DNA Migration Research Articles

Terminalia ivorensis demonstrates antioxidant properties and alters proliferation, genomic instability and migration of human colon cancer cells in vitro.

Colorectal cancer is a global killer that causes approximately 940 thousand deaths annually. Terminalia ivorensis (TI) is a tropical tree, the bark of which is used in African traditional medicine for the treatment of diabetes, malaria and ulcer. This study investigated TI as a potential anticancer agent in human colon cells in vitro. TI was extracted sequentially with petroleum ether, chloroform, ethyl acetate and ethanol. Antioxidant activity was assessed by DPPH and FRAP, and differential effects on cell viability, growth, DNA damage, DNA repair, and migration were measured in human colon cancer cells (CaCo-2) and/or non-cancerous human colonocytes (NCM460). The TI phytochemicals most strongly associated with these effects were identified by partial least-squares discriminant analysis. DPPH and FRAP activity were highest in TI ethyl acetate and ethanol extracts (p=0.001). All TI extracts significantly inhibited cell viability and growth and induced DNA damage and inhibited DNA repair in both cell models. The majority of TI extracts were significantly (p=0.01) more toxic to cancer cells than non-cancerous colonocytes. DNA repair was significantly (p=0.001) inhibited in CaCo-2 cells by ethyl acetate extract compared with NCM460 cells. Migration was also significantly inhibited (p<0.001) in CaCo-2 by ethyl acetate (80%) and ethanol extracts (75%). Specific benzoic acids, flavonoids and phenols were identified to be strongly associated with these effects. TI displayed strong antioxidant activity and specific anticancer effects by inducing cell death and DNA damage, and by inhibiting DNA repair, cell proliferation and migration.

A new herbal anesthetic agent for common carp (Cyprinus carpio) sedation and anesthesia: nutmeg (Myristica fragrans) essential oil.

In aquaculture, interest in natural essential oils is increasing alongside synthetic anesthetic agents. In this context, the anesthetic efficacy of nutmeg essential oil, which had not been previously tested, was investigated in common carp (Cyprinus carpio). The study, conducted using three different concentrations (800 μL/L "LC", 1,200 μL/L "MC", and 1,400 μL/L "HC"), found that induction times were <3 min for MC and HC, while LC had a longer induction time, exceeding acceptable levels. Within the first 4 h, white blood cells, red blood cells, hemoglobin, and hematocrit levels increased to >5 103/μL, >1 106/μL, >6 g/dL, and >12%, respectively. However, they returned to control levels after 8 h. Histological signs were more severe with higher concentrations, and necrosis was only observed in the HC group. Alkaline comet assay results showed DNA migration only in the HC group. According to the PROMETHEE multi-criteria decision-making model, the LC concentration is suitable for sedation, while the MC concentration should be used for deep anesthesia. The current study demonstrates that nutmeg essential oil can be used as an alternative to commercial synthetic anesthetic agents.

Novel role of circRNAs in the drug resistance of gastric cancer: regulatory mechanisms and future for cancer therapy.

Cancer, including gastric cancer, has become a serious disease that jeopardizes public life. Currently, the main treatment methods are surgery, radiation therapy, and chemotherapy. One of the primary causes of death for patients with gastric cancer is drug resistance. Several mechanisms of anticancer drugs resistance have been reported, including changes in drugs transport and metabolism, mutations in drug targets, changes in DNA repair systems, inhibition of cell apoptosis and autophagy, gastric cancer stem cells, invasion and migration. It is becoming more widely known that non-coding RNAs, like circRNAs, play a critical role in the resistance of drugs used to treat gastric cancer. CircRNAs have a unique structure and function that is related to gastric cancer resistance, cell proliferation, apoptosis, autophagy, DNA repair systems, migration, and invasion. A clear understanding of the molecular mechanism of circRNAs mediated the resistance of gastric cancer drugs will open a new window for the treatment and management of gastric cancer. Therefore, in this review, we will summarize the current mechanism of drug resistance, and finally discuss the molecular mechanism of circRNAs in regulating the development of drug resistance in gastric cancer.

