Cell Deoxyribonucleic Acid Research Articles

Toxicological responses of A549 and HCE-T cells exposed to fine particulate matter at the air–liquid interface

Fine particulate matter (PM2.5) can enter the human body in various ways and have adverse effects on human health. Human lungs and eyes are exposed to the air for a long time and are the first to be exposed to PM2.5. The “liquid immersion exposure method” has some limitations that prevent it from fully reflecting the toxic effects of particulate matter on the human body. In this study, the collected PM2.5 samples were chemically analyzed. An air–liquid interface (ALI) model with a high correlation to the in vivo environment was established based on human lung epithelial cells (A549) and immortalized human corneal epithelial cells (HCE-T). The VITROCELL Cloud 12 system was used to distribute PM2.5 on the cells evenly. After exposure for 6 h and 24 h, cell viability, apoptosis rate, reactive oxygen species (ROS) level, expression of inflammatory factors, and deoxyribonucleic acid (DNA) damage were measured. The results demonstrated significant dose- and time-dependent effects of PM2.5 on cell viability, cell apoptosis, ROS generation, and DNA damage at the ALI, while the inflammatory factors showed dose-dependent effects only. It should be noted that even short exposure to low doses of PM2.5 can cause cell DNA double-strand breaks and increased expression of γ-H2AX, indicating significant genotoxicity of PM2.5. Increased abundance of ROS in cells plays a crucial role in the cytotoxicity induced by PM2.5 exposure These findings emphasize the significant cellular damage and genotoxicity that may result from short-term exposure to low levels of PM2.5.

The Impact of Dose Rate on the Tumor Microenvironment Using Flattening-filter-free Beams

AimsRecently, dose delivery technology has rapidly evolved with flattening filter-free beams (FFF), and the biological effects of high dose rates are a matter of interest. We hypothesized that FFF beams at different dose rates obtained with modern linear accelerators have different effects on the TME. Materials and methodsThe B16–F10 melanoma syngeneic tumor model was established, and mice were randomized to 2 different doses (2 Gy and 10 Gy) and 3 different dose rates (1 Gy/min, 6 Gy/min, and 14 Gy/min) along with the control group. Euthanasia was performed on the seventh day after RT, and intracardiac blood was collected for a comet assay. Tumors were harvested and examined histomorphologically and immunohistochemically. Statistical analyses were performed using SPSS software version 23 (SPSS Inc., Chicago, IL, USA). ResultsThe daily growth rate was uniform, and no difference was observed between tumor volumes across all three dose rates for each dose. Deoxyribonucleic acid (DNA) damage in blood mononuclear cells was not affected by dose or dose rate. In the TME histomorphological examination, the number of mitosis is less in the 10 Gy arm, whereas the pleomorphism score was greater. Nevertheless, varying dose rates had no effect on the number of mitosis or the pleomorphism score. The severity of the inflammation, cell densities in the TME, and expression of immunohistochemical markers were comparable across all doses and dose rates. ConclusionIn our study involving the B16–F10 syngeneic tumor model, varying dose rates obtained with FFF beams had no effect on tumor volume, blood mononuclear cell DNA damage, or TME parameters. However, in order to fully understand the biological impacts of novel techniques, our study should be validated with alternative preclinical setups.

Malondialdehyde: A Toxic Stress Marker for Periodontitis

Recently, it has been discovered that one of the primary causes of a variety of inflammatory illnesses, including periodontitis, is an excess of Reactive Oxygen Species (ROS). It is recognised that the host’s response to pathogens plays an equal or even bigger part in determining how connective tissue breaks down. The process of Polymorphonuclear (PMN) phagocytosis greatly increases ROS formation through the metabolic pathway known as the “respiratory burst.” Because the antioxidant defense system is unable to neutralise these high levels or activities of ROS, Oxidative Stress (OS) and tissue damage ensue. ROS can directly affect tissue through Lipid Peroxidation (LPO), Deoxyribonucleic Acid (DNA) damage, protein breakdown, and the oxidation of essential enzymes. Numerous aldehydes can be produced as byproducts of LPO, including Malondialdehyde (MDA), propanal, hexanal, and 4-Hydroxynonenal (4-HNE). They can be detected in bodily fluids and suggest a pro-oxidant state. MDA is a by-product created by the enzymatic or non enzymatic breakdown of Arachidonic Acid (AA) and larger Polyunsaturated Fatty Acids (PUFAs). MDA is the most studied OS tissue damage indicator. After it is formed, MDA can either be broken down by enzymes or combine with DNA or proteins in cells and tissues to form adducts that can cause harm to biological molecules.

