Paternal p,p'-DDE exposure and pre-pubertal high-fat diet increases the susceptibility to fertility impairment and sperm Igf2 DMR2 hypo-methylation in male offspring.

The hypothesis of paternal origins of health and disease (POHaD) indicates that paternal exposure to adverse environment could alter the epigenetic modification in germ line, increasing the disease susceptibility in offspring or even in subsequent generations. p,p'-Dichlorodiphenyldichloroethylene (p,p'-DDE) is an anti-androgenic chemical and male reproductive toxicant. Gestational p,p'-DDE exposure could impair reproductive development and fertility in male offspring. However, the effect of paternal p,p'-DDE exposure on fertility in male offspring remains uncovered. From postnatal day (PND) 35 to 119, male rats (F0) were given 10 mg/body weight (b.w.) p,p'-DDE or corn oil by gavage. Male rats were then mated with the control females to generate male offspring. On PND35, the male offspring were divided into 4 groups according whether to be given the high-fat diet (HF): corn oil treatment with control diet (C-C), p,p'-DDE treatment with control diet (DDE-C), corn oil treatment with high-fat diet (C-HF) or p,p'-DDE treatment with high-fat diet (DDE-HF) for 35 days. Our results indicated that paternal p,p'-DDE exposure did not affect the male fertility of male offspring directly, but decreased sperm quality and induced testicular apoptosis after the high-fat diet treatment. Further analysis demonstrated that paternal exposure to p,p'-DDE and pre-pubertal high-fat diet decreased sperm Igf2 DMR2 methylation and gene expression in male offspring. Hence, paternal exposure to p,p'-DDE and pre-pubertal high-fat diet increases the susceptibility to male fertility impairment and sperm Igf2 DMR2 hypo-methylation in male offspring, posing a significant implication in the disease etiology.

DNA methylation reprogramming mediates transgenerational diabetogenic effect induced by early-life p,p'-DDE exposure.

Increasing evidence shows that an adverse environment during the early fetal development can affect the epigenetic modifications on a wide range of diabetes-related genes, leading to an increased diabetic susceptibility in adulthood or even in subsequent generations. p,p'-Dichlorodiphenoxydichloroethylene (p,p'-DDE) is a break-down product of the pesticide dichlorodiphenyltrichloroethane (DDT). p,p'-DDE has been associated with various health concerns, such as diabetogenic effect. However, the precise molecular mechanism remains unclear. In this study, p,p'-DDE was given by gavage to pregnant rat dams from gestational day (GD) 8 to GD15 to generate male germline to investiagate the transgenerational effects. We found that early-life p,p'-DDE exposure increased the transgenerational diabetic susceptibility through male germline inheritance. In utero exposure to p,p'-DDE altered the sperm DNA methylome in F1 progeny, and a significant number of those differentially methylated genes could be inherited by F2 progeny. Furthermore, early-life p,p'-DDE exposure altered DNA methylation in glucose metabolic genes Gck and G6pc in sperm and the methylation modification were also found in liver of the next generation. Our study demonstrate that DNA methylation plays a critical role in mediating transgenerational diabetogenic effect induced by early-life p,p'-DDE exposure.

Mesenchymal stem cell-derived exosomes attenuate DNA damage response induced by cisplatin and bleomycin.

