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As a common heavy metal contaminant, Cd2+ has adverse effects on food safety and consumer health. It is very important for human health to realize highly sensitive Cd2+ detection methods. The self-powered sensing system based on enzyme biofuel cells (EBFCs) does not need an external power supply, which can simplify the experimental equipment and has great application value in portable detection. Thus, the biosensor is innovatively integrated into the screen-printed electrode to construct a new type of portable sensor suitable for on-site and real-time Cd2+ detection. Hybridization chain reaction (HCR) combined with the Cd2+-dependent deoxyribose (DNAzyme) signal amplification strategy is used to enhance the detection sensitivity while specifically recognizing the Cd2+. Moreover, the self-powered sensor combines with smartphones to realize quantitative Cd2+ detection without other instruments and has the characteristic of Effectively improving the hazard detection technology is essential to ensure food safety. Portability, simplicity, and speed are suitable for real-time Cd2+ detection in the field. The dual mechanism and three quantitative modes combining colorimetric and two electrical signals output modes are adopted to realize the visualization and accurate detection. A series of research results confirm that this strategy is of great significance to strengthen the development of intelligent Cd2+ technology, expand the application of self-powered sensing technology, and improve the safety detection system. • Plug and play self-powered chip facilitates precision heavy metal detection. • Dual mechanism and three quantitative mode for quantitative analysis. • High sensitive detection chip can be used for Cd detection in tea samples. [ABSTRACT FROM AUTHOR]

We study transitions in intrinsically disordered regions (IDRs) upon complex formation, utilizing X-ray-solved structural dataset of protein-DNA and protein-RNA complexes, along with their available unbound protein forms. The identified IDRs are categorized into three classes: Disordered-to-Ordered (D-O), Disordered-to-Partial Ordered (D-PO) and Disordered-to-Disordered (D-D) after comparing them in unbound and complex forms. In the D-O class, IDRs form secondary structures like coils, helices, and strands upon binding to nucleic acids. Though a majority of these IDRs are present at the surface of the complexes, a significant number of IDRs are also observed at the interfaces and are involved in polar interactions. The hydrogen bonds made by the interface IDRs (B_IDRs) with phosphates and bases of nucleic acids are comparatively more than those formed with sugars. B_IDRs form more H-bonds with the ribose in protein-RNA than with the deoxyribose in protein-DNA. Among the B_IDRs, Arg and Lys prefer to interact with the major and minor grooves of DNA and RNA, respectively. Ser, however, prefers the minor groove in both the nucleic acids. Interestingly, we report 61 and 48 IDRs in 31 protein-DNA and 22 protein-RNA complexes, respectively, suggesting nucleic acid binding to proteins may also result in ordered-to-disordered transitions. [ABSTRACT FROM AUTHOR]

We propose a microscopic model describing the process of DNA thermal denaturation. The process consists of the splitting of DNA base pairs, or nucleotides, resulting in the separation of two complementary DNA strands. In contrast to the previous modelling attempts we take into account the states of all base pairs of DNA which in fact imposes the microscopic nature of the approach. The model is a linear integro-differential non-autonomous equation describing the dynamics of probability density which characterizes the distances between the bases within individual base pairs. The non autonomous structure of the equation comes from the dependence on temperature that may be in the general case a (given) function of time. To our knowledge it is the first model taking into account not only the strength of double and triple hydrogen bonds between the complementary bases but also the stacking interactions between neighborhood base pairs. In the present paper the basic preliminary properties of the model including the existence, uniqueness and stability results in various cases are discussed. Moreover the symmetrized version of the model is considered and the "macroscopic limit" is studied. The performed numerical simulations reproduce the sigmoid shape of DNA melting curves and reveal the appearance of experimentally observed denaturation bubbles. [ABSTRACT FROM AUTHOR]

