DNA Fragment Patterns Research Articles

A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos

BackgroundCell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming.ResultsHere we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding.ConclusionsSRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.

Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection

Deregulation of transcription factors (TFs) is an important driver of tumorigenesis, but non-invasive assays for assessing transcription factor activity are lacking. Here we develop and validate a minimally invasive method for assessing TF activity based on cell-free DNA sequencing and nucleosome footprint analysis. We analyze whole genome sequencing data for >1,000 cell-free DNA samples from cancer patients and healthy controls using a bioinformatics pipeline developed by us that infers accessibility of TF binding sites from cell-free DNA fragmentation patterns. We observe patient-specific as well as tumor-specific patterns, including accurate prediction of tumor subtypes in prostate cancer, with important clinical implications for the management of patients. Furthermore, we show that cell-free DNA TF profiling is capable of detection of early-stage colorectal carcinomas. Our approach for mapping tumor-specific transcription factor binding in vivo based on blood samples makes a key part of the noncoding genome amenable to clinical analysis.

Pulsed-Field Gel Electrophoresis Typing of Staphylococcus aureus Strains.

Pulsed-field gel electrophoresis (PFGE) is one of the most effective genotyping methods for the study of genetic relatedness of microorganisms as well as for epidemiologic investigation of outbreaks caused by pathogenic bacteria, including Staphylococcus aureus. The technique relies on the size patterns of large DNA fragments generated by the cleavage of intact bacterial chromosomes with a rare cutting restriction enzyme, and subsequently resolved by pulsed-field electrophoresis with periodic changes of the orientation of the electrical field across the gel. The high discriminatory power, improved reproducibility by standardization of experimental protocols and data analysis guidelines, and establishment of a national PFGE database of S. aureus profiles have made it a valuable means for global tracking of S. aureus infection sources and determination of genetic relatedness of the strains.

Heat induced differential pattern of DNA fragmentation in male germ cells of rats.

Spermatogenesis being a highly dynamic processes is highly vulnerable to various stresses including heat stress. Though, the relationship between physiological temperature and male germ cells is certainly immense, the magnitude of spermatozoal damage after exposure to heat is evidently degree and dose dependent. Further, there are contradictory reports related to germ cells apoptosis in relation to temperatures. Thus, currently the dynamics of temperature and time dependence on germ cell apoptosis were studied by modulating the heat treatment strategies. It was observed that the rate of apoptosis increased initially then decreased with time. The DNA fragmentation in the 10,000×g supernatant of testis homogenate of rats that received heat treatment for 15-min, 30-min as well as 45-min treatment with 15-min intermittent period was found to be almost equal. In various heat treated animals, the apoptosis was found to be maximum after day-1 of treatments, which then followed a decreased pattern. These results indicate that there may be an initial induction of apoptosis in the germ cells, which later primed or programmed the other germ cells to activate protective mechanisms against heat induced DNA damage and thus protecting germ cell population to undergo apoptosis at later durations.

Induction of Non-Apoptotic Cell Death by Adrenergic Agonists in Human Oral Squamous Cell Carcinoma Cell Lines.

Although adrenergic agonists have been used in dental treatments and oral surgery for general anesthesia, their cytotoxicity against human oral malignant and non-malignant cell has not been well- understood. The present study was undertaken to investigate the cytotoxicity of five adrenergic agonists against human oral squamous cell carcinoma (OSCC), glioblastoma, promyelocytic leukemia, and normal oral mesenchymal cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) and normal epidermal keratinocytes. Tumor-specificity (TS) was calculated by the ratio between the mean 50% cytotoxic concentration against normal cells to that of tumor cells. Internucleosomal DNA fragmentation was detected using agarose gel electrophoresis. Caspase-3 activity was measured by substrate cleavage. Both cytotoxicity and tumor-specificity of adrenergic agonists against OSCC cell lines was in the order of isoprenaline>dexmedetomidine> adrenaline>clonidine and phenylephrine. Isoprenaline and dexmedetomidine did not induce apoptosis markers, such as internucleosomal DNA fragmentation and caspase-3 activation, but induced a smear pattern of DNA fragmentation in OSCC cell lines. Their cytotoxicity was not reduced by pretreatment with autophagy inhibitors, or by adrenoceptors antagonists. Addition of superoxide dismutase and catalase significantly reduced the cytotoxicity of isoprenaline, but not that of dexmedetomidine. Isoprenaline and dexmedetomidine induce non-apoptotic cell death by different mechanisms.

