DNA damage repair lies at the core of all cells’ survival strategy, including cancerous. Therefore, targeting such repair mechanisms forms the major goal of cancer therapeutics. The mechanism of DNA repair has been tousled with the discovery of multiple kinases. Recent studies on Tousled like Kinases have brought significant clarity on the effectors of these kinases which stands to regulate DSB repair. In addition to their well-established role in the DDR and cell cycle checkpoint mediation after DNA damage or inhibitors of replication, their suspected involvement in the actual DSB repair process has more recently been strengthened by the important finding that TLK1 phosphorylates RAD54 and regulates some of its activities and localization in the cell. Earlier findings of its regulation of RAD9 during checkpoint deactivation as well as defined steps during NHEJ ends processing were earlier hints of its important involvement broadly in DSB repair. All this has opened up new avenues to target cancer cells in combination therapy with genotoxins and TLK inhibitors.

The DNA Damage Response (DDR) network and the mitogen-activated protein kinase (MAPK) signaling pathway are crucial mechanisms for the survival of all living beings. An accumulating body of evidence suggests that there is a crosstalk between these two systems, thus favoring the appropriate functioning of multi-cellular organisms. On the other hand, aberrations within these mechanisms are thought to play a vital role in the onset and progression of several diseases, including cancer, as well as in the emergence of drug resistance. Here, we provide an overview of the current knowledge regarding alterations in the DDR machinery and the MAPK signaling pathway, as well as abnormalities in the DDR-MAPK functional crosstalk in multiple myeloma, the second most common hematologic malignancy. We also present the latest advances in the development of anti-myeloma drugs targeting crucial DDR- and MAPK-associated molecular components. These data could potentially be exploited to discover new therapeutic targets and effective biomarkers, as well as for the design of novel clinical trials. Interestingly, they might provide a new approach to increase the efficacy of anti-myeloma therapy by combining drugs targeting the DDR network and the MAPK signaling pathway.

It has been more than three decades since the discovery of multifunctional factors, Non-POU Domain-Containing Octamer-Binding Protein, NonO and Splicing Factor, Proline-and Glutamine-Rich, SFPQ. Some of their functions, including their participations in transcriptional and posttranscriptional regulation as well as their contribution to paraspeckle subnuclear body organization have been well-documented. In this review, we focus on the roles of NonO and SFPQ in cell cycle with particular attention to their participation in nonhomologous end joining (NHEJ) and homologous recombination (HR) of DNA damage responses induced by irradiation. In these contexts, absence or malfunction of either or both lead to genome instability and ultimately to apoptosis, cellular senescence, and tumor development. This review also will shed light on NonO and SFPQ function in DNA/RNA/protein binding and their association with Ku heterodimer of the NHEJ pathway required for repair of DNA double-strand breaks (DSBs).

Abstract Background In tumors, somatic mutagenesis presumably drives DNA damage response (DDR) via altered regulatory pathways increasing genomic instability and proliferative activity. These considerations led to the standard therapeutic strategy against cancer: the disruption of mutation activated DNA repair pathways of tumors. Purpose Justifying that cancer cells are not enemies to be killed, but rather they are ill human cells having the remnants of physiologic regulatory pathways. Results 1. Genomic instability and cancer development may be originated from the flaw of estrogen signal rather than excessive estrogen signaling. 2. Healthy cells with genomic instability exhibit somatic mutations helping DNA restitution. 3. Somatic mutations in tumor cells aim the restoration of DNA damage rather than further genomic derangement. 4. In tumors, estrogen signal drives the pathways of DNA stabilization leading to apoptotic death. 5. In the peritumoral cellular infiltration, the genomic damage of tumor induces inflammatory cytokine secretion and increased estrogen synthesis. In the inflammatory cells, increased growth factor receptor (GFR) signal confers unliganded activation of estrogen receptors (ERs). 6. In tumor cells responsive to genotoxic therapy, constitutive mutations help the upregulation of estrogen signal and consequential apoptosis. In tumors non responsive to genotoxic therapy, the possibilities for ER activation via either liganded or unliganded pathway, are exhausted leading to farther genomic instability and unrestrained proliferation. Conclusion Understanding the real character and behavior of human tumors at molecular level suggests that we should learn the genome repairing methods of tumors and follow them by supportive therapy rather than provoking additional genomic damages.

Base excision repair (BER) is a cellular process that removes damaged bases arising from exogenous and endogenous sources including reactive oxygen species, alkylation agents, and ionizing radiation. BER is mediated by the actions of multiple proteins that work in a highly concerted manner to resolve DNA damage efficiently to prevent toxic repair intermediates. During the initiation of BER, the damaged base is removed by one of 11 mammalian DNA glycosylases resulting in abasic sites. Many DNA glycosylases are product inhibited by binding to the abasic site more avidly than the damaged base. Traditionally apurinic/apyrimidinic endonuclease, APE1, was believed to help turnover the glycosylases to undergo multiple rounds of damaged base removal. However, in a series of papers from our laboratory we have demonstrated that UV-damaged DNA binding protein (UV-DDB) stimulates the glycosylase activities of human 8-oxoguanine glycosylase (OGG1), MUTY DNA glycosylase (MUTYH), alkyladenine glycosylase/N-methylpurine DNA glycosylase (AAG/MPG), and single-strand selective monofunctional glycosylase (SMUG1), between three-to-five-fold. Moreover, we have shown UV-DDB can assist chromatin decompaction facilitating access of OGG1 to 8-oxoguanine damage in telomeres. This review summarizes the biochemistry, single-molecule, and cell biology approaches that our group used to directly demonstrate the essential role of UV-DDB in BER.

DNA double-strand breakage is the most lethal damage to chromosomal DNA. It activates a series of cellular DNA damage response pathways, including DNA damage sensing, control of cell cycle arrest and apoptosis, and DNA repair. DNA damage response pathways are regulated by complex signaling machineries. Of the intracellular signaling cascades, diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Because both DG and PA serve as second messengers, DGK activity induces a shift of signaling pathways from DG-mediated to PA-mediated cascades, thereby implicating DGK in the regulation of widely various functions. Reportedly, one member of the DGK family, DGKζ, is intimately involved in the regulation of stress responses through p53 and NF-κB. Stresses such as ischemia and infarction cause DGKζ downregulation. Experimental DGKζ depletion renders cells and mice vulnerable to various stressors such as chemotherapeutic agents and ionizing irradiation. Nevertheless, how DGKζ is involved in DNA repair, a critical event of DNA damage response for survival remains unknown. For this study, we examined how DGKζ depletion affects DNA repair mechanisms. We demonstrated that DGKζ depletion causes attenuation of Akt activation and DNA-PK protein expression upon DNA damage, which might engender downregulated BRCA1 protein synthesis and stability. Results suggest that DGKζ depletion attenuates BRCA1-mediated DNA repair machinery, thereby conferring vulnerability to DNA damage.

Chromothripsis is a mutational mechanism leading to complex and relatively clustered chromosomal rearrangements resulting in diverse phenotypic outcomes depending on the involved genomic landscapes. It may occur both in the germ and the somatic cells resulting in congenital and developmental disorders and cancer, respectively. Asymptomatic individuals may be carriers of chromotriptic rearrangements and experience recurrent reproductive failures when two or more chromosomes are involved. Several mechanisms are postulated to underly chromothripsis. The most attractive hypothesis involves chromosome pulverization in micronuclei followed by incorrect reassembly of fragments through DNA repair to explain the clustered nature of the observed complex rearrangements. Moreover, exogenous or endogenous DNA damage induction and dicentric bridge formation may be involved. Chromosome instability is commonly observed in the cells of patients with DNA-repair disorders, such as ataxia telangiectasia, Nijmegen breakage syndrome and Bloom syndrome. In addition, germline variations of TP53 have been associated with chromothripsis in Sonic-Hedgehog medulloblastoma and acute myeloid leukemia. In the present review, we focus on the underlying mechanisms of chromothripsis and the involvement of defective DNA-repair genes resulting in chromosome instability and chromothripsis-like rearrangements.

The enterohemorrhagic Escherichia coli (EHEC) O157: H7 EspF is known to be a multifunctional effector that triggers several damage processes in the host cells. However, in the process of EHEC O157: H7 infection, the interactions between EspF, its N- or C-terminus, and host proteins are still unclear. In this work, we use co-immunoprecipitation combined with mass spectrometry (CoIP-MS) to screen the interactions between EspF/EspF-N/EspF-C terminus and host proteins. A total of 311 host proteins are analyzed. The N-terminus of EspF is found to interact with 192 proteins, whereas 205 proteins interact with the C-terminus. These proteins are mainly involved in RNA splicing, endoplasmic reticulum stress, and a variety of metabolic signaling pathways. Surprisingly, MS results reveal that EspF can also phosphorylate H2AX, suggesting that EspF may directly mediate DNA damage. Here, by western blot and immunofluorescence (IF), we verified that EspF can cause phosphorylation of H2AX and mediates cell multi-nuclearation and cell hypertrophy. Furthermore, we verify here for the first time that SMC1 interacts with EspF -C-terminus, and provide evidence that EspF increases p-SMC1 levels. p-SMC1 is known to influence S-phase cell cycle arrest and usually increases during periods of DNA damage. Our work revealed a novel interaction between EspF and the host protein SMC1 and lays a foundation for further research on EspF-mediated host DNA damage, apoptosis, and even colorectal carcinogenesis.

Track-structure Monte Carlo simulations are useful tools to evaluate initial DNA damage induced by irradiation. In the previous study, we have developed a Gean4-DNA-based application to estimate the cell surviving fraction of V79 cells after irradiation, bridging the gap between the initial DNA damage and the DNA-rejoining kinetics by means of the two-lesion kinetics (TLK) model. However, since the DNA repair performance depends on cell line, the same model parameters cannot be used for the different cell lines. Thus, we extended the Geant4-DNA application with an updated TLK model for the evaluation of DNA damage repair performance in HSGc-C5 carcinoma cells which are typically used for evaluating proton/carbon radiation treatment effects. For this evaluation, we also performed experimental measurements for cell surviving fractions and DNA-rejoining kinetics of the HSGc-C5s cells. Concerning fast- and slow-DNA rejoining, the TLK model parameters were adequately optimized with the simulated initial DNA damage. Using the optimized TLK model, the Geant4-DNA simulation is now able to predict cell survival and DNA-rejoining kinetics for HSGc-C5s cells.

The Ramazzottius varieornatus tardigrade is an extremotolerant terrestrial invertebrate belonging to the phylum of Tardigrada. At a length of 0.1-1.0 mm, tardigrades are small animals with an exceptional tolerance to extreme conditions such as high pressure, chemicals and irradia-tion. These properties have been attributed to the recently-discovered Dsup protein. Dsup is a nucleosome-binding protein that prevents DNA damage against X-ray and oxidative stress without impairing cell life, also in Dsup-transfected animal and plant cells. However, the precise “protective” role of this protein is still under study. We performed experiments on human cells and shows that, as compared to control cells, Dsup+ cells are more resistant to UV-C exposure and H2O2. Real-time PCR identified different expression patterns of endogenous genes involved in apoptosis, cell survival and DNA damage repair in Dsup+ cells in response to H2O2 and UV-C. While H2O2 treatment in Dsup+ cells only marginally involved the activation of pathways responsible for DNA repair reinforcing the idea of a direct protective effect of the protein on DNA, in UV-C exposed cells, Dsup efficiently upregulates DNA damage repair genes. In conclusion, our data may help to delineate the different mechanisms by which the Dsup protein operates in response to different insults.

Cells respond to DNA double-strand breaks by initiating DSB repair and ensuing a cell cycle checkpoint. The primary responder to DSB repair is non-homologous end joining, an error prone repair pathway. However, when DSBs are generated after DNA replication in the G2 phase of the cell cycle, a second DSB repair pathway, homologous recombination, can come into action. Both ATM and ATR are important for DSB-induced DSB repair and checkpoint responses. One method of ATM and ATR working together is through the DNA end-resection of DSBs. As a readout and marker of DNA end-resection, RPA is phosphorylated at Ser4/Ser8 of the N-terminus of RPA32 in response to DSBs. Here, the significance of RPA32 Ser4/Ser8 phosphorylation in response to DNA damage, specifically in S phase to G2 phase of the cell cycle is examined. RPA32 Ser4/Ser8 phosphorylation in G2 synchronized cells is necessary for increases in TopBP1 and Rad9 accumulation on chromatin and full activation of the ATR-dependent G2 checkpoint. In addition, our data suggest RPA Ser4/Ser8 phosphorylation modulates ATM-dependent KAP-1 phosphorylation and Rad51 chromatin loading in G2 cells.

DNA double-strand breaks (DSBs) are deleterious lesions that are generated in response to ionizing radiation or replication fork collapse that can lead to genomic instability and cancer. Eukaryotes have evolved two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ) to repair DSBs. Whereas the roles of protein-DNA interactions in HR and NHEJ have been fairly well defined, the functions of small and long non-coding RNAs and RNA-DNA hybrids in the DNA damage response is just beginning to be elucidated. This review summarizes recent discoveries on the identification of non-coding RNAs and RNA-mediated regulation of DSB repair

Cancer-associated fibroblasts (CAF) form the basis of tumor microenvironment and possess immunomodulatory functions by interacting with other cells surrounding tumor, including T lymphocytes, macrophages, dendritic cells and natural killer cells. Ionizing radiation is a broadly-used method in radiotherapy to target tumors. In mammalian cells, ionizing radiation induces various types of DNA damages and DNA damage response. Being unspecific, radiotherapy affects all the cells in tumor microenvironment, including the tumor itself, CAFs and immune cells. CAFs are extremely radio-resistant and do not initiate apoptosis even at high doses of radiation. However, following radiation, CAFs become senescent and produce a distinct combination of immunoregulatory molecules. Radiosensitivity of immune cells varies depending on the cell type due to inefficient DNA repair in, for example, monocytes and granulocytes. In this minireview, we are summarizing recent findings on the interaction between CAF, ionizing radiation and immune cells in the tumor microenvironment.