PEITC Induces DNA Damage and Inhibits DNA Repair-Associated Proteins in Human Retinoblastoma Cells In Vitro.

Phenethyl isothiocyanate (PEITC), a natural product, exists in biological activities, including anticancer activity in many human cancer cells. No information shows that PEITC affects DNA damage in human retinoblastoma (RB) cells in vitro. In this study, the aim of experiments was to determine whether PEITC decreased total viable cell number or not by inducing protein expressions involved in DNA damage and repair in Y79 RB cells in vitro. Total cell viability was measured by PI exclusion assay, and PEITC reduced the total Y79 viable cell numbers in a dose-dependent manner. DNA condensation and DNA impairment were conducted by DAPI staining and comet assays, respectively, in Y79 cells. The findings show that PEITC induced DNA condensation dose-dependently based on the brighter fluorescence of cell nuclei stained by DAPI staining. PEITC-induced DNA damage showed a more extended DNA migration smears than that of the control, which was performed by a comet assay. Western blotting was performed to measure the protein expressions involved in DNA damage and repair, which showed that PEITC at 2.5-10 μM increased NRF2, HO-1, SOD (Mn), and catalase; however, it decreased SOD (Cu/Zn) except 10 μM PEITC treatment, and decreased glutathione, which were associated with oxidativestress. Furthermore, PEITC increased DNA-PK, MDC1, H2A.XpSer139, ATMpSer1981, p53, p53pSer15, PARP, HSP70, and HSP90, but decreased TOPIIα, TOPIIβ, and MDM2pSer166 that were associated with DNA damage and repair mechanism in Y79 cells. The examination from confocal laser microscopy shows that PEITC increased H2A.XpSer139 and p53pSer15, and decreased glutathione and TOPIIα in Y79 cells. In conclusion, the cytotoxic effects of PEITC on reducing the number of viable cells may be due to the induction of DNA damage and the alteration of DNA repair proteins in Y79 cells in vitro.

Hsa_circ_0004776 regulates the retina neovascularization in progression of diabetic retinopathy via hsa-miR-382-5p/BDNF axis

The aim of this work was to identify the regulatory function of hsa_circ_0004776 in the progression of diabetic retinopathy (DR). The direct interactions between hsa_circ_0004776 and hsa-miR-382-5p and between hsa-miR-382-5p and BDNF, were confirmed via dual-luciferase reporter assays. Quantitative Real-Time PCR analysis indicated that hsa_circ_0004776 was highly expressed in aqueous humour samples of DR patients and human retinal microvascular epithelial cells (hRECs) under a high-glucose environment, whereas hsa-miR-382-5p showed the opposite trend. Overexpressed hsa_circ_0004776 significantly enhanced DNA synthesis, proliferation, migration, and tube formation in hRECs in hyperglycaemia, while hsa-miR-382-5p mimics reversed these changes. Additionally, in a streptozotocin-induced Sprague-Dawley rat model of DR, vitreous microinjection of rno-miR-382-5p agomir reversed the pathologic features in the progression of DR, including retinal vascular leakage, capillary decellularization, loss of pericytes, fibrosis, and gliosis. Our results indicated that under hyperglycaemic conditions, hsa_circ_0004776 influences the progression of DR via hsa-miR-382-5p and thus represents a potential therapeutic target.

Proper application of DNA dyes in agarose gel electrophoresis.