Genome-wide cell-free DNA fragmentation in patients with cancer.

Cell-free DNA (cfDNA) in the blood provides a noninvasive diagnostic avenue for patients with cancer1. However, characteristics of the origins and molecular features of cfDNA are poorly understood. We developed an approach to evaluate fragmentation patterns of cfDNA across the genome and found that cfDNA profiles of healthy individuals reflected nucleosomal patterns of white blood cells, while patients with cancer had altered fragmentation profiles. We applied this method to analyze fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric, or bile duct cancer and 245 healthy individuals. A machine learning model incorporating genome-wide fragmentation features had sensitivities of detection ranging from 57% to >99% among the seven cancer types at 98% specificity, with an overall AUC of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation based cfDNA analyses detected 91% of cancer patients. The results of these analyses highlight important properties of cfDNA and provide a proof of principle approach for screening, early detection, and monitoring of human cancer.

Circulating cell-free DNA (cfDNA) levels and fragmentation pattern can distinguish nonmuscle invasive (NMI) from muscle-invasive (MI) and metastatic (met) bladder cancer (BC).

4523 Background: Occult MI and met BC may be under-staged. Circulating cfDNA may be a dynamic, low-cost and minimally invasive biomarker. We evaluated correlations between total circulating cfDNA and presence of MIBC and met BC. We hypothesized that the relative abundance of circulating low molecular weight cfDNA would correlate with BC stage. Methods: Peripheral blood from pts with BC was collected in Streck BCT tubes and processed to obtain cf nucleic acid extracts. Total cfDNA quantity (ng/ml) was assessed by fluorimetry. cfDNA fragment size was measured by Bioanalyzer DNA analysis. Wilcoxon rank sum test and Fisher’s Exact test were used to compare cfDNA quantity and fragmentation pattern among pts with NMIBC, MIBC, met BC. Results: Blood was obtained from 58 pts with BC (20% women, 34% never smokers, median age 71 (29-89). There was no significant difference in cfDNA between MIBC and met BC, however, it was significantly lower in pts with NMIBC vs MIBC and met BC (table). The concentration of low molecular weight fragments (LMW-frags) (100 - 400) base pairs and the ratio of LMW-Frag to cfDNA were significantly different between pts with NMIBC and pts with MIBC or met BC (table). Using median values as the cutoff, there was a significantly higher proportion of pts with cfDNA > 7 ng/ml and LMW-frags > 1.6 ng/mL, in MIBC & met BC vs NMIBC (p < 0.001). The % of pts with LMW-frags to cfDNA > 30%, was significantly different among NMIBC, MIBC and met BC groups: 16%, 53%, 78%, respectively (p < 0.001). Conclusions: This exploratory study suggests that cfDNA levels may correlate with BC stage. Measuring the relative abundance of LMW-frags with the expected size of cf DNA can enhance the specificity of cfDNA analysis for distinction between MIBC and met BC. Further studies are needed to confirm findings and define the optimal cut-points for optimal BC staging. [Table: see text]

Phycocyanin Enhances the Cytotoxic Profile of Cisplatin in Platinum‐Resistant Ovarian Cancer Cells by Targeting Focal Adhesion Kinase

