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The ubiquitin-proteasome system (UPS) plays a key role in maintaining cellular protein homeostasis and participates in modulating various cellular functions. Target of rapamycin (TOR), a highly conserved Ser/Thr kinase found across species from yeasts to humans, forms two multi-protein complexes, TORC1 and TORC2, to orchestrate cellular processes crucial for optimal growth, survival, and stress responses. While UPS-mediated regulation of mammalian TOR complexes has been documented, the ubiquitination of yeast TOR complexes remains largely unexplored. Here we report a functional interplay between the UPS and TORC2 in Saccharomyces cerevisiae. Using avo3-2ts, a temperature-sensitive mutant of the essential TORC2 component Avo3 exhibiting TORC2 defects at restrictive temperatures, we obtained evidence for UPS-dependent protein degradation and downregulation of the TORC2 component Avo2. Our results established the involvement of the E3 ubiquitin ligase Ubr1 and its catalytic activity in mediating Avo2 degradation in cells with defective Avo3. Coimmunoprecipitation revealed the interaction between Avo2 and Ubr1, indicating Avo2 as a potential substrate of Ubr1. Furthermore, depleting Ubr1 rescued the growth of avo3-2ts cells at restrictive temperatures, suggesting an essential role of Avo2 in sustaining cell viability under heat stress and/or TORC2 dysfunction. This study uncovers a role of UPS in yeast TORC2 regulation, highlighting the impact of protein degradation control on cellular signaling.

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INTRODUCTION: The clinical presentations of dry eye disease (DED) and depression (DEP) often comanifest. However, the robustness and the mechanisms underlying this association were undetermined. OBJECTIVES: To this end, we set up a three-segment study that employed multimodality results (meta-analysis, genome-wide association study [GWAS] and Mendelian randomization [MR]) to elucidate the association, common pathways and causality between DED and DEP. METHODS: A meta-analysis comprising 26 case-control studies was first conducted to confirm the DED-DEP association. Next, we performed a linkage disequilibrium (LD)-adjusted GWAS and targeted phenotype association study (PheWAS) in East Asian TW Biobank (TWB) and European UK Biobank (UKB) populations. Single-nucleotide polymorphisms (SNPs) were further screened for molecular interactions and common pathways at the functional gene level. To further elucidate the activated pathways in DED and DEP, a systemic transcriptome review was conducted on RNA sequencing samples from the Gene Expression Omnibus. Finally, 48 MR experiments were implemented to examine the bidirectional causation between DED and DEP. RESULTS: Our meta-analysis showed that DED patients are associated with an increased DEP prevalence (OR = 1.83), while DEP patients have a concurrent higher risk of DED (OR = 2.34). Notably, cross-disease GWAS analysis revealed that similar genetic architecture (rG = 0.19) and pleiotropic functional genes contributed to phenotypes in both diseases. Through protein-protein interaction and ontology convergence, we summarized the pleiotropic functional genes under the ontology of immune activation, which was further validated by a transcriptome systemic review. Importantly, the inverse variance-weighted (IVW)-MR experiments in both TWB and UKB populations (p value <0.001) supported the bidirectional exposure-outcome causation for DED-to-DEP and DEP-to-DED. Despite stringent LD-corrected instrumental variable re-selection, the bidirectional causation between DED and DEP remained. CONCLUSION: With the multi-modal evidence combined, we consolidated the association and causation between DED and DEP.

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Gene therapy is an innovative approach in the field of regenerative medicine. This therapy entails the transfer of genetic material into a patient's cells to treat diseases. In particular, gene therapy for neurological diseases has recently achieved significant progress, with numerous studies investigating the use of adeno-associated viruses for the targeted delivery of therapeutic genetic fragments. This approach has potential applications for treating incurable diseases, including paralysis and motor impairment caused by spinal cord injury and Parkinson's disease, and it is characterized by dopaminergic neuron degeneration. Recently, several studies have explored the potential of direct lineage reprogramming (DLR) for treating incurable diseases, and highlighted the advantages of DLR over conventional stem cell therapy. However, application of DLR technology in clinical practice is hindered by its low efficiency compared with cell therapy using stem cell differentiation. To overcome this limitation, researchers have explored various strategies such as the efficiency of DLR. In this study, we focused on innovative strategies, including the use of a nanoporous particle-based gene delivery system to improve the reprogramming efficiency of DLR-induced neurons. We believe that discussing these approaches can facilitate the development of more effective gene therapies for neurological disorders.