Various dyes are used to visualize DNA bands in agarose gel electrophoresis (AGE) by the methods of pre- or post-staining. The DNA dye user's guides generally state that the binding of the dye to DNA will affect DNA mobility in electrophoresis, thus recommending post-staining for accurate measurement of DNA size. However, many AGE performers prefer pre-staining procedures for reasons such as convenience, real-time observation of DNA bands, and/or the use of a minimal amount of dye. The detrimental effect of the dye on DNA mobility and the associated risk for inaccurate measurement of DNA size are often overlooked by AGE performers. Here we quantitatively determine the impact on DNA migration imposed by frequently used dyes, including GelRed, ethidium bromide (EB), and Gold View. It was observed that pre-staining with GelRed and EB significantly slowed down DNA migration to cause as much as 39.1% overestimation on the size of sample DNA, whereas Gold View had little effect. The slowdown of DNA migration increased with dye concentration until it plateaued when the dye concentration reached a saturated level. Thus, to take advantage of pre-staining, saturated levels of DNA dyes should always be applied for both DNA samples and DNA markers to ensure a fair comparison of DNA sizes. In addition, GelRed and EB display much higher sensitivity than Gold View in the detection of DNA bands in post-staining. The saturated concentrations, cost considerations, and other useful features of these frequently used dyes are summarized for the information of AGE performers.

Impact of high (1950 MHz) and extremely low (50 Hz) frequency electromagnetic fields on DNA damage caused by occupationally relevant exposures in human derived cell lines

Epidemiological studies indicate that electromagnetic fields (EMF) are associated with cancer in humans. Exposure to mobile phone specific high frequency fields (HF-EMF) may lead to increased glioma risks, while low frequency radiation (LF-EMF) is associated with childhood leukemia. We studied the impact of HF-EMF (1950 MHz, UMTS signal) on DNA stability in an astrocytoma cell line (1321N1), and the effect of LF-EMF (50 Hz) in human derived lymphoma (Jurkat) cells. To find out if these fields affect chemically induced DNA damage, co-exposure experiments were performed. The cells were exposed to HF-EMF or LF-EMF and treated simultaneously and sequentially with mutagens. The compounds cause DNA damage via different molecular mechanisms, i.e. pyrimidine dimers which are characteristic for UV light (4-nitroquinoline 1-oxide, 4NQO), bulky base adducts (benzo[a]pyrene diolepoxide, BPDE), DNA-DNA and DNA-protein cross links and oxidative damage (NiCl2, CrO3). DNA damage was measured in single cell gel electrophoresis (comet) assays. We found a moderate reduction of basal and 4NQO-induced DNA damage in the astrocytoma line, but no significant alterations of chemically induced DNA migration by the HF and LF fields under all other experimental series. The biological consequences of the moderate reduction remain unclear, but our findings indicate that acute mobile phone and power line specific EMF exposures do not enhance genotoxic effects caused by occupationally relevant chemical exposures.

Comparison of DNA damage in fresh and frozen blood samples: implications for the comet assay in human biomonitoring studies.

The use of the comet assay in large biomonitoring studies may present logistical and technical challenges because of the processing of numerous samples. Proper sample preservation becomes imperative to prevent spurious DNA breakage. Previous research has shown the feasibility of conducting the comet assay on frozen blood samples, highlighting the potential of freezing at - 80°C in preserving DNA integrity. Nonetheless, this approach presents challenges, including potential DNA damage during freezing and thawing, variability in processing, and the need for standardized protocols. Our objective was to evaluate whether there are comparable results in DNA migration assessed by the comet assay between fresh and frozen blood samples on a larger scale (N = 373). In our findings, elevated DNA migration was evident in frozen samples relative to fresh ones. Additionally, smoking, alcohol consumption, and season were linked to increased DNA damage levels in whole blood cells. Based on our results and available literature, conducting the comet assay on frozen blood samples emerges as a practical and efficient approach for biomonitoring and epidemiological research. This method enables the assessment of DNA damage in large populations over time, with samples, if properly cryopreserved, that may be used for years, possibly even decades. These observations hold significant implications for large-scale human biomonitoring and long-term epidemiological studies, particularly when samples are collected during fieldwork or obtained from biobanks. Continued method optimization and validation efforts are essential to enhance the utility of this approach in environmental and occupational health studies, emphasizing caution when comparing data obtained between fresh and frozen blood samples.

A novel antimicrobial peptide S24 combats serious wound infections caused by Pseudomonas aeruginosa and Acinetobacter baumannii.