Simple and complex clustered DNA damage represent critical initial damage caused by radiation. In this paper a multinomial probability model of clustered damage is developed with probabilities dependent on the energy imparted to DNA and surrounding water molecules. The model consists of four probabilities: A) direct damage of sugar-phosphate moieties leading to SSB, B) OH^- radical formation with subsequent SSB and BD formation, C) direct damage to DNA bases, and D) energy imparted to histone proteins and other molecules in a volume not leading to SSB or BD. These probabilities are augmented by introducing probabilities for relative location of SSB using a <10 bp criteria for double strand break (DSB), and for the possible success of radical attack to lead to SSB or BD. Model predictions for electrons, ⁴He and ¹²C ions are compared to experimental data and show good agreement. Thus, the developed model allows an accurate and rapid computational method to predict simple and complex clustered DNA damage as a function of radiation quality and to explore the resulting challenges to DNA repair.

Astronauts in space are subject to a continuous bombardment of ionizing radiation. The Earth's magnetic field and the ISS shield some biologically damaging particles traveling through. Still, travel beyond low-Earth orbit and extravehicular activities are exceedingly more dangerous and there is a concern for the acute and late-occurring adverse health effects befalling astronauts. So, it is vital to consider the current tools and models used to describe and study the organic consequences of ionizing radiation exposure. It is equally important to see where these models may improve. This article reviews the historical development and current state of knowledge of radiation effects impacting astronauts in orbit. We explain the space radiation environment, cellular microenvironment, and how these may be incorporated in radiobiological models to aid in our understanding of the influence space travel may have on astronaut health. The topics discussed in this paper include a review of DNA damage and repair mechanisms and the numerical models that aim to explain the biological effects resulting from ionizing radiation damage. Historically, radiobiological models focused on how radiation damages nuclear DNA, built upon the hypotheses of Crowther and Lea in the 1940s and 1960s, and neglected other sub-cellular targets outside of nuclear DNA.

Natural and synthetic small molecules from the NCI Developmental Therapeutics Program (DTP) were employed in molecular dynamics-based docking with DNA repair proteins whose RNA-Seq based expression was associated with overall cancer survival (OS) after adjustment for the PCNA metagene. The compounds employed were required to elicit a sensitive response (vs. resistance) in more than half of the cell lines tested for each cancer. Methodological approaches included peptide sequence alignments and homology modeling for 3D protein structure determination, ligand preparation, docking, toxicity and ADME prediction. Docking was performed for unique lists of DNA repair proteins which predict OS for AML, cancers of the breast, lung, colon, and ovaries, GBM, melanoma, and renal papillary cancer. Results indicate hundreds of drug-like and lead-like ligands with best-pose binding energies less than -6 kcal/mol. Ligand solubility for the top 20 drug-like hits approached lower bounds, while lipophilicity was acceptable. Most ligands were also blood-brain barrier permeable with high intestinal absorption rates. While the majority of ligands lacked positive prediction for Herg channel blockage and Ames carcinogenicity, there was considerable variation for predicted fathead minnow, honey bee, and Tetrahymena pyriformis toxicity. The computational results suggest the potential for new targets and mechanisms of repair inhibition and can be directly employed for in vitro and in vivo confirmatory laboratory experiments to identify new targets of therapy for cancer survival.

Removal of the proliferation component of gene expression by PCNA adjustment has been addressed in numerous survival prediction studies for breast cancer and all cancers in the TCGA. These studies indicate that widespread co-regulation of proliferation upwardly biases survival prediction when gene selection is performed on a genome-wide basis. In addition, removal of the correlative effects of proliferation does not reduce the random bias associated with survival prediction using random gene selection. Since most cancers become addicted to DNA repair as a result of forced cellular replication, increased oxidation, and repair deficiencies from oncogenic loss or genetic polymorphisms, we pursued an investigation to remove the proliferation component of expression in DNA repair genes to determine survival prediction. This translational hypothesis-driven focus on DNA repair genes is directly amenable to finding new sets of DNA repair genes that could potentially be studied for inhibition therapy. Overall survival (OS) prediction was evaluated in 18 cancers by using normalized RNA-Seq data for 126 DNA repair genes with expression available in TCGA. Transformations for normality and adjustments for age at diagnosis, stage, and PCNA metagene expression were performed for all DNA repair genes. We also analyzed genomic event rates (GER) for somatic mutations, deletions, and amplification in driver genes and DNA repair genes. After performing empirical p-value testing with use of randomly selected gene sets, it was observed that OS could be predicted significantly by sets of DNA repair genes for 61% (11/18) of the cancers. Interestingly, PARP1 was not a significant predictor of survival for any of the 11 cancers. Results from cluster analysis of GERs indicates that the most opportunistic cancers for inhibition therapy may be AML, colorectal, and renal papillary, because of potentially less confounding due to lower GERs for mutations, deletions, and amplifications in DNA repair genes. However, the most opportunistic cancer for inhibition therapy is likely to be AML, since it showed the lowest GERs for mutations, deletions, and amplifications in DNA repair genes. In conclusion, our hypothesis-driven focus to target DNA repair gene expression adjusted for the PCNA metagene as a means of predicting OS in various cancers resulted in statistically significant sets of genes.

The sperm heads of frozen/thawed horse spermatozoa were injected into ovulated mouse oocytes where they decondensed and formed paternal pronuclei in which the DNA damage has been evaluated. The motile spermatozoa exhibit none or minor level of DNA damage and the xenogenic zygotes cleave to the two-cell embryonic stage with regular nuclei and no micronuclei (MNs). On the other hand, the immotile spermatozoa exhibit typically high levels of DNA damage and blastomeres in two-cell stage embryos contain multiple MNs. Moreover, extremely high sperm DNA damage prevents even the cleavage to the two-cell stage.Keywords: keyword 1; keyword 2; keyword 3 (List three to ten pertinent keywords specific to the article yet reasonably common within the subject discipline.)

Tdp1 and Tdp2 are DNA repair enzymes that repair DNA damages caused by various agents, including anticancer drugs. Thus, these enzymes resist anticancer therapy and could be the reasons of resistance to such widely used drugs as topotecan and etoposide. In the present work, we found compounds capable of inhibiting both enzymes among derivatives of (-)-usnic acid. Both (+)- and (-)-enantiomers of compounds act equally effectively against Tdp1, and only (-)-enantiomers inhibited Tdp2. Surprisingly, the compounds protect HEK293FT wild type cells from the cytotoxic effect of etoposide but potentiate it against Tdp2 knockout cells. We assume that the sensitizing effect of the compounds in the absence of Tdp2 is associated with effective inhibition of Tdp1, which could take over the functions of Tdp2.

In archaea, the interaction of HerA helicase and NurA nuclease is required for 3' end resection during DNA double-stranded break repair, and its disruption results in unfaithful DNA repair. Interactions transpire by the four conserved domains, including the N-terminal HAS domain (HerA-ATP synthase), which serves as a binding site for the exonuclease NurA. As a HerA hexamer between its two subunits, the conserved C-terminal brace extension functions as an adjacent brace. DBD serves as a location for DNA binding, which causes the DNA strands to be propelled into the HerA pocket to begin unwinding. The putative pore entrance is made up of four helix coils, which provide a suitable charged surface for guiding DNA into the pore. The crystal structure of the complex HerA-NurA allows for the identification of key residues for protein-protein interaction. This allows us to predict that most archaeal HerA orthologs will be able to interact with one another. Archaeal HerA is a homo-hexameric assembly featuring a DNA-binding domain that is conserved. It also features distinctive ATP and NurA binding domains. Following the structure and sequence conservation criteria, we find one HerA ortholog in Pyrobaculum calidifontis and multiple HerA paralogs, but the role of none is established yet.Interestingly, orthologous HerA is part of an Mre11-Rad50 gene neighborhood while paralogous HerA is not, which could be misleading. As a result, it sought to describe the criteria that may be used as a possible fingerprint for identifying orthologous genes, as well as the cloning procedures and tactics that would aid in the confirmation of the HerA helicase in P. calidifontis. Their confirmation is based on PCR cloning and characterization following HerA construct creation.

N6-methyladenosine (M6A) modification is the most abundant internal chemical modification on RNA molecules in eukaryotes and can affect mRNA metabolism and regulate RNA transcription, nuclear export, splicing, degradation, translation, ets, and has a significant impact on various aspects of physiology or pathobiology. Radiotherapy is the most common measure of tumor treatment, variety of cellular intrinsic mechanisms affect the responses of cells to ionizing radiation (IR) and effectiveness of cancer radiotherapy. In this review, we summarize and discuss the recent advances about the roles and mechanisms of RNA M6A methylation in cellular responses to radiation-induced DNA damage response, and outcomes of cancer radiotherapy. Insights into the RNA M6A methylation in radiation biology may facilitate the improvement of therapeutic strategies for cancer radiotherapy as well as radioprotection on normal tissues.

Relative contribution of different repair pathways to the radiation-induced DNA damage responses remains a challenging issue of studying the radiation injury endpoints. Comparative manifestation of homologous recombination (HR) after application of different radiation doses greatly determines an overall effectiveness of recovery in dividing cell after irradiation, since HR is an error-free mechanism intended for repair of DNA double strand breaks (DSB) during S/G2 phases of cell cycle. In this article, we present an experimentally observed evidence of dose-dependent shift in relative contribution of HR in human fibroblasts after X-ray exposure at doses 20-1000 mGy, which is also supported by quantitative modeling of DNA DSB repair. Our findings indicate that the radiation dose increase leads to dose-dependent decrease in relative contribution of HR into the entire repair process.

More than 50% of patients with non-small cell lung cancer (NSCLC) are treated with radiotherapy (RT) during different phases of treatment. However, radiation pneu-monitis (RP) and resistance often lead to RT failure in NSCLC patients. JWA, a tumor suppressor gene, is known to enhance DNA damage in gastric cancer cells while protect normal cells from DNA damage induced by cisplatin. Recently, we have re-ported that JWA agonist compound 4 (JAC4) effectively protects intestinal epithelium from RT triggered damage in mice. However, the potential synergistic and attenuated effects of JAC4 in chest RT of lung cancer are not been illuminated. The aim of this study was to investigate the effects of JAC4 on the radiotoxicities of both NSCLC and normal lung tissue. CCK-8 and colony formation assays showed that JAC4 played a bidirectional role in radiation-treated SPCA-1 and BEAS-2B cells. Western blotting and immunofluorescence assays showed that JAC4 in combination with RT increased DNA damage and apoptosis in SPCA-1 cells, while the opposite effect was observed in BEAS-2B cells. Mechanistically, JAC4 inhibited homologous recombination repair (HR) and non-homologous end joining (NHEJ) in SPCA-1 cells, but not in normal cells. JAC4 increased antioxidant capacity, and reduced oxidative stress and inhibited nu-clear factor Kappa-B (NF-κB, P65) translocation to the nucleus in BEAS-2B cells. Im-portantly, the bidirectional roles of JAC4 on RT were reversed by siJWA in both SPCA-1 and BEAS-2B cells. Finally, the bidirectional effects of JAC4 in combination with RT were further validated in NSCLC xenograft model mice. In conclusion, JAC4 enhanced effect of RT on tumor growth while alleviated RP and lung injury. Our re-sults may provide new strategy for optimizing RT regimen for NSCLC.

Often, in biology, we are faced with the problem of exploring relevant unknown biological hypotheses in the form of myriads of combinations of factors/genes/proteins that might be affecting the pathway under certain conditions. In colorectal cancer (CRC) cells treated with ETC-1922159, many genes were found up and down regu- lated, individually. A recently developed search engine ranked combinations of DNA repair gene-X (X, a particular gene/protein) at 2nd order level after drug administration. These rankings reveal which Wnt-X combinations might be working synergistically in CRC. If found true, oncologists can further test the combination of interest in wet lab and determine the mechanism of functioning between the Wnt and X. In this re- search work, we cover combinations of RAD with X-ray repair cross complementing (XRCC) family, 5’-3’ exoribonuclease 2 (XRN2), NFKB repressing factor (NKRF), B cell CLL/lymphoma (BCL) family, exosome component (EXOSC) family, FA comple- mentation group (FANC) family and XRCC with EXOSC.

Abstract: Background: Gender dimorphism and marital status on colorectal cancer mortality have 9 been previously documented, but the relationship between these factors and DNA mismatch repair 10 protein (MMRP) expression status is unknown. Methods: Colectomy specimens were reviewed ret- 11 rospectively for patients between 2018 to 2023 with demographics including race/ethnicity, gender, 12 marital status, faith, body mass index, pathologic staging, and MMRP expression status. Statistical 13 analyses were performed by using baseline characteristics tables and various programs in R-pack- 14 age. Results: A total 1018 colectomies were reviewed, and the tumor stages were significantly higher 15 in right colon (stage 3 and 4) than those in left colon and rectosigmoid colon (p<0.01). Marital status 16 was significantly associated with patients’ gender, age, tumor size and tumor stages (all p<0.01). 17 MMRP status were available in 775 cases, and 139 (17.9%) were MMRP deficient, and 636 (82%) 18 MMRP proficient. MMRP deficiency was significantly associated with older female patients, larger 19 tumor in size, higher tumor stages, higher histologic grade and more common in right colon (all 20 p<0.01). In addition, MMRP deficiency was statistically associated with higher percentage of di- 21 vorced and widowed patients (p<0.01). Multivariate linear regression analysis revealed persistent 22 association of MMRP deficiency with tumor size, tumor grade, tumor stage and nodal metastasis, 23 but the associations with gender and marital status no longer existed. Conclusion: The differences 24 in prevalence of CRC by gender and marital status and tumor MMRP status illustrate the im- 25 portance of these factors on tumor stages and nodal metastasis but these associations were more 26 complex with other confounding factors.