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Partial cellular reprogramming via transient expression of Oct4, Sox2, Klf4, and c-Myc induces rejuvenation and reduces aged-cell phenotypes. In this study, we found that transcriptional activation of the endogenous Oct4 gene by using the CRISPR/dCas9 activator system can efficiently ameliorate hallmarks of aging in a mouse model of Hutchinson-Gilford progeria syndrome (HGPS). We observed that the dCas9-Oct4 activator induced epigenetic remodeling, as evidenced by increased H3K9me3 and decreased H4K20me3 levels, without tumorization. Moreover, the progerin accumulation in HGPS aorta was significantly suppressed by the dCas9 activator-mediated Oct4 induction. Importantly, CRISPR/dCas9-activated Oct4 expression rescued the HGPS-associated vascular pathological features and lifespan shortening in the mouse model. These results suggest that partial rejuvenation via CRISPR/dCas9-mediated Oct4 activation can be used as a novel strategy in treating geriatric diseases.

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High-efficiency particulate air (HEPA) filters is a potential tool used to remove fine particles and improve indoor air quality. This study aims to analyze the real-world efficacy of portable HEPA air cleaners in a household environment. Laser light dispersion PM2.5 sensors are used to continuously monitor the indoor and outdoor PM2.5 level before and after HEPA air cleaner filtration. Overall, HEPA air cleaners significantly reduce the indoor PM2.5 level (33.5 ± 10.3 vs. 17.2 ± 10.7 µg/m3, mean difference (MD) = -16.3 µg/m3, p < 0.001) and indoor/outdoor PM2.5% (76.3 ± 16.8 vs. 38.6 ± 19.8%, MD = -37.7%, p < 0.001). The efficacy to reduce PM2.5 is strongest in three machines with medium-flow setting group (indoor PM2.5 MD: -26.5 µg/m3, indoor/outdoor PM2.5 percentage MD: -56.4%). Multiple linear regression demonstrates that outdoor PM2.5, machine number, airflow speed, and window ventilation are significant factors associated with indoor PM2.5 concentrations (R = 0.879) and percentage of the indoor/outdoor PM2.5 ratio (R = 0.808). HEPA air cleaners can effectively improve indoor PM2.5 air pollution. Adequate air cleaner machine numbers, appropriate airflow, and window ventilation limitations are important to achieve the best efficacy of the HEPA air cleaner.

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Non-medical use of ketamine as an adulterant to ecstasy is more prevalent than amphetamine in Taiwan. Ketamine's effect on immunosuppression might play some functional role in tumor growth, while it is still controversial whether ketamine abuse could increase tumor growth or not. This study aimed to investigate the influence of ketamine addiction in breast tumors and related gene expressions. The effect of ketamine treatment on proliferation, colony formation, migration, and invasion of triple-negative breast cancer cell line EO771 was examined. In addition, a ketamine addiction mice model was established by intraperitoneal injection (IP) of ketamine in mice and used to investigate the effects of ketamine addiction on tumor growth and the possible mechanisms. In the in vitro studies, ketamine treatment at different concentrations did not affect EO771 cell proliferation and colony formation. But ketamine did enhance migration and invasion of EO771 cells. The in vivo experiments showed significantly increased breast tumor volume and weight in ketamine-addicted mice than in normal saline groups. miR-27b-3p level, human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor (EGFR) significantly increased in tumors of ketamine addiction mice compared to control mice. In vivo evidence showed that Ketamine might increase tumor growth on the tumor microenvironment, and miR-27b-3p, HER2, and EGFR might play a role in the process.

