DNA-controlled protein fluorescence: Design of aptamer-split peptide hetero-modulator for GFP to respond to intracellular ATP levels.

Enabling the precise control of protein functions with artificially programmed reaction patterns is beneficial for investigating biological processes. Although several strategies have been established that employ the programmability of nucleic acid, they have been limited to DNA hybridization without external stimuli or target binding. Here, we report an approach for the DNA-mediated control of the tripartite split-GFP assembly via aptamers with responsiveness to intracellular small molecules as stimuli. We designed a novel structure-switching aptamer-peptide conjugate as a hetero modulator for split GFP in response to ATP. By conjugating two peptides (S10/11) derived from the tripartite split-GFP to ATP aptamer, we achieved GFP reassembly using only ATP as a trigger molecule. The response to ATP at ≥4 mM concentrations indicated that it can be applied to respond to intracellular ATP in live cells. Furthermore, our hetero-modulator exhibited high and long-term stability, with a half-life of approximately four days in a serum stability assay, demonstrating resistance to nuclease degradation. We validated that our aptamer-modulator split GFP was successfully reconstituted in the cell in response to intracellular ATP levels. Our aptamer-modulated split GFP platform can be utilized to monitor a wide range of intracellular metabolites by replacing the aptamer sequence.

Elucidating TH301's influence on the torsion angle of CRY1 W399 using replica exchange with solute tempering (REST) molecular dynamics (MD) simulations.

Cryptochrome 1 (CRY1) is a protein involved in the circadian clock and associated with various diseases. Targeting CRY1 for drug development requires the discovery of competitive inhibitors that target its FAD binding site through ubiquitination. During the development of compounds to regulate CRY1, an intriguing compound called TH301 was identified. Despite binding to CRY1, TH301 does not induce the expected reaction and is considered an inactive compound. However, it has been observed that TH301 affects the torsion angle of CRY1's W399 residue, which plays a crucial role in the regulation of ubiquitination by influencing the movement of the lid loop. In our research, we aimed to understand how TH301 induces the torsion angle of CRY1's W399 to shift to an "out-form" by performing REST-based MD simulations. The cyclopentane of TH301 tends to align parallel with W292, creating a repulsive force when W399 is in the "in-form", leading to a flip. In the "out-form", W399's side chain interacts with TH301's chlorobenzene through a π-π interaction, stabilizing this pose. This analysis helps identify compounds binding to CRY1 and filter out inactive ones. We found that assessing the interaction energy between TH301 and W399 is crucial to evaluate whether W399 flips or not. These findings contribute to the development of drugs targeting CRY1 and enhance our understanding of its regulatory mechanisms.

Curcumin Ameliorates Particulate Matter-Induced Pulmonary Injury through Bimodal Regulation of Macrophage Inflammation via NF-κB and Nrf2.

The direct effects of particulate matter (PM) on lung injury and its specific molecular mechanisms are unclear. However, experimental evidence has shown that oxidative stress-mediated inflammation in macrophages is the main pathological outcome of PM exposure. Curcumin has been reported to protect organs against the disturbance of homeostasis caused by various toxic agents through anti-inflammatory and antioxidative effects. However, the protective action of curcumin against PM-induced pulmonary inflammation and the underlying mechanism have not been thoroughly investigated. In this study, we established a PM-induced pulmonary inflammation mouse model using the intratracheal instillation method to investigate the protective ability of curcumin against PM-induced pulmonary inflammation. Compared to the mice treated with PM only, the curcumin-treated mice showed alleviated alveolar damage, decreased immune cell infiltration, and reduced proinflammatory cytokine production in both lung tissue and BALF. To evaluate the underlying mechanism, the mouse macrophage cell line RAW264.7 was used. Pretreatment with curcumin prevented the production of PM-induced proinflammatory cytokines by deactivating NF-κB through the suppression of MAPK signaling pathways. Furthermore, curcumin appears to attenuate PM-induced oxidative stress through the activation of Nrf2 and downstream antioxidant signaling. Our findings demonstrate that curcumin protects against PM-induced lung injury by suppressing oxidative stress and inflammatory activation in macrophages.

Down-regulation of IDH2 sensitizes cancer cells to erastin-induced ferroptosis.