Esophageal cancer detection framework based on time series information from smear images

The gold standard for esophageal cancer diagnosis and treatment is the Thinprep Cytologic Test (TCT) of suspected sections. TCT refers to analyze the features of the lesion areas using tissue regions stained with hematoxylin and eosin. However, pathologists are currently subjected to high workloads and limited experiences. Moreover, automatic esophageal cancer detection methods typically screen the smears by focusing on suspicious lesions identified by cytotechnicians, cell misclassification often results in inaccurate and weak robust diagnoses, smear utilization rate is also reduced. Computer-assisted diagnosis also faces challenges such as information loss, non-quantitative analysis, and nonstandard diagnosing processes. To address these issues, this article proposes a deep learning based detection framework called the Esophageal Time Series Thinprep Cytologic Test (ETS-TCT) to provide a quantitative and robust screening of Whole Slide Imaging (WSI) for esophageal cancer. Our system is divided into three modules: Coarse–fine detection model, quantitative analysis model, and Fusion Long Short Term Memory-Support Vector Machine (FLSTM-SVM) model. These modules are organized to map the pathological information from cell-level to smear-level and offer interpretable predictions to pathologists. For each smear, we design the coarse–fine segmentation model for adaptively locating and classifying target cells, give mathematical descriptions for cell Deoxyribonucleic Acid (DNA) values using the quantitative analysis model, establish an early risk screening model for esophageal cancer using the optimized FLSTM-SVM network. To validate the methodology, we use two clinical datasets containing 580 samples, achieving the accuracy, sensitivity, specificity, and area under ROC (AUC) of 92.0%, 93.4%, 94.4%, and 0.913, respectively. In short, the framework provides robust and accurate diagnosis of esophageal cancer WSIs using the time series features. This screening can assist the pathologists in strengthening the diagnosis by robustly presenting the quantitative intermediate results as well as visual interpretations. Moreover, we clearly present and expatiate the concepts of time series features, highlighting the relationship between these features to physiological states of patients. Our results indicate the possibility of using the framework as a second system to the computer-assisted esophageal cancer screening.

Azadirachta indica A. Juss (neem) phenolic extract inhibits human B-lymphoblastoid cells growth via cell cycle arrest, apoptosis induction, and DNA damage

Aim: As far as is known, the pharmaceutical effects of neem on human B-lymphoblastoid (TK6) cells have not been studied until now. Hence, the present study aimed to obtain neem phenolic extracts for inhibits the proliferation of TK6 cells and explore some possible underlying mechanisms involved in these effects. Methods: Hexane extract (HE) was obtained in the first step. After that, the residual hexane was removed from the neem. The dried neem sample was used in a new extraction for obtaining the ethyl acetate extract (EAE). Total phenolic compounds (TPC) and total flavonoid contents (TFC) were determined by spectrophotometric methods. Lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were used to evaluate the cytotoxicity in TK6 cells. The stop at G0/G1 cell cycle and inducing apoptosis in the TK6 cells were analyzed by flow cytometry. For deoxyribonucleic acid (DNA) damage evaluation, the alkaline comet test was used. Results: The higher TFC (65.50 mg/g of extract ± 1.17 mg/g of extract) and TPC (52.08 mg of extract ± 0.88 mg of extract) were obtained in EAE compared to HE that was obtained TFC of 14.61 mg/g of extract ± 0.60 mg/g of extract and TPC of 3.20 mg/g of extract ± 1.20 mg/g of extract. EAE was more significantly cytotoxic to TK6 cells than HE. The apoptosis induction was higher after exposure to 15.0 µg/mL of EAE (11.29%) in comparison to 15.0 µg/mL of HE (2.52%). The G0/G1 phase increased from 72% negative control (NC) to 83% after treatment with neem extracts (15 µg/mL). Neem extracts were also able to cause DNA strand breaks in TK6 cells. Conclusions: The extraction residue from neem leaf after hexane extraction is a source important of cytotoxic and genotoxic molecules against TK6 cells, the results also can suggest that the toxic effects in TK6 cells can be provided most likely due to the presence of high content of TPC from neem extracts.