Stem cell-derived exosomes (SC-Exos) have been shown to protect cells from chemical-induced deoxyribonucleic acid (DNA) damage. However, there has been no systematic comparison of the efficacy of exosomes against different types of DNA damage. Therefore, in this study, we assessed the protective effect of exosomes derived from human embryonic stem cell-induced mesenchymal stem cells (hESC-MSC-Exos) on two types of DNA damage, namely, intra-/inter-strand crosslinks and DNA double-strand breaks induced by cisplatin (Pt) and bleomycin (BLM), respectively, in HeLa cells. The alkaline comet assay demonstrated that hESC-MSC-Exos effectively inhibited Pt- and BLM-induced DNA damage in a dose-dependent manner. When the concentration of hESC-MSC-Exos reaches 2.0 × 106 and 4.0 × 106 particles/mL in Pt- and BLM-treated groups, respectively, there was a significant decrease in tail DNA percentage (Pt: 20.80 ± 1.61 vs 9.40 ± 1.14, p < 0.01; BLM: 21.80 ± 1.31 vs 6.70 ± 0.60, p < 0.01), tail moment (Pt: 10.00 ± 1.21 vs 2.08 ± 0.51, p < 0.01; BLM: 12.00 ± 0.81 vs 2.00 ± 0.21, p < 0.01), and olive tail moment (Pt: 6.01 ± 0.55 vs 2.09 ± 0.25, p < 0.01; BLM: 6.03 ± 0.37 vs 1.53 ± 0.13, p < 0.01). Phospho-histone H2AX (γH2AX) immunofluorescence and western blotting showed an over 50 % decrease in γH2AX expression when the cells were pretreated with hESC-MSC-Exos. As reactive oxygen species (ROS) are important mediators of Pt- and BLM-induced DNA damage, dichloro-dihydro-fluorescein diacetate staining indicated that hESC-MSC-Exos inhibited the increase in intracellular ROS in drug-treated cells. In conclusion, our findings suggest that hESC-MSC-Exos can protect cells from the two types of DNA-damaging drugs and that reduced intracellular ROS is involved in this effect.

Dibazol-induced relaxation of ophthalmic artery in C57BL/6J mice is correlated with the potency to inhibit voltage-gated Ca2+ channels.

We aimed to explore the effect of dibazol on the ophthalmic artery (OA) and ophthalmic artery smooth muscle cells (OASMCs) of C57BL/6J mice as well as the underlying mechanisms. The OA of C57BL/6J mice was isolated under a dissecting microscope for primary OASMCs culture and myogenic tests. OASMCs were identified through morphological and immunofluorescence analyses. Morphology changes in the OASMCs were examined by staining using rhodamine-phalloidin. We performed a collagen gel contraction assay to measure the contractile and relaxant activities of the OASMCs. The molecular probe Fluo-4 AM was used to examine intracellular free Ca2+ levels ([Ca2+]in). The myogenic effects of OA were examined using wire myography. Additionally, the whole-cell patch-clamp technique was used to investigate the mechanisms underlying the relaxant effect of dibazol on L-type voltage-gated Ca2+ channels (LVGC) in isolated cells. 10-5 M dibazol significantly inhibited the contraction of OASMCs and increased the [Ca2+]in response to 30 mM KCl in a concentration-dependent manner. Dizabol had a more significant relaxant effect than 10-5 M isosorbide dinitrate (ISDN). Similarly, dibazol showed a significant dose-dependent relaxant effect on OA contraction induced by 60 mM KCl or 0.3 µM 9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U46619). The current-voltage (I-V) curve revealed that dibazol decreased Ca2+ currents in a concentration-dependent manner. In conclusion, dibazol exerted relaxant effects on the OA and OASMCs, which may involve the inhibition of the Ca2+ influx through LVGC in the cells.

Endogenous Ca2+ release was involved in 50-Hz MF-induced proliferation via Akt-SK1 signal cascade in human amniotic epithelial cells.