Ferroptosis is a form of regulated cell death induced by lipid peroxidation that is dependent on iron. This pathway is being considered as an alternative anticancer therapeutic strategy, and the chemoreagent erastin induces ferroptosis by blocking system Xc-, which causes a cysteine shortage that depletes intracellular GSH. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) is major enzyme that produces NADPH, which is a crucial source for mitochondrial GSH turnover. Therefore, we hypothesized that down-regulation of IDH2 would have a synergic effect on erastin-induced ferroptosis. Here, we investigated the effect of IDH2 knockdown on ferroptosis in human HT1080 fibrosarcoma and murine Hepa1-6 hepatoma cells cultured in vitro as well as in an in vivo model of allografted Hepa1-6 cells in nude mice. Our results show that susceptibility to ferroptosis was substantially increased when IDH2 was down-regulated. This study supports that IDH2 has protective effect against ferroptotic cell death, and that the enzyme could be targeted to sensitize cancer cells to ferroptosis.

IDH2 Deficiency Is Critical in Myogenesis and Fatty Acid Metabolism in Mice Skeletal Muscle.

Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) catalyzes the oxidative decarboxylation of isocitrate into α-ketoglutarate with concurrent reduction of NADP+ to NADPH. However, it is not fully understood how IDH2 is intertwined with muscle development and fatty acid metabolism. Here, we examined the effects of IDH2 knockout (KO) on skeletal muscle energy homeostasis. Calf skeletal muscle samples from 10-week-old male IDH2 KO and wild-type (WT; C57BL/6N) mice were harvested, and the ratio of skeletal muscle weight to body and the ratio of mitochondrial to nucleic DNA were measured. In addition, genes involved in myogenesis, mitochondria biogenesis, adipogenesis, and thermogenesis were compared. Results showed that the ratio of skeletal muscle weight to body weight was lower in IDH2 KO mice than those in WT mice. Of note, a noticeable shift in fiber size distribution was found in IDH2 KO mice. Additionally, there was a trend of a decrease in mitochondrial content in IDH2 KO mice than in WT mice (p = 0.09). Further, mRNA expressions for myogenesis and mitochondrial biogenesis were either decreased or showed a trend of decrease in IDH2 KO mice. Moreover, genes for adipogenesis pathway (Pparg, Znf423, and Fat1) were downregulated in IDH2 KO mice. Interestingly, mRNA and protein expression of uncoupling protein 1 (UCP1), a hallmark of thermogenesis, were remarkably increased in IDH2 KO mice. In line with the UCP1 expression, IDH2 KO mice showed higher rectal temperature than WT mice under cold stress. Taken together, IDH2 deficiency may affect myogenesis, possibly due to impairments of muscle generation and abnormal fatty acid oxidation as well as thermogenesis in muscle via upregulation of UCP1.

Colon Transcriptomics Reveals Sex-Dependent Metabolic Signatures in Response to 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine Treatment in C57BL/6N Mice.

Diets high in red meats, particularly meats cooked at high temperature, increase the risk of colon cancer due to a production of heterocyclic aromatic amines (HAAs). Of the identified HAAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most mass abundant colon carcinogen in charred meat or fish. Here, we comprehensively examined sex-dependent colon transcriptome signatures in response to PhIP treatment to identify biological discrepancies. Eight-week-old male and female C57BL/6N mice were intraperitoneally injected with PhIP (10 mg/kg of body weight) and colon tissues were harvested 24 h after PhIP injection, followed by colon transcriptomics analysis. A list of differentially expressed genes (DEGs) was utilized for computational bioinformatic analyses. Specifically, overrepresentation test using the Protein Analysis Through Evolutionary Relationships tool was carried out to annotate sex-dependent changes in transcriptome signatures after PhIP treatment. Additionally, the most significantly affected canonical pathways by PhIP treatment were predicted using the Ingenuity Pathway Analysis. As results, male and female mice presented different metabolic signatures in the colon transcriptome. In the male mice, oxidative phosphorylation in the mitochondrial respiratory chain was the pathway impacted the most; this might be due to a shortage of ATP for DNA repair. On the other hand, the female mice showed concurrent activation of lipolysis and adipogenesis. The present study provides the foundational information for future studies of PhIP effects on underlying sex-dependent mechanisms.

Hepatic transcriptomics analysis reveals that fructose intervention down-regulated xenobiotics-metabolising enzymes through aryl hydrocarbon receptor signalling suppression in C57BL/6N mice.