Harnessing single fluorescent probe to image deoxyribonucleic acid and ribonucleic acid in cells

The roles of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in cells are closely related. However, the absence of molecular tools for simultaneous imaging of the two nucleic acids has prevented scientists from elucidating the regulatory mechanisms of nucleic acid interaction. The nucleic acid probes developed in recent years have ignored the regulatory relationship between DNA and RNA. Simultaneously imaging RNA and DNA in cells through a single small-molecule fluorescent probe is important. In this study, we propose a strategy for developing fluorescent probes localized to DNA and RNA to investigate their detection and imaging characteristics. The novel probe Bptp-RD has been successfully used for DNA and RNA imaging in cells. We investigated the detection and imaging characteristics of this nucleic acid probe and discovered the following: 1) the differences in the detection results of this nucleic acid probe for DNA and RNA come from the structural differences of the nucleic acids rather than chemical composition differences; 2) through using small-molecule probes to image a nucleic acid in cells, another nucleic acid can be visualized by reducing the fluorescence signal caused by DNA or RNA; 3) the order of response of the small-molecule fluorescent probe with intercalation and binding mechanisms to the type of nucleic acid structure is single chain, double chain, and ring. This work will help improve the understanding of RNA and DNA probes, and the novel probe has high potential to explore the interaction between RNA and DNA in cells.

Radiosensitization of Breast Cancer Cells with a 2-Methoxyestradiol Analogue Affects DNA Damage and Repair Signaling In Vitro.

Radiation resistance and radiation-related side effects warrant research into alternative strategies in the application of this modality to cancer treatment. Designed in silico to improve the pharmacokinetics and anti-cancer properties of 2-methoxyestradiol, 2-ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) disrupts microtubule dynamics and induces apoptosis. Here, we investigated whether pre-exposure of breast cancer cells to low-dose ESE-16 would affect radiation-induced deoxyribonucleic acid (DNA) damage and the consequent repair pathways. MCF-7, MDA-MB-231, and BT-20 cells were exposed to sub-lethal doses of ESE-16 for 24 h before 8 Gy radiation. Flow cytometric quantification of Annexin V, clonogenic studies, micronuclei quantification, assessment of histone H2AX phosphorylation and Ku70 expression were performed to assess cell viability, DNA damage, and repair pathways, in both directly irradiated cells and cells treated with conditioned medium. A small increase in apoptosis was observed as an early consequence, with significant repercussions on long-term cell survival. Overall, a greater degree of DNA damage was detected. Moreover, initiation of the DNA-damage repair response was delayed, with a subsequent sustained elevation. Radiation-induced bystander effects induced similar pathways and were initiated via intercellular signaling. These results justify further investigation of ESE-16 as a radiation-sensitizing agent since pre-exposure appears to augment the response of tumor cells to radiation.