Graphical abstract We developed a quantitative and selective DNA detection biosensor requiring only one step for DNA recognition and signal reporter generation using a loop-based DNA competitive hybridization assay. Highlights • One-step sensitive DNA detection by the personal gulucose meter using loop-based DNA competitive hybridization assay. • There was a linear relationship between the PGM signals and the target DNA concentration in the range of 100 pM to 100 nM. • The sensor exhibits excellent sequence selectivity and excellent anti-interference ability. • The biosensor is easy to use. Abstract A portable, quantitative and selective DNA detection biosensor requiring only one step for target recognition and signal reporter generation using a loop-based DNA competitive hybridization assay and personal glucose meter (PGM) was developed. In the presence of target DNA, due to its competitive binding, the invertase-DNA conjugate was released which could be collected with the help of a magnet subsequently. The released invertase-DNA was used to catalyze the hydrolysis of sucrose into glucose with millions of turnovers which was target concentration dependent. We found that there was a linear relationship between the PGM signals and the target DNA concentration in the range of 100 pM to 100 nM with a detection limit of 100 pM. In addition, the sensor exhibits excellent sequence selectivity, being able to differentiate a single mismatch in the target DNA, and excellent anti-interference ability, having almost no effect in serum on the detection performance. The biosensor reported here is easier to operate, owning great potential in point of care testing in environments with limited resources and skilled personnel for rapid and sensitive detection of specific DNA sequence in real biological samples. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A vitamin D2 analog (D2) combined with a plant-derived antioxidant carnosic acid (CA) can potentiate the therapeutic effect of AraC on AML blasts. • D2/CA activates apoptotic signals in cells with DNA damage resulting from AraC treatment. • Vitamin D-upregulated protein TXNIP is a regulator of the apoptotic cascade triggered by the differentiation agents (D2/CA) in this system. • TXNIP targets the apoptosis signal regulating kinase ASK1, which then sequentially activates MKK4 and JNK and the executioners of apoptosis. • These data may provide a paradigm for future improvements of Cytarabine-based therapy of patients with AML. Abstract Standard therapy for Acute Myeloid Leukemia (AML) is rarely curative, and several suggested improvements have had little success so far. We have reported that in an in vitro model of a potential therapeutic regimen for AML, the activity of cytarabine (AraC) is enhanced by a sequential treatment with a combination of the vitamin D2 analog Doxercalciferol (D2) and the plant-derived antioxidant carnosic acid (CA). Importantly, the enhancement occurred selectively in patient-derived AML blasts, but not in the normal bone marrow cells. We now demonstrate that TXNIP, previously known as Vitamin D up-regulated protein 1 (VDUP1) [PMID 808674] plays a part in signaling cell death (CD) in this regimen. This is shown by the reduced CD when TXNIP protein levels are decreased by the CRISPR/CAS9 or RNAi technology. Further, we show that direct activation of ASK1 kinase by TXNIP is required for the optimal transmission of the CD signal to apoptotic machinery, regulated by JNK and BIM. These studies provide a rationale for a projected clinical trial of this vitamin D -based new therapeutic regimen for AML. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • (Citrate coated gold nanoparticles + plasmid DNA) are exposed to ultra-soft X-rays. • The radiosensitizing effect of GNPs is verified. • The radiosensitizing effect is inversely proportional to the size of the nanoparticles. • Reactive oxygen species are specifically quenched. • Hydrogen peroxide is converted into OH radicals at the surface of GNPs. Abstract Plasmid DNA in aerated aqueous solution is used as a probe to determine whose of the reactive oxygen species (ROS) generated after absorption of ultra-soft X-rays (USX) take part in biomolecule damage in the presence and in absence of Gold Nano-Particles (GNP) and specific scavengers. Citrate-coated GNPs with core sizes of 6, 10 and 25 nm are synthetized and characterized, especially in terms of plasmon band shift, ζ-potential and hydrodynamic radii (respectively 9, 21 and 30 nm). We confirm the radiosensitizing effect of GNP and show that the SSB number per plasmid increases when, for a same mass of gold element, the core size of the gold nanoparticles decreases. Hydroxyl radicals (OH) are scavenged using the positively-charged 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) and the neutral dimethyl sulfoxide (DMSO) molecules. Due to both negatively-charged environments of DNA and GNP, at identical scavenging capacity, TRIS is more effective at quenching OH than DMSO. The strong radiosensitizing effect of hydroxyl radicals is confirmed. Methanoate anions are then used to transform OH into hydrogen peroxide; the latter being known to be non-aggressive regarding DNA in the absence of easily oxidable metallic ions (Fenton reactions). Surprisingly, in the presence of GNP, high DNA damage yields are observed even though hydrogen peroxide might not be hold as responsible. Conversely, the radiosensitizing effect of GNP is not observed anymore when H 2 O 2 is scavenged using pyruvate ions. We demonstrate that hydrogen peroxide constitutes quite unexpectedly a hidden stock of OH which are activated at the surface of the GNP by decomposition of H 2 O 2 molecules. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Synthetic DNA lattices and natural DNA thin films doped with Ln3+ are constructed. • he optimum concentration of Ln3+ is estimated by analysis of the topological data. • The significances of optical and electrical characteristics are discussed. • Advantages and applications of Ln3+‒doped DNA nanostructures are addressed. Abstract Deoxyribonucleic acid (DNA) and lanthanide ions (Ln3+) exhibit exceptional optical properties that are applicable to the development of nanoscale devices and sensors. Although DNA nanostructures and Ln3+ ions have been investigated for use in the current state of technology for more than a few decades, researchers have yet to develop DNA and Ln3+ based ultra-violet (UV) photodetectors. Here, we fabricate Ln3+ (such as holmium (Ho3+), praseodymium (Pr3+), and ytterbium (Yb3+))‒doped double crossover (DX)‒DNA lattices through substrate-assisted growth and salmon DNA (SDNA) thin films via a simple drop-casting method on oxygen (O 2) plasma-treated substrates for high performance UV photodetectors. Topological (AFM), optical (UV–vis absorption and FTIR), spectroscopic (XPS), and electrical (I ‒ V and photovoltage) measurements of the DX‒DNA and SDNA thin films doped with various concentrations of Ln3+ ([Ln3+]) are explored. From the AFM analysis, the optimum concentrations of various Ln3+ ([Ln3+] O) are estimated (where the phase transition of Ln3+‒doped DX‒DNA lattices takes place from crystalline to amorphous) as 1.2 mM for Ho3+, 1.5 mM for Pr3+, and 1.5 mM for Yb3+. The binding modes and chemical states are evaluated through optical and spectroscopic analysis. From UV–vis absorption studies, we found that as the [Ln3+] was increased, the absorption intensity decreased up to [Ln3+] O , and increased above [Ln3+] O. The variation in FTIR peak intensities in the nucleobase and phosphate regions, and the changes in XPS peak intensities and peak positions detected in the N 1 s and P 2p core spectra of Ln3+‒doped SDNA thin films clearly indicate that the Ln3+ ions are properly bound between the bases (through chemical intercalation) and to the phosphate backbone (through electrostatic interactions) of the DNA molecules. Finally, the I ‒ V characteristics and time-dependent photovoltage of Ln3+‒doped SDNA thin films are measured both in the dark and under UV LED illuminations (λ LED = 382 nm) at various illumination powers. The photocurrent and photovoltage of Ln3+‒doped SDNA thin films are enhanced up to the [Ln3+] O compared to pristine SDNA due to the charge carriers generated from both SDNA and Ln3+ ions upon the absorption of light. From our observations, the photovoltages as function of illumination power suggest higher responsivities, and the photovoltages as function of time are almost constant which indicates the stability and retention characteristics of the Ln3+‒doped SDNA thin films. Hence, our method which provides an efficient doping of Ln3+ into the SDNA with a simple fabrication process might be useful in the development of high-performance optoelectronic devices and sensors. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A troponin I biosensor composed of DNA 3WJ on Au nanospike was developed. • The DNA 3WJ was rolled to recognition, EC signal transduction and immobilization. • Au nanospike was prepared to increase the detection limit of troponin I. • This study showed label-free EC detection elements for troponin I detection. Abstract Acute myocardial infarction (AMI) is one of the most serious diseases affecting human beings. In this study, in order to rapidly detect AMI disease, the authors fabricated a label-free electrochemical biosensor composed of a multi-functional DNA structure on Au nanospike (AuNS) with a fabricated Au micro-gap electrode which was incorporated with a PCB chip in order to detect cardiac troponin I (cTnI). As a bioprobe, the DNA 3 way-junction (3WJ) was introduced, because the DNA 3WJ has three arms for embodying the multi-functionality. Each piece of DNA was assembled to simultaneously form the DNA 3WJ for cTnI detection, signal transduction, and immobilization, respectively. The assembled DNA 3WJ structure was confirmed by Native-TBM PAGE. Moreover, in order to increase the electrochemical signal sensitivity, AuNS was prepared. The Au micro-gap array is fabricated with a printed circuit board (PCB) chip in order to control each micro-gap electrode panel selectively so as to detect low volumes of cTnI. Then, the DNA strucuture on pAuNS-modified electrode was prepared using the layer-by-layer (LbL) assembly method. FE-SEM and AFM were used to investigate the modified-surface morphology. The cyclic voltammetry (CV) was measured to confirm the cTnI binding to DNA 3WJ-modified electrode. cTnI was detected in the HEPES solution and human serum, respectively. The LOD result exhibited 1.0 pM in HEPES solution and 1.0 pM in 20% diluted human serum, respectively. In addition, the selectivity test was carried out with various proteins as the control experiment. The present study showed label-free, simple fabrication, and easy-to-tailor detection elements for cTnI. [ABSTRACT FROM AUTHOR]