The mechanism underlying the biological effects caused by an extremely low-frequency electromagnetic field (ELF-EMF) is still unclear. Previously, we found that L-type calcium channel and sphingosine kinase 1 (SK1) were involved in 50-Hz MF exposure-induced cell proliferation. In the present study, the role of intracellular Ca2+ and signal molecules related to SK1 in cell proliferation induced by 50-Hz MF was investigated in human amniotic epithelial (FL) cells. Results showed that the intracellular Ca2+ chelator, BAPTA, could completely inhibit 50-Hz MF-induced cell proliferation, whereas NIF, the inhibitor of L-type calcium channel, only partly blocked it. When cells were cultured in calcium-free medium, MF exposure also increased intracellular Ca2+, activated SK1 and promoted cell proliferation although all of those increasing levels were lower than those in complete medium. Moreover, MF-activated SK1 could be completely inhibited by BAPTA, and MF-induced cell proliferation was abolished by SKI II, the specific inhibitor of SK1. Additionally, a 50-Hz MF exposure did not affect the activation of ERK and PKCα under the condition of calcium-free medium, but activated the Akt, which could be precluded entirely by BAPTA, but not be inhibited by NIF. Treatment of FL cells with LY294002, the inhibitor of Akt, could delete the MF-induced SK1 activation under the condition of calcium-free medium. Based on the data from the present experiment, it is concluded that endogenous Ca2+ release was involved in 50-Hz MF-induced cell proliferation via Akt-SK1 signal cascade.

The pharmacological properties and corresponding mechanisms of farrerol: a comprehensive review.

CONTEXT: Farrerol, a typical natural flavanone isolated from the traditional Chinese herb 'Man-shan-hong' [Rhododendron dauricum L. (Ericaceae)] with phlegm-reducing and cough-relieving properties, is widely used in China for treating bronchitis and asthma. OBJECTIVE: To present the anti-inflammatory, antioxidant, vasoactive, antitumor, and antimicrobial effects of farrerol and its underlying molecular mechanisms. METHODS: The literature was reviewed by searching PubMed, Medline, Web of Knowledge, Scopus, and Google Scholar databases between 2011 and May 2021. The following key words were used: 'farrerol,' 'flavanone,' 'anti-inflammatory,' 'antioxidant,' 'vasoactive,' 'antitumor,' 'antimicrobial,' and 'molecular mechanisms'. RESULTS: Farrerol showed anti-inflammatory effects mainly mediated via the inhibition of interleukin (IL)-6/8, IL-1ß, tumour necrosis factor(TNF)-α, NF-κB, NO, COX-2, JNK1/2, AKT, PI3K, ERK1/2, p38, Keap-1, and TGF-1ß. Farrerol exhibited antioxidant effects by decreasing JNK, MDA, ROS, NOX4, Bax/Bcl-2, caspase-3, p-p38 MAPK, and GSK-3ß levels and enhancing Nrf2, GSH, SOD, GSH-Px, HO-1, NQO1, and p-ERK levels. The vasoactive effects of farrerol were also shown by the reduced α-SMA, NAD(P)H, p-ERK, p-Akt, mTOR, Jak2, Stat3, Bcl-2, and p38 levels, but increased OPN, occludin, ZO-1, eNOS, CaM, IP3R, and PLC levels. The antitumor effects of farrerol were evident from the reduced Bcl-2, Slug, Zeb-1, and vimentin levels but increased p27, ERK1/2, p38, caspase-9, Bax, and E-cadherin levels. Farrerol reduced α-toxin levels and increased NO production and NF-κB activity to impart antibacterial activity. CONCLUSIONS: This review article provides a theoretical basis for further studies on farrerol, with a view to develop and utilise farrerol for treating of vascular-related diseases in the future.

Combined effect of co-exposure to di (2-ethylhexyl) phthalates and 50-Hz magnetic-fields on promoting human amniotic cells proliferation.

Di (2-ethylhexyl) phthalate (DEHP) and extremely low-frequency electromagnetic fields (ELF-EMFs) exist far and wide in our surroundings. Studies have reported that both of DEHP and ELF-EMFs could promote cell proliferation which is related with adverse bioeffects. In this study, we investigated whether there is the combined effect between DEHP and 50-Hz magnetic fields (MFs) on cell proliferation in human amniotic (FL) cells. Results revealed that the low-concentration DEHP (1 µM) could promote FL cell proliferation, whereas the high-dose DEHP (100 µM) inhibited cell proliferation. When FL cells were treated jointly by a 50-Hz, 0.2-mT MF and 0.1 µM DEHP, the proliferation rate of cells was significantly higher than that of single factor exposure. Additionally, co-exposure to under-threshold MF and DEHP could cooperatively activate protein kinase B (Akt), sphingosine kinase 1 (SphK1) and extracellular signal regulated kinase (ERK) in a cascade manner, and finally mediate cell proliferation. Taken together, the findings of this study indicated that the co-exposure to under-threshold MF and DEHP could jointly promote cell proliferation through activating proliferation-related signal pathway, which warned us that it should be cautious about assessing the underlying health hazards of co-exposure to MFs and DEHP at under-threshold levels.