Lactobacillus spp. inhibits the growth of HCT-116 and reduces IL-8 secretion by Salmonella typhimurium-infected HCT-116 colorectal carcinoma cells

Salmonella typhimurium causes symptoms resembling typhoid fever and gastroenteritis in humans. Its toxicity is due to an outer membrane consisting largely of lipopolysaccharides (LPS) which is responsible for the host immune response. The aim of this study is to evaluate the antimicrobial, anti-apoptotic ability of Lactobacillus plantarum and reduce Salmonella-induced pro-inflammatory cytokine IL-8 secretion. Adhesive tests were performed using lactobacilli co-cultured with the colon cancer cell line HCT-116 for 2 hours. The strains displaying the highest adhesion were selected for downstream 3- (4, 5- Dimethylthiazol -2-yl) -2, 5- diphenyltetrazolium bromide (MTT) tests to assess cytotoxicity. The supernatants of Lactobacillus cultured with HCT-116 cells for 24 and 48 h to evaluate the inhibitory effect. To determine Interleukin 8 (IL-8) secretion in colon cancer induced by S. typhimurium, we stimulated HCT-116 cells with S. typhimurium and co-cultured with lactobacilli for 24 h. Lactobacilli had the most significant inhibitory effects on cell growth, and their inhibitory effects were time-dependent. Strain No. 03-03-026 caused cancer cell deoxyribonucleic acid (DNA) fragmentation, and the anti-apoptosis protein (B-cell lymphoma 2) was reduced in the HCT-116 cells as determined. IL-8 production in colon cancer cells was significantly reduced by these lactobacilli. Our results suggested that lactobacilli maybe effectively reduce the numbers of S. typhimurium, IL-8 levels and the anti-apoptotic phosphorylated-p38 mitogen-activated protein kinase and B-cell lymphoma 2 proteins. Lactobacillus can be added to the diet as a food additive to prevent colorectal cancer and used to be the prophylactic agent against S. typhimurium.

Facial skin ageing: Key concepts and overview of processes.

IntroductionThe face is a cosmetically sensitive region where the process of ageing is most clearly manifested. With increased focus on anti‐ageing and longevity, more anti‐senescent treatments are being proposed despite limited evidence. This study outlines the pathways and mechanisms underpinning the biological process of ageing in the face.MethodsComprehensive searches of MEDLINE, EMBASE, Cochrane Library and CINAHL from inception to 2020. Inclusion criteria included all empirical human research studies specific to facial ageing features, written in the English language.ResultsA total of 65 papers met inclusion criteria for analysis. Pathways were subdivided into intrinsic and extrinsic senescence mechanisms. Intrinsic pathways included genetics, generation of reactive oxygen species and hormonal changes. Extrinsic pathways included photoageing and damage to skin layers. The combined intrinsic and extrinsic pathway alterations result in wrinkles, higher laxity, slackness and thinning of the skin. Skin functions such as barrier immune function, wound healing, thermoregulation and sensory function are also impaired.ConclusionThe ageing process is unique to the individual and depends on the interplay between an individual's genetics and external environmental factors. Through understanding the molecular and cellular mechanisms, an appreciation of the consequent structural and functional changes can be achieved. Based on this knowledge, further research can focus on how to slow or impede the ageing process and identify specific targets to develop and evolve new treatment strategies.

Silver-coated silicon nanowire platform discriminates genomic DNA from normal and malignant human epithelial cells using label-free Raman spectroscopy

Genomic deoxyribonucleic acid (DNA) stores and carries the information required to maintain and replicate cellular life. While much efforts have been devoted in decoding the sequence of DNA basis to detect the genetic mutations related to cancer disease, it is becoming clear that physical properties, like structural conformation, stiffness and shape, can play an important role to recognize DNA modifications. Here, silver-coated silicon nanowires (Ag/SiNWs) are exploited as Raman spectroscopic platform to easily discriminate healthy and cancer genomic DNA, extracted from human normal skin and malignant melanoma cells, respectively. In particular, aqueous DNA droplets are directly deposited onto a forest of Ag/SiNWs and Raman maps are acquired after sample dehydration. By applying principal component analysis (PCA) to the Raman spectra collected within the droplets, healthy and cancer cell DNA can be distinguished without false negative identifications and with few false positive results (< 2%). The discrimination occurs regardless the analysis of specific DNA sequencing, but through Raman bands strictly related to the interfacing of the DNA and the NWs. The observed phenomenon can be ascribed to conformational differences and/or diverse charge properties between healthy and cancer cell DNA determining a different arrangement of the molecules adsorbed onto the NWs upon water evaporation. The unique interaction with DNA and facile fabrication technology make Ag/SiNWs an effective platform for a robust, rapid and label-free cancer diagnosis, as well as a potential tool to investigate physical properties of DNA.