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The efficient production of dopaminergic neurons via the direct conversion of other cell types is of interest as a potential therapeutic approach for Parkinson's disease. This study aimed to investigate the use of elongated porous gold nanorods (AuNpRs) as an enhancer of cell fate conversion. We observed that AuNpRs promoted the direct conversion of fibroblasts into dopaminergic neurons in vivo and in vitro. The extent of conversion of fibroblasts into dopaminergic neurons depended on the porosity of AuNpRs, as determined by their aspect ratio. The mechanism underlying these results involves specific AuNpR-induced transcriptional changes that altered the expression of antioxidant-related molecules. The generation of dopaminergic neurons via the direct conversion method will open a new avenue for developing a therapeutic platform for Parkinson's disease treatment. STATEMENT OF SIGNIFICANCE: In this study, we applied modified gold nanoporous materials (AuNpRs) to the direct lineage reprogramming of dopaminergic neurons. The cell reprogramming process is energy-intensive, resulting in an excess of oxidative stress. AuNpRs facilitated the direct conversion of dopaminergic neurons by ameliorating oxidative stress during the reprogramming process. We have found this mechanistic clue from high throughput studies in this research work.

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BACKGROUND: The inconsistency in decisions to commence oral feeding indicates that health professionals require clearer guidelines to determine when to initiate oral feeding in preterm infants. This study applied the Taiwan version of Preterm Oral Feeding Readiness Assessment Scale (TW-POFRAS) to clinical decision-making, especially for preterm infants with a birth weight less than 1,500 g or gestational age (GA) less than 32 weeks. METHODS: This was a single-center observational cross-sectional study and 81 preterm infants were recruited. Lengths of stay from admission to initial one-meal oral feeding, to one-day all-meal oral feeding, and to discharge were analyzed. Scale scores, physician orders, and smooth oral intake of 5 mL of milk were analyzed. Kappa coefficients were examined to determine concordances within the results. RESULTS: At least moderate concordance was evident (k = 0.492). Most preterm infants can begin to consume one meal of the least 5 mL of milk smoothly and proceed to consume a full day of meals with a week; they are typically discharged from the hospital within a month, except for those with a birth weight less than 1,500 g or a GA less than 32 weeks. For 17 of 81 participants, assessment results for physician orders, 5-mL milk consumption, and scale scores were inconsistent. Participants with a birth weight less than 1,500 g or GA less than 32 weeks were able to meet the 5-mL standard by the postmenstrual age of 35 weeks, at latest. CONCLUSION: We recommend that TW-POFRAS should be used in conjunction with physicians' clinical decision-making for oral feeding readiness for preterm infants in the NICU.

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Adult neurogenesis is the lifelong process by which new neurons are generated in the dentate gyrus. However, adult neurogenesis capacity decreases with age, and this decrease is closely linked to cognitive and memory decline. Our study demonstrated that electromagnetized gold nanoparticles (AuNPs) promote adult hippocampal neurogenesis, thereby improving cognitive function and memory consolidation in aged mice. According to single-cell RNA sequencing data, the numbers of neural stem cells (NSCs) and neural progenitors were significantly increased by electromagnetized AuNPs. Additionally, electromagnetic stimulation resulted in specific activation of the histone acetyltransferase Kat2a, which led to histone H3K9 acetylation in adult NSCs. Moreover, in vivo electromagnetized AuNP stimulation efficiently increased hippocampal neurogenesis in aged and Hutchinson-Gilford progeria mouse brains, thereby alleviating the symptoms of aging. Therefore, our study provides a proof-of-concept for the in vivo stimulation of hippocampal neurogenesis using electromagnetized AuNPs as a promising therapeutic strategy for the treatment of age-related brain diseases.