Up-regulation of nPKC contributes to proliferation of mice pulmonary artery smooth muscle cells in hypoxia-induced pulmonary hypertension.

This study is designed to investigate the role of novel protein kinases C (nPKC) in mediating pulmonary artery smooth muscle cells (PASMCs) proliferation in pulmonary hypertension (PH) and the underlying mechanisms. Mouse PASMCs was isolated using magnetic separation technology. The PASMCs were divided into 24 h group, 48 h group and 72 h group according to different hypoxia treatment time, then detected cell proliferation rate and nPKC expression level in each group. We treated PASMCs with agonists or inhibitors of PKCdelta (PKCδ) and PKCepsilon (PKCε) and exposed them to hypoxia or normoxia for 72 h, then measured the proliferation of PASMCs. We also constructed a lentiviral vector containing siRNA fragments for inhibiting PKCδ and PKCε to transfected PASMCs, then examined their proliferation. PASMCs isolated successfully by magnetic separation method and were in good condition. Hypoxia promoted the proliferation of PASMCs, and the treatment for 72 h had the most significant effect. Hypoxia upregulated the expression of PKCδ and PKCε in mouse PASMCs, leading to PASMCs proliferation. Moreover, Our study demonstrated that hypoxia induced upregulation of PKCδ and PKCε expression resulting to the proliferation of PASMCs via up-regulating the phosphorylation of AKT and ERK. Our study provides clear evidence that increased nPKC expression contributes to PASMCs proliferation and uncovers the correlation between AKT and ERK pathways and nPKC-mediated proliferation of PASMCs. These findings may provide novel targets for molecular therapy of pulmonary hypertension.

A 50-Hz magnetic-field exposure promotes human amniotic cells proliferation via SphK-S1P-S1PR cascade mediated ERK signaling pathway.

Extremely low-frequency electromagnetic fields (ELF-EMFs) present a kind of common non-ionizing radiation in public and occupational environments. Previous studies have suggested that ELF-EMF exposure might have a potential impact on co-carcinogenesis and the progression of tumorigenesis by inducing cell proliferation. However, the underlying mechanisms remain largely unknown. In this study, we investigated the possible role of the sphingosine-1-phosphate (S1P)-related pathway in regulating cell proliferation induced by 50-Hz, 0.4-mT magnetic-field (MF) exposure. The results showed that MF exposure significantly promoted sphingosine kinase 1 (SphK1) activity, and that inhibition of the SphK1-S1P-S1P receptor (S1PR) pathway could remarkably reverse MF-induced cell proliferation. Additionally, we could infer indirectly from an exogenous-S1P experiment that MF-induced S1P might act on S1PR1/3 in a paracrine and/or autocrine manner to mediate the proliferation effect. Notably, although the MF activated the extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) pathways, the SphK1-S1P-S1PR1/3 cascade regulated MF-induced proliferation by activating the ERK rather than the Akt pathway. Taken together, the findings of this study indicated that the SphK1-S1P-S1PR1/3 cascade played an important role in MF-induced proliferation by mediating the ERK signaling pathway, which could bring new insights into understanding and preventing the adverse effects of MFs.

An Integrated Next-Generation Sequencing System for Analyzing DNA Mutations, Gene Fusions, and RNA Expression in Lung Cancer.