The selective progesterone receptor modulator, telapristone acetate, is a mixed antagonist/agonist in the human and mouse endometrium and inhibits pregnancy in mice

To investigate the effect of the selective progesterone receptor modulator, telapristone acetate (CDB-4124), on endometrial biology and reproductive outcomes. Ovariectomized and hormone-treated CD1 female mice, CD1 female mice with xenotransplants of reconstructed human endometrial tissue, mated wildtype female mice, and cultured human endometrial stromal cells (hESCs) were treated with CDB-4124, followed by the assessment of endometrial cell deoxyribonucleic acid (DNA) proliferation, stromal decidual response, and embryo implantation. Experimental study. Academic research laboratory. Healthy volunteer women from the community were recruited for endometrial biopsies. CD1 out-bred mice (Charles River Laboratories) and nude mice, NU/J (Jackson Laboratories, Bar Harbor, ME). Treatment of mice and hESCs with CDB-4124. The effect of CDB-4124 on endometrial cell morphology and DNA synthesis, decidual response, and mouse embryo implantation. CDB-4124 inhibited estradiol-induced epithelial DNA synthesis in the mouse uterus and xenotransplanted human endometrium. This antiproliferative effect was less than that of progesterone (P4) and was observed when CDB-4124 was administered alone or concomitantly with P4. In the uterine epithelium, CDB-4124 acted as a P4 agonist and partial antagonist. In contrast, CDB-4124 acted as a complete P4 antagonist in the uterine stroma, where it blocked P4's action to induce a decidual response in the pseudopregnant mouse uterus and wildtype mouse uterus after copulation. In mated female mice, CDB-4124 impaired embryo implantation. Similarly, CDB-4124 inhibited the morphological and biochemical transformations of hESCs to decidual cells invitro. CDB-4124 exerts mixed P4 antagonistic/agonistic effects in the human and mouse endometrium, which result in failed embryo implantation because of the absence of stromal decidualization.

Prolonged Posttreatment Virologic Control and Complete Seroreversion After Advanced Human Immunodeficiency Virus-1 Infection

Possible human immunodeficiency virus (HIV)-1 clearance has rarely been reported. In this study, we describe a unique case of an HIV-positive, combination antiretroviral therapy (cART)-experienced woman with prior acquired immunodeficiency syndrome (AIDS) who has not experienced viral rebound for over 12 years since discontinuing cART. Leukapheresis, colonoscopy, and lymph node excision were performed for detailed examination of virologic (including HIV reservoir) and immunologic features. Comparisons were made with chronically infected patients and healthy controls. No HIV-specific antibodies were detected in serum. Plasma HIV ribonucleic acid (RNA) levels were <0.2 copies/mL, and, except for low-frequency HIV deoxyribonucleic acid (DNA)+ cells in lymph node tissue (1 copy/3 × 106 cells), HIV antigen could not be detected by quantitative virus outgrowth (<0.0025 infectious units/106 CD4+ T cells) or by most measurements of HIV RNA or DNA in blood, lymph node, or gut-associated mononuclear cells. Human immunodeficiency virus-specific T-cell responses were detectable but low. Brain imaging revealed a prior biopsy site and persistent white matter disease since 1996. Human immunodeficiency virus DNA+ cells in the 1996 brain biopsy specimen confirmed her identity and initial HIV diagnosis. This represents the first report of complete seroreversion, prolonged posttreatment virus suppression, a profoundly small HIV reservoir, and persistent HIV-specific T cells in an adult with prior AIDS.

