Intrapopulation analysis of longitudinal lifespan in Caenorhabditis elegans identifies W09D10.4 as a novel AMPK-associated healthspan shortening factor.

Caenorhabditis elegans is a model organism widely used for longevity studies. Current advances have been made in the methods that allow automated monitoring of C. elegans behavior. However, ordinary manual assays as well as automated methods have yet to achieve qualitative whole-life analysis of C. elegans longevity based on intrapopulation variation. Here, we utilized live-cell analysis system to determine the parameters of nematode lifespans. Image-based superposition method enabled to determine not only frailty in worms, but also to measure individual and longitudinal lifespan, healthspan, and frailspan. Notably, k-means clustering via principal component analysis revealed four clusters with distinct longevity patterns in wild-type C. elegans. Physiological relevance of clustering was confirmed by assays with pharmacological and/or genetic manipulation of AMP-activated protein kinase (AMPK), a crucial regulator of healthspan. Finally, we focused on W09D10.4 among the possible regulators extracted by integrative expression analysis with existing data sets. Importantly, W09D10.4 knockdown increased the high-healthspan populations only in the presence of AMPK, suggesting that W09D10.4 is a novel AMPK-associated healthspan shortening factor in C. elegans. Overall, the study establishes a novel platform of longitudinal lifespan in C. elegans, which is user-friendly, and may be a useful pharmacological tool to identify healthspan modulatory factors.

Combined effects of cisplatin and photon or proton irradiation in cultured cells: radiosensitization, patterns of cell death and cell cycle distribution.

The purpose of the current study was to investigate the biological effects of protons and photons in combination with cisplatin in cultured cells and elucidate the mechanisms responsible for their combined effects. To evaluate the sensitizing effects of cisplatin against X-rays and proton beams in HSG, EMT6 and V79 cells, the combination index, a simple measure for quantifying synergism, was estimated from cell survival curves using software capable of performing the Monte Carlo calculation. Cell death and apoptosis were assessed using live cell fluorescence imaging. HeLa and HSG cells expressing the fluorescent ubiquitination-based cell cycle indicator system (Fucci) were irradiated with X-rays and protons with cisplatin. Red and green fluorescence in the G1 and S/G2/M phases, respectively, were evaluated and changes in the cell cycle were assessed. The sensitizing effects of ≥1.5 µM cisplatin were observed for both X-ray and proton irradiation (P < 0.05). In the three cell lines, the average combination index was 0.82-1.00 for X-rays and 0.73-0.89 for protons, indicating stronger effects for protons. In time-lapse imaging, apoptosis markedly increased in the groups receiving ≥1.5 µM cisplatin + protons. The percentage of green S/G2/M phase cells at that time was higher when cisplatin was combined with proton beams than with X-rays (P < 0.05), suggesting more significant G2 arrest. Proton therapy plus ≥1.5 µM cisplatin is considered to be very effective. When combined with cisplatin, proton therapy appeared to induce greater apoptotic cell death and G2 arrest, which may partly account for the difference observed in the combined effects.

A novel condition of mild electrical stimulation exerts immunosuppression via hydrogen peroxide production that controls multiple signaling pathway.

Different modes of exogenous electrical stimulation at physiological strength has been applied to various diseases. Previously, we extensively demonstrated the usability of mild electrical stimulation (MES) with low frequency pulse current at 55 pulses per second (MES55) for several disease conditions. Here we found that MES with high frequency pulse-current (5500 pulse per second; MES5500) suppressed the overproduction of pro-inflammatory cytokines induced by phorbol myristate acetate/ionomycin in Jurkat T cells and primary splenocytes. MES5500 also suppressed the overproduction of inflammatory cytokines, improved liver damage and reduced mouse spleen enlargement in concanavalin-A-treated BALB/c mice. The molecular mechanism underlying these effects included the ability of MES5500 to induce modest amount of hydrogen peroxide and control multiple signaling pathways important for immune regulation, such as NF-κB, NFAT and NRF2. In the treatment of various inflammatory and immune-related diseases, suppression of excessive inflammatory cytokines is key, but because immunosuppressive drugs used in the clinical setting have serious side effects, development of safer methods of inhibiting cytokines is required. Our finding provides evidence that physical medicine in the form of MES5500 may be considered as a novel therapeutic tool or as adjunctive therapy for inflammatory and immune-related diseases.

Taurine Inhibits TRPV-Dependent Activity to Overcome Oxidative Stress in Caenorhabditis elegans.

Taurine has important physiological roles as well as a wide range of pharmacological effects. Studies have suggested that taurine ameliorates diabetes, hypertension, oxidative stress, and inflammatory diseases. However, its mechanisms of action are still unclear. It has been reported that N-acyl taurine activates transient receptor potential vanilloid-1 (TRPV1), which is related to the pathogenesis of many inflammatory diseases. In this study, we hypothesized that taurine has a regulatory effect on TRPV1 activation via N-acyl taurine. To evaluate this hypothesis, we assessed the calcium influx activated by a TRPV1 agonist in human keratinocyte (HaCaT) cells and paraquat-induced oxidative stress in Caenorhabditis elegans. Our results indicate that taurine inhibits TRPV-dependent activity to overcome oxidative stress in cultured cell lines and in C. elegans.