Platinum‐based therapy is one of the major treatment approaches in ovarian cancer. Although greater than 80% of advanced stage ovarian cancer patients initially achieve remission with platinum therapy; the majority of this cohort eventually relapse, and develop drug‐resistant disease. Resistance to chemotherapeutic drugs such as cisplatin has been attributed in part to the overexpression of Focal Adhesion Kinase (FAK), which is known to promote cancer cell survival and metastasis. In recent years, there has been heightened interest in targeting focal adhesion kinase in platinum resistant cancer as a new modality in ovarian cancer treatment. In previous studies, we have reported that Phycocyanin (C‐PC), a major accessory pigment in the Limnothrix sp. 37‐2‐1, demonstrates cytotoxicity towards multiple cancer cell lines. Therefore, in the present study we evaluated the ability of C‐PC to sensitize platinum resistant OVCAR3 cells to cisplatin treatment. We also examined the response of FAK activation by phosphorylation, to treatment with cisplatin and CP‐C alone, and in combination (C‐PC +cisplatin). Cell viability was determined by MTT assay, and DNA fragmentation was used to confirm apoptotic cell death. The expression of phosphorylated FAK (pFAK), total FAK, pAKT, as well as the key apoptotic regulators p21, p53, Bax, and Bcl‐2 in response to the treatments were determined through western blotting. The results from our study confirm that C‐PC significantly enhances the cytotoxic profile of cisplatin in OVCAR‐3 cells through the induction of apoptosis as confirmed by the presence of DNA fragmentation patterns in all three treatment groups. The expression of activated pFAK was also significantly reduced in the CPC and CPC + cisplatin treatment groups and this corresponded with increased p53, p21 and Bax expression, while pAkt and Bcl‐2 expression was downregulated. These results confirm a novel effect of CPC on FAK phosphorylation in platinum resistant ovarian cancer, which can be further explored towards the treatment of advanced stage disease through combination strategies.Support or Funding InformationThis project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, FloridaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Indicators of the cell cycle in the thyroid gland in rats when applying infusion of 0.9% solution of NaCl, lactoprotein with sorbitol or HAES-LX 5%

The thyroid gland is an important organ that is involved in the regulation of homeostasis and adaptation in various pathological conditions. However, the question of the study of the proliferative activity of thyroid cells by flow cytometry is still poorly understood. Purpose of study: to investigate the indices of the cell cycle and DNA fragmentation of thyroid cells in rats against the background of infusion of 0.9% NaCl solution, lactoprotein with sorbitol or HAES-LX 5%. Experimental studies were performed on 90 white male rats weighing 160-180 g. Infusion of 0.9% NaCl solution, lactoprotein with sorbitol or HAES-LX 5% was performed in the inferior vena cava after its catheterization in aseptic conditions through the femoral vein. The infusions were performed once a day for the first 7 days. Trunk catheterization and decapitation of animals (after 1, 3, 7, 14, 21, and 30 days) were performed under propofol anesthesia (60 mg/kg i/v). Within the framework of the agreement on scientific cooperation between the Research Center of National Pirogov Memorial Medical University, Vinnytsya and the Department of Histology, Cytology and Embryology of the Odessa National Medical University (from 01/01/2018), DNA content in the nuclei of thyroid cells of rats was determined by flow DNA cytometry. Cell cycle analysis was performed using the software FloMax (Partec, Germany) in full digital accordance with the mathematical model, which determined: G0G1 – the percentage of cells of the phase G0G1 to all cells of the cell cycle (DNA content = 2c); S – the percentage of the phase of DNA synthesis to all cells of the cell cycle (DNA content > 2c and < 4c); G2+M – the percentage ratio of the G2+M phase to all cells in the cell cycle (DNA = 4c). Determination of DNA fragmentation (SUB-G0G1, apoptosis) was performed by isolating the RN2 region on DNA histograms before the G0G1 peak, indicating nuclei of cells with a DNA content < 2c. The statistical processing of the obtained results was carried out in the license package “STATISTICA 6.1” using nonparametric estimation methods. The data obtained showed a virtually identical pattern of rat cell cycle and DNA fragmentation of the thyroid gland cells at all study times against the use of 0.9% NaCl solution, lactoprotein with sorbitol or HAES-LX 5%. Thyroid cells in rats are predominantly in the inactive phase of DNA synthesis (G0G1) (90.32% – 91.88%), significantly fewer cells are in the G2+M phase (7.56% – 9.17%), and there is a small percentage of cells in the S-phase (DNA synthesis) (0.52% – 0.67%) and the SUB-G0G1 interval (DNA fragmentation, apoptosis) (2.23% – 2.81%). We can state that the activity of the main part of the thyroid gland is rather low without pathological irritation.

Cell-bound Exopolysaccharide Extract from Indigenous Probiotic Bacteria Induce Apoptosis in HT-29 cell-line.