Abstract Most of the multiple sclerosis (MS) patients are initially diagnosed with relapsing remitting multiple sclerosis (RRMS). Th17 cells and macrophages have been shown to play critical roles in pathogenesis of MS and initiation of CNS tissue damage. MiR-301a have recently been exposed as an activator of STAT3 in Th17 cells as well as an activator of NF-κB in macrophages by targeting PIAS3 and NKRF correspondingly. However, the possible role of miR-301a in RRMS has not yet been elucidated. Herewith, for the first time, we have studied the expression of miR-301a, NKRF and PIAS3 by quantitative real-time PCR and western blotting method in peripheral blood mononuclear cells (PBMCs) of 71 RRMS patients, including two groups of patients in relapse phase (n = 44) and a group of remitting phase patients (n = 28) in comparison to healthy volunteers (n = 28). In this work, we demonstrate a significant upregulation of miR-301a in relapse phase of MS patients compared to healthy controls and remitting phase patients (P <.05). Our findings also showed a striking decrease of NKRF and PIAS3 expression in relapse phase patients, in contrast to miR-301a and, NF-κB and STAT3 downstream genes (SKA2 and RORc) (P <.05). Subsequently, using luciferase reporter system we confirmed that miR-301a directly targets the mRNA encoding PIAS3 and NKRF proteins. We also showed that miR-301a increasing expression is correlated with down-regulation of PIAS3 and NKRF expression in RRMS patients. Our findings suggest that miR-301a, PIAS3 and NKRF play crucial roles in RRMS and could be considered as promising therapeutic targets. Graphical abstract MiR-301a in a positive feedback loop promotes persistent NF-κB activation; miR-301a gene is located between exons 1 and 2 of SKA2 gene in the human genome which contains an authentic κB site. NF-κB coordinately up-regulates the miR-301a and SKA2. NKRF mRNA, which encodes a repressor of NF-κB transcriptional activity, is targeted by miR-301a. Consequently, miR-301a in a positive feedback loop mediates induction of persistent NF-κB activation. MiR-301a promotes Th17 cells differentiation; Th17 cells are differentiated from human naïve CD4+ T-cells in IL-6/23-STAT3 pathway. PIAS3 is able to suppress activation of STAT3-induced gene RORc by blocking the DNA-binding activity of STAT3. MiR-301a promotes Th17 cells differentiation through negative regulation of PIAS3 expression. Unlabelled Image Highlights • miR-301a, SKA2 and RORc expression levels are up-regulated at relapse phase of RRMS. • Showing an increase expression of miR-301a, NKRF and PIAS3 expression levels are reduced at relapse phase of RRMS. • miR-301a directly interacts and suppresses NKRF and PIAS3 transcript sequences. • miR-301a is a pathogenic factor in RRMS and can modulate inflammatory responses. [ABSTRACT FROM AUTHOR]

Highlights • We carry out a comparison of likelihood ratios obtained with either a continuous or a discrete method on a published set of DNA mixtures. • We compute likelihood ratios for contributors as well as for non-contributors. • For mixtures with replicates almost the same LR's were found for all contributors. • For mixtures without replicates the information increase for contributors obtained by the continuous method is larger, unless the contributors is one of several minor donors. • False positive rates were very low with both methods. Abstract We present the results of a comparison of likelihood ratios obtained from DNA mixture data, obtained either by a peak height model (EuroForMix) and by a discrete model, using probabilities of dropout and integrating over them (MixKin). We use the mixture data that have been published in [1] and were made publicly available. We show that, for mixtures for which replicate analyses were available in this set, there is almost no difference in weight of evidence, suggesting that the additional information in the peak heights is limited. On the other hand, for mixtures for which only a single replicate is available, the weight of evidence for true donors can be substantially higher with the peak height model, especially so for major donors and also for minor donors provided they are the only minor contributor to the mixture. False positive rates were very low with both methods, but for related non-contributors the risk of a false positive is much higher with the discrete method unless kinship is taken into account in the likelihood ratio calculation. [ABSTRACT FROM AUTHOR]

Highlights • Postmortem swabs transferred to FTA® cards can streamline disaster victim identifications. • Total Body Score (TBS) can be a quick field assessment to evaluate the degree of body decomposition. • No relationship exists between Accumulated Degree Days (ADD) and DNA degradation. • A TBS of 13 and below consistently yielded a full profile regardless of the sampling season or ADD. Abstract In mass fatality events, the need to identify large numbers of deceased persons using DNA can be a significant drain on already overburdened forensic practitioners, both in the field setting and the laboratory. The laboratory may be required to extract DNA from a variety of postmortem sample types, family or direct reference samples related to the missing, and perform matching of these results in a short period of time. While most forensic institutions are well equipped to handle both family and direct reference samples, postmortem samples such as bone or heterogeneous tissue samples can be difficult for labs to analyze. We have devised an easily deployable, efficient, and inexpensive method for collecting postmortem DNA samples on commercially available DNA preservation cards (“FTA®” cards). FTA® cards are already widely used in forensic labs and are convenient for shipping due to their small volume and stability at room temperature. We evaluated the suitability of a protocol involving swabbing of incisions made on cadavers and sample deposition onto FTA® cards over various postmortem intervals and under different environmental conditions. Each trial took place during a different point in the calendar year to evaluate the effects of seasonal weather patterns and temperature on decomposition, DNA yield, and rates of DNA degradation. To further account for the effects of seasonality (temperature and humidity), the progression of body decomposition was recorded following the Total Body Score (TBS) method [1]. DNA degradation was assessed either through STR amplification of 1.2 mm FTA punches or DNA extraction from 3.0 mm punches followed by real-time PCR quantification and STR amplification and genotyping. No consistent relationship was observed between postmortem interval and DNA degradation. Instead, the TBS score, which captures the stage of body decomposition, was shown to correlate well with DNA quantity. A TBS of 15 and below consistently yielded strong partial or full profiles (20 STR loci and Amelogenin using the PowerPlex 21 System) from all individuals from either 1.2 mm or 3.0 mm punches. Transfer of sample swabs to FTA cards is shown to be a simple and effective method for both field and laboratory operations over a range of conditions that can be evaluated by field forensic practitioners based on a body decomposition score. The approach could be beneficially integrated into mass fatality response plans. [ABSTRACT FROM AUTHOR]