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PURPOSE: A successful transition from gavage to full oral feeding is a decisive indicator for discharging premature infants from the neonatal intensive care unit. A clinically useful measure of oral feeding readiness would help nurses initiate implementation of the cue-based feeding model in Taiwan. The study aimed to assess the validity and reliability of the Traditional Chinese Preterm Oral Feeding Readiness Assessment Scale (TC-POFRAS). DESIGN AND METHODS: 81 preterm infants were enrolled and assessed by TC-POFRAS regarding their oral feeding readiness. This study included two phases. Phase 1 conducted a cross language validation procedure and item-level content validity indices (I-CVIs) for content validity were estimated. In phase 2, Cronbach's alpha for internal consistency at each category and total scale levels were estimated. A receiver operating characteristic (ROC) curve was estimated to explore the scale's performance. The optimal cut-off value of TC-POFRAS was identified by the best Youden's Index [maximum (sensitivity + specificity - 1)]. RESULTS: All of the I-CVIs were 1.00. The whole Cronbach's alpha for internal consistency was 0.804 (95% CI = 0.736-0.862), and Cronbach's alpha values were between 0.538 (95% = 0.332-0.689) and 0.687 (95%CI = 0.572-0.781) for categories. The area under ROC was 92.2%, and an optimal cut-off value of TC-POFRAS was 29 (sensitivity: 0.938, specificity: 0.941). CONCLUSIONS: The TC-POFRAS has been verified to be an effective and accurate instrument to determine the initiation of oral feeding in preterm infants. PRACTICE IMPLICATIONS: The TC-POFRAS is an appropriate and complementary assessment instrument for professionals to conveniently use in clinical practice.

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Increased ROS proto-oncogene 1 (ROS1) expression has been implicated in the invasiveness of human oral squamous cell carcinoma (OSCC). The cellular distribution of ROS1 has long-been assumed at the plasma membrane. However, a previous work reported a differential cellular distribution of mutant ROS1 derived from chromosomal translocation, resulting in increased carcinogenesis. We thus hypothesized that cellular distribution of upregulated ROS1 in OSCC may correlate with invasiveness. We found that ROS1 can localize to mitochondria in the highly invasive OSCC and identified a mitochondria-targeting signal sequence in ROS1. We also demonstrated that ROS1 targeting to mitochondria is required for mitochondrial fission phenotype in the highly invasive OSCC cells. OSCC cells expressing high levels of ROS1 consumed more oxygen and had increased levels of cellular ATP levels. Our results also revealed that ROS1 regulates mitochondrial biogenesis and cellular metabolic plasticity. Together, these findings demonstrate that ROS1 targeting to mitochondria enhances OSCC invasion through regulating mitochondrial morphogenesis and cellular respiratory.

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N6-methyladenosine (m6A), a conserved epitranscriptomic modification of eukaryotic mRNA (mRNA), plays a critical role in a variety of biological processes. Here, we report that m6A modification plays a key role in governing direct lineage reprogramming into induced neuronal cells (iNs). We found that m6A modification is required for the remodeling of specific mRNAs required for the neuronal direct conversion. Inhibition of m6A methylation by Mettl3 knockdown decreased the efficiency of direct lineage reprogramming, whereas increased m6A methylation by Mettl3 overexpression increased the efficiency of iN generation. Moreover, we found that transcription factor Btg2 is a functional target of m6A modification for efficient iN generation. Taken together, our results suggest the importance of establishing epitranscriptomic remodeling for the cell fate conversion into iNs.

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Isoplumbagin (5-hydroxy-3-methyl-1,4-naphthoquinone), a naturally occurring quinone from Lawsonia inermis and Plumbago europaea, has been reported to have anti-inflammatory and antimicrobial activity. Inflammation has long been implicated in cancer progression. In this study, we examined the anticancer effect of chemically synthesized isoplumbagin. Our results revealed that isoplumbagin treatment suppressed cell viability and invasion of highly invasive oral squamous cell carcinoma (OSCC) OC3-IV2 cells, glioblastoma U87 cells, non-small cell lung carcinoma H1299 cells, prostate cancer PC3 cells, and cervical cancer HeLa cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Boyden chamber assays. In vivo studies demonstrate the inhibitory effect of 2 mg/kg isoplumbagin on the growth of orthotopic xenograft tumors derived from OSCC cells. Mechanistically, isoplumbagin exerts its cytotoxic effect through acting as a substrate of reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] dehydrogenase quinone 1 (NQO1) to generate hydroquinone, which reverses mitochondrial fission phenotype, reduces mitochondrial complex IV activity, and thus compromises mitochondrial function. Collectively, this work reveals an anticancer activity of isoplumbagin mainly through modulating mitochondrial dynamics and function.