We developed and characterized a next-generation sequencing (NGS) technology for streamlined analysis of DNA and RNA using low-input, low-quality cancer specimens. A single-workflow, targeted NGS panel for non-small cell lung cancer (NSCLC) was designed covering 135 RNA and 55 DNA disease-relevant targets. This multiomic panel was used to assess 219 formalin-fixed paraffin-embedded NSCLC surgical resections and core needle biopsies. Mutations and expression phenotypes were identified consistent with previous large-scale genomic studies, including mutually exclusive DNA and RNA oncogenic driver events. Evaluation of a second cohort of low cell count fine-needle aspirate smears from the BATTLE-2 trial yielded 97% agreement with an independent, validated NGS panel that was used with matched surgical specimens. Collectively, our data indicate that broad, clinically actionable insights that previously required independent assays, workflows, and analyses to assess both DNA and RNA can be conjoined in a first-tier, highly multiplexed NGS test, thereby providing faster, simpler, and more economical results.

Involvement of calcium in 50-Hz magnetic field-induced activation of sphingosine kinase 1 signaling pathway.

Previously, we found that exposure to a 50-Hz magnetic field (MF) could induce human amniotic epithelial (FL) cell proliferation and sphingosine kinase 1 (SK1) activation, but the mechanism was not clearly understood. In the present study, the possible signaling pathways which were involved in SK1 activation induced by 50-Hz MF exposure were investigated. Results showed that MF exposure increased intracellular Ca2+ which was dependent on the L-type calcium channel, and induced Ca2+ -dependent phosphorylation of extracellular regulated protein kinase (ERK), SK1, and protein kinase C α (PKCα). Also, treatment with U0126, an inhibitor of ERK, could block MF-induced SK1 phosphorylation, but had no effect on PKCα phosphorylation. Also, the inhibitor of PKCα, Gö6976, had no effect on MF-induced SK1 activation in FL cells. In addition, the activation of ERK and PKCα could be abolished by SKI II, the inhibitor of SK1. In conclusion, the intracellular Ca2+ mediated the 50-Hz MF-induced SK1 activation which enhanced PKCα phosphorylation, and there might be a feedback mechanism between SK1 and ERK activation in responding to MF exposure in FL cells. Bioelectromagnetics. 9999:XX-XX, 2019. © 2019 Bioelectromagnetics Society.

S1P mediates human amniotic cells proliferation induced by a 50-Hz magnetic field exposure via ERK1/2 signaling pathway.

Extremely low frequency electromagnetic field (ELF-EMF) is a kind of physical stimulus in public and occupational environment. Numerous studies have indicated that exposure of cells to ELF-EMF could promote cell proliferation. But the detailed mechanisms implicated in these proliferative processes remain unclear. In the present experiment, the possible roles of sphingosine-1-phosphate (S1P) in 50-Hz magnetic field (MF)-induced cell proliferation were investigated. Results showed that exposure of human amniotic (FL) cells to a 50-Hz MF with an intensity of 0.4 mT significantly enhanced ceramide metabolism, increased S1P production, activated extracellular signal regulated kinase 1/2 (ERK1/2), and promoted cell proliferation. All of these effects induced by MF exposure could be inhibited by SKI II, an inhibitor of sphingosine kinase (SphK). In addition, both the cell proliferative response and the ERK1/2 activation induced by MF exposure were blocked completely by U0126, a specific inhibitor of MEK (ERK kinases 1 and 2). Taken together, the findings in present study suggested that S1P mediated 50-Hz MF-induced cell proliferation via triggering ERK1/2 signal pathway.

Reactive oxygen species mediates 50-Hz magnetic field-induced EGF receptor clustering via acid sphingomyelinase activation.