Free radical scavenging, α-glucosidase inhibitory and lipase inhibitory activities of eighteen Sudanese medicinal plants.

BACKGROUND: Lifestyle-related diseases such as diabetes are steadily increasing worldwide. In Sudan, there are a variety of plant species used traditionally for the treatment of diabetes, obesity and other symptoms which need to be validated through scientific studies for their claimed traditional uses. Therefore, in the current study, the free radical scavenging activity, α-glucosidase inhibitory and pancreatic lipase inhibitory activities of 70% ethanol and water extracts of eighteen Sudanese medicinal plants were investigated using various in vitro assays. Moreover, the cytotoxicity and genotoxicity were assessed for the bioactive plant extracts. METHODS: Eighteen plants were selected on the basis of their traditional uses and extracted with 70% ethanol and water to obtain thirty-six extracts. The obtained extracts were screened using different in vitro bioassays namely, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, α-glucosidase inhibitory and pancreatic lipase inhibitory assays. Furthermore, the active plant extracts were investigated for their cytotoxicity and genotoxicity on HeLa cell line using HCS DNA Damage Assay. RESULTS: Both 70% ethanol and water extracts of Acacia nilotica, Ziziphus spina-christi, Abrus precatorius, and Geigeria alata along with the 70% ethanol extract of Martynia annua showed potent free radical scavenging activity. Regarding the α-glucosidase inhibition assay, both extracts of Acacia nilotica, Ziziphus spina-christi, Geigeria alata, and Cyperus rotundus showed potent activity. In general, 70% ethanol extracts were more potent compared to water extracts with exception of Cordia sinensis and Cymbopogon proximus, for which water extracts also showed potent enzyme inhibitory activity. Similarly, water extracts of Acacia nilotica and Ziziphus spina-christi showed potent inhibitory activity against pancreatic lipase enzyme. Some of the extracts also showed significant genotoxicity and cytotoxicity at the concentration range used for bioactivities. CONCLUSION: The extracts of Acacia nilotica, Ziziphus spina-christi, Geigeria alata, Martynia annua and Abrus precatorius exhibited an appreciable range of activity on antioxidant and enzyme inhibitory assays.

Mild electrical stimulation with heat shock reduces inflammatory symptoms in the imiquimod-induced psoriasis mouse model.

Psoriasis is a chronic skin disease caused by immune disorder. The chronic skin inflammation involves inflammatory molecules that are released from T lymphocytes and keratinocytes. Therefore, developing an anti-inflammatory therapy that is suitable for long-term treatment is needed. Electrical stimulation induces biological responses by modulating intracellular signaling pathways. Our previous studies showed that the optimized combination treatment of mild electrical stimulation (MES, 0.1-millisecond; ms, 55-pulses per second; pps) and heat shock (HS, 42°C) modulates inflammatory symptoms of metabolic disorders and chronic kidney disease in mice models and clinical trials. Here, we investigated the effect of MES+HS treatment on imiquimod-induced psoriasis mouse model. Topical application of imiquimod cream (15 mg) to mice ear induced keratinocyte hyperproliferation and psoriasis-like inflammation. In MES+HS-treated mice, imiquimod-induced skin hyperplasia was significantly decreased. MES+HS treatment reduced the protein expression of IL-17A and the infiltration of CD3-positive cells in lesioned skin. In addition, MES+HS-treated mice had decreased mRNA expression level of antimicrobial molecules (S100A8 and Reg3γ) which aggravate psoriasis. In IL-17A-stimulated HaCaT cells, MES+HS treatment significantly lowered the mRNA expression of aggravation markers (S100A8, S100A9 and ß-defensin2). Taken together, our study suggested that MES+HS treatment improves the pathology of psoriasis via decreasing the expression of inflammatory molecules.

Mild electrical stimulation increases stress resistance and suppresses fat accumulation via activation of LKB1-AMPK signaling pathway in C. elegans.

Electrical current at physiological strength has been applied as a therapeutic approach for various diseases. Several of our works showed that mild electrical stimulation (MES) at 0.1-ms pulse width has positive impact on organisms. But despite the growing evidence of the beneficial effects of MES, its effects on individual animals and the molecular underpinnings are poorly understood and rarely studied. Here, we examined the effects of MES on individual animal and its mechanisms by mainly using Caenorhabditis elegans, a powerful genetic model organism. Interestingly, MES increased stress resistance and suppressed excess fat accumulation in wild-type N2 worms but not in AMPK/AAK-2 and LKB1/PAR-4 mutant worms. MES promoted the nuclear localization of transcription factors DAF-16 and SKN-1 and consequently increased the expression of anti-stress genes, whereas MES inhibited the nuclear localization of SBP-1 and suppressed the expression of lipogenic genes. Moreover, we found that MES induced the activation of LKB1/PAR4-AMPK/AAK2 pathway in C. elegans and in several mammalian cell lines. The mitochondrial membrane potential and cellular ATP level were slightly and transiently decreased by MES leading to the activation of LKB1-AMPK signaling pathway. Together, we firstly and genetically demonstrated that MES exerts beneficial effects such as stress resistance and suppression of excess fat accumulation, via activation of LKB1-AMPK signaling pathway.