Highlights • A new method to determine the shedder status of an individual is presented that is simple to perform. • The use of a DNA staining dye can visualise the presence of cellular material and allow real-time collection of the cellular material to a swab head. • Heavy, intermediate and light shedders were with little variation between marks deposited by an individual’s left and right thumbs. • Correlations can be drawn between the amount of cellular material deposited and the resulting DNA profiles. • By comparing the cellular material in a thumbprint and the resulting DNA profile, an individual’s shedder status can be more accurately determined than previously possible. Abstract All previous examinations of the shedder status of individuals have been based on conclusions inferred from the amount of DNA deposited by donors after they have held an object for a fixed period of time. In all interpretations of shedder status experiments have involved a range uncertainties, especially in regards to results arising from studies carried out in different laboratories. These apply to the efficiency of the swab collecting DNA from the item touched, the amount of DNA left on the swab after attempts to recover it, and the percentage loss of DNA during the lysis and extraction processes. No previous study has attempted to mitigate these uncertainties or verify how much of the DNA deposited was collected through swabbing, how much DNA present on the swab was recovered or how much DNA is lost during the extraction process. We present a study that accurately measures the deposition, collection and amplification of DNA deposited by a range of donors allowing for an accurate determination of the shedder status of individuals. Eleven donors were asked to wash their hands and then deposit a thumbprint onto glass slides by making pressure for 15 seconds 0, 15, 60 and 180 minutes after handwashing. Both left and right thumbs were used and all testing was performed in triplicate. Measurement of the quantity of cellular material deposited on the slides was carried out using DiamondTM Nucleic Acid Dye and fluorescence microscopy on each of 264 thumbprints. Fluorescence microscopy was then used to demonstrate that all the DNA present on the slides was recovered by the swabbing operations and then direct PCR, using the Identifiler™ Plus kit, was used to ensure that none of the DNA present on swabs was lost during DNA profiling. The combination of using a DNA binding dye and direct PCR allowed an accurate means of measuring the extent to which individuals exhibit different extents of shedding. This small study, 11 donors, showed that individuals fell into one of three distinct groups: heavy, intermediate, and light shedders, regardless of the hand used. [ABSTRACT FROM AUTHOR]

The effect of oligomer length on the vibrational mode coupling and energy relaxation mechanisms of AT-rich DNA oligomers in double- and single-stranded conformations has been investigated using two-dimensional infrared spectroscopy. Vibrational coupling of modes of the DNA bases to the symmetric stretching vibration of the backbone phosphate group was observed for oligomers long enough to form duplex-DNA structures. The coupling was lost upon melting of the duplex. No significant effect of oligomer length or DNA secondary structure was found on either the timescale for vibrational relaxation of the base modes or the mechanism, which was consistent with a cascade process from base modes to intermediate modes, some of which are located on the deoxyribose group, and subsequently to the phosphate backbone. The study shows that vibrational coupling between base and backbone requires formation of the double-helix structure while vibrational energy management is an inherent property of the nucleotide. [ABSTRACT FROM AUTHOR]

Radiotherapy has been used for more than a hundred years as a local tumor treatment. The occurrence of systemic antitumor effects manifesting as regression of tumors outside of the irradiated field (abscopal effect) was occasionally observed but deemed too rare and unpredictable to be a therapeutic goal. This has changed with the advent of immunotherapy. Remarkable systemic effects have been observed in patients receiving radiotherapy to control tumors that were progressing during immune checkpoint blockade, stimulating interest in using radiation to overcome primary and acquired cancer resistance to immunotherapy. Here, we review the immunological mechanisms that are responsible for the ability of focal radiation to promote antitumor T cell responses that mediate tumor rejection and, in some cases, result in systemic effects. [ABSTRACT FROM AUTHOR]

In this work, we have successfully designed a novel biosensor based on DNA hybridization for ultrasensitive detection of NOS terminator gene sequences (NOSt). This biosensor was synthesized by connecting single-stranded capture DNA (sDNA)-labeled graphene oxide quantum dots (GOQDs) (QDs-sDNA) as fluorescent probe and graphene oxide (GO) as quencher. The detection principle based on hybridization combinations can occur between QDs-sDNA and complementary target DNA; moreover, QDs-sDNA can bind to GO with significantly higher affinity than QDs-dsDNA. In the absence of complementary target DNA, QDs-sDNA was absorbed onto the surface of GO, and the fluorescence of QDs-sDNA was quenched due to fluorescent resonant energy transfer. In the presence of a complementary target DNA, its hybridization with QDs-sDNA formed QDs-dsDNA, which cannot be adsorbed to the GO surface and this leads to reduced quenching. By comparing the fluorescence intensity of QDs-sDNA and QDs-dsDNA in the presence of GO, we can achieve target DNA detection. Thus, rapid, simple, sensitive, efficient, and eco-friendly detection of NOSt was realized. This biosensor had a detection limit of 0.008 nM and a linear range of 0.05–50 nM. Moreover, this sensor can selectivity detect target DNA compared with random and single-base-mismatched sequences, and was successfully applied to the determination target DNA sequences in biological fluids directly. This sensor can be applied to detect other target DNA sequences by simply changing the types of sDNA coupled to the GOQDs. [ABSTRACT FROM AUTHOR]

Three ruthenium complexes containing a bidentate piq ligand, [(piq)Ru(bpy) 2 ] 2+ ( 1 ), [(piq)Ru(phen) 2 ] 2+ ( 2 ), and [(piq)Ru(DIP) 2 ] 2+ ( 3 ) (piq = phenylisoquinolinate, bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline, DIP = 4,7-diphenyl-1,10-phenanthroline), were prepared. The DNA binding properties of complexes 1 – 3 to double-stranded DNA were studied. The binding of 1 – 3 to calf-thymus DNA (ct-DNA) yielded lower emission intensities than those observed with the corresponding Ru complexes alone. To explore potential interactions of complexes 1 – 3 with lipid-rich organs in live cells, the emission properties of the Ru probes were studied with liposomes. The emission intensities of complexes 1 – 3 were enhanced to similar extents upon interaction with liposomes. The cytotoxic activities of the complexes against MDA-MB-231 and HUVECs were evaluated in vitro. The effects of complexes 1 – 3 on the survival of MDA-MB-231 cells were examined and compared with that of cis -platin. Complexes 2 and 3 were more cytotoxic to cancer cells than cis -platin. Complexes 1 – 3 showed cellular uptakes of 1.1, 10.6, and 76.6%, respectively, indicating that the greatest amount of complex 3 entered the cancer cells. Inhibition of cell migration by complexes 1 – 3 was also evaluated by the wound healing assay. [ABSTRACT FROM AUTHOR]