For decades, fructose intake has been recognised as an environmental risk for metabolic syndromes and diseases. Here we comprehensively examined the effects of fructose intake on mice liver transcriptomes. Fructose-supplemented water (34 %; w/v) was fed to both male and female C57BL/6N mice at their free will for 6 weeks, followed by hepatic transcriptomics analysis. Based on our criteria, differentially expressed genes (DEG) were selected and subjected to further computational analyses to predict key pathways and upstream regulator(s). Subsequently, predicted genes and pathways from the transcriptomics dataset were validated via quantitative RT-PCR analyses. As a result, we identified eighty-nine down-regulated and eighty-eight up-regulated mRNA in fructose-fed mice livers. These DEG were subjected to bioinformatics analysis tools in which DEG were mainly enriched in xenobiotic metabolic processes; further, in the Ingenuity Pathway Analysis software, it was suggested that the aryl hydrocarbon receptor (AhR) is an upstream regulator governing overall changes, while fructose suppresses the AhR signalling pathway. In our quantitative RT-PCR validation, we confirmed that fructose suppressed AhR signalling through modulating expressions of transcription factor (AhR nuclear translocator; Arnt) and upstream regulators (Ncor2, and Rb1). Altogether, we demonstrated that ad libitum fructose intake suppresses the canonical AhR signalling pathway in C57BL/6N mice liver. Based on our current observations, further studies are warranted, especially with regard to the effects of co-exposure to fructose on (1) other types of carcinogens and (2) inflammation-inducing agents (or even diets such as a high-fat diet), to find implications of fructose-induced AhR suppression.

Disruption of IDH2 attenuates lipopolysaccharide-induced inflammation and lung injury in an α-ketoglutarate-dependent manner.

Acute lung injury (ALI) is an acute failure of the respiratory system with unacceptably high mortality, for which effective treatment is urgently necessary. Infiltrations by immune cells, such as leukocytes and macrophages, are responsible for the inflammatory response in ALI, which is characterized by excessive production of pro-inflammatory mediators in lung tissues exposed to various pathogen-associated molecules such as lipopolysaccharide (LPS) from microbial organisms. α-Ketoglutarate (α-KG) is a key metabolic intermediate and acts as a pro-inflammatory metabolite, which is responsible for LPS-induced proinflammatory cytokine production through NF-κB signaling pathway. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) has been reported as an essential enzyme catalyzing the conversion of isocitrate to α-KG with concurrent production of NAPDH. Therefore, we evaluated the role of IDH2 in LPS-induced ALI using IDH2-deficient mice. We observed that LPS-induced inflammation and lung injury is attenuated in IDH2-deficient mice, leading to a lengthened life span of the mice. Our results also suggest that IDH2 disruption suppresses LPS-induced proinflammatory cytokine production, resulting from an inhibition of the NF-κB signaling axis in an α-KG-dependent manner. In conclusion, disruption of IDH2 leads to a decrease in α-KG levels, and the activation of NF-κB in response to LPS is attenuated by reduction of α-KG levels, which eventually reduces the inflammatory response in the lung during LPS-induced ALI. The present study supports the rationale for targeting IDH2 as an important therapeutic strategy for the treatment of systemic inflammatory response syndromes, particularly ALI.

Cruciferous vegetables and colorectal cancer prevention through microRNA regulation: A review.

Diets rich in fruits and vegetables may lower colorectal cancer risk. In particular, a number of in vitro and in vivo studies demonstrated that cruciferous vegetables and their active compounds elicit chemopreventive potency through multiple mechanisms. However, it is relatively unexplored whether these vegetables modulate the risk of cancer development through epigenetic mechanisms including noncoding RNAs. Therefore, the objective of the present review is to report and discuss existing evidence with regards to modulation of microRNAs (miRNAs), one variety of noncoding RNAs, by cruciferous vegetables and their chemo-preventive effects against colorectal cancers. As results, it seems clear, considering accumulating evidence regarding their interactions with cancer related genes and relevant signaling pathways, that miRNA modulation via cruciferous vegetables is an attractive target for the prevention of colorectal cancer. In addition, miRNAs have been characterized as diagnostic and prognostic biomarkers and utilized in cancer therapeutics. Thus, it is very possible that natural agents (not limited to those in cruciferous vegetables) enhance cancer therapeutic efficacy and elicit chemopreventive effects through modulating key miRNAs.

Lactobacillus plantarum LRCC 5273 isolated from Kimchi ameliorates diet-induced hypercholesterolemia in C57BL/6 mice.

This study was designed to select potent cholesterol-lowering probiotic strains on HepG2 cell and investigate the effect of selected strain, Lactobacillus plantarum LRCC 5273 and LRCC 5279 in hypercholesterolemic mice. In the results, LP5273 group showed significantly reduced total and LDL cholesterol compared to HCD group. In addition to significantly up-regulated hepatic mRNA expression of LXR-α and CYP7A1, intestinal LXR-α and ABCG5 were significantly up-regulated in LP5273 group. With activation of hepatic and intestinal LXR-α and its target genes, fecal cholesterol and bile acid excretion were increased in LP5273 fed mice. These results suggest that LP5273 ameliorates hypercholesterolemia in mice through the activation of hepatic and intestinal LXR-α, resulting in enhancement of fecal cholesterol and bile acids excretion in the small intestine. The results of present study suggest mechanistic evidences for hypocholesterolemic effects of L. plantarum spp., and may contribute to future researches for prevention of hypercholesterolemia and cardiovascular disease.