2-Monokloropropandiol (2-MCPD)’nin Sitotoksik Etkilerinin Fare TM3 Leydig ve TM4 Sertoli Hücreleri Üzerinde in vitro Değerlendirilmesi

Amaç: 2-monokloropropandiol (2-MCPD), 3-monokloropropandiol (3-MCPD) ve esterleri ile glisidil yağ asidi esteri (GE), gıda veya gıda işlem kaynaklı kontaminantlar olarak adlandırılmaktadır. Bu gıda kontaminantlarının başta palmiye yağı olmak üzere bitkisel yağlar, margarinler, pasta ve kek gibi işlem görmüş gıdalarda ve bebek mamalarında tespit edilmesi, güvenlikleri konusundaki ilgiyi arttırmıştır. Avrupa Gıda Güvenliği Otoritesi’nin (European Food Safety Authority, EFSA) Mayıs 2016 yılında yayınlamış olduğu bilimsel görüşe göre bitkisel yağ ve işlenmiş gıdalarda bulunan GE, 3-MCPD, 2-MCPD ve bunların yağ asidi esterlerinin insan sağlığı için zararlı olduğu raporlanmıştır. Ancak 2-MCPD toksisitesi hakkında yeterli veri olmadığı için tam bir değerlendirme yapılamamıştır. Bu amaçla 2-MCPD’nin, TM3 Leydig ve TM4 Sertoli hücre hatları üzerinde in vitro sitotoksik, apoptotik etkileri ve deoksiribonükleik asit (DNA) hasarı yapma potansiyeli değerlendirilmiştir.&#x0D; Gereç ve Yöntem: Hücre canlılığı, 3-(4,5-dimetiltiyazol-2-il)-2,5-difeniltetrazolyum bromid (MTT) ve nötral kırmızısı alım (NRU) yöntemleri ile değerlendirilmiştir. Akış sitometrisi ile hücre ölüm tipinin belirlenmesinde aneksin V-FITC/PI ikili boyama kiti kullanılmıştır. Genotoksisite değerlendirmesinde komet yöntemi uygulanmıştır. &#x0D; Bulgular: Hücre canlılık yöntemleri sonucu 2-MCPD’nin test edilen konsantrasyonlarda 1000 µM’a kadar sitotoksik olmadığı tespit edilmiştir. Aynı zamanda 2-MCPD’nin, sub-toksik konsantrasyonlarda (1-1000 µM) her iki hücre hattında da (TM3 Leydig ve TM4 Sertoli hücreleri) DNA hasarını indüklemediği bulunmuştur. &#x0D; Sonuç: 2-MCPD, her ne kadar test edilen konsantrasyonlarda (1-1000 µM) sitotoksik ve apoptotik etki göstermese de daha yüksek konsantrasyonlarda hücre ölümüne ve DNA hasarına neden olabilir. Ancak bu yüksek konsantrasyonlar günlük maruziyet dozlarından çok uzaktır.

The Potential Genotoxic Effects of Antineoplastic Drugs in Occupationally Exposed Nurses

Background: Antineoplastic drugs are commonly used globally. They directly interact with the tumor cell deoxyribonucleic acid and inhibit their growth. These actions are non-selectiveaffecting both tumor cells and healthy cells producing toxic effects. The present study was designed to evaluate oxidative stress and genotoxic effects of antineoplastic drugs in occupationally exposed nurses under routine working conditions. Methods: The studied groups included 28 nurses working in Hematology and Oncology unit, Benha University Hospitals and 20 non exposed subjects that work in academic jobs in Benha Faculty of Medicine, matched in age, gender & socioeconomic status. Malondoaldehyde (MDA) & reduced glutathione (GSH) were measured for all participants as markers of oxidative stress. Extent of DNA damage in leucocytes was also evaluated by comet assay as a biomarker of genotoxicity. Results: There were a significant increase in MDA & reduction in GSH in occupationally exposed subjects than control. Also, extent of DNA damage in the lymphocytes of occupationally exposed participants has a high significance than the controls. The work duration and age had a significant impact on degree of DNA damage. Conclusion: occupationally exposed nurses to antineoplastic agents are at greater risk of oxidative stress with consequent DNA damage and potential genotoxic damage.