The detection of transgenic animals in food and environmental samples will require the development of reliable screening assays. The identification of junction regions between genetic elements is the unequivocal way to prove the presence of transgene constructs. However, a sample from the transgenic organism or genetic construct is not always available for analysis. Here, we describe an easy-to-implement method to create junctions of genetic elements that can serve as DNA template or positive control for the development and validation of detection methods. The new methodology was successfully used to test several pairs of PCR primers intended for the detection of the first transgenic animal approved for human consumption (the AquAdvantage salmon). The junction region was created by the PCR amplification of a genetic element (antifreeze protein gene) with a tailed primer containing part of the adjacent element (growth hormone gene), yielding a PCR product with both regions fused. This strategy can be adapted to create junctions of genetic elements in any transgenic organism if reliable sequence data is available. [ABSTRACT FROM AUTHOR]

2-Deoxy- d -ribose-5-phosphate aldolase (DERA) belongs to the family of lyases and can form C–C bonds to generate multiple chiral centers, thus providing an interesting route to produce key chiral compounds. However, several problems, such as low activity and poor stability (poor tolerance to high aldehyde concentrations), limit the practical application of DERA in large-scale production. Many approaches have been introduced to address these issues. Specifically, in the last decade, many new DERAs, with high catalytic activity or excellent aldehyde tolerance, have been cloned from various extremophilic microorganisms. In addition, on the basis of the analysis of the catalytic mechanism of DERA, rational design engineering and computational design have been used to reconstruct DERA enzymes to alter their stability and catalytic activity. [ABSTRACT FROM AUTHOR]

Radioresistance remains the major obstacle to the failure of radiotherapy for ESCC. SOCS6 was first reported in our previous study to enhance the radiosensitivity of ESCC. Recent studies have shown that various materials were applied as radiosensitizer. Nevertheless, these radiosensitizer still suffer from inadequate tumor uptake of radiosensitizers and radiation sickness. The aim of our study was to generate a tumor targeted virus-inspired hollow mesoporous manganese-bismuth (vHMMn-Bi-PEG) loaded with SOCS6 plasmid and to explore the effect for radiosensitization and safety in vitro and in vivo. We hypothesize that SOCS6@vHMMn-Bi-PEG could enhance ESCC radiosensitivity The vHMMn-Bi-PEG were obtained by the traditional hard template synthesis. SEM and TEM were analyzed for the characterization of vHMMn-Bi-PEG. The expression of SOCS6 were detected by RT-PCR and western blot. MTT was detected cell cytotoxicity. Flow cytometry was used to detect the cellular uptake of SOCS6@vHMMn-Bi-PEG. Colony formation and γ-H2AX foci were assessed the radiosensitization effect of the SOCS6@vHMMn-Bi-PEG. The effect of SOCS6@vHMMn-Bi-PEG on radiosensitization and biosafety were analyzed by subcutaneous xenograft models in vivo. By using SEM observation, the vHMMn-Bi-PEG exhibited the rough surface morphology, which was similar to the spike on the surface of natural virus. Subsequently, TEM results exhibited that the building blocks of the interpenetrated ultrasmall MnO 2 /Bi 2 O 3 nanoparticles were constructed to form the bimetallic oxide shells. RT-PCR and western blot results revealed that pCDNA3.1-SOCS6 were successfully loaded into the vHMMn-Bi-PEG. After incubation with vHMMn-Bi-PEG or SOCS6@vHMMn-Bi-PEG, the cell survival rates of TE-10 were greater than 90%, indicating their excellent biocompatibility. Besides, flow cytometry results confirmed that SOCS6@ vHMMn-Bi-PEG could be easily taken up by TE-10 cells. Colony formation results revealed that SOCS6@vHMMn-Bi-PEG (8.90%) was significantly lower than that in the control group (21.90%) and pCDNA3.1-SOCS6 group (18.32%). Moreover, a significantly stronger fluorescence density of γ-H 2 AX foci was found in the SOCS6@vHMMn-Bi-PEG + X-ray than that in the control and pCDNA3.1-SOCS6 + X-ray, indicating the effective DNA breakage induced by the vHMMn -Bi-PEG nano-system. Finally, in vivo results found that SOCS6@vHMMn-Bi-PEG with X-ray exhibited a stronger inhibitory effect on tumor growth compared with the pCDNA3.1-SOCS6 + X-ray and vHMMn-Bi-PEG + X-ray group, indicating that SOCS6@ vHMMn-Bi-PEG has the potential to significantly enhance radiosensitivity. We successfully constructed a novel biomimetic-gene-loaded nano-system SOCS6@vHMMn-Bi-PEG as a radio-sensitizer. Moreover, SOCS6@vHMMn-Bi-PEG exhibited superior antitumor performance of ESCC without systemic toxicity. [ABSTRACT FROM AUTHOR]

Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV–visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 10 3 M − 1 , which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results. [ABSTRACT FROM AUTHOR]

In targeting DNA repair pathways of the most genomic instable cancer, poly-(adenosine diphosphate [ADP])-ribose polymerase inhibitors (PARPi) have been demonstrated as the most effective drug since platinum in high grade serous or endometrioid ovarian cancer. Immunotherapy is strongly pushing the door of ovarian cancer and has the ambition to change the fate of this deadly disease when combined with chemotherapy, vascular endothelial growth factor inhibitor or PARPi. The activity of PARPi could also be improved by modulators of the cell cycle, which are required to give time enough for DNA repair. Even more ambitious are drug targeting the driver p53 mutation or the pathway which inhibit tumor cell apoptosis. Some original approaches still give life to new chemotherapy compounds such as the marine derivative lurbinectedin or the immunoconjugate mirvetuximab soravtansine including a monoclonal antibody targeting the folate receptor. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was to analyse the frequencies of genotypes and alleles of Single Nucleotide Polymorphism (SNP) -2548 G/A (rs12112075) of leptin gene (LEP) and an attempt to evaluate the effect this DNA marker has on endometrial cancer (EC) and uterine leiomyomas (UL).Material and Methods: The study comprised 120 patients treated for endometrial cancer and 90 patients treated for uterine leiomyomas. 90 disease-free individuals were used as controls. In total, 300 patients were investigated in this research.Results: In this paper we have demonstrated that genotype AG of SNP -2548 G/A of LEP may reduce the risk of developing endometrial cancer, whereas allele A itself may be a risk factor of this malignancy. No association was found between the studied polymorphism and uterine leiomyomas.Conclusions: -2548 G/A SNP of LEP may play a significant role in the development of EC, however, uterine leiomyomas are not associated with this DNA marker. [ABSTRACT FROM AUTHOR]