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Neurodegenerative diseases are incurable and debilitating conditions characterized by the deterioration of brain function. Most brain disease models rely on human post-mortem brain tissue, non-human primate tissue, or in vitro two-dimensional (2D) experiments. Resource limitations and the complexity of the human brain are some of the reasons that make suitable human neurodegenerative disease models inaccessible. However, recently developed three-dimensional (3D) brain organoids derived from pluripotent stem cells (PSCs), including embryonic stem cells and induced PSCs, may provide suitable models for the study of the pathological features of neurodegenerative diseases. In this review, we provide an overview of existing 3D brain organoid models and discuss recent advances in organoid technology that have increased our understanding of brain development. Moreover, we explain how 3D organoid models recapitulate aspects of specific neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, and explore the utility of these models, for therapeutic applications.

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Mortality caused by age-related bone fractures or osteoporosis is steadily increasing worldwide as the population ages. The pace of the development of bone regeneration engineering to treat bone fractures has consequently increased in recent years. A range of techniques for bone regeneration, such as immunotherapy, allografts, and hydrogel therapy, have been devised. Cell-based therapies using bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells derived from somatic cells are considered to be suitable approaches for bone repair. However, these cell-based therapies suffer from a number of limitations in terms of efficiency and safety. Somatic cells can also be directly differentiated into osteoblasts by several transcription factors. As osteoblasts play a central role in the process of bone formation, the direct reprogramming of fibroblasts into osteoblasts may hence be a new way to treat bone fractures in elderly individuals. Here, we review recent developments regarding the therapeutic potential of the direct reprogramming of cells for bone regeneration.

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BACKGROUND: With the rapid increase in genome sequencing projects for non-model organisms, numerous genome assemblies are currently in progress or available as drafts, but not made available as satisfactory, usable genomes. Data quality assessment of genome assemblies is gaining importance not only for people who perform the assembly/re-assembly processes, but also for those who attempt to use assemblies as maps in downstream analyses. Recent studies of the quality control, quality evaluation/ assessment of genome assemblies have focused on either quality control of reads before assemblies or evaluation of the assemblies with respect to their contiguity and correctness. However, correctness assessment depends on a reference and is not applicable for de novo assembly projects. Hence, development of methods providing both post-assembly and pre-assembly quality assessment reports for examining the quality/correctness of de novo assemblies and the input reads is worth studying. RESULTS: We present SQUAT, an efficient tool for both pre-assembly and post-assembly quality assessment of de novo genome assemblies. The pre-assembly module of SQUAT computes quality statistics of reads and presents the analysis in a well-designed interface to visualize the distribution of high- and poor-quality reads in a portable HTML report. The post-assembly module of SQUAT provides read mapping analytics in an HTML format. We categorized reads into several groups including uniquely mapped reads, multiply mapped, unmapped reads; for uniquely mapped reads, we further categorized them into perfectly matched, with substitutions, containing clips, and the others. We carefully defined the poorly mapped (PM) reads into several groups to prevent the underestimation of unmapped reads; indeed, a high PM% would be a sign of a poor assembly that requires researchers' attention for further examination or improvements before using the assembly. Finally, we evaluate SQUAT with six datasets, including the genome assemblies for eel, worm, mushroom, and three bacteria. The results show that SQUAT reports provide useful information with details for assessing the quality of assemblies and reads. AVAILABILITY: The SQUAT software with links to both its docker image and the on-line manual is freely available at https://github.com/luke831215/SQUAT .