PURPOSE: Exposure to extremely low frequency electromagnetic fields (ELF-EMFs) could elicit biological effects including carcinogenesis. However, the detailed mechanisms by which these ELF-EMFs interact with biological system are currently unclear. Previously, we found that a 50-Hz magnetic field (MF) exposure could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell membranes. In the present experiment, the possible roles of reactive oxygen species (ROS) in MF-induced EGFR clustering were investigated. MATERIALS AND METHODS: Human amnion epithelial (FL) cells were exposed to a 50-Hz MF with or without N-acetyl-l-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC). EGFR clustering on cellular membrane surface was analyzed using confocal microscopy after indirect immunofluorescence staining. The intracellular ROS level and acid sphingomyelinase (ASMase) activity were detected using an ROS assay kit and an Amplex® Red Sphingomyelinase Assay Kit, respectively. RESULTS: Results showed that exposure of FL cells to a 50-Hz MF at 0.4 mT for 15 min significantly enhanced the ROS level, induced EGFR clustering and increased ASMase activity. However, pretreatment with NAC or PDTC, the scavenger of ROS, not only counteracted the effects of a 50-Hz MF on ROS level and AMS activity, but also inhibited the EGFR clustering induced by MF exposure. CONCLUSIONS: The present and previous data suggest that ROS mediates the MF-induced EGFR clustering via ASMase activation.

Study of acetylcholinesterase activity and apoptosis in SH-SY5Y cells and mice exposed to ethanol.

Ethanol is one of the most commonly abused psychotropic substances with deleterious effects on the central nervous system. Ethanol exposure during development results in the loss of neurons in brain regions and when exposed to ethanol cultured cells undergo apoptosis. To date no information is available on whether abnormally high AChE activity is characteristic of apoptosis in animals exposed to ethanol. The aims of the present study were to determine whether induction of AChE activity is associated with ethanol-induced apoptosis and to explore the mechanism of enhanced AChE activity induced by ethanol. For this purpose, in vitro and in vivo experiments were performed. AChE activity was quantified by spectrophotometry and apoptosis by flow cytometer in SH-SY5Y cells exposed to ethanol. The results showed that cells treated with 500mM ethanol for 24h had a 9-fold increase in apoptotic cells and a 6-fold increase in AChE activity compared with controls. Mice exposed acutely to 200µl of 20% ethanol daily on days 1-4 had elevated AChE activity in plasma on days 3-7. On day 4, plasma AChE activity was 2.4-fold higher than pretreatment activity. More apoptotic cells were found in the brains of treated mice compared to controls. Cells in brain sections that were positive in the TUNEL assay stained for AChE activity. In conclusion, AChE activity and apoptosis were induced in SH-SY5Y cells and mice treated with ethanol, which may indicate that increased AChE may related to apoptosis induced by ethanol. Unusually high AChE activity may be an effect marker of exposure to ethanol. The relationship between AChE and apoptosis might represent a novel mechanism of ethanol-associated neuronal injury.

NADPH oxidase-produced superoxide mediated a 50-Hz magnetic field-induced epidermal growth factor receptor clustering.

PURPOSE: A 50-Hz magnetic field (MF) was found to induce epidermal growth factor receptor (EGFR) clustering in our previous study. The aim of this work was to investigate the molecular mechanisms that mediated MF-induced EGFR clustering. MATERIALS AND METHODS: Human amniotic epithelial (FL) cells were exposed to a 50-Hz MF. Total reactive oxygen species (ROS), cytoplasmic and mitochondrial superoxide production were detected by DCFH-DA, DHE and MitoSOX, respectively. EGFR clustering was analyzed using confocal microscopy after indirect immunofluorescence staining. RESULTS: Results showed that exposing FL cells to MF at intensity higher than 0.2 mT for 15 min enhanced total ROS production. Additionally, enhanced total ROS and cytoplasmic superoxide production were observed after exposing cells to MF at 0.4 mT for 5, 15, or 30 min, while mitochondrial superoxide production for 15 or 30 min. Pretreatment with Nox inhibitor, DPI, effectively inhibited MF-induced cytoplasmic superoxide production and subsequent EGFR clustering while mitochondrial superoxide production was not affected. CONCLUSIONS: Nox-produced superoxide mediated a 50-Hz magnetic field-induced EGFR clustering.