A metformin containing palladium complex, [Pd(Met) 2 ](OAc) 2 ( 1 ), and several cyclopalladated complexes of the general formula [Pd(benzylamine)(Met)](NO 3 ), in which Met is metformin drug and benzylamine is dl -α-methylbenzylamine ( 2 ), N , N -dimethylbenzylamine ( 3 ), N -ethylbenzylamine ( 4 ) and N - t -buthylbenzylamine ( 5 ), have been synthesized and characterized using elemental analysis, FT-IR and NMR spectroscopy. Spectroscopic titrations, displacement experiments, DNA melting and circular dichroism (CD) spectroscopy have been applied in the investigation of complexes ( 1 ), ( 2 ) and free metformin, DNA binding properties. The complexes and free metformin bind to CT-DNA (Calf thymus DNA) efficiently through groove binding and intercalation modes, respectively, according to the results obtained. The corresponding intrinsic binding constants, K b , of the complexes and free metformin are as follows: metformin > complex ( 2 ) > complex ( 1 ). Strong association of complexes ( 1 ), ( 2 ) and free metformin with BSA via a static mechanism with intrinsic binding constants (K b ) in 1.18 × 10 4 –4.81 × 10 4 M −1 concentration range is indicated by absorption and emission measurements. The binding sites of complexes and free metformin on BSA were located at sites I and II of BSA, respectively, as demonstrated by competitive experiments. The in vitro cytotoxic activities of complexes ( 1 ), ( 2 ) and free metformin were evaluated against Hela (cervical cancer), MCF7 (breast cancer) and A549 (lung cancer) cellular lines. [ABSTRACT FROM AUTHOR]

Circulating tumour DNA (ctDNA) has emerged as a promising blood-based biomarker for monitoring disease status of patients with advanced cancers. In melanoma, ctDNA has been shown to have clinical value as an alternative tumour source for the detection clinically targetable mutations for the assessment of response to therapy. This review provides a critical summary of the evidence that gives credence to the utility of ctDNA as a biomarker for monitoring of disease status in advanced melanoma and the steps required for its implementation into clinical settings. [ABSTRACT FROM AUTHOR]

Infectious pancreatic necrosis virus (IPNV) is the etiological agent of a contagious viral disease causing remarkable mortalities in different fish species. Despite the availability of commercial vaccines against IPN, the disease still constitutes one of the main threats to the aquaculture industry worldwide. In this study, we developed a DNA vaccine encoding the VP2 gene of IPNV and evaluated its ability to induce protective immunity in rainbow trout fry (3 g) at doses of 10 and 25 μg/fish and boosting with the same doses two weeks later through the oral route using chitosan/tripolyphosphate (CS-TPP) nanoparticles and alginate microparticles incorporated into fish feed. The distribution of the administered vaccines in different organs and transcription of VP2 gene were confirmed by RT-PCR assay at day 30 post boost-vaccination. Transcript levels of IFN-1, Mx-1, IgM, IgT and CD4 genes was dependent on vaccine dose and was significantly up-regulated in head kidney of all orally vaccinated fish groups compared to controls (pcDNA3.1). Cumulative mortalities post-challenge with virulent isolate of the virus were lower in the vaccinated fish and a relative percentage survival (RPS) of 59% and 82% were obtained for the 10 and 25 μg/fish pcDNA3.1-VP2 groups, respectively. Vaccination with the same amount of pcDNA3.1-VP2 encapsulated with CS-TPP nanoparticles resulted in RPS of 47 %and 70%, respectively. Detectable anti-IPNV antibodies were shown until 90 days postvaccination. The orally administrated vaccines significantly decreased VP4 transcripts thus contributing to reducing viral load in surviving fish on day 45 post-challenge. In conclusion, these results show good to high protection post-vaccination alongside with significant up-regulation of key immune genes and detectable levels of circulating antibodies after oral administration of the DNA vaccine formulated in CS-TPP nanoparticles and alginate microparticles in fish feed. [ABSTRACT FROM AUTHOR]

Several mitochondrial tRNA mutations have been associated with hypertension, but their pathophysiology remains poorly understood. In this report, we identified a novel homoplasmic 3253T3C mutation in the mitochondrial tRNALeu(UUR) gene in a Han Chinese family with maternally inherited hypertension. The m.3253T→C mutation affected a highly conserved uridine at position 22 at the D-stem of tRNALeu(UUR), introducing a G-C base pairing (G13-C22) at the D-stem and a tertiary base pairing (C22-G46) between the D-stem and the variable loop.Wetherefore hypothesized that the m.3253T→C mutation altered both the structure and function of tRNALeu(UUR). Using cytoplasmic hybrid (cybrid) cell lines derived from this Chinese family, we demonstrated that the m.3253T→C mutation perturbed the conformation and stability of tRNALeu(UUR), as suggested by faster electrophoretic mobility of mutated tRNA relative to the wild-type molecule. Northern blot analysis revealed an ∼45% decrease in the steady-state level of tRNALeu(UUR) in the mutant cell lines carrying the m.3253T→C mutation, as compared with control cell lines. Moreover, an ∼35% reduction in aminoacylation efficiency of tRNALeu(UUR) was observed in the m.3253T→C mutant cells. These alterations in tRNALeu(UUR) metabolism impaired mitochondrial translation, especially for those polypeptides with a high proportion of Leu(UUR) codons, such as ND6. Furthermore, we demonstrated that the m.3253T→C mutation decreased the activities of mitochondrial complexes I and V, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in the cells. In conclusion, our findings may provide new insights into the pathophysiology of maternally inherited hypertension. [ABSTRACT FROM AUTHOR]