The aim of this study was to compare the cytotoxiceffects of local probiotic bacteria, including Lactobacillus paracasei, Lactobacillus brevis, while isolated from "Tarkhine" food and the induction of apoptosis in the HT-29 human colon adenocarcinoma cell line and normal fibroblasts. HT-29 and L-929 cell lines were treated with cell-bound exopolysaccharide extract (cb-EPS) from L. paracasei and L. brevis. The MTT assay was used to analyze cell viability. Cellular apoptosis was examined by flow cytometry and DNA fragmentation assay. The cb-EPS from both probiotic bacteria prevented the proliferation of HT-29 colon cancer cells. In addi- tion, the cytotoxic and anti-proliferative effects of the exopolysaccharide extract from both bacteria in L-929 fibro- blasts were much lower than HT-29 cells. The induction of apoptosis in HT-29 cells was observed at 48h compared with 72h. It seems that the exopolysaccharides extracted from both bacteria have a greater effect on the induction of apoptosis at 48h. The cb-EPS of L. brevis showed more potent anti-proliferative and apoptotic properties than the cb- EPS of L. paracasei. The ladder pattern of DNA fragmentation confirmed the induction of apoptosis in cancer cells. The results of the MTT assay and apoptosis indicate that the induction of apoptosis by the exopolysac- charide from bacteria depends on the dose, time, and strain of bacteria. Further studies may contribute toward the understanding of using these probiotic bacteria as biological products to treat and prevent cancers.

Effects of human sperm cryopreservation on apoptotic markers in normozoospermic and non-normozoospermic patients

SummaryThe negative effects of cryopreservation on sperm parameters are well documented but little information is known about molecular basis of the process. The aim of the present study was to investigate the possible effects of sperm cryopreservation on main apoptotic signs including DNA fragmentation and caspase-3 activation and to determine if these effects vary according to sperm parameters. Sperm samples of 72 patients were cryopreserved. The patients were sub-grouped as normozoospermic or non-normozoospermic patients according to their semen parameters. DNA fragmentation rates and caspase-3 activation levels were analyzed before and after cryopreservation in both groups. Mean DNA fragmentation rate was increased significantly from 23.98% in neat semen samples to 27.34% after cryopreservation (P = 0.03). DNA fragmentation rates were slightly higher in non-normozoospermic patients compared with the normozoospermic patients in both the neat semen and after cryopreservation (23.25 and 24.71% vs. 26.32 and 28.36%, respectively) although the difference obtained were not statistically significant. An increasing trend for caspase-3 activations (0.093 vs. 0.116) was observed after cryopreservation but the differences were not statistically significant. Caspase-3 activation was found to be slightly higher in non-normozoospermic patients both in the neat semen and after cryopreservation compared with the normozoospermic patients but the differences were not statistically significant. Caspase-3 expression was also shown using immunocytochemistry in both fresh ejaculated sperm and thawed sperm after cryopreservation but at different localizations. The cryopreservation process had detrimental effects on sperm quality but the quality of the sperm samples was not adversely effective for the apoptotic markers including DNA fragmentation and caspase-3 activation patterns. In fact, it was the cryopreservation process itself that adversely effected the above apoptotic markers and apoptosis. It was concluded therefore that sperm cell cryopreservation triggers apoptosis after thawing and this process adversely affects semen parameters.

Differential preservation of endogenous human and microbial DNA in dental calculus and dentin

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007–0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003–70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute > 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure.

Berberis aristata Ameliorates Testicular Toxicity Induced by Combination of First-Line Tuberculosis Drugs (Rifampicin + Isoniazid + Pyrazinamide) in Normal Wistar Rats

ABSTRACTFirst-line antituberculosis drugs, namely, isoniazid (INH), rifampicin (RIF), and pyrazinamide (PZA), contribute to diverse pathological complications. Testicular toxicity is one such complication. Berberis aristata DC is an herb with potentially curative characteristics. The aim of this study was to test whether extract of Berberis aristata DC (Berberidaceae) has curing potential against testicular toxicity. Characterization of extract was done using ultra-performance liquid chromatography along with acute toxicity testing. Antioxidant activity of extract was checked by DPPH inhibition assay and H2O2 scavenging assay. Rats were dosed once daily for 28 days in groups: control group (saline), toxicant group (30.85 mg/kg body weight INH + 61.7 mg/kg body weight RIF + 132.65 mg/kg body weight PZA), treatment groups (TB drugs + 150/300 mg/kg body weight extract) and standard group (TB drugs +100 mg/kg body weight silymarin). Spectrophotometric evaluations of lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD), glutathione-S-transferase (GST), and catalase (CAT) content in testes were done using standard protocols. DNA fragmentation and histopathological studies were performed to check the damage at the cellular level. Acute toxicity studies revealed LD50 > 5 g/Kg body weight of B. aristata extract. IC50 for DPPH free-radical scavenging activity and H2O2 scavenging assay were 44.78 µg/mL and 85.28 µg/mL, respectively. Results revealed significant increase in thiobarbituric acid reactive substances, decrease in glutathione and different antioxidants levels, DNA fragmentation pattern, and changes in histology in toxicant group. All the changes were absent in high-dose (300 mg/kg body weight) extract treatment group. This work proved that B. aristata extract has protective efficacy against testicular damage caused by anti-TB drugs.