Mitochondrial ROS Release and Subsequent Akt Activation Potentially Mediated the Anti-Apoptotic Effect of a 50-Hz Magnetic Field on FL Cells.

BACKGROUND/AIMS: Our previous study showed that exposure to a 50-Hz magnetic field (MF) could induce transient mitochondrial permeability transition (MPT) in cells. In the present study, the aim was to explore the possible biological implications of MF-induced transient MPT. MATERIALS AND METHODS: Human amniotic (FL) cells were exposed to MF for different durations or intensities followed by incubation with staurosporine for 4 h. After MF exposure, cell early apoptosis, cell viability mitochondrial ROS and the level of phosphorylated Akt were assessed. After MF exposure followed by incubation with staurosporine, cell early apoptosis was assessed. RESULTS: MF exposure had a protective effect against early apoptosis induced by staurosporine, which could be abolished by MPT inhibitors, although MF exposure alone had no significant effect on early apoptosis or viability of cells. In addition, exposing cells to MF increased the level of mitochondrial ROS which were released into cytoplasm through mitochondrial permeability transition pores (mPTP), and induced ROS-dependent phosphorylation of Akt. Furthermore, the anti-apoptotic effect of MF exposure was completely eliminated when Akt was inhibited. CONCLUSIONS: The present study indicated a possibility that mitochondrial ROS release through mPTP and subsequent Akt activation were necessary for the anti-apoptotic effect of MF.

Exposure to a 50-Hz magnetic field induced mitochondrial permeability transition through the ROS/GSK-3ß signaling pathway.

PURPOSE: To investigate the biological effects of a 50-Hz magnetic field (MF) on mitochondrial permeability. MATERIALS AND METHODS: Human amniotic epithelial cells were exposed to MF (50 Hz, 0.4 mT) for different durations. Mitochondrial permeability, mitochondrial membrane potential (ΔΨm), cytochrome c (Cyt-c) release and the related mechanisms were explored. RESULTS: Exposure to the MF at 0.4 mT for 60 min transiently induced mitochondrial permeability transition (MPT) and Cyt-c release, although there was no significant effect on mitochondrial membrane potential (ΔΨm). Other than decreasing the total Bcl-2 associated X protein (Bax) level, MF exposure did not significantly affect the levels of Bax and B-cell lymphoma-2 (Bcl-2) in mitochondria. In addition, cells exposed to the MF showed increased intracellular reactive oxidative species (ROS) levels and glycogen synthase kinase-3ß (GSK-3ß) dephosphorylation at 9 serine residue (Ser(9)). Moreover, the MF-induced MPT was attenuated by ROS scavenger (N-acetyl-L-cysteine, NAC) or GSK-3ß inhibitor, and NAC pretreatment prevented GSK-3ß dephosphorylation (Ser(9)) caused by MF exposure. CONCLUSION: MPT induced by MF exposure was mediated through the ROS/GSK-3ß signaling pathway.

Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

PURPOSE: The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. METHODS: We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. RESULTS: Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. CONCLUSION: AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies.

The formalin-fixed, paraffin-embedded (FFPE) biopsy is a challenging sample for molecular assays such as targeted next-generation sequencing (NGS). We compared three methods for FFPE DNA quantification, including a novel PCR assay ('QFI-PCR') that measures the absolute copy number of amplifiable DNA, across 165 residual clinical specimens. The results reveal the limitations of commonly used approaches, and demonstrate the value of an integrated workflow using QFI-PCR to improve the accuracy of NGS mutation detection and guide changes in input that can rescue low quality FFPE DNA. These findings address a growing need for improved quality measures in NGS-based patient testing.

Targeted, high-depth, next-generation sequencing of cancer genes in formalin-fixed, paraffin-embedded and fine-needle aspiration tumor specimens.

Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at <10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.