Correction: Development of a DNA damage assay system using stable human hepatocytes.

Validation of advanced tools to evaluate sperm function in barramundi (Lates calcarifer).

The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish ( Fundulus heteroclitus)

Antimony (Sb) and its compounds are negative in gene mutation assays in bacteria and cultured mammalian cells but positive in some assays for clastogenicity and/or DNA damage. In order to better understand the modes of action for antimony genotoxicity, we assessed reporter gene activation by antimony and antimony compounds in the new expanded ToxTracker assay. ToxTracker evaluates the activation of biomarkers for different cellular defense mechanisms using a series of green fluorescent protein reporters inserted into mouse embryonic stem cell lines. The assay responds to: 1) DNA damage and inhibition of DNA replication; 2) oxidative stress; 3) unfolded protein response (protein damage); and 4) p53-dependent cellular stress. Sb metal powder, six trivalent (Sb(III)) compounds, and five pentavalent antimony (Sb(V)) compounds were assessed. Sb powder and all six Sb(III) compounds activated oxidative stress ToxTracker reporters at non-toxic doses. Of the five Sb(V) compounds, antimony pentachloride and potassium hexahydroantimonate induced a robust oxidative stress response while sodium antimonate induced only a weak oxidative stress response. At higher concentrations (up to either 75 % toxicity or the highest dissolved concentration tested), Sb powder and all Sb(III) compounds except for antimony trichloride induced the unfolded protein response. Of the five Sb(V) compounds tested, only potassium hexahydroantimonate induced weak activation of the unfolded protein response and was also the only pentavalent compound to yield modest (30 %) cytotoxicity. None of the compounds tested activated the DNA damage/inhibition of DNA replication reporters, nor did they activate the p53-dependent response. All Sb(III) compounds, Sb powder, and three of the five Sb(V) compounds activated the oxidative stress reporters, but there was no activation of reporters associated with DNA damage and repair or p53-dependent cellular stress. The consistent activation of reporters for oxidative stress suggests this mode of action may underlie genotoxicity responses for antimony and its compounds.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract. Sea conch peptide hydrolysate (CPH) was produced by enzymatic digestion of fresh conch meat with trypsin enzyme. To analyze the molecular composition, functional groups, and structural morphology of the hydrolysate, we employed liquid chromatography-mass spectrometry (LC-MS), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Results confirmed that crude protein could be effectively digested by enzymes to generate peptides. In this study, we evaluated the bioactivities of CPH on dextran sulfate solution (DSS)-induced colitis in mice. The findings demonstrated that CPH supplementation improved body weight, food and water intake, and colon length. The therapeutic efficacy and immunoregulatory effect of CPH were further determined. Our results exhibited that CPH treatment significantly ameliorated pathological symptoms by enhancing intestinal integrity, mucin production, and goblet cell count. Moreover, the immunoregulatory effect of CPH on mRNA expression levels of different pro- and anti-inflammatory cytokines was determined. Results exhibited a decrease in the expression of pro-inflammatory cytokines and an increase in anti-inflammatory cytokines in the colon. Additionally, the CPH administration modulates the nuclear factor kappa B (NF-κB) pathway, preventing DNA damage and cell death. Assays for apoptosis and DNA damage revealed that CPH reduced oxidative DNA damage and apoptosis. These findings highlight the immunomodulatory and treatment amelioration effect of CPH in reducing the severity of colitis.