Cellular aging has drawn the attention of researchers, scientists, and biotech businesses for the treatment of a number of medical conditions. Cellular aging is primarily defined by consistent cessation of proliferative development in response to internal and external stressors, including DNA damage, telomere shortening, mitochondrial dysfunction, and regulation of the senescence-associated secretory phenotype (SASP). Disturbances involving these factors may contribute to age-related disease development. Therefore, the current review aims to explore anti-aging factors targeting DNA damage response and SASP regulation and their detailed signaling networks. In addition, it provides an opportunity for researchers to identify not only anti-aging targets, but also anti-aging therapeutic strategies based on identified and non-identified targets.

Bone injury is a common side effect of radiotherapy to tumors, which is a long-term response after damage to osteoblasts, especially reducing osteoblasts proliferation and differentiation. Currently, there are few studies on radiation-induced bone injury, the molecules involved in the ionizing radiation (IR) induced osteoblasts damage remain need to be excavated. In this study, the optimal IR damage conditions (8Gy, 2.22Gy/min) were firstly determined by measuring cell proliferation, cell cycle, cell apoptosis and further cell differentiation and mineralization abilities and the related genes (p-AKT, p-ERK1/2, cyclinB, BAX, BCL2, ALP, OPN, RUNX2, Collagen1) expression level changes in radiation-induced osteoblast injury model. Then, we screened 26 differentially expressed genes after the RNA-sequencing of the 8 Gy-irradiated MC3T3-E1 cells, and they were mainly involved in DNA damage and repair, cell apoptotic progress and cell cycle regulation, meanwhile, participated in several main pathway including PI3K-AKT signaling pathway, p53 signaling pathway and signaling pathway involved in cell cycle and cell senescence. We focused on verifying the differential expression genes and confirmed the MDM2, NOTCH1, CDKN1A and GCLC were upregulated after IR treatment, suggesting the key roles in the response of the IR. In addition, regarding on our sequencing results, the DNA damage and repair were also verified and the results suggested that IR induced DNA damage and repair in MC3T3-E1 cells. In summary, IR damaged MC3T3-E1 cells by inhibiting cell proliferation, impacting cell cycle process, inducing cell apoptosis and affecting the osteoblasts differentiation, which maybe due to the DNA damage and the differential expression of the key genes (MDM2, NOTCH1, CDKN1A and GCLC ).

In this study, Clostridium butyricum TO-A culture supernatant (CBCS) or butyric acid was added to a culture medium of human cervical carcinoma HeLa S3 cells, and changes in DNA repair-related gene promoter activi-ties were investigated. HeLa S3 cells were transfected with a luciferase (Luc) expression vector containing ap-proximately 500-bp of the 5′-upstream region of several human DNA repair-related genes and cultured with a medium containing CBCS (10%) or butyric acid (2.5 mM). Cells were harvested after 19 to 42 h of incubation. Luc assay revealed that the human ATM, PARG, PARP1, and RB1 gene promoter activities were significantly increased. Western blot analysis showed that amounts of the proteins encoded by those genes markedly in-creased. Furthermore, 8, 24, and 48 h after the addition of the CBCS (10%), total RNA was extracted and subjected to RNAseq analysis. The result showed that the expression of several DNA replication/repair-related genes, including NFKB, and the MCM gene groups, decreased markedly after 8 h. However, the expression of the histone genes increased after 24 h. Elucidation of the mechanism by which CBCS and butyrate affect DNA-repair associated protein-encoding gene expression may contribute to the prevention of carcinogenesis, of which the risk rises in accordance with aging.

This work describes practical immunization of European sea bass (Dicentrarchus labrax) juveniles against viral nervous necrosis virus (VNNV), a betanodavirus causing worldwide mortalities in many fish species. Protection was obtained with the so called spinycterin vehicles consisting in irreversibly DNA-damaged DNA-repair-less E.coli displaying at their surface a downsized antigen. In this work we, i) maximized bacterial expression levels by downsizing the C protein to a fragment (frgC91-220) containing most of its antigenicity, ii) developed an scalable autoinduction bacterial media based in soy-bean increasing membrane display and reproducibility, iii) enriched surface expression by screening different anchors from several prokaryotic origins (anchor+frgC91-220), iv) preserved frgC91-220 antigenicity by inactivating bacteria by irreversible DNA-damage by means of Ciprofloxacin, and v) increased safety using a repair-less E.coli strain as spinycterin chassis. These second generation of spinycterins protected fish against VNNV challenge with partial (Nmistic+frgC91-220) or 100 % (YBEL+frgC91-220 ) protection, in contrast to those fish immunized with frgC91-220 spinycterins. The proposed spinycterin platform has high levels of environmental safety and cost effectiveness, thus providing potential for small fish vaccines for sustainable aquaculture.

1) Background: We tested whether AHR activation induces DNA damage, whether polymorphisms in genes related to risk of Non-Hodgkin lymphoma are associated with DNA damage, and whether the two conditions do interact with each other. 2) Methods: Our study population included 36 subjects, randomly selected among the population controls participating in a case-control study on lymphoma in Sardinia, Italy, who donated a blood sample. We investigated 47 single nucleotide polymorphisms (SNPs) previously reported to convey risk of lymphoma; the Dual-Glo® Luciferase Assay System to detect activation of the aryl hydrocarbon receptor (AhR) by the serum of study subjects; and the COMET Assay to detect DNA damage. 3) Results: Activation of the aryl hydrocarbon receptor did not increase DNA damage in our study population. On the other hand, the mutant allele (G) of rs1056932/BCL6 increased the occurrence of DNA damage (p = 0.045); such association was confirmed among AhR negative, but not AhR positive subjects. 4) Conclusions: We observed excess DNA damage associated with a gene polymorphism, namely rs1056932/ BCL6, previously reported in association with risk of lymphoma. No increase in DNA damage was associated with AhR activation per se, nor with the other gene polymorphisms we investigated.

Minute Virus of Mice (MVM) is an autonomous parvovirus of the Parvovirus family that replicates in mouse cells and transformed human cells. MVM genomes localize to cellular sites of DNA damage with the help of their essential non-structural phosphoprotein NS1 to establish viral replication centers. MVM replication induces a cellular DNA damage response that is mediated by signaling through the ATM kinase pathway, while inhibiting induction of the ATR kinase signaling pathway. However, the cellular signals regulating virus localization to cellular DNA damage response sites has remained unknown. Using chemical inhibitors to DNA damage response proteins, we have discovered that NS1 locallization to cellular DDR sites is independent of ATM or DNA-PK signaling, but is dependent on ATR signaling. Pulsing cells with an ATR inhibitor after S-phase entry leads to attenuated MVM replication. These observations suggest that the initial localization of MVM to cellular DDR sites depends on ATR signaling before it is inactivated by vigorous virus replication.

Gastric cancer is a heterogeneous group of diseases with only a fraction of patients responds to immunotherapy. The relationships between tumor DNA damage response, the immune system and immunotherapy have recently attracted attention. Accumulating evidence indicate that DNA repair landscape is a significant factor in driving response to immune checkpoint blockade (ICB) therapy. In this study, to explore new prognostic and predictive biomarkers for gastric cancer patients who are sensitive and responsible to immunotherapy, we developed a novel 15-DNA repair gene signature (DRGS) and its related scoring system and evaluated the efficiency of DRGS in discriminating different molecular and immune characteristics and therapeutic outcomes of gastric adenocarcinoma. The results showed that DRGS high score patients showed significantly better therapeutic outcomes compared to DRGS low score patients (P < 0.001). Integrated analysis of multi-omics data demonstrated that the patients with high DRGS score were characteristic of high levels of anti-tumor lymphocytes infiltration, tumor mutation burden (TMB) and PD-L1 expression, and these patients exhibited a longer overall survival and may benefit more from ICB therapy, as compared to the low-score patients. Therefore, the DRGS and its scoring system may have implications in tailoring immunotherapy in gastric cancers.

Radiation therapy (RT) is one of the main approaches of cancer therapy, including non-small cell lung cancer (NSCLC). Radiation-induced DNA double-stranded breaks (DSBs), single-stranded DNA breaks (SSBs) and oxidized nucleobases cause replication fork stalling, chromosomal instability or point mutagenesis and can lead to cell death. Translesion DNA synthesis (TLS) is one of the DNA damage tolerance (DDT) pathways promoting replication of damaged DNA. Here, we explored the effects of the knockout of primase-polymerase PRIMPOL (PRIMPOL −/−) and TLS polymerase POLI (POLI −/−) genes in two different monoclonal lines derived from human NSCLC A549 cells. Our study aimed to investigate the impact of their absence on the susceptibility of these cells to ionizing radiation (IR)-induced stress. Without IR, cells that do not have PRIMPOL or POLI experience a considerable rise in apoptosis by day 7 of continuous cultivation and a noteworthy decline in cell migration under spatial confinement when compared to the parental wild-type cells. Despite this, we have found that PRIMPOL −/− exhibited higher clonogenic survival compared to wild-type cell lines in response to IR stress. Both of these cell lines demonstrated remarkable resilience to radiation stress, surpassing the survival rate of the original A549 cells. POLI deficiency reduces cellular γH2AX foci accumulation, while the presence of highest number of γH2AX foci in PRIMPOL−/− cells was ATM-independent. We report for the first time that increased post-irradiation clonogenic survival of PRIMPOL −/− A549 cells might be associated with the increase of CD133+ but not CD44+ populations of cancer stem-like cells.

DNA damage causes the mutations that are the principal source of genetic variation. DNA damage detection and repair mechanisms therefore play a determining role in generating the genetic diversity on which natural selection acts. Speciation, it is commonly assumed, occurs at a rate set by the level of standing allelic diversity in a population. The process of speciation is driven by a combination of two evolutionary forces: genetic drift and ecological selection. Genetic drift takes place under conditions of relaxed selection, and results in a balance between rates of mutation and rates of genetic substitution. These two processes, drift and selection, are necessarily mediated by a variety of mechanisms guaranteeing genome stability in any given species. One of the outstanding questions in evolutionary biology concerns the origin of the widely varying phylogenetic distribution of biodiversity across the tree of life, and how the forces of drift and selection contribute to shaping that distribution. The following examines some of the molecular mechanisms underlying genome stability and the adaptive radiations that are associated with biodiversity and the widely varying species richness and evenness in the different eukaryotic lineages.

Furazolidone (FZD) is a synthetic nitrofuran with the antiprotozoal and antibacterial activity. The proper mechanism of FZD induced toxicity is still unclear. This study aimed to investigate the protective effect of curcumin on FZD induced oxidative stress, DNA injury and apoptosis in human hepatocyte L02 cells. The results showed that curcumin treatment significantly ameliorated FZD induced cytotoxicity, characterized by decreasing the production of reactive oxygen species (ROS) and malondialdehyde, as well as increasing superoxide dismutase, catalase activities and glutathione contents. Moreover, curcumin pretreatment significantly inhibited FZD induced the loss of mitochondrial membrane potential, the activation caspase-9 and -3 and apoptosis. Comet assay showed that curcumin attenuated FZD induced DNA injury in a dose-dependent manner. Correspondingly, curcumin markedly reversed the up-regulation of p53, Bax, caspase-9 and -3 mRNA expressions and the down-regulation of Bcl-2 mRNA (all p<0.05 or 0.01). These results reveal that curcumin protects against FZD induced oxidative stress, DNA injury and cell apoptosis via inhibiting oxidative stress and mitochondrial pathway, which may be attributed to ROS scavenging and anti-oxidative ability of curcumin. Importantly, our study highlights that curcumin may be a potential way to prevent FZD-mediated oxidative DNA injury and apoptosis in human or animals.

Previously, we reported that MTH1 inhibitors TH588 and TH1579 selectively induce oxidative damage and kill Ras expressing or transforming cancer cells, as compared to non-transforming immortalized or primary cells. While this explains the impressive anti-cancer properties of the compounds, the molecular mechanism remains elusive. Oncogenes induce replication stress, re-sulting in under replicated DNA and replication continuing into mitosis, where TH588 and TH1579 treatment cause toxicity and incorporation of oxidative damage. Hence, we hypothesized that oncogene-induced replication stress explains the cancer selectivity. To test this, we overex-pressed c-Myc in human epithelial kidney cells (HA1EB), resulting in increased proliferation, polyploidy and replication stress. TH588 and TH1579 selectively kill c-Myc overexpressing clones, enforcing the cancer cell selective killing of these compounds. Moreover, the toxicity of TH588 and TH1579 in c-Myc overexpressing cells is rescued by transcription, proteasome or CDK1 inhibitors, but interestingly not by nucleoside supplementation. This suggest that cancer selectivity is unrelated oncogene-induced replication stress. We conclude that the molecular toxi-cological mechanisms how TH588 and TH1579 kill c-Myc overexpressing cells have several com-ponents and involves MTH1-independent proteosomal degradation of c-Myc itself, c-Myc driven transcription and CDK activation, and is likely unrelated to oncogene-induced replication stress.

