological and molecular changes, resulting in HA-related illnesses. HA may impact oxidative stress, antioxidant capacity, and iron homeostasis, yet it is unclear how both repeated exposure and HA acclimatization may modulate such effects. Therefore, we assessed the effects of weeklong repeated daily HA exposure (2,900-5,050 m) in altitude-naïve individuals (n = 21 individuals, 13 females, mean ± SD, 25.3 ± 3.7 yr) to mirror the working schedule of HA workers (n = 19 individuals, all males, 41.1 ± 9.4 yr) at the Atacama Large Millimeter Array (ALMA) Observatory (San Pedro de Atacama, Chile). Markers of oxidative stress, antioxidant capacity, and iron homeostasis were measured in blood plasma. Levels of protein oxidation (P < 0.001) and catalase activity (P = 0.023) increased and serum iron (P < 0.001), serum ferritin (P < 0.001), and transferrin saturation (P < 0.001) levels decreased with HA exposure in both groups. HA workers had lower levels of oxidative stress, and higher levels of antioxidant capacity, iron supply, and hemoglobin concentration as compared with altitude-naïve individuals. On a second week of daily HA exposure, changes in levels of protein oxidation, glutathione peroxidase, and nitric oxide metabolites were lower as compared with the first week in altitude-naïve individuals. These results indicate that repeated exposure to HA may significantly alter oxidative stress and iron homeostasis, and the degree of such changes may be dependent on if HA is visited naïvely or routinely. Further studies are required to fully elucidate differences in HA-induced changes in oxidative stress and iron homeostasis profiles among visitors of HA.

Self-recovery study of fluoride-induced ferroptosis in the liver of zebrafish (Danio rerio).

Ferroptosis plays a key role in fluorosis in aquatic organisms, but whether it is involved in fluoride-induced liver damage remains unclear. Previous studies have indicated that fluoride toxicity has the reversible tendency, but the mechanism of self-recovery after fluorosis in aquatic animals has not been elucidated. In this study, adult zebrafish and embryos were exposed to 0, 20, 40, 80 mg/L of fluoride for 30, 60 and 90 d and 3, 4 and 5 d post-fertilization (dpf), respectively. After 90 d, adult zebrafish were transferred to clean water for self-recovery of 30 d. The results showed that fluoride induced the prominent histopathologial changes in liver of adults, and the developmental delay and dark liver area in larvae. Fluoride significantly increased the iron overload, while decreased the expression levels of transferrin (tf), transferrin receptor (tfr), ferroportin (fpn), membrane iron transporter (fpn), and ferritin heavy chain (fth) in adults and larvae. Fluoride also induced the oxidative stress in adults and larvae by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), while decreasing the glutathione (GSH) content and the levels of glutathione peroxidase 4 (gpx4) and solute carrier family 7 member 11 (slc7a11). Self-recovery relieved fluoride-induced ferroptosis by reducing the histopathological damage and oxidative stress, reversing the expression levels of fth and slc7a11, Fe2+ metabolism and GSH synthesis. Lipid peroxidation and Fe2+ metabolism may be the key factor in alleviating effects of self-recovery on fluoride toxicity. Moreover, males are more sensitive than females. Our results provide a theoretical basis for studying the alleviating effects of self-recovery on fluoride toxicity and the underlying mechanism of its protective effect.

Lycopene supplementation to serum-free embryo culture medium and its effect on development and quality of bovine blastocysts produced in vitro.

Selenium is commonly used as an antioxidant in a serum-free culture medium setting. However, lycopene has emerged as a potent antioxidant being twice as efficient as ß-carotene and 10 times as efficient as α-tocopherol with beneficial effects when supplemented in a serum-free maturation medium. Here, we aimed to evaluate the effect of lycopene supplementation in a serum-free culture medium on blastocyst development and quality. After in vitro maturation and fertilization, presumed zygotes were cultured in groups of 25 in 50 µl droplets of synthetic oviductal fluid. Culture medium supplementation was done using four experimental groups: insulin, transferrin, selenium (ITS, control); ITS + DMSO (diluent control); ITS + DMSO-lycopene 0.1 µM (ITSL); and IT + DMSO-lycopene 0.1 µM (ITL). DMSO was used as a diluent for lycopene. Blastocyst development among experimental groups was fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. The cleavage (85.3 ± 2.4, 82.6 ± 2.7, 86 ± 2.3 and 86.4 ± 2.3% for control, diluent control, ITSL and ITL, respectively) and day 8 blastocyst rates (37.4 ± 3.3, 36.9 ± 3.4, 39.7 ± 3.3 and 46.2 ± 3.4% for control, diluent control, ITSL and ITL, respectively) were not different (p > .1) among experimental groups. Embryos produced in the ITL group resulted in blastocysts with higher total cell numbers (TCN; 141 ± 19.2), inner cell mass (ICM; 65.3 ± 11.6) and trophectoderm cells (TE; 75.2 ± 8.8) compared with the control (129 ± 19.2, 56.3 ± 11.6 and 72.7 ± 8.8, for TCN, ICM and TE; p < .01, respectively). Lycopene-supplemented groups (ITSL and ITL) resulted in blastocysts with similar TCN, ICM and TE (p > .2). The number of apoptotic cells was not different among experimental groups (p > .1). Lycopene supplementation to the culture medium only produced a numerical increase in the blastocyst rate but replacing selenium with lycopene in a serum-free culture medium resulted in blastocysts with more cells.