Background: EMT (Epitherial-Mesenchymal transition) is a significant event in tumor metastasis and malignancy. Cancer cells are stimulated by EMT-inducible agonists from the surrounding stromal cells, and cause transformation such as weakening of cell-cell adhesion and the accompanying acceleration of cell migration and invasion. Inhibition of EMT is considered to enable controlling of malignant transformation. In this study, we developed a high-throughput assay system of EMT inhibitor screen in 3D cell culture. Method: We adopted 3D cell culture method on NanoCulture® Plate (NCP), a scaffold type spheroid culture plate, as a culture method which can confirm spheroid morphologies. TGF-β and its inhibitor, SB431542, were used as an EMT inducer and as a positive control for EMT inhibitor, respectively. Expressions of E-cadherin, N-cadherin, vimentin and zeb-1, represent EMT maker genes, were evaluated by qRT-PCR. As hypoxia inside the spheroids reflected the spheroid size and cell-cell adhesion strength, we visualized the intra-spheroid hypoxia, and used it as an indicator for EMT induction and inhibition. Result: A549 cell spheroids gradually collapsed, by the weakening of cell-cell adhesion, and cells migrating out from the spheroids, by treating with TGF-β. This collapse of the spheroids was inhibited by SB431542 treatment. SB431542 treatment also accompanied inhibition of E-cadherin down-regulation, and N-cadherin, vimentin and zeb-1 up-regulation. These results indicate that this method is clearly reproducing EMT and its inhibition. And we elucidated that intra-spheroid hypoxicity was closely correlated with spheroid morphology. From this, it is demonstrated that this method is simple but high-precision assay. Conclusion: We developed a high-throughput assay system for EMT inhibitor screen, by evaluating intra-spheroid hypoxicity as an indicator for alternation of the spheroid morphology. This method is an unique method which enables to evaluate EMT condition, by monitoring the collapse of spheroids directly. We are now running a screen for EMT inhibitor candidate compounds, by using this assay system. Citation Format: Kazuya Arai, Ruriko Sakamoto, Manabu Itoh, Hiromi Miura, Manami Shimomura, Tetsuya Nakatsura. Development of novel high-throughput screening assay system for exploring EMT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5525. doi:10.1158/1538-7445.AM2013-5525

A Novel Immunocompetent Rat Model of HCV Infection and Hepatitis

ObjectiveThe aim of our study was to investigate the effect of Transforming growth factor beta-1 (TGF-β1) gene therapy on the surface markers, multilineage differentiation, viability, apoptosis, cell cycle, DNA damage and senescence of human Dental Pulp-derived Mesenchymal Stromal Cells (hDPSC).MethodshDPSCs were isolated from human teeth, and were cultured with 20% Fetal Bovine Serum (FBS) in minimum essential media-alpha (α-MEM). TGF-β1 gene transfer into hDPSCs was performed by electroporation method after the plasmid was prepared. The transfection efficiency was achieved by using western blot and flow cytometry analyses and GFP transfection. Mesenchymal stem cell (MSC) markers, multilineage differentiation, cell proliferation, apoptosis, cell cycle, DNA damage and cellular senescence assays were performed by comparing the transfected and non-transfected cells. Statistical analyses were performed using GraphPad Prism.ResultsStrong expression of TGF-β1 in pCMV-TGF-β1-transfected hDPSCs was detected in flow cytometry analysis. TGF-β1 transfection efficiency was measured as 95%. Western blot analysis showed that TGF-β1 protein levels increased at third and sixth days in pCMV-TGF-β1-transfected hDPSCs. The continuous TGF-β1 overexpression in hDPSCs did not influence the immunophenotype and surface marker expression of MSCs. Our results showed that TGF-β1 increased osteogenic and chondrogenic differentiation, but decreased adipogenic differentiation. Overexpression of TGF-β1 increased the proliferation rate and decreased total apoptosis in hDPSCs (p<0.05). The number of cells at “S” phase was higher with TGF-β1 transfection (p<0.05). Cellular senescence decreased in TGF-β1 transfected group (p<0.05).ConclusionsThese results reflect that TGF-β1 has major impact on MSC differentiation. TGF-β1 transfection has positive effect on proliferation, cell cycle, and prevents cellular senescence and apoptosis.