ReaxFF-based molecular dynamics simulation of DNA molecules destruction in cancer cells by plasma ROS

Medical applications of plasma have been a hot topic of research recently. Studies have shown that cold atmospheric pressure plasma (CAP) plays a beneficial role in cancer treatments and can selectively kill cancer cells. Some reactive oxygen species (ROS) in plasma react with cancer cells, altering the cellular structure and inducing apoptosis. Currently, however, it is difficult to determine the microscopic mechanism of the reaction between plasma and cancer cells. In this work, a reactive force field was used to model the deoxyribonucleic acid (DNA) damage caused by three ROS (O, OH, and H2O2) at the atomic level. The simulation showed that the ROS of CAP reacted with the DNA of cancer cells, breaking important chemical bonds in DNA molecules and damaging the genes of cancer cells. Therefore, the reaction between ROS and DNA molecules affects gene transcription, replication, and reproduction of cancer cells and inhibits their unlimited proliferation. O atoms, OH radicals, and H2O2 can damage DNA molecules at different structural positions, causing major irreversible damage to the molecular structure of the DNA.

A novel mitochondria-targetable probe for imaging endogenous deoxyribonucleic acid in biological systems

Mitochondria is a main organelle for conducting aerobic respiration in the process of metabolism. Deoxyribonucleic acid (DNA) plays central roles in the genetic encoding, regulation and expression in the living normal and cancer cells. However, imaging of mitochondria and DNA in biological system is restricted due to lack of functional molecular tools. To address this critical challenge, herein, we developed a single small-molecular fluorescent probe BISEI. This novel probe possessed large Stokes shift, high fluorescence quantum yield, prominent photostability, and excellent two-photon properties. Owing to its excellent properties, the probe BISEI is successfully used to image mitochondria and DNA in the cancer cells, tissues and zebrafish. For the cell imaging, the results showed distribution of the mitochondrial reticular structure; For the tissue imaging, the 3D imaging results firstly exhibited distribution of nuclei in living tissues; For the in vivo imaging, the probe displayed a strong red fluorescence in the chest of zebrafish.

Host Cell Deoxyribonucleic Acid Methylation Markers for the Detection of High-grade Anal Intraepithelial Neoplasia and Anal Cancer.

BackgroundHigh-grade anal intraepithelial neoplasia (AIN2/3; HGAIN) is highly prevalent in human immunodeficiency virus positive (HIV+) men who have sex with men (MSM), but only a minority will eventually progress to cancer. Currently, the cancer risk cannot be established, and therefore all HGAIN is treated, resulting in overtreatment. We assessed host cell deoxyribonucleic acid (DNA) methylation markers for detecting HGAIN and anal cancer.MethodsTissue samples of HIV+ men with anal cancer (n = 26), AIN3 (n = 24), AIN2 (n = 42), AIN1 (n = 22) and HIV+ male controls (n = 34) were analyzed for methylation of 9 genes using quantitative methylation-specific polymerase chain reaction. Univariable and least absolute shrinkage and selection operator logistic regression, followed by leave-one-out cross-validation, were used to determine the performance for AIN3 and cancer detection.ResultsMethylation of all genes increased significantly with increasing severity of disease (P < 2 × 10-6). HGAIN samples revealed heterogeneous methylation patterns, with a subset resembling cancer. Four genes (ASCL1, SST, ZIC1,ZNF582) showed remarkable performance for AIN3 and anal cancer detection (area under the curve [AUC] > 0.85). ZNF582 (AUC = 0.89), detected all cancers and 54% of AIN3 at 93% specificity. Slightly better performance (AUC = 0.90) was obtained using a 5-marker panel.ConclusionsDNA methylation is associated with anal carcinogenesis. A marker panel that includes ZNF582 identifies anal cancer and HGAIN with a cancer-like methylation pattern, warrantingvalidation studies to verify its potential for screening and management of HIV+ MSM at risk for anal cancer.