In humans, nonhomologous DNA end-joining (NHEJ) is the major pathway by which DNA double-strand breaks are repaired. Recognition of each broken DNA end by the DNA repair protein Ku is the first step in NHEJ, followed by the iterative binding of nucleases, DNA polymerases and the XRCC4-DNA ligase IV (X4-LIV) complex in an order influenced by the configuration of the two DNA ends at the break site. The endonuclease Artemis improves joining efficiency by functioning in a complex with DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) that carries out endonucleolytic cleavage of 5' and 3' overhangs. Previously, we observed that X4-LIV alone can stimulate Artemis activity on 3' overhangs, but this DNA-PKcsindependent endonuclease activity of Artemis awaited confirmation. Here, using in vitro nuclease and ligation assays, we find that stimulation of Artemis nuclease activity by X4-LIV and the efficiency of blunt-end ligation are determined by structural configurations at the DNA end. Specifically, X4-LIV stimulated Artemis to cut near the end of 3' overhangs without the involvement of other NHEJ proteins. Of note, this ligase complex is not able to stimulate Artemis activity at hairpins or at 5' overhangs. We also found that X4-LIV and DNA-PKcs interfere with one another with respect to stimulating Artemis activity at 3' overhangs, favoring the view that these NHEJ proteins are sequentially rather than concurrently recruited to DNA ends. These data suggest specific functional and positional relationships among these components that explain genetic and molecular features of NHEJ and V(D)J recombination within cells. [ABSTRACT FROM AUTHOR]

Elucidation of the function of synaptonemal complex (SC) in Saccharomyces cerevisiae has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Consequently, significant gaps remain in the mechanistic understanding of the activities of various SC proteins and the functional relationships among them. S. cerevisiae Hop1 and Red1 are essential structural components of the SC axial/lateral elements. Previous studies have demonstrated that Hop1 is a structure-selective DNA-binding protein exhibiting high affinity for the Holliday junction and promotingDNAbridging, condensation, and pairing between double-stranded DNA molecules. However, the exact mode of action of Red1 remains unclear, although it is known to interact with Hop1 and to suppress the spore viability defects of hop1 mutant alleles. Here, we report the purification and functional characterization of the full-length Red1 protein. Our results revealed that Red1 forms a stable complex with Hop1 in vitro and provided quantitative insights into their physical interactions. Mechanistically, Red1 preferentially associated with the Holliday junction and 3-way junction rather than with single- or double-strandedDNAwith overhangs. Although Hop1 and Red1 exhibited similar binding affinities toward several DNA substrates, the two proteins displayed some significant differences. Notably, Red1, by itself, lacked DNA-pairing ability; however, it potentiated Hop1-promoted intermolecular pairing between double-stranded DNA molecules. Moreover, Red1 exhibited nonhomologousDNAend-joining activity, thus revealing an unexpected role for Red1 in recombination-based DNA repair. Collectively, this study presents the first direct insights into Red1's mode of action and into the mechanism underlying its role in chromosome synapsis and recombination. [ABSTRACT FROM AUTHOR]

5-Bromo-2′-deoxyuridine (BrdU) photosensitizes DNA to strand break formation. However, this type of photodamage is completely quenched by the presence of triethylamine (TEA) which originates from RP-HPLC purification commonly employed by oligonucleotide providers. While the presence of TEA in oligonucleotide samples does not interfere with PCR or other molecular biology applications, the mechanism of photochemical reaction proceeding in the labeled DNA is dramatically changed due to the photoinduced electron transfer (PET) between the photoexcited BrdU and the ground state TEA. For the first time, we demonstrated that the latter process produces 2′-deoxyuridne2′-deoxyuridine (debromination) in the labeled DNA instead of the expected strand break. PET between TEA and BrdU was additionally confirmed by the UV irradiations of aqueous solutions containing both species. Indeed, the efficient formation of 2′-deoxyuridine was observed in the studied photolytes. Moreover, we showed the formation of an additional product in these binary mixtures, i.e. imidazole derivative, that is not formed in DNA and was reported in the literature in the context of dark rather than photochemical processes. Using mass spectrometry we demonstrated that the amount of TEA impurity in the commercial samples of oligos exceeds up to 3 orders of magnitude that of the purchased DNA. [ABSTRACT FROM AUTHOR]

The fidelity of DNA replication relies on three error avoidance mechanisms acting in series: nucleotide selectivity of replicative DNA polymerases, exonucleolytic proofreading, and post-replicative DNA mismatch repair (MMR). MMR defects are well known to be associated with increased cancer incidence. Due to advances in DNA sequencing technologies, the past several years have witnessed a long-predicted discovery of replicative DNA polymerase defects in sporadic and hereditary human cancers. The polymerase mutations preferentially affect conserved amino acid residues in the exonuclease domain and occur in tumors with an extremely high mutation load. Thus, a concept has formed that defective proofreading of replication errors triggers the development of these tumors. Recent studies of the most common DNA polymerase variants, however, suggested that their pathogenicity may be determined by functional alterations other than loss of proofreading. In this review, we summarize our current understanding of the consequences of DNA polymerase mutations in cancers and the mechanisms of their mutator effects. We also discuss likely explanations for a high recurrence of some but not other polymerase variants and new ideas for therapeutic interventions emerging from the mechanistic studies. [ABSTRACT FROM AUTHOR]

DNA double strand breaks need to be repaired in an organized fashion to preserve genomic integrity. In the organization of faithful repair, histone ubiquitination plays a crucial role. Recent findings suggest an integrated model for DNA repair regulation through site-specific histone ubiquitination and crosstalk to other posttranslational modifications. Here we discuss how site-specific histone ubiquitination is achieved on a molecular level and how different multi-protein complexes work together to integrate different histone ubiquitination states. We propose a model where site-specific H2A ubiquitination organizes the spatio-temporal recruitment of DNA repair factors which will ultimately contribute to DNA repair pathway choice between homologous recombination and non-homologous end joining. [ABSTRACT FROM AUTHOR]

DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases. [ABSTRACT FROM AUTHOR]

The Base Excision Repair (BER) pathway, initially studied as a mere DNA repair pathway, has been later found to be implicated in the expression of cancer related genes in human. For several years, this intricate involvement in apparently different processes represented a mystery, which we now are starting to unveil. The BER handles simple alkylation and oxidative lesions arising from both endogenous and exogenous sources, including cancer therapy agents. Surprisingly, BER pathway involvement in transcriptional regulation, immunoglobulin variability and switch recombination, RNA metabolism and nucleolar function is astonishingly consolidating. An emerging evidence in tumor biology is that RNA processing pathways participate in DNA Damage Response (DDR) and that defects in these regulatory connections are associated with genomic instability of cancers. In fact, many BER proteins are associated with those involved in RNA metabolism, ncRNA processing and transcriptional regulation, including within the nucleolus, proving a substantial role of the interactome network in determining their non-canonical functions in tumor cells. Maybe these new insights of BER enzymes, along with their emerging function in RNA-decay, may explain BER essential role in tumor development and chemoresistance and may explain the long-time mystery. Here, we would like to summarize different roles of BER pathway in human cells. First, we will give a short description of the classical BER pathway, which has been covered in detail in recent reviews. We will then outline potential new roles of BER in gene expression and RNA metabolism. Although recent works have provided tremendous amount of data in this field, there are still lot of open questions. [ABSTRACT FROM AUTHOR]