Idh2 deficiency accelerates renal dysfunction in aged mice.

The free radical or oxidative stress theory of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species (ROS) that are produced as by-products of normal metabolic processes in mitochondria. The oxidative stress may arise as a result of either increased ROS production or decreased ability to detoxify ROS. The availability of the mitochondrial NADPH pool is critical for the maintenance of the mitochondrial antioxidant system. The major enzyme responsible for generating mitochondrial NADPH is mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2). Depletion of IDH2 in mice (idh2-/-) shortens life span and accelerates the degeneration of multiple age-sensitive traits, such as hair grayness, skin pathology, and eye pathology. Among the various internal organs tested in this study, IDH2 depletion-induced acceleration of senescence was uniquely observed in the kidney. Renal function and structure were greatly deteriorated in 24-month-old idh2-/- mice compared with wild-type. In addition, disruption of redox status, which promotes oxidative damage and apoptosis, was more pronounced in idh2-/- mice. These data support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice, and thus support the oxidative stress theory of aging.

Saponin-Based Nanoemulsification Improves the Antioxidant Properties of Vitamin A and E in AML-12 Cells.

Our work aimed to investigate the protective effects of saponin-based nanoemulsions of vitamin A and E against oxidative stress-induced cellular damage in AML-12 cells. Saponin nanoemulsions of vitamin A (SAN) and vitamin E (SEN) were prepared by high-pressure homogenization and characterized in terms of size, zeta potential, and polydispersity index. SEN and SAN protect AML-12 cells against oxidative stress-induced cellular damage more efficiently via scavenging reactive oxygen species (ROS), and reducing DNA damage, protein carbonylation, and lipid peroxidation. These results provide valuable information for the development of nanoemulsion-based delivery systems that would improve the antioxidant properties of vitamin A and E.

Circadian clock, cancer, and chemotherapy.

The circadian clock is a global regulatory system that interfaces with most other regulatory systems and pathways in mammalian organisms. Investigations of the circadian clock-DNA damage response connections have revealed that nucleotide excision repair, DNA damage checkpoints, and apoptosis are appreciably influenced by the clock. Although several epidemiological studies in humans and a limited number of genetic studies in mouse model systems have indicated that clock disruption may predispose mammals to cancer, well-controlled genetic studies in mice have not supported the commonly held view that circadian clock disruption is a cancer risk factor. In fact, in the appropriate genetic background, clock disruption may instead aid in cancer regression by promoting intrinsic and extrinsic apoptosis. Finally, the clock may affect the efficacy of cancer treatment (chronochemotherapy) by modulating the pharmacokinetics and pharmacodynamics of chemotherapeutic drugs as well as the activity of the DNA repair enzymes that repair the DNA damage caused by anticancer drugs.

Retraction: Protective effects of bazedoxifene paired with conjugated estrogens on pancreatic ß-cell dysfunction.

This article has been retracted by the Editorial Committee of The Pharmaceutical Society of Japan because it contains scientific misconduct. Although the data published in this article were generated in part by the first author, the authors violated authorship and sponsorship protocol.

Acetic acid enhances endurance capacity of exercise-trained mice by increasing skeletal muscle oxidative properties.

Acetic acid has been shown to promote glycogen replenishment in skeletal muscle during exercise training. In this study, we investigated the effects of acetic acid on endurance capacity and muscle oxidative metabolism in the exercise training using in vivo mice model. In exercised mice, acetic acid induced a significant increase in endurance capacity accompanying a reduction in visceral adipose depots. Serum levels of non-esterified fatty acid and urea nitrogen were significantly lower in acetic acid-fed mice in the exercised mice. Importantly, in the mice, acetic acid significantly increased the muscle expression of key enzymes involved in fatty acid oxidation and glycolytic-to-oxidative fiber-type transformation. Taken together, these findings suggest that acetic acid improves endurance exercise capacity by promoting muscle oxidative properties, in part through the AMPK-mediated fatty acid oxidation and provide an important basis for the application of acetic acid as a major component of novel ergogenic aids.

Mitochondrial NADP(+)-dependent isocitrate dehydrogenase knockdown inhibits tumorigenicity of melanoma cells.