Nigella sativa (NS) is an effective medicinal plant possessing noteworthy antioxidant property. In NS, there are more than hundred phyto-chemicals reported, out of which thymoquinone is the utmost active phyto-constituent having sturdy antioxidative property. Thymoquinone is a cyclicdione, when reacts with sodium azide, converts into α-azido ketones i.e its analogs which are handy with extensive range of reactions. Sodium azide induces stress in plants thereby, modulating the antioxidant system. The present investigation was planned to elucidate the effect of sodium azide at different concentrations (5µM, 10µM, 20µM, 50µM, 100µM and 200µM) on its secondary metabolites (mainly thymoquinone) in NS callus culture extract (NSE). The results showed sodium azide effect on thymoquinone content and a concentration dependent boost in antioxidant property. It was also observed that thymoquinone content and percent yield (analyzed by RP-HPLC; Reverse Phase- High Performance Liquid Chromatography) were minimum (0.033±0.006% and 0.420±0.045%, respectively) at 200 µM sodium azide used. Whereas, antioxidant activity (analyzed by DPPH; 2, 2-diphenyl-1-picrylhydrazyl) was found to be maximum (3.873±0.402%) at same dose. Further, analysis was done for inhibition of oxidative DNA damage at different concentrations of sodium azide on NSE, maximum inhibition of DNA damage (0.243±0.017%) was found at 200 µM concentration of sodium azide. When correlated, strong positive correlation was observed between percent yield and percent thymoquinone, antioxidant property and inhibition of DNA damage. Whereas, strong negative correlation was observed between percent yield and antioxidant property, percent thymoquinone and antioxidant property, percent thymoquinone and inhibition of DNA damage. The findings evidently point out that the content of thymoquinone, antioxidant property and inhibition of DNA damage was affected by sodium azide.

Chromatin is dynamically remodeled to adapt to all DNA-related processes, including DNA damage responses (DDR). This adaptation requires DNA and histone epigenetic modifications, which are mediated by several types of enzymes; among them are lysine methyltransferases (KMTs). Methods: KMT inhibitors, chaetocin and tazemetostat (TZM), were used to study their role in the DDR induced by ionizing radiation or doxorubicin in two human sarcoma cells lines. The effect of these KMT inhibitors was tested by the analysis of chromatin epigenetic modifications, H4K16ac and H4K20me2. DDR was monitored by the formation of γH2AX, MDC1, NBS1 and 53BP1 foci, and the induction of apoptosis. Results: Chaetocin and tazemetostat treatments caused a significant increase of H4K16 acetylation, associated with chromatin relaxation; and increased DNA damage, detected by the labeling of free DNA-ends. These inhibitors significantly reduced H4K20 dimethylation levels in response to DNA damage and impaired the recruitment of 53BP1, but not of MDC1 and NBS1, at DNA damaged sites. This modification of epigenetic marks prevents DNA repair by the NHEJ pathway and leads to cell death. Conclusion: KMT inhibitors can function as sensitizers to DNA damage-based therapies and be used in novel synthetic lethality strategies for sarcoma treatment.

Epithelial-mesenchymal transition (EMT) networks are essential in acquiring the drug resistance and cancer malignant features in cancer stem cells (CSCs). In this regard, gene expression profiles in diffuse- and intestinal-type gastric cancer (GC) have been analyzed to reveal the network pathways in EMT and CSCs, since the diffuse-type GC has much more mesenchymal features than intestinal-type GC that has the intestinal features. The study results revealed that the activation state of several canonical pathways related to cell cycle regulation was altered. The canonical pathway on Cell cycle: G1/S checkpoint regulation was activated in diffuse-type GC, and canonical pathways on Cell cycle control of chromosomal replication and Cyclins and cell cycle regulation were activated in intestinal-type GC. Canonical pathway related to Role of BRCA1 in DNA damage response was activated in intestinal-type GC, where BRCA1, which is related to G1/S phase transition was up-regulated in intestinal-type GC. Several microRNAs (miRNAs), including mir-10, mir-17, mir-19, mir-194, mir-224, mir-25, mir-34, mir-451, and mir-605, were identified to have direct relationships of RNA-RNA interaction in Cell cycle: G1/S checkpoint regulation pathway. Additionally, cell cycle regulation may be altered in EMT conditions. The alterations in activation states of the pathways related to cell cycle regulation in diffuse- and intestinal-type GC would indicate the significance of cell cycle regulation in EMT.

DNA damage response (DDR) pathways in keto-enol genotoxicity have not been characterized, and few studies have reported genotoxic effects in non-target organisms. The present study shows that concentrations of 11.2, 22.4, 37.3 mg/L of Movento® 240SC and 12.3, 24.6, 41.1 mg/L of Envidor® 240SC for 72 h oral exposure induced DSBs by significantly increasing the percentage of γH2AV expression in regions 2b and 3 from the germarium of wildtype females of Drosophila melanogaster Oregon R, compared to the control group (0.0 mg/L of insecticides), by confocal immunofluorescence microscopy. The comparison between both insecticides’ revels that only the Envidor® 240SC induces concentration-dependent DNA damage, as well as structural changes in the germarium. We determined that the DDR induced by Movento® 240SC depends on the activation of the ATMtefu, Chk1grp and Chk2lok kinases by significantly increasing the percentage of expression of γH2AX in regions 2b and 3 of the germarium, and that ATRmei-29D and p53dp53 kinases only respond at the highest concentration of 37.3 mg/L of Movento® 240SC. With the Envidor® 240SC insecticide, we determined that the DDR depends on the activation of the ATRmei-29D/Chk1grp and ATMtefu/Chk2lok kinases, and p53dp53 by significantly increasing the percentage of expression of γH2AX in the germarium.

Background: Increased DNA damage and the propension to cancer development, depend on the modulation of the mechanisms to control and maintain genomic integrity. Poly(ADP-Ribose)Polymerase activation and automodification are early responses to genotoxic stress. Upon binding to DNA strand breaks, the enzyme, a molecular DNA nick sensor, is hyperactivated: this is the first step in a series of events leading to either DNA repair or apoptosis. Enzyme hyperactivation and automodification can be easily measured and are widely used to look at DNA damage extent in the cell. We investigated whether these two markers (increased catalytic activity and auto modification), could help to monitor DNA damage in lymphocytes of flower growers from Southern Italy, occupationally exposed to pesticides. Methods: Peripheral lymphocyte lysates were analysed for Poly(ADP-Ribose) Polymerase activity, and by SDS-PAGE and anti-Poly(ADP-Ribose)Polymerase 1-antibody to measure automodified anti-Poly(ADP-Ribose) Polymerase levels by densitometry. Results: Poly(ADP-Ribose)Polymerase activity levels were consistent with those of enzyme auto-modification. Growers daily exposed to pesticides, showed both biomarkers very high, either in the presence or in the absence of pathologies. Conclusions: PARP activity and auto-modification in peripheral blood lymphocytes are possible,non-invasive, and routinar tools to monitor the healthy conditions of floricoltorists.

Homologous recombination deficiency (HRD) has become an important prognostic and predictive biomarker for patients with high-grade serous ovarian cancer who may benefit from poly-ADP ribose polymerase inhibitors (PARPi) and platinum-based therapies. HRD testing provides relevant information to personalize patients’ treatment options, and it has been progressively incorporated in diagnostic laboratories. In this study, we evaluated the feasibility of in-house HRD testing deployable in a diagnostic clinical setting, comparing results from two commercially available next-generation sequencing (NGS)-based tumor tests (SOPHiA DDMTM HRD Solution and AmoyDx HRD Focus Panel) with the reference assay from Myriad MyChoice CDx. A total of 85 ovarian cancer samples were subjected to HRD testing. An overall strong correlation was observed across the three assays evaluated, regardless of the different underlying methods employed to assess genomic instability, with the highest pairwise correlation between Myriad and SOPHiA (R=0.87, p-value=3.39x10-19). The comparison of the assigned HRD status to the reference Myriad’s test revealed a positive predictive value (PPV) and negative predictive value (NPV) of 90.9% and 96.3% for SOPHiA’s test, while AmoyDx’s test achieved 75% PPV and 100% NPV. This is the largest HRD testing evaluation using different methodologies and provides a clear picture of the robustness of NGS-based tests currently offered in the market. Our data show that the implementation of in-house HRD testing in diagnostic laboratories is technically feasible and it can be reliably performed with commercial assays. Also, the turnaround time is compatible with clinical needs, being an ideal alternative to offer to a broader number of patients, while still maintaining high-quality standards at more accessible price tiers.

Chronic obstructive pulmonary disease (COPD) is a respiratory disease associated with airways inflammation and lung parenchyma fibrosis. The primary goals of COPD treatment are to re-duce symptoms and risk of exacerbations, therefore pulmonary rehabilitation is considered the key component of managing COPD patients. Oxidative airway damage, inflammation and re-duction of endogenous antioxidant enzymes are known to play a crucial role in the pathogenesis of COPD. Natural antioxidants have also recently been considered as they play an important role in metabolism, DNA repair and fighting the effects of oxidative stress. In this paper we evaluated the response of 105 elderly COPD patients to pulmonary rehabilitation (PR), based on high or low vegetable consumption, by analyzing clinical parameters and biological measure-ments at baseline and after completion of the three weeks PR. We found that high vegetable in-take in normal diet, without any specific intervention, can increase the probability to success-fully respond to rehabilitation (65.4% of responders ate vegetables daily vs. 40.0% of Non-Responders, p=0.033). Three weeks of pulmonary rehabilitation are probably too short to reveal a reduction of the oxidative stress and DNA damage, but are enough to show an im-provement in the patient's inflammatory state.

Unavoidable exposure to radiosensitive normal tissues around cancerous tumor during the radiotherapy can cause side effects such as self-limited acute toxicities, mild chronic symptoms, or severe organ dysfunction. Nevertheless, clinical use of currently available radiation protective agents is limited because of their generic cytotoxicity. A study on radiation protective effect of delphinidin was conducted with proton-beam-exposed human colon cells (CCD-18Co). The measurement in changes of survival fractions of CCD-18Co with/without delphinidin administration at different radiation doses were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The changes in expression of reactive oxygen species (ROS) and in activities of antioxidant enzymes were measured by colorimetric assays using pertinent assay kits. The measurement of pro-apoptosis/pro-survival protein expressions using Western blot assay and the measurement of DNA damage using comet assay were also fulfilled to evaluate the molecular level of radiation damages in CCD-18Co cells. The experimental results revealed that the pre-administration of delphinidin regulated antioxidant enzymes, reduced ROS, decreased DNA damage, regulated pro-apoptosis/pro-survival proteins, and eventually reduced apoptosis of CCD-18Co cells. In conclusion, it is claimed that delphinidin is nontoxic natural radiation protective compound, and thus delphinidin can be used to protect normal colon tissues during the proton beam therapy.

Chronic obstructive pulmonary disease (COPD) is a respiratory disease associated with airways inflammation and lung parenchyma fibrosis. The primary goals of COPD treatment are to re-duce symptoms and risk of exacerbations, therefore pulmonary rehabilitation is considered the key component of managing COPD patients. Oxidative airway damage, inflammation and re-duction of endogenous antioxidant enzymes are known to play a crucial role in the pathogenesis of COPD. Natural antioxidants have also recently been considered as they play an important role in metabolism, DNA repair and fighting the effects of oxidative stress. In this paper we evaluated the response of 105 elderly COPD patients to pulmonary rehabilitation (PR), based on high or low vegetable consumption, by analyzing clinical parameters and biological measure-ments at baseline and after completion of the three weeks PR. We found that high vegetable in-take in normal diet, without any specific intervention, can increase the probability to success-fully respond to rehabilitation (65.4% of responders ate vegetables daily vs. 40.0% of Non-Responders, p=0.033). Three weeks of pulmonary rehabilitation are probably too short to reveal a reduction of the oxidative stress and DNA damage, but are enough to show an im-provement in the patient's inflammatory state.

Radiotherapy and cisplatin-based chemotherapy belong to the main treatment modalities for head and neck squamous cell carcinoma (HNSCC), and induce cancer cell death by generating DNA damage, including the most severe double strand breaks (DSBs). Alterations in DSB response and repair genes may affect individual DNA repair capacity and treatment sensitivity, contributing to therapy resistance and poor prognosis often observed in HNSCC. In this study, we investigated the association of a panel of single nucleotide polymorphisms (SNPs) in 20 DSB signaling and repair genes with therapy results and prognosis in 505 HNSCC patients treated non-surgically with DNA damage-inducing therapies. In the multivariate analysis, there were a total of 14 variants associated with overall, locoregional recurrence-free or metastasis-free survival. Moreover, we identified 10 of these SNPs as independent predictors of therapy failure and unfavorable prognosis in the whole group or in two treatment subgroups. They were MRE11A rs2155209, XRCC5 rs828907, RAD51 rs1801321, rs12593359, LIG4 rs1805388, CHEK1 rs558351, TP53 rs1042522, ATM rs1801516, XRCC6 rs2267437 and NBS1 rs2735383. Only CHEK1 rs558351 remained statistically significant after correction for multiple testing. These results suggest that specific germline variants related to DSB response and repair may be potential genetic modifiers of therapy effects and disease progression in HNSCC treated with radiotherapy and cisplatin-based chemoradiation.

Obesity and environmental toxins are risk factors for breast cancer, however there is limited knowledge on how these risk factors interact to promote breast cancer. Acrylamide, a probable carcinogen and obesogen, is a biproduct in foods prevalent in the obesity-inducing Western diet. Acrylamide is metabolized by cytochrome P450 2E1 (CYP2E1) to the genotoxic epoxide, glycidamide, and is associated with increased risk for breast cancer. To investigate how acrylamide and obesity interact to increase breast cancer risk, female mice were fed a low-fat (LFD) or high-fat diet (HFD) and 0.7mM acrylamide-supplemented water or control water. While HFD significantly enhanced weight gain in mice, the addition of acrylamide did not significantly alter body weights compared to respective controls. Mammary epithelial cells from obese, acrylamide-treated mice had increased DNA strand breaks and oxidative DNA damage compared to all other groups. In vitro, glycidamide-treated COMMA-D cells showed significantly increased DNA strand breaks while acrylamide-treated cells demonstrated significantly higher levels of intracellular reactive oxygen species. Knockdown of CYP2E1 rescued the acrylamide-induced oxidative stress. These studies suggest that long-term acrylamide exposure through foods common in the Western diet may enhance DNA damage and CYP2E1-induced generation of oxidative stress in mammary epithelial cells, potentially enhancing obesity-induced breast cancer risk.