&lt;div&gt;Abstract&lt;p&gt;This study aims to explore whether E545K, the most common hotspot mutation of &lt;i&gt;PIK3CA&lt;/i&gt; in cervical cancer, confers radioresistance to cervical cancer cells, to demonstrate the underling mechanism, and to develop the effective targets. SiHa and MS751 cells with &lt;i&gt;PIK3CA&lt;/i&gt;-WT and &lt;i&gt;PIK3CA&lt;/i&gt;-E545K were established by lentiviral transfection. The radiosensitivity was assessed by colony formation, cell cycle, cell apoptosis, DNA damage, and repair assay. The growth and immunohistochemical assay of xenograft tumor–related toxicity were evaluated &lt;i&gt;in vivo&lt;/i&gt;. It was indicated that more cells with &lt;i&gt;PIK3CA&lt;/i&gt;-E545K arrested in S phase. Irradiation (IR) led to more survival percentage, less apoptosis, fewer pH2A.X foci, and higher expression of Chk1/2 in SiHa and MS751 cells bearing &lt;i&gt;PIK3CA&lt;/i&gt;-E545K. Mechanically, AKT/GSK3β/β-catenin pathway was highly activated, and more β-catenin was found accumulated in nucleus in cells with &lt;i&gt;PIK3CA&lt;/i&gt;-E545K after IR. Furthermore, targeting β-catenin by shRNA or XAV939 enhanced IR sensitivity in cells with &lt;i&gt;PIK3CA&lt;/i&gt;-WT and &lt;i&gt;PIK3CA&lt;/i&gt;-E545K, whereas it was more notably in the latter. β-Catenin shRNA and XAV939 increased IR-mediated inhibition of colony formation with highly activated p53/bcl2/bax pathway. XAV939 enhanced IR-caused apoptosis, DNA damage, overcame S-phase arrest, DNA repair and reversed β-catenin nuclear accumulation in MS751 cells with &lt;i&gt;PIK3CA&lt;/i&gt;-E545K. &lt;i&gt;In vivo&lt;/i&gt;, XAV939 enhanced the radiosensitivity of cervical cancer xenografts with &lt;i&gt;PIK3CA&lt;/i&gt;-E545K with invisible viscera toxicity. The findings demonstrate that cervical cancer cells with &lt;i&gt;PIK3CA&lt;/i&gt;-E545K are resistant to IR by enhancing the expression and nuclear accumulation of β-catenin. Targeting β-catenin reverses the radioresistance, which suggests possible areas for preclinical research on β-catenin inhibition for strengthening the radiosensitivity of cervical cancer.&lt;/p&gt;&lt;/div&gt;

Effects of an integrated yoga program in modulating perceived stress levels, anxiety, as well as depression levels and radiation-induced DNA damage were studied in 68 breast cancer patients undergoing radiotherapy. Two psychological questionnaires--Hospital Anxiety and Depression Scale (HADS) and Perceived Stress Scale (PSS)--and DNA damage assay were used in the study. There was a significant decrease in the HADS scores in the yoga intervention group, whereas the control group displayed an increase in these scores. Mean PSS was decreased in the yoga group, whereas the control group did not show any change pre- and postradiotherapy. Radiation-induced DNA damage was significantly elevated in both the yoga and control groups after radiotherapy, but the postradiotherapy DNA damage in the yoga group was slightly less when compared to the control group. An integrated approach of yoga intervention modulates the stress and DNA damage levels in breast cancer patients during radiotherapy.

Comparative toxicity of silver nanoparticles on oxidative stress and DNA damage in the nematode, Caenorhabditis elegans

Pathway-centric analysis of the DNA damage response to chemotherapeutic agents in two breast cell lines

Several human syndromes are described with which have been associated a deficiency in the ability to repair damage to cellular DNA. This deficiency is generally manifested as a sensitivity to DNA-damaging agents such as u. v. light, ionizing radiation, or psoralen photosensiti­zation (PUVA). In xeroderma pigmentosum (XP) a high frequency of light-induced skin cancers is correlated with hypermutability of fibro­blasts following u. v. irradiation in vitro, providing telling support for the somatic mutation theory of cancer. Closer inspection, however, reveals that a pseudopromoting action of light or PUVA may be equally import­ant in skin carcinogenesis in both XP and normal individuals. It is suggested that impairment of cell-mediated immune response in the skin (possibly by DNA damage) may be responsible for this pseudopromoting action. That more than an enhanced frequency of somatic mutation is necessary for early skin neoplasms is illustrated by Cockayne syndrome (and possibly patient 11961), where XP-like changes in the skin are not observed despite an enhanced u. v. mutability of cultured fibroblasts. Xeroderma pigmentosum, ataxia-telangiectasia and Cockayne syn­drome all show progressive neurological disease, suggesting that common factors are involved in DNA repair and the normal development and function of the nervous system. Fibroblasts from certain Huntington’s disease patients also show some sensitivity to ionizing radiation but this cannot at present be attributed to a DNA-repair deficiency. Patients with ataxia-telangiectasia also show a severely depressed immune response, and a search for other individuals with impaired im­munity revealed patient 46BR, whose cells are sensitive to a wide range of DNA-damaging agents. It is suggested that common factors may be involved in DNA repair and the proper development and functioning of the immune system. One possibility is (by analogy with the recA mutation in bacteria) that common steps exist between some types of DNA repair and the somatic recombination events that are involved in the generation of immunoglobulin genes. Human DNA-repair-deficient mutants may not yet have proved their value in yielding an understanding of the molecular mechanisms of DNA repair, but they have shown that several unexpected aspects of human health may be linked to functional DNA-repair processes.