Extract of Penicillium sclerotiorum an endophytic fungus isolated from Cassia fistula L. induces cell cycle arrest leading to apoptosis through mitochondrial membrane depolarization in human cervical cancer cells.

Seventeen endophytic fungi were isolated from various tissues of Cassia fistula and the ethyl acetate extracts obtained from 21-day cultures of all the endophytic fungal isolates were initially screened for their cytotoxicity against HeLa (human cervical carcinoma) cells using MTT assay. Of these, Penicillium sclerotiorum extract (PSE), significantly affected the viability of HeLa cells in a dose-dependent manner. The extract of P. Sclerotiorum was further analyzed by GC-MS, which showed three compounds, hexadecanoic acid, oleic acid and benzoic acid to be the major active principles in the extracts.The extract was further tested for invitro cytotoxicity against five cancer cell lines. Of the cell lines tested, HeLa cells showed maximum sensitivity followed by A549, while A431 and U251 were moderately sensitive and MCF-7 was insensitive to the treatment. In addition, normal human embryonic kidney cells, HEK293 remained insensitive to the treatment. Furthermore, the mechanism of cytotoxic activity exhibited by PSE was investigated by evaluating cell cycle progression and apoptotic induction in HeLa cells. Cell cycle analysis revealed that the PSE arrested cells at S and G2/M phase of the cell cycle in a dose-dependent manner. Annexin V- Propidium iodide double staining showed that, the extract potentiates apoptosis rather than necrosis in cells. This was supported by the down regulation in the proapoptotic protein BCL2 and up regulation of BAX (BCL2 Associated X), tumor suppressor protein, p53 and Apaf-1 [Apoptotic Peptidase Activating Factor 1]. Loss of mitochondrial membrane potential and a distinct DNA fragmentation pattern observed following the treatment, suggest that the PSE treatment leads to activation of mitochondrial pathway of apoptosis. Further, the extract also exhibited both antioxidant and anti-angiogenic properties. These results indicate that endophytic fungi isolated from medicinal plants may serve as potential sources of the anti-cancerous compounds.

Luteolin induces myelodysplastic syndrome‑derived cell apoptosis via the p53‑dependent mitochondrial signaling pathway mediated by reactive oxygen species.

Luteolin, a common dietary flavonoid, induces the apoptosis of cells in several types of cancer. However, its role in myelodysplastic syndrome (MDS) and the potential underlying mechanisms remain to be elucidated. To evaluate the potential benefit and underlying mechanisms of luteolin in MDS cells, the viability of SKM‑1 cells and primary bone marrow (PBM) mononuclear cells from patients with intermediate‑ or high‑risk MDS were assessed using a Cell Counting Kit‑8 assay. The apoptotic features of cell morphology were assessed using Wright‑Giemsa staining, DNA fragmentation was analyzed by agarose gel electrophoresis, and the extent of apoptosis was quantified by flow cytometry (FCM). Reactive oxygen species (ROS) were measured by FCM with 2,7‑dichlorodihydrofluorescein diacetate staining and mitochondrial membrane potential (ΔΨm) was determined using 5,5',6,6'‑tetrachloro‑1,1',3,3'‑tetraethylbenzimidazolylcarbocyanine iodide staining. Caspase activity was detected using a fluorometric protease assay. Furthermore, the effects of luteolin on the expression of apoptosis‑related proteins were analyzed using western blot analysis. The resulting data revealed that luteolin significantly inhibited the proliferation of SKM‑1 cells invitro, and its half maximal inhibitory concentration was 139.41µM at 24h and 23.95µM at 72h. Luteolin also markedly inhibited the proliferation of mononuclear cells from patients with intermediate‑ or high‑risk MDS. Luteolin suppressed cell proliferation, mainly as a result of the induction of apoptosis, as demonstrated by typical apoptotic morphological features, the ladder pattern of genomic DNA fragmentation, and the results of FCM using Annexin V‑FITC/PI double staining. It was also found that short‑term exposure of SKM‑1 cells to luteolin led to a marked increase in the accumulation of ROS. The increased intracellular level of ROS appeared to induce the activation of p53 and elevate the B‑cell lymphoma 2 (Bcl‑2)‑associated X protein/Bcl‑2 ratio, which modulates ΔΨm and triggers the release of cytochrome c, and may increase the activities of apoptotic protease activating factor 1, caspase‑3, ‑8 and ‑9 to further trigger the destruction of structural and specific proteins and thereby cell apoptosis. Notably, the inhibition of ROS generation by the antioxidant N‑acetyl‑L‑cysteine significantly attenuated the luteolin‑induced loss of ΔΨm and activities of caspase‑3, ‑8 and ‑9. These data suggested that luteolin exerts its pro‑apoptotic action partly through the p53‑dependent mitochondrial signaling pathway mediated by intracellular ROS, which provides a promising therapeutic candidate for patients with MDS.