DNA repair helps in maintaining the proper and healthy functioning for the cells in the human body. Failure in DNA repair process can lead to aberrations as well as tumorous stages. There are various types of damages that a DNA can go through, one of which is the DNA double strand breaks (DSB) that can be repaired via homologous recombination (HR). RAD51 plays a central role in HR and has been implicated as a negative/poor prognostic marker for colorectal adenocarcinoma, with high expression in colorectal cancer. Mechanistically, RAD51AP1 facilitates RAD51 during the repairing process by binding with RAD51 via two DNA binding sites, thus helping in the D-loop formation in the HR process. Often, in biology, we are faced with the problem of exploring relevant unknown biological hypotheses in the form of myriads of combination of factors that might be affecting the pathway under certain conditions. For example, RAD51AP1-XRCC2 is one such 2^nd order combination whose relation needs to be tested under the influence recently developed Porcupine-WNT inhibitor ETC-1922159. The x-ray repair cross complementing XRCC family is known to work as a mediator or stabilizer for RAD51 during the HR process. The inhibitor is known to suppress Porcupine and thus inhibit a range of oncogenes known to be directly or indirectly affected by the Wnts. In a recent unpublished work in bioRxiv, we had the opportunity to rank these unknown biological hypotheses for down regulated genes at 2^nd order level after the drug was administered. The in silico observations showed that the combination of RAD51AP1-XRCC2 was assigned a relatively lower rank, thus validating the pipeline's efficacy with the confirmed wet lab experiment that indicate that both RAD51AP1 and XRCC2 were down regulated after treatment in cancer cells. Here, we take one step further by in silico analysis of the 3^rd order combinations of RAD51-X-X & RAD51AP1-X-X (X can be known or unknown factor), from a range of 100 randomly picked down regulated genes after ETC-1922159 treatment. The pipeline uses the density based HSIC (Hibert Schmidth Information Criterion) sensitivity index with an rbf (radial basis function) kernel, which is known to be highly effective in sensitivity analysis. Various unknown/unexplored/untested RAD51/RAD51AP1 related 3^rd order biological hypotheses emerge some of which are confirmed in wet lab, while others need to be tested.

The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process. Quantification guarantees maximum efficiency and avoids repeated analyses, over amplified samples, or unnecessary examinations. Here, we present our experience using an extremely sensitive real-time-based method with the Quantifiler® Trio DNA quantification kit, which we have explored in the ISO/IEC17025 accredited test validation process conducted in our laboratory. In this study, our focus was on establishing a minimum threshold beyond which DNA typing is unnecessary, by defining the correlation of negative quantification results with the absence of genetic profiles resulting from DNA typing. We investigated the results in the range in which the technique is used (50-0.005 ng/μl). In the validation process of the internally accredited method, we used data from the commercial quantification system, based on the average values of the standard curve using a wide range of samples, including artificially degraded samples. This method was extensively used continuously for nine years to quantify the DNA extract coming from a wide range of samples from criminal cases. The validation allowed us to verify the limits of the technique, especially in the use of the minimum quantities necessary to obtain genetic profiles acceptable for sending to the Italian DNA database. This optimization of the protocol for analysing forensic samples allows us to optimise the protocol for analysing forensic samples. However, the technique is used in our laboratory with a completely manual preparative phase, and we have decided to investigate the contribution of human error more accurately. Thus, in our experience, we have found that it is preferable to use quantification ranges rather than exact limits before deciding how to analyse the extracts via PCR. In addition, here we present some preliminary data related to the analysis of certain samples that initially appeared negative during quantification, applying a concentration method for maximum yield from forensic samples of judicial interest. Our aim is to ensure that no source of evidence is lost in the reconstruction of a criminal event.

Background: Telomere length (TL) shortening process is associated with several known environment and individual determinants. DNA methylation is the most studied epigenetic process and may be associated with TL. We investigated the associations between DNA methylation and TL in peripheral blood. Methods: Methylation wide association study was conducted in 47 women (37.1±8.8 years) with different nutritional status. Association between TL and DNA methylation levels were explored by univariate and multiple linear regression models, corrected by age and Body Mass Index. Corrections for multiple comparisons by Benjamini‐Hochberg test was also performed. WEBGestalt was used to identify pathways that are responsible for regulating TL. Results: We found negative correlations between TL and BMI (r = -0.641; p = 0.001), abdominal circumference (r = -0.622; p = 0.001) and fat mass (r = -0.656; p = 0.001). 44 CpGs sites were associated with TL, independent of age and BMI. The most of these sites were negatively correlated with TL. For the 7 remained sites, DNA hypomethylation were associated with shorter TL. These CpGs were related to nine different pathways, including thermogenesis, cancer, glutamatergic and serotonergic synapse. Conclusion: There is an epigenetic contribution in TL, independent of nutritional status and age. Genes related to TL are involved in important metabolic pathways.

Replication protein A (RPA) is a heterotrimeric protein complex and the main single-stranded DNA (ssDNA) binding protein in eukaryotes. RPA has key functions in most of the DNA-associated metabolic pathways and DNA damage signalling. Its high affinity for ssDNA helps to stabilise ssDNA structures and prevents the DNA sequence from nuclease attacks. RPA consists of multiple DNA-binding domains which are oligonucleotide/oligosaccharide-binding (OB)-folds, responsible for DNA binding, and interactions with proteins. These RPA-ssDNA and RPA-protein interactions are crucial for DNA replication, DNA repair, DNA damage signalling, and the conversation of the genetic information of cells. Proteins such as ATR use RPA to locate to regions of DNA damage for DNA damage signalling. Recruitment of nucleases and DNA exchange factors to sites of double strand breaks are also an important RPA function to ensure effective DNA recombination to correct these DNA lesions. Due to its high affinity to ssDNA, RPA’s removal from ssDNA is of central importance to allow these metabolic pathway to proceed and processes to exchange RPA against downstream factors are established in all eukaryotes. These facetted and multi-layered functions of RPA will be discussed in detail in the review. RPA is also a major player in a variety of human diseases, which will be discussed.

Non-viral mediated plasmid DNA transfection by electroporation (EP) is an established method for gene transfection. In this study, the usefulness of direct EP at an intradermal (i.d.) site (D_EP) with implanted electrodes to achieve a high protein expression level was investigated. In addition, D_EP application with various intervals with a low application voltage was also evaluated to confirm its effect on protein expression. A higher protein expression level was observed after green fluorescent protein (GFP)- and luciferase-encoding DNA were delivered by i.d. injection followed by D_EP application. When luciferase expression was observed with an in vivo imaging system, continuous expression was confirmed over 21 days after i.d. injection followed by D_EP at 100 V. This approach provided increased gene expression levels compared with conventional EP methods via the stratum corneum layer. In addition, the effect of application voltage on luciferase expression was investigated; two-time applications (repeated D_EP) at 20 V with 5 min intervals showed similar luciferase expression level to single D_EP application with 100 V. Histological observations showed the skin became thicker after a single D_EP at 100 V, whereas no apparent thickness changes was confirmed after repeated D_EP at 20 V with 5 min intervals. This study revealed that direct i.d. voltage application achieved high protein expression levels even at low voltages. Skin is a promising administration site for DNA vaccines, so this approach may be effective in DNA vaccine delivery into skin tissue.

Covalent DNA protein crosslinks (DPCs) are common lesions that block replication. We examine here the consequence of DPCs on mutagenesis involving replicational template-switch reactions in Escherichia coli. 5-azacytidine (5azaC) is a potent mutagen for template-switching, dependent on DNA cytosine methylase (Dcm), implicating the trapped Dcm-DNA covalent complex as the initiator for mutagenesis. The leading strand of replication is more mutable than the lagging strand, explained by blocks to the replicative helicase and/or fork regression. We find that template-switch mutagenesis induced by 5-azaC does not require DSB repair via RecABCD. The ability to induce the SOS response is anti-mutagenic by an unknown mechanism. Mutants in recB, but not recA, exhibit high constitutive rates of template-switching and we suggest that RecBCD-mediated DNA degradation prevents template-switching associated with fork regression. A mutation in the DnaB fork helicase also promotes high levels of template-switching. We also find that other DPC-inducers, formaldehyde (a non-specific crosslinker) and ciprofloxacin (a topoisomerase II poison) are also strong mutagens for template-switching. Induction of mutations and genetic rearrangements that occur by template-switching may constitute a previously unrecognized component of the genotoxicity and genetic instability promoted by DPCs.

It is well known that X-ray crystallography is based on X-ray diffraction (XRD) by atoms and molecules. The diffraction pattern arises as a result of scattering of incident radiation, which makes it possible to determine the structure of the scattering substance. With the advent of ultrashort radiation sources, the theory and interpretation of X-ray diffraction analysis remained the same. This work shows that when an attosecond laser pulse is scattered on a DNA molecule, including during its nicking and bending, the pulse duration is an important characteristic of the scattering. In this case, the diffraction pattern changes significantly compared to the previously known scattering theory. The results obtained must be used in XRD theory to study DNA structures, their mutations and damage, since the previously known theory can produce large errors and, therefore, the DNA structure can be “deciphered” incorrectly.

DNA methylation is an epigenetic mechanism for gene expression modulation and can be used as a predictor of future disease risks. A prospective birth cohort study was performed to clarify the effects of neurotoxicants on child development, namely, the Tohoku Study of Child Development, in Japan. This study aimed to evaluate the association of prenatal exposure to five toxic metals—arsenic, cadmium, mercury, lead (Pb), antimony (Sb), and polychlorinated biphenyls (PCBs, N = 166)—with global DNA methylation in umbilical cord blood DNA. DNA methylation markers, 5-methyl-2’-deoxycytidine (mC) and 5-hydroxymethyl-2’-deoxycytidine (hmC), were determined using liquid chromatography-tandem mass spectrometry. The mC content in cord blood DNA was positively correlated with Pb and Sb levels (r = 0.442 and 0.288, respectively) but not with cord blood PCBs. We also observed significant positive correlations among Pb levels, maternal age, and hmC content (r = 0.159 and 0.243, respectively). The multiple regression analysis among the potential predictors demonstrated consistent positive associations between Pb and Sb levels and mC and hmC content. Our results suggest that global DNA methylation is a promising biomarker for prenatal exposure to Pb and Sb.

Abstract5′,8-Cyclo-2′-deoxyadenosine (cdA), in the 5′R and 5′Sdiastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the diastereomeric cdA lesions could induce specific PARP1 binding. Here we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions 5′S-cdA and 5′R-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism,fluorescence spectroscopy, immunoblotting analysis and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1were characterized. Remarkably, PARP1 efficiently binds to both cdA lesions in the double stranded (ds)-oligonucleotides. In particular, PARP1 proved to bind 5′S-cdAwith a higher affinity constant for the 5'S lesion in a model of ds DNA than 5′R-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion.

Recent advances in DNA sequencing technology have shown that the human milk microbiota of healthy women varies substantially. The gDNA extraction method may influence the observed variation, biasing the microbiological reconstruction after all. In this study, a genomic DNA extraction method for DNA isolation from human milk samples was standardized and compared with commercial and standard hose make methods. Spectrophotometric measurements, gel electrophoresis, and PCR amplifications were used as criteria for evaluating the quantity, quality, and functionality of the extracted DNA. Furthermore, the standardized method of extracting gDNA from human milk was evaluated for its ability to isolate functional DNA from gram-positive, and gram-negative bacteria and fungi, to improve the reconstruction of microbiological profiles. The novel DNA extraction method increased the quantity and quality of the gDNA extracted compared with commercial and standard house-make protocols. This method even allowed PCR amplification of the V3-V4 regions of the 16S ribosomal gene in all samples, and the ITS-1 region of the fungal 18S ribosomal gene in 95 % of the samples as well. It is concluded that the proposed method provides better performance for the extraction of gDNA from complex samples such as human milk.

DNA methylation, one of the major epigenetic mechanisms, plays critical roles in regulating gene expression, genomic stability and cell lineage commitment. Establishment and maintenance of DNA methylation in mammals is achieved by two groups of DNA methyltransferases: DNMT3A and DNMT3B, which are responsible for installing DNA methylation patterns during gametogenesis and early embryogenesis, and DNMT1, which is essential for propagating DNA methylation patterns during replication. Both groups of DNMTs are multi-modular proteins, containing a large N-terminal regulatory region in addition to the C-terminal methyltransferase domain. Recent structure-function investigations of the individual domains or large fragments of DNMT1 and DNMT3A have revealed the molecular basis for their substrate recognition and specificity, intramolecular domain-domain interactions, as well as their crosstalk with other epigenetic mechanisms. These studies highlight a multifaceted regulation for both DNMT1 and DNMT3A/3B, which is essential for the precise establishment and maintenance of lineage-specific DNA methylation patterns in cells. This review summarizes current understanding of the structure and mechanism of DNMT1- and DNMT3A-mediated DNA methylation, with emphasis on the functional cooperation between the methyltransferase and regulatory domains.