Triallate is a selective herbicidal chemical used for control of wild oats in wheat. It has an extensive genotoxicity database that includes a variety of in vitro and in vivo studies. The chemical has produced mixed results in in vitro assay systems. It was genotoxic in bacterial mutation Ames assays, predominantly in Salmonella typhimurium strains TA100 and TA1535 in the presence of S9. Weaker responses have been observed in TA100 and TA1535 in the absence of S9. Mixed results have been observed in strain TA98, whereas no genotoxicity has been observed in strains TA1537 and TA1538. The presence and absence of S9 and its source seem to play a role in the bacterial response to the chemical. There have also been conflicting results in other test systems using other bacterial genera, yeast, and mammalian cells. Chromosome effects assays (sister-chromatid exchange and cytogenetics assays) have produced mixed results with S9 but no genotoxicity without S9. Triallate has not produced any genotoxicity in in vitro DNA damage or unscheduled DNA synthesis assays using EUE cells, human lymphocytes, and rat and mouse hepatocytes. In a series of in vivo genotoxicity assays (cytogenetics, micronucleus, dominant lethal, and unscheduled DNA synthesis), there has been no indication of any adverse genotoxic effect. Metabolism data indicate that the probable explanation for the differences observed between the in vitro studies with S9 and without S9 and between the in vitro and the in vivo studies is the production of a mutagenic intermediate in vitro at high doses of triallate is expected to be at most only transiently present in in vivo studies. The weight of evidence strongly suggests that triallate is not likely to exert mutagenic activity in vivo due to toxicokinetics and metabolic processes leading to detoxification.

Phytomedicines are potential immunity-boosting components with effective anticystic properties, minimal side effects, and biomedical applications, making them valuable for combating various diseases. India is renowned globally for Ayurveda, an ancient treatment methodology known for its holistic approach in identifying the root cause of diseases. Tulsi (Ocimum sanctum) is a common household medicine in India. While essential oils from plants like Tulsi have long been recognized for their medicinal properties, there is a gap in understanding their potential in synthesizing gold nanoparticles (AuNPs) and their efficacy against breast carcinoma, particularly in the context of immunosuppressive conditions. We investigated the potential application of essential oils isolated from O. sanctum in the synthesis of AuNPs and their efficacy against MCF-7 breast carcinoma. Gas chromatography-mass spectroscopy identified compounds with potential anticancer effects against breast cancer cells. Synthesised AuNPs displayed high hemocompatibility and antimicrobial activity against nosocomial Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, and Bacillus subtilis strains. Os-AuNPs induced chromosomal instability and mitotic arrest in the G2/M cell cycle phase. Subsequent fluorescence and cell cytometry studies demonstrated the systemic release of ROS, depolarisation of mitochondrial membrane potential, and production of apoptotic bodies. DNA damage and comet assays confirmed the anticancer potential of synthesised AuNPs. This study illuminates the potential of O. sanctum-derived AuNPs in breast carcinoma treatment, paving the way for future AuNP-based therapies in biomedicine.

Persistent infection with hepatitis B virus (HBV) is a major worldwide health problem, and chronically infected individuals are at high risk for developing liver cirrhosis and hepatocellular carcinoma (1,2). Despite the availability of an HBV vaccine, there are still more than 350 million chronically infected people worldwide, and the few antiviral treatments currently available have a limited rate of efficacy. As for other infectious diseases in humans, advances in the study of viral hepatitis have been critically dependent on the development of suitable experimental systems. Replication of HBV can be successfully achieved by transfecting hepatoma cell lines with cloned HBV DNA genomes. However, the lack of culture systems permissive for hepadnavirus infection and the narrow host range of HBV have hampered the understanding of some crucial events of the hepadnavirus life cycle, as well as the development of more effective antiviral drugs aimed at eradicating the virus from chronic carriers. Although HBV has been grown successfully in cultures of human hepatocytes, primary human hepatocytes are difficult to maintain in culture and become nonpermissive for HBV very soon after plating. In addition, they normally differ in function and gene expression from hepatocytes integrated in the liver architecture. Therefore, any technique that would allow keeping human hepatocytes in an environment that supports both cell growth and liver-specific differentiation would offer unique opportunities to study human hepatotropic viral infections and to evaluate the efficacy of novel antiviral drugs in a system closely mimicking the in vivo situation.KeywordsHuman Serum AlbuminHuman HepatocytePrimary Human HepatocyteLuer LockIntrasplenic InjectionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.