Pseudomonas aeruginosa and Acinetobacter baumannii are ranked as top-priority organisms by WHO. Antimicrobial peptides (AMPs) are promising antimicrobial agents that are highly effective against serious bacterial infections. In our previous study, a series of α-helical AMPs were screened using a novel multiple-descriptor strategy. The current research suggested that S24 exhibited strong antimicrobial activity against major pathogenic bacteria, and displayed minimal haemolysis, good serum stability and maintained salt resistance. We found that S24 exerted an antimicrobial effect by destroying outer membrane permeability and producing a strong binding effect on bacterial genomic DNA that inhibits genomic DNA migration. Furthermore, S24 exerted a strong ability to promote healing in wound infected by P. aeruginosa, A. baumannii and mixed strains in a mouse model. Overall, S24 showed good stability under physiological conditions and excellent antimicrobial activity, suggesting it may be a potential candidate for the development of serious bacterial infection treatment.

Toxic Effects of Pozzolanic Cement on Egg, Alevine, Fry and Juvenile Rainbow Trout (Oncorhynchus mykiss) Individuals and Some Water Quality Parameters

Current study aims to ascertain the harmful effects of pozzolanic cement (PC) on water quality and several indicators of aquatic life. Also, the effects of PC on different life stages of rainbow trout were investigated. The PC caused an increase in various water quality parameters including pH, suspended particles, turbidity, and conductivity. In rainbow trout, no significant differences were observed between control and experimental groups in terms of enzyme parameters. Also, this study revealed that the PC has impact on various hematological parameters. The PC was linked to histological changes in gills, DNA migration was observed in the rainbow trout exposed to PC. After 24 h, LC50 values of rainbow trout exposed to PC were 499.06, 324.66, and 361.26 mg/L for egg, alevine, and fry respectively. There was adverse relation between temperature and LC50. Consequently, cement pollution in the water and different stages of rainbow trout causes various negative effects.

Long-term exposure of endangered Danube sturgeon (Acipenser gueldenstaedtii) to bisphenol A (BPA): growth, behavioral, histological, genotoxic, and hematological evaluation.

Danube sturgeon (Acipenser gueldenstaedtii) which is identified as endangered species can be exposed to pollutants such as bisphenol A (BPA) that have a disruptive effect on the endocrine system at any time. Starting from this motivation, the current study focused on BPA toxicity in A. gueldenstaedtii juvenile individuals and its adverse effects in sub-lethal concentration. The median lethal concentration (LC50) of BPA was 5.03mg/L in 96th hour. In the chronic period, 0.625mg/L and 1.25mg/L BPA concentrations were evaluated based on the result of acute study. Accordingly, growth performance was significantly decreased in BPA groups (1.25mg/L BPA group was significantly lowest) compared to control (p < 0.05). In the acute period, behavioral disorders were standing at the bottom/corner of tank, slowing and stopping of gill movement, decreased response to stimuli, and death, respectively. While vacuolization was severe in the liver tissue of the fish in the acute period, intense necrosis and melanomacrophage centers were observed in the chronic period. In terms of genotoxicity, longer DNA migration was observed in all groups exposed to BPA than in the control group. In addition, lower erythrocyte and hemoglobin were observed in the BPA groups compared to control. As a result, the current study revealed toxic effect of BPA on A. gueldenstaedtii juvenile individuals and its negative results on fish physiology.

Identification of Anti-Cancer Compounds in Medicinal Plants Using Metabolomic Approaches: A Review