The term ‘Hallmarks of Cancer’ was coined by Hanahan and Weinberg in their influential reviews and they described genome instability as a property of cells enabling cancer development [1, 2]. Accurate DNA replication of genomes is central to diminish genome instability. Here, the understanding of the initiation of DNA synthesis in origins of DNA replication to start leading strand synthesis and the initiation of Okazaki fragment on the lagging strand are crucial to control genome instability. Recent findings have provided new insights into the mechanism of the remodelling of the prime initiation enzyme, DNA polymerase α-primase, during primer synthesis, how the enzyme complex achieves lagging strand synthesis, and how it is linked to replication forks to achieve optimal initiation of Okazaki fragments. Moreover, the central roles of RNA primer synthesis by Pol-prim in multiple genome stability pathways such as replication fork restart and protection of DNA against degradation by exonucleases during double-strand break repair is discussed.

Head and neck squamous cell carcinomas (HNSCCs) develop from the mucosa of the oral cavity, pharynx, and larynx. The methylation levels of Septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) genes in ccfDNA are considered pan-cancer biomarkers and have shown prognostic value in preliminary reports in HNSCC. Liquid biopsy is a non-invasive procedure that collects tumor-derived molecules including circulating cell-free DNA (ccfDNA). Here, we developed a ddPCR-based assay to detect the DNA methylation levels of plasma circulating SEPT9 and SHOX2 in patients with HNSCC. We first set up the assay on commercial methylated and un-methylated DNA. Then, the dynamic changes of the methylation levels of SEPT9 and SHOX2 were quantified in 20 patients during the follow-up. The results highlighted: i) the capability of the ddPCR-based assay in detecting very low copies of methylated molecules; ii) the significant decrease in methylation levels of SEPT9 and SHOX2 in plasma of HNSCC patients at the first time points of follow-up respect to T0; iii) a different trend of longitudinally DNA methylation variations associable to the clinicopathological features of the patients. The absolute quantification of the methylation levels of SEPT9 and SHOX2 in HNSCC may be used for risk stratification and disease monitoring.

Forensic genetic genealogy, a technique leveraging new DNA capabilities and public genetic databases to identify suspects, raises specific considerations in a law enforcement context. Use of this technique requires consideration of its scientific and technical limitations, including the composition of current online datasets, and consideration of its scientific validity. Additionally, forensic genetic genealogy needs to be considered in the relevant legal context to determine the best way in which to make use of its potential to generate investigative leads while minimising its impact on individual privacy. This article presents these issues from an Australian perspective, with the observations and conclusions likely to be applicable to other jurisdictions.

One of creators of quantum mechanics P. Jordan in his work on quantum biology claimed that life's missing laws were the rules of chance and probability of the quantum world. The article presents author’s results of studying frequencies (or probabilities) of nucleotides on so-called epi-chains of long DNA sequences of various eukaryotic and prokaryotic genomes. DNA epi-chains are algorithmically constructed subsequencies of DNA nucleotide sequences. According to the algorithm of construction of any epi-chain of the order n, the epi-chain is such nucleotide subsequence, in which the numerations of adjacent nucleotides differ by natural number n (n = 1, 2, 3, 4,…). Correspondingly each epi-chain of order n ≥ 2 contains n times less nucleotides than the original DNA sequence. The presented results unexpectedly discover that in long single-stranded and double-stranded DNA of any tested genome its DNA epi-chains of different orders n (values n are not too large) have practically identical frequencies (or probabilities) of each kind of nucleotides. These data allow considering DNA as a regular rich set of epi-chains, which can play a certain role in genetic and epigenetic phenomena as the author belives. Appropriate rules of nucleotide frequencies on epi-chains of long DNA sequences are formulated for further their tests on a wider set of genomes. These results testify on existence of long-range coherence in long DNA and remind the Fröhlich's theory of long-range coherence in biological systems. The phenomenological data are discussed from different standpoints: the DNA double helices and helical antennas with circular polarizations of electromagnetic waves; relations with the Fröhlich's theory; numerical analysis of DNA epi-chains under binary representations of nucleotides. Results are useful for developing quantum and algebraic biology.

Platinum(II) complexes have been found to be effective against cancer cells. Cisplatin curbs cell replication by interacting with the deoxyribonucleic acid (DNA), eventually leading to cell death and reducing cell proliferation. In order to investigate the ability of platinum complexes to affect cancer cells, two examples from the class of polyflurophenylorganoamidoplatinum(II) complexes were synthesised and tested on isolated DNA. The two compounds trans-[N,N’-bis(1,2,3,5,6-pentafluorophenyl)ethane-1,2-diaminato(1-)](2,3,4,5,6-pentafluorobenzoato)(pyridine)platinum(II) (PFB), and trans-[N,N’-bis(1,2,3,5,6-pentafluorophenyl)ethane-1,2-diaminato(1-)](2,4,6-trimethylbenzoato)(pyridine)platinum(II) (TMB) were compared with cisplatin through their reaction with DNA. Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy was applied to analyse the interaction of the Pt^(II) complexes with DNA in the hydrated, dehydrated and rehydrated state. These were compared with control DNA in acetone/water (PFB, TMB) and isotonic saline (cisplatin) under the same conditions. Principle Component Analysis (PCA) was applied to compare the ATR-FTIR spectra of the untreated control DNA with spectra of PFB and TMB treated DNA samples. Disruptions in the conformation of DNA treated with the Pt^(II) complexes upon rehydration were mainly observed by monitoring the position of the IR-band around 1711 cm^-1 assigned to the DNA base-stacking vibration. Furthermore, other intensity changes in the phosphodiester bands of DNA at ~1234 cm^-1 and 1225 cm^-1 and shifts in the dianionic phosphodiester vibration at 966 cm^-1 were observed. The isolated double stranded DNA (dsDNA) or single stranded DNA (ssDNA) showed different structural changes when incubated with the studied compounds. PCA confirmed PFB had the most dramatic effect by denaturing both dsDNA and ssDNA. Both compounds, along with cisplatin, induced changes in DNA bands at 1711, 1088, 1051 and 966 cm^-1 indicative of DNA conformation changes. The ability to monitor conformational change with infrared spectroscopy paves the way for a sensor to screen for new anticancer therapeutic agents.

Iron-sulfur (Fe-S) clusters are essential cofactors found in many proteins in the mitochondria, cytosol, and nucleus of the cell. These versatile cofactors may undergo reversible oxidation-reduction reactions to enable electron transfers; they may be structural and confer stability to a folded protein; they may be regulatory and transduce an iron signal that alters the function or stability of a recipient protein. Of the nearly 70 proteins described in mammalian cells that bind Fe-S clusters, about half localize exclusively or partially to the nucleus, where they are required for DNA replication and repair, telomere maintenance, transcription, mitosis, and cell cycle control. Most nuclear Fe-S cluster proteins interact with DNA, including DNA polymerases, primase, helicases, and glycosylases. However, the specific roles of the clusters in the enzymatic activities of these proteins and their interplay with DNA remain a matter of debate. Defects in the metallation of nuclear Fe-S proteins cause genome instability and alter the regulation of cell division and proliferation, which are hallmarks of various genetic diseases and cancers. Here, we provide an inventory of the nuclear Fe-S cluster binding proteins and discuss cluster types, binding sites, the process of cluster acquisition, and potential roles of the cluster in the function of the proteins. However, many questions remain unresolved. We highlight critical gaps in our understanding of cluster delivery to nuclear client proteins, the potential for cluster repair, and the mechanistic roles that clusters play in these enzymes. Taken together, this review brings the focus to the nucleus of the human cell as a hotspot for Fe-S cluster proteins and aims to inspire new research in the roles of iron in DNA metabolism and the maintenance of genome integrity.

Four 1,10-phenanthroline derivatives (1-4) have been synthesized as potential telomeric DNA binders, three substituted in their chains with thiosemicarbazones (TSCs), the neutral compound (1) and two ionic derivatives (PF6¯ and BF4¯ salts, 2 and 3, respectively), and a fourth corresponding to a 4-phenylthiazole derivative (4). Quadruplex and dsDNA interactions have been preliminarily studied, especially for the neutral derivative 1, by FRET-based DNA melting assays, equilibrium dialysis (both competitive and non-competitive), circular dichroism and viscosity titrations. The TSC derivatives bind and stabilize the telomeric Tel22 quadruplex more efficiently than dsDNA, with an estimated 24-fold selectivity determined by equilibrium dialysis for compound 1. In addition, cytotoxic activity against different tumor cells (PC-3, DU145, HeLa, MCF-7 and HT29), and two non-tumor cell lines (HFF-1 and RWPE-1) has been evaluated. Except for the 4-phenylthiazole derivative, which was inactive, the compounds show moderate cytotoxic properties, with the salts displaying lower IC50 values, compared to the neutral TSC. However, the neutral derivative was the only compound that exhibited a modest selectivity in the case of prostate cells (tumor PC-3 versus healthy RWPE-1). Cell cycle analysis and Annexin V/PI assays revealed that the compounds can produce cell death by apoptosis, an effect that has proven similar to that demonstrated by other known 1,10-phenanthroline G4 ligands endowed with antitumor properties, such as PhenDC3 or PhenQE8.

B chromosomes are supernumerary chromosomes which are found in addition to the normal standard chromosomes (A chromosomes). B chromosomes are well known to accumulate several types of repeats. Although the evolution of B chromosomes has been subject of numerous studies, the mechanisms of accumulation and evolution of repetitive sequences is not fully understood. Recently, new genomic approaches have shed light on the origin and accumulation of different classes of repetitive sequences in the process of B chromosome formation and evolution. Here we discuss the impact of repetitive sequences accumulation on the evolution of plant B chromosomes.

Cholangiocarcinoma is an epithelial malignancy arising in the region between the intrahepatic bile ducts and the ampulla of Vater at the distal end of the common bile duct. The effect of current chemotherapy regimens against cholangiocarcinoma is limited, and the prognosis of patients with cholangiocarcinoma is poor. Aberrant DNA methylation and histone modification induce silencing of tumor suppressor genes and chromosomal instability during carcinogenesis. Studies have shown that the tumor suppressor genes and microRNAs (miRNAs) including MLH1, p14, p16, DAPK, miR-370 and miR-376c are frequently methylated in cholangiocarcinoma. Silencing of these tumor suppressor genes and miRNAs plays critical roles in the initiation and progression of cholangiocarcinoma. In addition, recent studies have demonstrated that DNA methylation inhibitors induce expression of endogenous retroviruses and exert the anti-tumor effect of via an anti-viral immune response. Aberrant DNA methylation of tumor suppressor genes and miRNAs could be a powerful biomarker for diagnosis and treatment of cholangiocarcinoma. Epigenetic therapy with DNA methylation inhibitors hold considerable promise for the treatment of cholangiocarcinoma through re-activation of tumor suppressor genes and miRNAs as well as induction of an anti-viral immune response.

In the United States, the Federal Aviation Administration has officially classified flight crew (FC) consisting of commercial pilots, cabin crew or flight attendants as “radiation workers” since 1994 due to the potential for cosmic ionizing radiation (CIR) exposure at cruising altitudes originating from solar activity and galactic sources. Several epidemiological studies have documented elevated incidence and mortality for several cancers in FC, but it has not yet been possible to establish whether this is attributable to CIR. CIR and its constituents are known to cause a myriad of DNA lesions, which can lead to carcinogenesis unless DNA repair mechanisms remove them. But critical knowledge gaps exist with regard to CIR dosimetry, the role of other genotoxic exposures among FC, and possible biological mechanisms underlying higher cancer rates observed in FC exist. This review summarizes our understanding of the role of DNA damage and repair responses relevant to CIR exposure in FC. We aim to stimulate new research directions and provide information that will be useful for guiding regulatory, public health, and medical decision-making to protect and mitigate risks for those who travel by air.

Environmental DNA (eDNA) sampling has emerged as a powerful approach for monitoring biodiversity in the context of diverse terrestrial and aquatic ecosystems, and has broad potential for both environmental surveys and species management. eDNA studies focusing on plant and animal species have capitalized on analytical methodologies developed originally for metagenomic studies of microorganisms, and on technological improvements in sample collection. However, while eDNA studies have proliferated rapidly, efforts to standardize sampling and analytical methods are still nascent. Critically, fundamental data concerning the distribution and persistence of eDNA in different ecological contexts are lacking, complicating both confidence in species detection and comparability between studies. We propose that key deficits could be addressed by using well-designed synthetic DNA standards or tracers, which are widely used in fields ranging from medical diagnostics to hydrology. In-field application of a standardized mixture of barcoded synthetic DNA fragments of varying lengths and concentrations that are recovered concommitantly with eDNA sampling should provide critical yet missing data on environmental DNA distribution and turnover, as well as an internal molecular standard to enhance both species detection and comparability between eDNA studies.

Methylation of cytosines at CpG sites in DNA, carried out by de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed by physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduce Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices which have been classified as “possibly carcinogenic”, however their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (RE) may promote genomic instability, here we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of RE, such as long interspersed nuclear element1s (LINE-1), Alu short interspersed nuclear element and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE1 and ribosomal repeats in terms of both average profiles and organization of methylated and un-methylated CpG sites, in different ways in each of the three cell lines studied.

Here, our data provide the first evidence for the existence of a previously unknown receptive system formed by novel DNA- and RNA-based receptors in eukaryotes. This system, named the TR-system, is capable of recognizing and generating a response to different environmental factors and has been shown to orchestrate major vital functions of fungi, mammalian cells, and plants.Recently, we discovered the existence of a similar regulatory system in prokaryotes. These DNA- and RNA-based receptors are localized outside of the membrane forming a type of a network around cells that respond to a variety of chemical, biological, and physical factors and enabled the TR-system to regulate major aspects of eukaryotic cell life as follows: growth, including reproduction and development of multicellular structures; sensitivity to temperature, geomagnetic field, UV, light, and hormones; interaction with viruses; gene expression, recognition and utilization of nutrients. The TR-system was also implicated in cell memory formation and was determined to be responsible for its maintenance and the forgetting of preceding events. This system is the most distant receptive and regulatory system of the cell that regulates interactions with the outer environment and governs the functions of other receptor-mediated signaling pathways.