Phenolic compounds from Viscum album tinctures enhanced antitumor activity in melanoma murine cancer cells

Cancer is one of the biggest problems in public health worldwide. Plants have been shown important role in anticancer research. Viscum album L. (Santalaceae), commonly known as mistletoe, is a semi-parasitic plant that grows on different host trees. In complementary medicine, extracts from European mistletoe (Viscum album L.) have been used in the treatment of cancer. The study was conducted to identify chemical composition and antitumor potential of Viscum album tinctures. Chemical analysis performed by high resolution chromatography equipped with high resolution mass spectrometer identified caffeic acid, chlorogenic acid, sakuranetin, isosakuranetin, syringenin 4-O-glucoside, syringenin 4-O-apiosyl-glucoside, alangilignoside C and ligalbumoside A compounds. Some of these compounds are probably responsible for the reduction of tumoral cellular growth in a dose-dependent manner. It was observed that melanoma murine cells (B16F10) were more sensitive to V. album tinctures than human leukaemic cells (K562), besides non-tumoral cells (MA-104) had a much lower cytotoxicity to them. Apoptotic-like cells were observed under light microscopy and were confirmed by a typical DNA fragmentation pattern. Additionally, flow cytometry results using Annexin-V/FITC permitted to quantify increased expression of early and late apoptotic markers on tumoral cells, confirming augmented Sub G0 population, which was probably associated with a consistent decrease in G1, and an increase in S or G2/M populations. Results indicate the chemical composition of V. album tinctures influences the mechanisms of in vitro tumoral cell death, suggesting a potential use in cancer pharmacotherapy research.

Effects of Banxia Xiexin Decoction (半夏泻心汤) on Cisplatin-Induced Apoptosis of Human A549 Lung Cancer Cells

To examinie the synergistic effects of Banxia Xiexin Decoction (, Known as Banhasasim-tang in Korean) extract (BXDE) on cisplatin-induced cytotoxicity in the A549 human lung cancer cell lines. A549 cells were treated with varying concentrations (50-200 μg/mL) of cisplatin and BXDE alone or in combination for 96 h. We used 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay and flow cytometry to analyze cell viability and apoptosis, respectively. The exposure of cells to cisplatin and BXDE alone or in combination decreased cell viability dose- and time-dependently (P<0.05), which was found to be mediated by the apoptotic pathway as confirmed by the increase in the annexin V+/propidium iodide- stained cell population and a ladder pattern of discontinuous DNA fragments. Furthermore, the apoptosis was inhibited by the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (z-VAD-FMK). BXDE significantly potentiated apoptotic effects of cisplatin in A549 cells. Moreover, apoptosis induced by BXDE might be the pivotal mechanism mediating its chemopreventative action against cancer.