The Interaction of N-Acetylcysteine and Serum Transferrin Promotes Bacterial Biofilm Formation.

BACKGROUND/AIMS: N-acetylcysteine (NAC) is a novel and promising agent with activity against bacterial biofilms. Human serum also inhibits biofilm formation by some bacteria. We tested whether the combination of NAC and human serum offers greater anti-biofilm activity than either agent alone. METHODS: Microtiter plate assays and confocal laser scanning microscopy were used to evaluate bacterial biofilm formation in the presence of NAC and human serum. qPCR was used to examine expression of selected biofilm-associated genes. Extracellular matrix (ECM) was observed by transmission electron microscopy. The antioxidants GSH or ascorbic acid were used to replace NAC, and human transferrin, lactoferrin, or bovine serum albumin were used to replace serum proteins in biofilm formation assays. A rat central venous catheter model was developed to evaluate the effect of NAC on biofilm formation in vivo. RESULTS: NAC and serum together increased biofilm formation by seven different bacterial strains. In Staphylococcus aureus, expression of genes for some global regulators and for genes in the ica-dependent pathway increased markedly. In Pseudomonas aeruginosa, transcription of las, the PQS quorum sensing (QS) systems, and the two-component system GacS/GacA increased significantly. ECM production by S. aureus and P. aeruginosa was also enhanced. The potentiation of biofilm formation is due mainly to interaction between NAC and transferrin. Intravenous administration of NAC increased colonization by S. aureus and P. aeruginosa on implanted catheters. CONCLUSIONS: NAC used intravenously or in the presence of blood increases bacterial biofilm formation rather than inhibits it.

Selenium Augments microRNA Directed Reprogramming of Fibroblasts to Cardiomyocytes via Nanog.

We have recently shown that a combination of microRNAs, miR combo, can directly reprogram cardiac fibroblasts into functional cardiomyocytes in vitro and in vivo. However, direct reprogramming strategies are inefficient and slow. Moving towards the eventual goal of clinical application it is necessary to develop new methodologies to overcome these limitations. Here, we report the identification of a specific media composition, reprogramming media (RM), which augmented the effect of miR combo by 5-15-fold depending upon the cardiac marker tested. RM alone was sufficient to strongly induce cardiac gene and protein expression in neonatal tail-tip as well as cardiac fibroblasts. Expression of pluripotency markers Nanog, Oct4, Sox2, and Klf4 was significantly enhanced by RM, with miR combo augmenting the effect further. Knockdown of Nanog by siRNA inhibited the effect of RM on cardiac gene expression. Removal of insulin-transferrin-selenium completely inhibited the effect of reprogramming media upon cardiac gene expression and the addition of selenium to standard culture media recapitulated the effects of RM. Moreover, selenium enhanced the reprogramming efficiency of miR combo.

Holo and apo-transferrins interfere with adherence to abiotic surfaces and with adhesion/invasion to HeLa cells in Staphylococcus spp.

Staphylococcus aureus and Staphylococcus epidermidis are the major cause of infections associated with implanted medical devices. Colonization on abiotic and biotic surfaces is often sustained by biofilm forming strains. Human natural defenses can interfere with this virulence factor. We investigated the effect of human apo-transferrin (apo-Tf, the iron-free form of transferrin, Tf) and holo-transferrin (holo-Tf, the iron-saturated form) on biofilm formation by CA-MRSA S. aureus USA300 type (ST8-IV) and S. epidermidis (a clinical isolate and ATCC 35984 strain). Furthermore S. aureus adhesion and invasion assays were performed in a eukaryotic cell line. A strong reduction in biofilm formation with both Tfs was obtained albeit at very different concentrations. In particular, the reduction in biofilm formation was higher with apo-Tf rather than obtained with holo-Tf. Furthermore, while S. aureus adhesion to eukaryotic cells was not appreciably affected, their invasion was highly inhibited in the presence of holo-Tf, and partially inhibited by the apo form. Our results suggest that Tfs could be used as antibacterial adjuvant therapy in infection sustained by staphylococci to strongly reduce their virulence related to adhesion and cellular invasion.