Here we present 1. a model for amplicon sequencing 2. a definition of the best assignment of a read to a set of reference sequences 3. strategies and structures for indexing reference sequences and computing the best assign- ments of a set of reads efficiently, based on (ultra)metric spaces and their geometry The models, techniques, and ideas are particularly relevant to scenarios akin to 16S taxonomic profiling, where both the number of reference sequences and the read diversity is considerable.

Structural DNA nanotechnology provides an excellent foundation for diverse nanoscale shapes that can be used in various bioapplications and materials research. From all existing DNA assembly techniques, DNA origami has proven to be the most robust one for creating custom nanoshapes. Since its invention in 2006, building from the bottom up using DNA has drastically advanced, and therefore, more and more complex DNA-based systems have become accessible. So far, vast majority of the demonstrated DNA origami frameworks are static by nature, but interestingly, there also exist dynamic DNA origami devices that are increasingly coming into view. In this review, we discuss DNA origami nanostructures that perform controlled translational or rotational movement triggered by predefined DNA strands, various molecular interactions and/or other external stimuli such as light, pH, temperature and electromagnetic fields. The rapid evolution of such dynamic DNA origami tools will undoubtedly have a significant impact on molecular scale precision measurements, targeted drug delivery and diagnostics, but they can also play a role in development of optical/plasmonic sensors, nanophotonic devices and nanorobotics for numerous different tasks.

We present a numerical study in which large-scale bulk simulations of self-assembled DNA constructs have been carried out with a realistic coarse-grained model. The investigation aims at obtaining a precise, albeit numerically demanding, estimate of the free energy for such systems. We then, in turn, use these accurate results to validate a recently proposed theoretical approach that builds on a liquid-state theory, the Wertheim theory, to compute the phase diagram of all-DNA fluids. This hybrid theoretical/numerical approach, based on the lowest order virial expansion and a nearest-neighbor DNA model, can provide, in an undemanding way, a thermodynamic description of DNA associating fluids that is in semi-quantitative agreement with experiments. We show that the predictions of such scheme are as accurate as the ones obtained with more sophisticated methods. We also demonstrate the flexibility of the approach by incorporating non-trivial additional contributions that go beyond the nearest-neighbor model to compute the DNA hybridization free energy.

Many fundamental molecular techniques (PCR, Microarray, Southern and northern hybridization, siRNA, CRISPR/Cas9 etc.) developed so far shows errors. I wish to highlight these molecular techniques are developed on basis of Watson-Crick DNA model, ignoring the concept of parallel stranded DNA. Through this opinion article, I wish to highlight specificity and accuracy of these molecular techniques can be enhanced by considering both parallel and anti parallel hybridization of DNA. Hopefully my views will also solve issue of irreproducibility in life science research.

The National Institute of Justice reported that current methods for processing sexual assault samples have a high failure rate, with 60 to 80 percent of tested kits unable to produce usable DNA profiles. Even when samples test positive for male DNA, 34 percent of sexual assault kits (SAK) do not yield recovered male DNA after differential extraction. Less than 30% recovery of available sperm DNA contributes to this low success rate. The SpermX™ method (SX) has been shown to recover 80 percent or more of sperm DNA from sexual assault samples. An interlaboratory evaluation compared SX to standard differential extraction (DE) protocols. Mock samples with known ratios of female epithelial cells and sperm cells were processed using both methods. Results revealed that SX consistently provided CODIS up-loadable DNA profiles, even with as few as 25 sperm cells, whereas DE failed to produce usable results. On average, SX yielded a seven-fold increase in the recovery of unshared male alleles compared to DE. In conclusion, SX outperformed DE in recovering higher quantities of male DNA with minimal female carryover in sexual assault-type samples. This improved success rate in obtaining usable DNA profiles can significantly aid in solving sexual assault cases. Keywords: Differential extraction, Sexual Assault Kit (SAK), Sperm DNA, STRmix™, Combined DNA Index System (CODIS), Forensic DNA analysis, Low Sperm Samples, DNA Mixtures, Autosomal STR Analysis, SpermX.

Comprehensive studies into the various causes of this ubiquitous disease have been prompted by the worldwide increase in cancer incidence. There are many potential causes of cancer, but environmental factors are of particular concern now. This abstract explores the complex link between environmental factors and the growing cancer epidemic, shedding light on the role that our immediate environment plays in the emergence of cancers. The environment can contain carcinogenic agents that gradually influence the course of cancer, including air pollutants, water contaminants, workplace exposures, and industrial activities. The ways through which environmental carcinogens may cause DNA damage and unchecked cell proliferation are discussed in this abstract. Environmental justice and the moral necessity to reduce inequities in exposure and health effects are highlighted to underline the disproportionate impact on vulnerable groups. The study presented here highlights the need for preemptive action to reduce environmental risks. Society can reduce the hidden threat posed by environmental carcinogens through strict legislation, sustainable practices, and community-driven initiatives. In order to lessen the cancer burden and secure a healthier future for all, the authors of this abstract argue that there must be a concerted effort to create surroundings that promote health and well-being.

Topoisomerases, common targets for anti-tumor therapeutics, are crucial enzymes for DNA replication, transcription, and many other aspects of DNA metabolism. The potential anti-cancer effect of thiosemicarbazones (TSC) and metal-TSC complexes has been demonstrated by targeting several biological processes, including DNA metabolism. Human topoisomerases were discovered among the molecular targets for TSCs, and metal-chelated TSCs specifically displayed significant inhibition. The processes by which metal-TSCs or TSCs inhibit topoisomerases are still being studied. In this brief review, we summarize the TSCs and metal-TSCs that inhibit various types of human topoisomerases.

Phenotypes are necessary for genomic evaluations and management. Sometimes genomics can be used to measure phenotypes when other methods are difficult or expensive. Prolificacy of bulls used in multiple-bull pastures for commercial beef production is an example. A retrospective study of 79 bulls aged 2-year-old and older used 141 times in 4-5 pastures across 4 years was used to estimate repeatability from variance components. Traits available before each season’s use were tested for predictive ability. Sires were matched to calves using individual genotypes and evaluating exclusions. A lower cost method of measuring prolificacy was simulated for 5 pastures using the bulls’ genotypes and pooled genotypes to estimate average allele frequencies of calves and of cows. Repeatability of prolificacy was 0.62 ± 0.09. A combination of age-class and scrotal circumference accounted for less than 5 % of variation. Simulated estimation of prolificacy by pooling DNA of calves was accurate. Adding pooling of cow DNA or actual genotypes both increased accuracy about the same. Knowing a bull’s prior prolificacy would help predict future prolificacy for management purposes and could be used in genomic evaluations and research with coordination of breeders and commercial beef producers.

The ability to predict physical characteristics from DNA presents significant opportunities for forensic science. Giving scientists an ability to make predictions about the donor of genetic material at a crime scene can then give investigators new intelligence leads for cold cases where DNA evidence has not identified any person of interest. However, the interpretation of this new form of intelligence requires careful analysis. The responses to an online survey, conducted in 2018-19, were used to examine how actors in the criminal justice system assess and interpret different types of DNA evidence and intelligence. The groups of focus for the survey were investigators, legal practitioners and the general public (as potential jurors). Several statistically significant effects were identified based on occupation and whether an individual had prior exposure to new DNA technology. Monitoring how those involved in interpreting reports from different types of DNA evidence and intelligence interpret them helps to ensure that decisions are made based on a sound understanding of their capabilities and limitations and may inform broader training and awareness strategies.

A fully analytical treatment of the base-pair and codon dynamics in double-stranded DNA molecules is introduced, by means of a realistic treatment which considers different mass values for G, A, T, and C nucleotides and takes into account the intrinsic three-dimensional, helicoidal geometry of DNA in terms of a Hamitonian in cylindrical coordinates. Within the framework of the Peyrard-Dauxois-Bishop model we consider the coupling between stretching and stacking radial oscillations as well as the twisting motion of each base pair around the helix axis. By comparing the linearized dynamical equations for the angular and radial variables when going from the bp local scale to the longer triplet codon scale, we report an underlying hierarchical symmetry. The existence of synchronized collective oscillations of the base-pairs and their related codon triplet units are disclosed from the study of their coupled dynamical equations. The possible biological role of these correlated, long-range oscillation effects in double standed DNA molecules containing mirror-symmetric codons of the form XXX, XX’X, X’XX’, YXY, and XYX is discussed in terms of the dynamical equations solutions and their related dispersion relations.

There is a major clinical need for accurate biomarkers for prostate cancer prognosis, to better inform treatment strategies and disease monitoring. Current clinically recognised prognostic factors, including prostate-specific antigen (PSA) levels, lack sensitivity and specificity in distinguishing aggressive from indolent disease, particularly in patients with localised intermediate grade prostate cancer. There has therefore been a major focus on identifying molecular biomarkers that can add prognostic value to existing markers, including investigation of DNA methylation, which has a known role in tumorigenesis. In this review, we will provide a comprehensive overview of the current state of DNA methylation biomarker studies in prostate cancer prognosis, and highlight the advances that have been made in this field. We cover the numerous studies into well-established candidate genes, and explore the technological transition that has enabled hypothesis-free genome-wide studies and the subsequent discovery of novel prognostic genes.

Background: The presence of HIV residual replication markers was investigated among distinct subgroups of individuals on antiretroviral treatment (ART). Methods: One hundred sixteen patients were distributed into 5 treatment groups: first-line suppressive ART with a non-nucleoside analog reverse-transcriptase inhibitor (NNRTI) (n = 26), first-line suppressive ART with boosted protease inhibitors (PI-r) (n = 25), suppressive salvage therapy using PI-r (n = 27), suppressive salvage therapy with PI-r and raltegravir (n = 22) and virologic failure (n = 16). Episomal and total DNA quantitation was evaluated. HIV antibody and LPS quantitation was performed. Results: Episomal DNA was positive in 26% to 38% of individuals under suppressive ART, and it was higher among ART virologic failure group (p = 0.04). HIV proviral load was higher among patients with detectable episomal DNA (p = 0.01). Individuals receiving initial PI-r treatment presented lower HIV antibody (p = 0.027) and LPS (p = 0.029) levels than individuals receiving NNRTI. There was a negative correlation between episomal DNA quantitation and the duration of suppressive ART (p = 0.04), CD4+ T-cell count (p = 0.08), and CD8+ T-cell count (p = 0.07). Conclusions: Residual HIV replication has been inferred among individuals under suppressive ART according to episomal DNA detection. Residual replication may decrease with longer periods of suppressive ART and higher levels of CD4+ and CD8+ T cells. The relationship between episomal DNA and total DNA suggests there is a replenishment of the proviral reservoir. Lower antibody and LPS levels among patients with initial PI-r ART suggest these regimens may more effectively suppress HIV, more effectively decreasing HIV antigenic component.

High concentrations of Na⁺ or [Co(NH₃)₆]³⁺ can induce the B to Z conformational transition in alternating (dC-dG) oligo and polynucleotides. The use of short DNA oligomers (dC-dG)₄ and (dm⁵C-dG)₄ as models can allow a thermodynamic characterization of the transition. Both form right handed double helical structures (B-DNA) in standard phosphate buffer with 115 mM Na⁺ at 25 ^oC. However, at 2.0 M Na⁺ or 200 mM [Co(NH₃)₆]³⁺, (dm⁵C-dG)₄ assumes a left handed double helical structure (Z-DNA) while the unmethylated (dC-dG)₄ analogue remains right handed under those conditions. We have previously demonstrated that the enthalpy of the transition at 25 ^oC for either inducer can be determined using isothermal titration calorimetry (ITC) [Ferreira, J. M. & Sheardy, R. D., Biophys. J. 2006, 91, 1–7]. Here, ITC is used to investigate the linkages between temperature, water activity and DNA conformation. We found that the determined enthalpy for each titration varied linearly with temperature allowing determination of the heat capacity change (DC_p) between the initial and final states. As expected, the DC_p values were dependent upon the cation (i.e. Na⁺ vs [Co(NH₃)₆]³⁺) as well as the sequence of the DNA oligomer (i. e., methylated vs unmethylated). Osmotic stress experiments were carried out to determine the gain or loss of water by the oligomer induced by the titration. The results are discussed in terms of solvent accessible surface areas, electrostatic interactions and the role of water.

Oxidative and nitrosative stress are a common problem when manipulating gametes in vitro. In vitro development in mammalian embryos is highly affected by culture conditions, especially by reactive oxygen species (ROS) and reactive nitrogen species (RNS), because its absence or over production causes embryo arrest and changes in gene expression. Melatonin in gamete co-incubation during IVF has deleterious or positive effects depending on the concentration used in culture medium, demonstrating the delicate balance that must exist between antioxidant and pro-oxidant activity. Further research is needed to better understand the possible impact of melatonin on the different IVP steps in domestic animals, especially in seasonal breeds where this neuro-hormone system highly regulates its reproduction physiology.

In E. coli DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, PDIP46 and PDIP38, both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the splicesosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, Mdm2 alternative splicing and the regulation of the Nox4 NADPH oxidase.