The modern lifestyle despite its comfort has made cancer a second leading public health problem, following cardiovascular disease. As chemotherapeutic has many side effects including resistance, recent attention has focused on plants which may provide a good opportunity for complementary cancer treatment. Several studies have begun to focus on metabolomic research in order to discover the mechanisms of action of diverse medicinal plants, investigate biomarkers, and comprehend cancer progression at metabolic rates. This study aims to analyze and highlight related metabolomic investigations in medicinal plants that demonstrate the potential of biomarker compounds and their processes in cancer disease. The research methodology uses a literature review that is compiled from many connected journals. According to the findings, bioactive substances present in medicinal plants can be used as biomarkers to disrupt the metabolism of enzymes, transporters, and signaling pathways in cancer cells. Utilized analytical platforms include HPLC, GC-MS, FTIR and NMR. Statistical analysis employs the Anova, PCA, and OPLS-DA methods. Finally, we reviewed biomarkers of medicinal plants and metabolomic pathways, with the result that anticancer compounds may exert their activity by various methods affecting DNA replication, cell cycle, migration and apoptosis.The modern lifestyle despite its comfort has made cancer a second leading public health problem, following cardiovascular disease. As chemotherapeutic has many side effects including resistance, recent attention has focused on plants which may provide a good opportunity for complementary cancer treatment. Several studies have begun to focus on metabolomic research in order to discover the mechanisms of action of diverse medicinal plants, investigate biomarkers, and comprehend cancer progression at metabolic rates. This study aims to analyze and highlight related metabolomic investigations in medicinal plants that demonstrate the potential of biomarker compounds and their processes in cancer disease. The research methodology uses a literature review that is compiled from many connected journals. According to the findings, bioactive substances present in medicinal plants can be used as biomarkers to disrupt the metabolism of enzymes, transporters, and signaling pathways in cancer cells. Utilized analytical platforms include HPLC, GC-MS, FTIR and NMR. Statistical analysis employs the Anova, PCA, and OPLS-DA methods. Finally, we reviewed biomarkers of medicinal plants and metabolomic pathways, with the result that anticancer compounds may exert their activity by various methods affecting DNA replication, cell cycle, migration and apoptosis.

Polymorphisms in ERCC1, ERCC4 and ERCC5 genes as biomarkers of susceptibility for pesticide-induced DNA damage in North-West Indian agricultural workers

BackgroundOccupational pesticides exposure has raised health concerns due to genotoxicity and accumulation of DNA damage. Polymorphisms in genes encoding enzymes involved in nucleotide excision repair (NER) may affect the individual’s susceptibility to pesticide toxicity.MethodsThis study evaluates the association of excision repair cross complementation group 1 (ERCC1) (8092 C > A, 3’UTR, rs3212986) and ERCC1 (19007 C > T, Asn118Asn, rs11615), ERCC4 (1244 G > A, Arg415Gln, rs1800067) and ERCC5 (3507 G > C, Asp1104His, rs17655) polymorphisms with pesticide-induced DNA damage in North-West Indian agricultural workers. The study population comprised 225 agricultural workers exposed to pesticides and 225 non-exposed controls.ResultsOur study demonstrate that exposed workers carrying variant ERCC1 8092AA genotype showed higher total comet DNA migration (p = 0.015) as well as increased frequency of cells showing DNA migration (p = 0.027). Exposed agricultural workers with variant ERCC4 1244AA (415Gln/Gln) and ERCC5 3507CC (1104His/His) genotypes exhibited elevation in total comet DNA migration (p < 0.01). However, genotypes of ERCC1 19007 C > T (Asn118Asn) showed no association with total comet DNA migration (p = 0.963), frequency of cells showing DNA migration (p = 0.423) as well as mean tail length (p = 0.432).ConclusionERCC1, ERCC4 and ERCC5 polymorphisms influence DNA damage and can be used as biomarkers of susceptibility for pesticide-induced DNA damage in North-West Indian agricultural workers.

Slip-Coupled Electroosmosis and Electrophoresis Dictate DNA Translocation Speed in Solid-State Nanopores.