Potential chromosome damage, cell-cycle kinetics/and apoptosis induced by epoxiconazole in bovine peripheral lymphocytes in vitro

The epoxiconazole was tested in vitro for its potential on induction of chromosome damage and/or cell cycle kinetics in cultured bovine peripheral lymphocytes. Cytogenetic endpoints such as: Chromosome Aberrations (CA); Sister Chromatid Exchanges (SCE); Micronuclei (MN); Mitotic Index (MI); Proliferation Index (PI); and Cytokinesis Block Proliferation Index (CBPI) were investigated for 24 h and 48 h of incubation. The cultured lymphocytes were exposed to the epoxiconazole at concentrations of 2.5, 5, 10, 25, 50 and 100 μg mL−1. From our results is evident that treatment of bovine peripheral lymphocytes with the epoxiconazole was not related to DNA damage; no genotoxic effect and/or clastogenic/aneugenic effects were recorded. However, epoxiconazole has ability to significantly affect cell cycle kinetics/and induce apoptosis. A decrease of proliferation in the MI, CBPI and identically in the PI were observed; hence, cytostatic/cytotoxic effects of epoxiconazole have been recorded. The prolonged time of exposure at the highest concentration caused an inhibition of the replication. Electrophoretic analysis confirmed the epoxiconazole potential to induce ladder-like patterns of DNA fragments that are a hallmark of apoptosis.

A Study of Cell-free DNA Fragmentation Pattern and Its Application in DNA Sample Type Classification.

Plasma cell-free DNA (cfDNA) has certain fragmentation patterns, which can bring non-random base content curves of the sequencing data's beginning cycles. We studied the patterns and found that we could determine whether a sample is cfDNA or not by just looking into the first 10 cycles of its base content curves. We analysed 3189 FastQ files, including 1442 cfDNA, 1234 genomic DNA, 507 FFPE tumour DNA and 6 urinary cfDNA. By deep analysing these data, we find the patterns are stable enough to distinguish cfDNA from other kinds of DNA samples. Based on this finding, we build classification models to recognise cfDNA samples by their sequencing data. Pattern recognition models are then trained with different classification algorithms like k-nearest neighbours (KNN), random forest and support vector machine (SVM). The result of 1000 iteration .632+ bootstrapping shows that all these classifiers can give an average accuracy higher than 98%, indicating that the cfDNA patterns are unique and can make the dataset highly separable. The best result is obtained using random forest classifier with a 99.89% average accuracy (σ = 0.00068). A tool called CfdnaPattern (http://github.com/OpenGene/CfdnaPattern) has been developed to train the model and to predict whether a sample is cfDNA or not.

Abstract 3350: Cell-free DNA fragmentation patterns analyzed in over 15000 cancer patients reveal changes associated with tumor somatic mutations and result in improved sensitivity and specificity of somatic variant detection

Abstract Background: Cell-free DNA (cfDNA) isolated from plasma consists of DNA fragments surviving clearance of dying cells and bloodstream trafficking. In cancer, these fragments carry the footprint of tumor somatic variation as well as its microenvironment. Since genomic distribution of cell free DNA fragments was shown to reflect nucleosomal occupancy in hematopoietic cells, we hypothesized that (a) heterogeneous patterns of cfDNA positioning would be associated with distinct mutations in patient tumors and (b) integration of fragmentation patterns into analysis would allow increased sensitivity and specificity of somatic mutation detection. Methods: cfDNA fragment length and position distributions as well as associated somatic genomic profiles of over 15,000 patients with advanced-stage clinical cancer were determined by a highly accurate, deep-coverage (15,000x) ctDNA NGS test targeting 70 genes (Guardant360). An integrative data analysis of variant-free fragmentomics domain across different driver mutations was performed to identify patterns associated with detected somatic alterations. Results: We discovered distinct classes of fragmentomics subtypes significantly enriched in samples with different genomic subtypes. An independent cohort of samples with known HER2 immunohistochemistry status was interrogated to confirm discovered association between fragmentation patterns and HER2 status. Integrating fragmentomics amplification signature with ERBB2 copy number analysis has resulted in 42% increase in the sensitivity and 7% increase in specificity of detection. Observed lung adenocarcinoma fragmentomics subtypes co-occured with mutually exclusive genomic alterations and previously described intrinsic molecular subtypes of lung cancer. Conclusions: Fragmentomics classification of cancer cfDNA provides independent evidence for observed somatic variation and underlying tumor microenviroment, leading to higher sensitivity and accuracy of variant detection. Citation Format: Diana Abdueva, Helmy Eltoukhy, Darya Chudova, AmirAli Talasaz. Cell-free DNA fragmentation patterns analyzed in over 15000 cancer patients reveal changes associated with tumor somatic mutations and result in improved sensitivity and specificity of somatic variant detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3350. doi:10.1158/1538-7445.AM2017-3350