A simulation is performed to analyze the forces that arise in a conducting DNA-like double helix in the field of a microwave electromagnetic wave under half-wave resonance conditions. The helix is comprised of twenty-and-a-half turns and has geometric parameters proportional to the size of an actual DNA molecule. The forces acting on the strands of a double helix, both in the central region and at the edges of the helix, are investigated. It has been demonstrated that the afore-mentioned forces induce a change in the shape of the helix, specifically mutual repulsion of the strands, as well as their stretching and twisting in the field of electromagnetic waves. As opposed to a pair of parallel straight conductors, there is a radial repulsion of strands in all regions of a double DNA-like helix, including the center and the edges. This unique interaction of currents in a double DNA-like helix can be attributed to its pitch angle, a characteristic that has been estab-lished through empirical evidence. Consequently, exposure to an electromagnetic wave under half-wave resonance can damage the double helix. Conversely, the impact of electromagnetic waves has the potential to introduce novel avenues for controlling the shape of the double helix. These nonlinear effects must be considered for both actual DNA molecules and double DNA-like helices that serve as components of metamaterials and metasurfaces.

The study titled "Molecular Choreography of DNA: Insights and Innovations" explores the intricate dance of DNA, shedding light on its structure, function, and regulation. This comprehensive review delves into the historical significance of DNA research, from Watson and Crick's double helix discovery to recent innovations in genomics and molecular biology. The review highlights DNA's roles in replication, repair, gene regulation, and chromatin dynamics, emphasizing the precision and complexity of its molecular choreography. Innovations like CRISPR-Cas9 gene editing and advanced sequencing technologies have revolutionized our understanding of DNA, opening new avenues for scientific exploration. This study aims to provide readers with a deeper appreciation of DNA's central role in life's choreographed processes.

In our work, using the analysis of DNA barcodes and morphology (wing color, male genitalia, sphragis shape in females), we show that the group of species close to P. mnemosyne is represented by two phylogenetic lineages, the western and eastern ones. The eastern lineage includes P. stubbendorfii, P. glacialis and P. hoenei. The western lineage includes three morphologically similar species, P. mnemosyne (Western Eurasia), P. turatii (southwestern Europe) and P. nubilosus stat. nov. (Turkmenistan and NE Iran), as well as P. ariadne (Altai). The latter species differs from the rest of the group in the presence of red spots on the wings. Parnassius mnemosyne s.s. is represented by four differentiated mitochondrial clusters that show clear association with specific geographic regions. We propose to interpret them as subspecies: P. mnemosyne mnemosyne (Central and Eastern Europe, N Caucasus, N Turkey), P. mnemosyne adolphi (the Middle East), P. mnemosyne orientalis (Tian Shan) and P. mnemosyne gigantea (Gissar-Alai in Central Asia). We demonstrate that in P. ariadne the red spots on the wing evolved as a reversion to the ancestral wing pattern. This reversion is observed in Altai, where the distribution areas of the western lineage, represented by P. ariadne, and the eastern lineage, represented by P. stubbendorfii, overlap. These two species hybridize in Altai, and we hypothesize that the color change in P. ariadne is the result of reinforcement of prezygotic isolation in the contact zone. Lectotype of Parnassius mnemosyne var. nubilosus Christoph, 1873 is designated.

Although the APOBEC3 family of single-stranded DNA cytosine deaminases are well-known as antiviral factors, these enzymes are rapidly gaining attention as prominent sources of mutation in cancer. APOBEC3 signature single base substitutions, C-to-T and C-to-G in TCA and TCT motifs, are evident in over 70% of human malignancies and dominate the mutational landscape of numerous individual tumors. Recent murine studies have established cause-and-effect relationships, with both human APOBEC3A and APOBEC3B proving capable of promoting tumor formation in vivo. Here, we investigate the molecular mechanism of APOBEC3A-driven tumor development using the murine Fah liver complementation and regeneration system. First, we show that APOBEC3A alone is capable of driving tumor development (without Tp53 knockdown as in prior studies). Second, we show that the catalytic glutamic acid residue of APOBEC3A (E72) is required for tumor formation. Third, we show that an APOBEC3A separation-of-function mutant with compromised DNA deamination activity and wildtype RNA editing activity is defective in promoting tumor formation. Collectively, these results indicate that APOBEC3A is a “master driver” that fuels tumor formation through a DNA deamination-dependent mechanism.

The objective of this study was to evaluate the antifungal activity of eugenol against the genomic DNA of Candida albicans. A quantitative experimental study was conducted with the C. albicans INCQS 40006 strain. Eugenol was tested in concentrations of 12.5%, 25%, 50%, 75% and 100%. The test substance remained in contact with the fungus for 24 hours in a BHI medium in a microbio-logical greenhouse, then the DNA extraction was performed and part of the sample obtained was washed with TE to remove the Eugenol. The fungus was placed in BHI again and placed for 24 hours in a microbiological greenhouse. At the end, the samples with 100% eugenol went through seeds in Sabouraud Dextrose for confirmatory tests. Thus, from the electrophoresis, which admits evaluating the quality of the extracted material, preserving its characteristics and integrity, it was observed in the eugenol concentration test (test 1), in which the eugenol was in direct contact with the pathogen, that in concentrations of equal to or greater than 75% of the test substance the pathogen had no proliferative power. In which the fungus's ability to recover is evaluated (test 2), eugenol was removed from the sample, noting that in 100% of the test substance the fungus did not have the ability to recover, resulting in definitive cell death.

Dried blood spot (DBS) testing on confetti is one of the most common methods used to collect and extract DNA from field samples for molecular epidemiology of malaria studies. Here, we investigated whether ethanol or other solutions used to clean scissors are a source of sample contamination. DBS were prepared from 3 drops of blood from two Plasmodium falciparum cultures spotted on confetti, and 3 solutions (water, ethanol and DNase) were used to clean scissors between spots during DNA extraction. For each cleaning solution, two blank confetti were used as negative controls. Samples were analyzed by PCR-based genotyping of merozoite surface proteins 1 & 2 (msp-1 and msp-2), and P. falciparum chloroquine resistance transporter (Pfcrt). A total of 15 samples were analyzed. Based on msp-1 and msp-2 amplification, P. falciparum was detected on blank confetti when scissors were washed with ethanol. Based on Pfcrt amplification, DNA of P. falciparum was detected on blank confetti when scissors were cleaned with ethanol or water. No P. falciparum genes were detected on blank confetti when scissors were cleaned with DNase. Scissors cleaned with DNase prevented cross-contamination between samples during processing of dried blood spots, whereas ethanol (which is commonly used) fails to avert cross-contamination.

Two novel sizable multicharged cationic complexes, of the formulae [(η6-[12]CPP)[Ru(η5-Cp)]12]Χ12 and [(η6-[11]CPP)[Ru(η5-Cp)]11]Χ11, CPP= cycloparaphenylene, Cp = cyclopentadienyl, X = [PF6]-, (1), (3) and [Cl]-, (2), (4), were synthesized and characterized using NMR techniques, high-resolution mass spectrometry, and elemental analyses. The complexes, (1) and (3) were stable in acetone and acetonitrile solutions over 48 h. In contrast, the water-soluble, (2) and (4), begins to decompose in aqueous media after 1h, due to the [Cl]- tendency for nucleophilic attack on ruthenium of the {Ru(η5-Cp)} units. Fluorescence quenching experiments conducted during the stability window of (2) with the d(5′-CGCGAATTCGCG-3′)2-EtBr adducts, revealed remarkably high values for Ksv = 1.185 × 104 ± 0.025 M-1 and Kb = 3.162 × 105 ± 0.001 M-1. Furthermore, the cytotoxic activity of (2) against A2780, A2780res, and MCF-7 cancer cell lines shows that it is highly cytotoxic with IC50 values in the range of 4.76 ± 1.85 to 16 ± 0.81 μΜ.

Apurinic/apyrimidinic endonuclease 1 (APE1), also known as redox factor-1 (Ref-1), is a multifunctional protein which is widely existing in living organisms. It can specifically recognize and cleave the DNA in apurinic/apyrimidinic (AP) site in the base excision repair (BER) pathway, as well as regulate the expression of genes to activate some transcription factors. The abnormal ex-pression and disruptions in the biological functions of APE1 are linked to a number of diseases, including inflammation, immunodeficiency, and cancer. Hence, it is extremely desired to monitor the activity of APE1, acquiring a thorough understanding of the healing process of damaged DNA and making clinical diagnoses. Thanks to the advent of DNA nanotechnology, some nanodevices are used to image the activity of APE1 with great sensitivity and simplicity. In this review, we will summarize DNA nanotechnology-empowered fluorescence imaging in the past years for APE1 activity according to the types of DNA probe, which are classified into linear DNA probes, composite DNA nanomaterials and three-dimensional (3D) DNA nanostructures. We also highlight the future research directions in the field of APE1 activity imaging.

To activate gene expression, the initiation of transcription is a highly regulated process involving the interaction of proteins and DNA nucleotides at the promoter site, which consists of a small number of base pairs. As it involves interactions at the atomic scale, it is challenging to determine the mechanism of binding responsible for the great specificity between the amino acid residuals comprising the transcription binding protein and the DNA nucleotides comprising the promoter. Here, a new approach to characterize the transcription initiation process is developed and verified from analysis of comparative pharmacological efficacy data and elemental modeling. The newly developed description of a mechanism for transcription initiation involves the direct binding of small molecule ligands of approximately twenty carbon atoms, which are both structurally symmetric to DNA nucleotides, and also chemically complementary in its functional groups for interaction with the oxygen element at the carbon two position of thymine and with the phosphodiester chain. The results indicate that the activating ligands are transported to the DNA nucleotide promoter site by protein transcription factors, which serve as delivery vectors, for transfer of the ligand to the DNA nucleotide pairs. The ligands examined in this study include the steroid hormones, synthetic steroid molecules, derivatives of vitamin D, and prostaglandins, particularly PGJ2 and 15d-PGJ2. The transcription factors evaluated include glucocorticoid receptors, VDR, PPAR, and TBP. Through the developments, it is shown that because of the chemically complementary binding of the ligand to DNA nucleotide pairs, the resultant intermolecular complex produces three hydrogen bonds for the A-T and T-A configurations, which matches that of G-C and C-G. The orientation of the nucleotide base pairs is also seen to adjust as an inversion of the nominal position of the nucleobases to a dimer configuration presented via TBP transcription factor. The developments comprise a new approach to characterizing the initiation of the transcription process comprising the direct binding and interaction of ligands with DNA nucleotides as verified through comparative analysis of pharmacological activity and through perfect structural correspondence between the steroid hormone class as ligands with Watson-Crick DNA nucleotide pairings.

We apply hierarchical clustering (HC) of DNA k-mer counts on multiple Fastq files. The tree structures produced by HC may reflect experimental groups and thereby indicate experimental effects, but clustering of preparation groups indicates the presence of batch effects. Hence, HC of DNA k-mer counts may serve as an unspecific diagnostic device. In order to provide a simple applicable tool we implemented sequential analysis of Fastq reads with low memory usage in an R package (seqTools) available on Bioconductor. The approach is validated by analysis of Fastq file batches containing RNAseq data. Analysis of three Fastq batches downloaded from ArrayExpress indicated experimental effects. Analysis of RNAseq data from two cell types (dermal fibroblasts and Jurkat cells) sequenced in our facility indicate presence of batch effects. The observed batch effects were also present in reads mapped to the human genome and also in reads filtered for high quality (Phred > 30). We propose, that hierarchical clustering of DNA k-mer counts provides an unspecific diagnostic tool and a quality criterion and for RNAseq experiments.

This review article delves into the pivotal role of DNA in advancing criminal investigations, presenting a comprehensive analysis of its impact on modern justice systems. DNA, as the genetic blueprint of individuals, has revolutionized forensic science, augmenting the accuracy and reliability of criminal investigations. This study explores the historical evolution of DNA analysis, from its initial discovery to the development of sophisticated techniques like polymerase chain reaction (PCR), short tandem repeat (STR) profiling, and next-generation sequencing (NGS). The article elucidates the intricate process of DNA collection, preservation, and analysis, highlighting its significance in establishing identity, proving guilt or innocence, and resolving cold cases. The utilization of DNA databases and international cooperation in cross-border investigations are also examined, emphasizing their contributions to solving complex criminal cases. Moreover, the ethical, legal, and privacy considerations surrounding DNA usage in criminal justice are critically evaluated. Through a comprehensive literature review, this article synthesizes empirical evidence showcasing how DNA evidence has exonerated wrongfully convicted individuals and provided closure to victims' families. It further elucidates how technological advancements have extended DNA analysis to trace evidence, touch DNA, and forensic genealogy, thereby broadening its application in criminal investigations.

Background: Despite recent advances in epithelial ovarian carcinoma (EOC) treatment, recur-rence and mortality rates have not improved significantly. DNA hypermethylation has generally been associated with ominous prognosis and chemotherapy resistance, but the role of DNMTs in EOC remains to be investigated. Methods: In the current study, we systematically retrieved gene expression data from patients with EOC and studied the immunohistochemical expression of DNMTs in 108 primary and 26 relapsed tumors. Results: Our results showed that DNMT1, DNMT3A, DNMT3B and DNMT3L RNA levels were higher and DNMT2 lower in tumors compared to non-neoplastic tissue, and DNMT3A and DNMT2 expression decreased from Stage II to Stage IV carcinomas. Proteomic data also suggested that DNMT1 and DNMT3A levels were increased in tumors. Similarly, DNMT1, DNMT3A and DNMT3L protein levels were overex-pressed and DNMT2 expression was reduced in high grade carcinomas compared to non-neoplastic tissue and low-grade tumors. Moreover, DNMT1 and DNMT3L were increased in relapsed tumors compared to their primaries. DNMT3A, DNMT1 and DNMT3B mRNA lev-els were correlated with overall survival. Conclusions: Our study demonstrates that DNMT1 and DNMT3L are upregulated in primary high grade EOC and further increase in relapses, whereas DNMT3A is upregulated only in the earlier stages of cancer progression. DNMT2 downregulation highlights a presumptive tumor-suppressor activity of this gene in ovarian car-cinoma.