Controlling the DNA translocation speed is critical in nanopore sequencing, but remains rather challenging in practice, as attributable to a complex coupling between nanoscale fluidics and electrically mediated migration of DNA in a dynamically evolving manner. One important factor influencing the translocation speed is the DNA-liquid slippage stemming from the hydrophobic nature of the oligonucleotide, an aspect that has been widely ignored in the reported literature. In an effort to circumvent this conceptual deficit, here we first develop an analytical model to bring out the slip-mediated coupling between the electroosmosis and DNA-electrophoresis in a solid-state nanopore at low surface charge limits, ignoring the end effects. Subsequently, we compare these results with the numerical simulation data on electrokinetically modulated DNA translocation in such a nanopore, albeit of finite length with due accommodation of the end effects, connecting two end reservoirs by deploying a fully coupled Poisson-Nernst-Plank-Stokes flow model. Both the numerical and analytical results indicate that the DNA translocation speed is a linearly increasing function of the slip length, with more than four-fold increase being observed for a slip length as minimal as 0.5 nm as compared to the no-slip scenario. Considering specific strategies on demand for arresting high translocation speeds for accurate DNA sequencing, the above results establish a theoretical proposition for the same, premised on an analytical expression of the DNA-hydrophobicity modulated enhancement in the translocation speed for designing a nanopore-based sequencing platform─a paradigm that remained to be underemphasized thus far.

The hsa_circ_0004805/hsa_miR-149-5p/TGFB2 axis plays critical roles in the pathophysiology of diabetic retinopathy in vitro and in vivo

The aim of this study was to investigate the mechanism underlying the role of a recently identified hsa_circ_0004805/hsa_miR-149-5p/transforming growth factor beta 2 (TGFB2) axis in the progression of diabetic retinopathy (DR). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis suggested that hsa_circ_0004805 was highly expressed in aqueous humor samples of patients with DR, whereas hsa_miR-149-5p showed the opposite trend. Meanwhile, the results of a dual-luciferase reporter assay indicated that hsa_miR-149-5p directly interacted with both hsa_circ_0004805 and TGFB2. Using a variety of assays (Cell Counting Kit-8, EdU-labeling, Transwell, flow cytometric, wound healing, tube formation assays), we found that the overexpression of hsa_circ_0004805 significantly downregulated the level of hsa_miR-149-5p and promoted DNA synthesis, proliferation, migration, and tube formation in human retinal microvascular epithelial cells (hRECs) cultivated in a high-glucose environment. In contrast, hsa_miR-149-5p mimics inhibited DNA synthesis, proliferation, migration, and tube formation in hRECs by reducing the expression of its downstream target TGFB2 as well as the levels of phosphorylated SMAD2; however, these effects were reversed by the overexpression of hsa_circ_0004805. In a streptozotocin-induced Sprague-Dawley rat model of DR, retinal vascular leakage, capillary decellularization, loss of pericytes, fibrosis, and gliosis were evident, which could be reversed by vitreous microinjection of rat miR-149-5p mimics (rno-miR-149-5p agomir). Combined, our findings indicated that, under hyperglycemia, the hsa_circ_0004805/hsa_miR-149-5p/TGFB2 axis plays a critical role in the retinal pathophysiology associated with the development of DR, and has potential as a therapeutic target in the treatment of this condition.

Design, expression and characterization of lactiscin—A novel broad-spectrum peptidic bacteriocin

Bacteria-derived antimicrobial peptides known as peptidic bacteriocins offer a promising alternative to traditional antibiotics in the face of the emergence of multidrug-resistant bacteria. Here, a nucleotide sequence of the gene encoding Lactococcus lactis-derived peptidic bacteriocin designated as lactiscin selectively identified from the GenBank® database was synthesized with an added 6⋅His sequence and cloned into Escherichia coli. Upon low-temperature expression at 16 °C, the His-tagged peptide could be produced in both soluble form and insoluble inclusions. Efficient purification of the soluble His-tagged peptide was achieved via immobilized-Ni2+ affinity chromatography (IMAC) and its estimated molecular mass of ∼13.4 kDa was determined by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified peptide was highly active against both Gram-positive and Gram-negative bacteria as it exhibited a minimal inhibitory concentration of 0.45 mg/mL, 0.15 mg/mL, 0.35 mg/mL and 0.45 mg/mL against. Escherichia coli, Vibrio parachemolyticus, Staphylococcus aureus and Micrococcus luteus, respectively. In addition, the lactiscin peptide still retained antimicrobial activity over a pH range of 3.0–12.0 and heat stability of 100 °C for 30 min. A membrane integrity study revealed that this peptidic bacteriocin was able to induce E. coli membrane permeabilization in a concentration-dependent manner, albeit it showed a negligible toxic effect on erythrocytic cells. Gel retardation assay demonstrated that the lactiscin bacteriocin could suppress the migration of genomic DNA extracted from pathogenic bacteria, suggesting the presence of bacteriocin-responsive binding genomic. Our findings of lactiscin—a novel broad-spectrum bacteriocin would be a valuable additive for the application of food industry as a potential bio-preservative.