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In vivo gene editing in post-mitotic neurons of the adult brain may be a useful strategy for treating neurological diseases. Here, we develop CRISPR-Cas9 nanocomplexes and show they were effective in the adult mouse brain, with minimal off-target effects. Using this system to target Bace1 suppressed amyloid beta (Aß)-associated pathologies and cognitive deficits in two mouse models of Alzheimer's disease. These results broaden the potential application of CRISPR-Cas9 systems to neurodegenerative diseases.

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Recent advances in generating three-dimensional (3D) organoid systems from stem cells offer new possibilities for disease modeling and drug screening because organoids can recapitulate aspects of in vivo architecture and physiology. In this study, we generate isogenic 3D midbrain organoids with or without a Parkinson's disease-associated LRRK2 G2019S mutation to study the pathogenic mechanisms associated with LRRK2 mutation. We demonstrate that these organoids can recapitulate the 3D pathological hallmarks observed in patients with LRRK2-associated sporadic Parkinson's disease. Importantly, analysis of the protein-protein interaction network in mutant organoids revealed that TXNIP, a thiol-oxidoreductase, is functionally important in the development of LRRK2-associated Parkinson's disease in a 3D environment. These results provide proof of principle for the utility of 3D organoid-based modeling of sporadic Parkinson's disease in advancing therapeutic discovery.

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The reprogramming of induced cardiomyocytes (iCMs) has shown potential in regenerative medicine. However, in vivo reprogramming of iCMs is significantly inefficient, and novel gene delivery systems are required to more efficiently and safely induce in vivo reprogramming of iCMs for therapeutic applications in heart injury. In this study, we show that cationic gold nanoparticles (AuNPs) loaded with Gata4, Mef2c, and Tbx5 function as nanocarriers for cardiac reprogramming. The AuNP/GMT/PEI nanocomplexes show high reprogramming efficiency in human and mouse somatic cells with low cytotoxicity and direct conversion into iCMs without integrating factors into the genome. Importantly, AuNP/GMT/PEI nanocomplexes led to efficient in vivo conversion into cardiomyocytes after myocardial infarction (MI), resulting in the effective recovery of cardiac function and scar area. Taken together, these results show that the AuNP/GMT/PEI nanocarrier can be used to develop effective therapeutics for heart regeneration in cardiac disease patients.

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Exposure to 3,5-dimethylaminophenol (3,5-DMAP), the metabolite of the 3-5-dimethylaniline, was shown to cause high levels of oxidative stress in different cells. The aim of the present work was to observe whether this metabolite can lead to cytotoxicity, oxidative stress, DNA damage and cell cycle changes in non-small cell lung cancer A549 cells. 3,5-DMAP caused a dose-dependent increase in cytotoxicity, generation of superoxide (O2-.), inductions in the enzyme activities orchestrating cellular antioxidant balance, increases in lipid peroxidation as well as DNA damage. However, 3,5-DMAP showed significantly lower cytotoxicity towards human lung fibroblast (HLF) cells. 3,5-DMAP also led to molecular events, like inducing apoptotic markers (ie. p53, Bad, Bax and cytochrome c); decreasing anti-apoptotic proteins (Bcl-2) and alterations in cell cycle. Our findings indicate that the cytotoxicity caused by this particular alkylaniline metabolite led to initiation of caspase 3-mediated apoptosis. Furthermore, 3,5-DMAP attenuated carcinogenic properties like migration capacity of A549 cells and eventually inhibited growth of A549 cells in an in vivo mouse model. Tumor sections showed that 3,5-DMAP down-regulated c-Myc expression but up-regulated p53 and cytochrome c, all of which might result in tumor growth arrest. Co-treatment with N-acetylcysteine provided reductions in cytotoxicity and positively modulated genetic events induced by 3,5-DMAP in A549 cells. In conclusion, our findings demonstrate 3,5-DMAP may be a potential anti-cancer drug in cancer, due to its self redox cycling properties.

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