The potent cytotoxicity of reactive oxygen species (ROS) can cause various diseases but may also serve as a powerful weapon capable of destroying cancer cells. Although the balance between generation and elimination of ROS is maintained by the proper function of antioxidative systems, the severe disturbance of cellular redox status may cause various damages, leading to cell death. Mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2), an NADPH-generating enzyme, is one of the major antioxidant and redox regulators in mitochondria. To assess the effect of IDH2 knockdown in the malignancy process, we generated B16F10 melanoma cells stably transfected either with the cDNA for mouse IDH2 cloned in antisense orientation or with a control vector. Mice injected with B16F10 cells harboring IDH2 downregulation showed a dramatic reduction in tumor progression in comparison to mice administered control cells. This effect might be secondary to a shift from a reducing to an oxidative state in tumor cells. The tumor tissue of mice administered B16F10 cells transfected with the IDH2 cDNA exhibited induction of apoptosis and downregulation of angiogenesis markers. These observations demonstrate that reduction of IDH2 levels in malignant cells has anti-tumorigenic effects and suggest that IDH2 is a potential target for cancer therapy.

Circadian clock disruption improves the efficacy of chemotherapy through p73-mediated apoptosis.

The circadian clock in mammalian organisms is generated by a transcription-translation feedback loop that controls many biochemical pathways at the cellular level and physiology and behavior at the organismal level. Cryptochrome (Cry) is a key protein in the negative arm of the transcription-translation feedback loop. It has been found that Cry mutation in cells with p53-null genotype increased their sensitivity to apoptosis by genotoxic agents. Here we show that this increased sensitivity is due to up-regulation of the p53 gene family member p73 in response to DNA damage. As a consequence, when tumors arising from oncogenic Ras-transformed p53(-/-) and p53(-/-)Cry1(-/-)Cry2(-/-) cells are treated with the anticancer drug oxaliplatin, p53(-/-) tumors continue to grow whereas p53(-/-)Cry1(-/-)Cry2(-/-) tumors exhibit extensive apoptosis and stop growing. This finding provides a mechanistic foundation for overcoming the resistance of p53-deficient tumor cells to apoptosis induced by DNA-damaging agents and suggests that disruption of cryptochrome function may increase the sensitivity of tumors with p53 mutation to chemotherapy.

Regulation of apoptosis by the circadian clock through NF-kappaB signaling.

In mice and humans the circadian rhythm of many biochemical reactions, physiology, and behavior is generated by a transcriptional-translation feedback loop (TTFL) made up of the so-called core clock genes/proteins. The circadian system interfaces with most signaling pathways including those involved in cell proliferation and inflammation. Cryptochrome (CRY) is a core clock protein that plays an essential role in the repressive arm of the TTFL. It was recently reported that mutation of CRY in p53-null mice delayed the onset of cancer. It was therefore suggested that CRY mutation may activate p53-independent apoptosis pathways, which eliminate premalignant and malignant cells and thus delay overt tumor formation. Here we show that CRY mutation sensitizes p53 mutant and oncogenically transformed cells to tumor necrosis factor α (TNFα)-initiated apoptosis by interfacing with the NF-κB signaling pathway through the GSK3ß kinase and alleviating prosurvival NF-κB signaling. These findings provide a mechanistic foundation for the delayed onset of tumorigenesis in clock-disrupted p53 mutant mice and suggest unique therapeutic strategies for treating cancers associated with p53 mutation.

Optimized combination of Cervus nippon (Sika deer), Angelica (Dangui), and Rehmannia (Suk-jihwang) mitigates LPS-induced inflammation: exploring signaling pathways through plasma metabolomics.

This study aimed to determine the optimal combination of three anti-inflammatory materials [i.e., Cervus nippon Temminck (CT), Angelica gigas Nakai (AN), and Rehmannia glutinosa (RG)] for the strongest anti-inflammatory potential. Eighteen combinations of the three materials were tested in LPS-stimulated RAW264.7 cells via assessing nitric oxide (NO). The best combination from in vitro studies was administered to LPS-treated C57BL/6J mice for five days. Subsequently, plasma metabolites were profiled by bioinformatics analyses and validations. As results, 2, 20, and 50 µg/mL of CT, AN, and RG (TM) were the most effective combination suppressing inflammation. In mice, TM mitigated hepatic inflammatory markers. Similarly, the metabolomics indicated that TM may suppress NF-κB signaling pathway, thereby alleviating hepatic inflammation. TM also decreased systemic and hepatic pro-inflammatory cytokines. Collectively, we found the optimal combination of TM for mitigating inflammation; thus further studies on safety, mechanisms, and clinical models are warranted for human applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01476-x.