Backscatter from therapeutic doses of ionizing irradiation does not impair cell migration on titanium implants in vitro

ObjectiveThe influence of radiation backscatter from titanium on DNA damage and migration capacity of human osteoblasts (OBs) and mesenchymal stem cells (MSCs) may be critical for the osseointegration of dental implants placed prior to radiotherapy. In order to evaluate effects of radiation backscatter, the immediate DNA damage and migration capacity of OBs and MSCs cultured on titanium or plastic were compared after exposure to ionizing irradiation. Materials and methodsHuman OBs and MSCs were seeded on machined titanium, moderately rough fluoride-modified titanium, or tissue culture polystyrene, and irradiated with nominal doses of 2, 6, 10, or 14 Gy. Comet assay was performed immediately after irradiation, while a scratch wound healing assay was initiated 24 h post-irradiation. Fluorescent live cell imaging documented the migration.ResultsDNA damage increased with higher dose and with backscatter from titanium, and MSCs were significantly more affected than OBs. All doses of radiation accelerated the cell migration on plastic, while only the highest dose of 10 Gy inhibited the migration of both cell types on titanium.ConclusionsHigh doses (10 Gy) of radiation inhibited the migration capacity of both cell types on titanium, whereas lower doses (2 and 6 Gy) did not affect the migration of either OBs or MSCs.Clinical relevanceFractionated doses of 2 Gy/day, as distributed in conventional radiotherapy, appear not to cause severe DNA damage or disturb the migration of OBs or MSCs during osseointegration of dental implants.

Involvement of ATR-CHK1 pathway in fish megalocytivirus infection induced DNA-damage response in vitro

Increasing evidence has demonstrated that megalocytiviruses cause severe disease with high mortality in both freshwater and marine species. However, the molecular events employed in megalocytivirus infection in vitro still remained largely uncertain. Here, using an in vitro infection model of sea perch iridovirus (SPIV), we investigated the transcriptomic profiles of sea perch fry (SPF) cells upon SPIV infection, and elucidated the roles of crucial molecular events during infection using the specific inhibitors. Our data showed that almost all the differentially expressed genes (DEGs) associated with DNA-damage response (DDR), including DNA replication, mismatch repair, homologous recombination, nucleotide excision repair and Fanconi anemia (FA) pathway exhibited the up-regulation expression patterns during SPIV infection. As expected, DNA damage was detected in infected SPF cells, as evidenced by the DNA migration and the increased phosphorylation levels of H2AX along with the infection time. Furthermore, the key components of DDR, ataxia telangiectasia mutated and Rad3 related (ATR) and checkpoint kinase 1 (CHK1) kinases were elucidated to be essential for SPIV replication. Meanwhile, we also found that SPIV infection induced cell cycle arrest at G2/M phase, and 45 genes related to cell cycle pathway were up-regulated during infection. Inhibition of ATR activity induced cell cycle arrest of the infected cells at S phase, perhaps leading to the inhibitory effects on SPIV replication. Together, our data demonstrated for the first time that fish megalocytivirus induced DDR in vitro, and the ATR-CHK1 pathway exerted crucial roles in viral replication via regulating the cell cycle progression. These findings also provided new insights into the understanding of the interactions between fish megalocytivirus and host cells in vitro.

Visual comet scoring revisited: a guide to scoring comet assay slides and obtaining reliable results.

Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20%-25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8%, and 15.2% in training sets I-III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I-III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9%-9.6%) as compared to 1 week (CV = 1.3%-6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18%-20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets.

Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial.

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.