Roles of cell cycle phases and DNA end structures in determining requirements for the Yku, Mrx and Ligase IV complexes in nonhomologous end‐joining repair of plasmid DNA

Deoxyribonucleic acid (DNA) can be damaged through a variety of different damaging agents such as free radicals, radiation and environmental chemicals. Types of damage include single‐strand lesions such as base or sugar modifications and more deleterious lesions such as interstrand crosslinks and DNA double‐stranded breaks (DSBs). Unrepaired or misrepaired DSBs in a cell can lead to mutations and chromosome rearrangements, potentially leading to loss of cell function and even cell death. There are two independent pathways used to repair DSBs in DNA; these pathways are homologous recombination (HR) and nonhomologous end‐joining (NHEJ). NHEJ involves the functions of three major protein complexes. In the budding yeast Saccharomyces cerevisiae, these assemblages include the Yku, Mrx and DNA ligase IV complexes. Each complex has a specific function. Yku (Yku70 and Yku80) protects the broken ends from degradation and recruits other repair proteins. Mrx, composed of Mre11, Rad50 and Xrs2, may act to tether the ends together and perform nuclease processing of the ends if necessary. The ligase IV complex, containing Dnl4 and Lif1 and possibly Nej1, performs the final step, ligating the two broken ends of DNA thus ending the NHEJ process.Repair of DSBs by NHEJ is frequently analyzed using plasmid‐based assays. In these experiments, a plasmid with a single DSB is transformed into cells and the efficiency of repair/recircularization inside cells is monitored. The region around the DSB site is designed so that it lacks homology with the host cell's chromosomal DNA to prevent repair by HR. An uncut plasmid is usually also transformed into the cells to normalize repair efficiencies to simple cell transformation efficiencies. Many past studies involving NHEJ repair using plasmid assays have observed that repair in yku, mrx, and dnl4 mutants is reduced similarly, typically 10–30 fold, suggesting equal importance of the three major complexes in the pathway. Using a high efficiency yeast transformation method involving treatment with dithiothreitol (DTT) and inclusion of a rich broth recovery step, we observed that repair of broken plasmids with 5′ single‐stranded DNA overhangs (4 nt long) was strongly reduced in yku mutants (yku70 or yku80) and DNA ligase IV mutants (dnl4, lif1, or nej1), but not in mrx strains (mre11, rad50 or xrs2). The accuracy of repair was also strikingly different among mrx cells vs. yku and dnl4 mutants. By contrast, efficiency and accuracy of repair of plasmids containing 3′ overhangs was reduced to similar levels in yku70, mre11 and dnl4 mutants. Since NHEJ repair efficiency is most active in G1 phase, we have also assessed the differential repair of broken plasmids in yku, mrx and dnl4 mutants in cultures with different proportions of G1 cells. These experiments have included mid‐log phase, early stationary phase, stationary phase and alpha factor‐arrested G1 phase cells. Work utilizing linear plasmids containing different end sequences and structures will be described.Support or Funding InformationNational institute of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

5-Hydroxymethylcytosine is a predominantly stable DNA modification.

5-Hydroxymethylcytosine (hmC) is an oxidation product of 5-methylcytosine which is present in the deoxyribonucleic acid (DNA) of most mammalian cells. Reduction of hmC levels in DNA is a hallmark of cancers. Elucidating the dynamics of this oxidation reaction and the lifetime of hmC in DNA is fundamental to understanding hmC function. Using stable isotope labelling of cytosine derivatives in the DNA of mammalian cells and ultrasensitive tandem liquid-chromatography mass spectrometry, we show that the majority of hmC is a stable modification, as opposed to a transient intermediate. In contrast with DNA methylation, which occurs immediately during replication, hmC forms slowly during the first 30 hours following DNA synthesis. Isotopic labelling of DNA in mouse tissues confirmed the stability of hmC in vivo and demonstrated a relationship between global levels of hmC and cell proliferation. These insights have important implications for understanding the states of chemically modified DNA bases in health and disease.

CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.