Programmable biomolecules, such as DNA strands, deoxyribozymes, and restriction enzymes, have been used to solve computational problems, construct large-scale logic circuits, and program simple molecular games. Although studies have shown the potential of molecular computing, the capability of computational learning with DNA molecules, i.e., molecular machine learning, has yet to be experimentally verified. Here, we present a novel molecular learning in vitro model in which symmetric internal loops of double-stranded DNA are exploited to measure the differences between training instances, thus enabling the molecules to learn from small errors. The model was evaluated on a data set of twenty dialogue sentences obtained from the television shows Friends and Prison Break . The wet DNA-computing experiments confirmed that the molecular learning machine was able to generalize the dialogue patterns of each show and successfully identify the show from which the sentences originated. The molecular machine learning model described here opens the way for solving machine learning problems in computer science and biology using in vitro molecular computing with the data encoded in DNA molecules. [ABSTRACT FROM AUTHOR]

The skeleton is a metabolically active organ throughout life where specific bone cell activity and paracrine/endocrine factors regulate its morphogenesis and remodeling. In recent years, an increasing number of reports have used multi-omics technologies to characterize subsets of bone biological molecular networks. The skeleton is affected by primary and secondary disease, lifestyle and many drugs. Therefore, to obtain relevant and reliable data from well characterized patient and control cohorts are vital. Here we provide a brief overview of omics studies performed on human bone, of which our own studies performed on trans-iliacal bone biopsies from postmenopausal women with osteoporosis (OP) and healthy controls are among the first and largest. Most other studies have been performed on smaller groups of patients, undergoing hip replacement for osteoarthritis (OA) or fracture, and without healthy controls. The major findings emerging from the combined studies are: 1. Unstressed and stressed bone show profoundly different gene expression reflecting differences in bone turnover and remodeling and 2. Omics analyses comparing healthy/OP and control/OA cohorts reveal characteristic changes in transcriptomics, epigenomics (DNA methylation), proteomics and metabolomics. These studies, together with genome-wide association studies, in vitro observations and transgenic animal models have identified a number of genes and gene products that act via Wnt and other signaling systems and are highly associated to bone density and fracture. Future challenge is to understand the functional interactions between bone-related molecular networks and their significance in OP and OA pathogenesis, and also how the genomic architecture is affected in health and disease. [ABSTRACT FROM AUTHOR]

We characterized molecularly the first freshwater member ever reported for the family Bathycoccaceae in Lake Musters (Argentinean Patagonia). Members of this family are extremely numerous and play a key ecological role in marine systems as primary producers. We cloned a fragment comprising the SSU rRNA gene + ITS region from environmental DNA using specific mamiellophyte primers. The unique SSU rRNA gene sequence obtained clustered robustly with Bathycoccus prasinos . Analysis of the two-dimensional structure of the ITS region showed the presence of a typical supplementary helix in the ITS-2 region, a synapomorphy of Bathycoccaceae, which confirmed further its phylogenetic placement. We finally discuss the possible causes for the presence of this organism in Lake Musters. [ABSTRACT FROM AUTHOR]

Bacterial RNA polymerase (RNAP) requires σ factors to recognize promoter sequences. Domain 1.1 of primary σ factors (σ1.1) prevents their binding to promoter DNA in the absence of RNAP, and when in complex with RNAP, it occupies the DNA-binding channel of RNAP. Currently, two 3D structures of σ1.1 are available: from Escherichia coli in complex with RNAP and from T. maritima solved free in solution. However, these two structures significantly differ, and it is unclear whether this difference is due to an altered conformation upon RNAP binding or to differences in intrinsic properties between the proteins from these two distantly related species. Here, we report the solution structure of σ1.1 from the Gram-positive bacterium Bacillus subtilis. We found that B. subtilis σ1.1 is highly compact because of additional stabilization not present in σ1.1 from the other two species and that it is more similar to E. coli σ1.1. Moreover, modeling studies suggested that B. subtilis σ1.1 requires minimal conformational changes for accommodating RNAP in the DNA channel, whereas T. maritima σ1.1 must be rearranged to fit therein. Thus, the mesophilic species B. subtilis and E. coli share the same σ1.1 fold, whereas the fold of σ1.1 from the thermophile T. maritima is distinctly different. Finally, we describe an intriguing similarity between σ1.1 and δ, an RNAP-associated protein in B. subtilis, bearing implications for the so-far unknown binding site of δ on RNAP. In conclusion, our results shed light on the conformational changes of σ1.1 required for its accommodation within bacterial RNAP. [ABSTRACT FROM AUTHOR]

Non-viral gene therapy and DNA vaccination currently hold great potential for treatment and prevention of genetic and acquired diseases. The large-scale manufacturing of plasmid DNA (pDNA) vectors, and the downstream processing in particular, is one of the key aspects of process development required to move non-viral gene therapy to the clinic. To address the problematic isolation and purification of pDNA molecules, an alternative and cost effective process based on multimodal chromatography was developed. In particular, the possibility of using a cationic multimodal ligand (Capto™ adhere) to remove RNA impurities from E. coli pre-purified lysates and to isolate supercoiled (sc) pDNA isoforms from open circular (oc) pDNA was explored. The process involves E. coli culture, cell harvesting and alkaline lysis followed by isopropanol, ammonium acetate and PEG-8000 precipitation. Finally, sc pDNA is isolated by multimodal chromatography using a stepwise NaCl elution method that includes washing of unbound oc pDNA at 830 mM NaCl, sc pDNA elution at 920 mM and removal of RNA at 2 M. The method provided baseline separation of isoforms and yielded sc-rich fractions (>90%) that are virtually free from RNA and have levels of gDNA (1.3 ± 0.3% µg gDNA/µg sc pDNA) and protein (4.97 ± 1.34 µg/mL) impurities within specifications. Approximately 376 μg of sc pDNA were obtained from 200 mL of bacterial cell culture (OD 600nm ≈ 19). The process was reproducible and performed similarly with differently sized plasmids (2686 bp, 3696 bp and 10,410 bp). [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a molecular technique to determine the level of human originated DNA contamination in unhygienic food products. In the study, four model foods were prepared under both hygienic (H) and non-hygienic (NH) conditions and the human originated microbial loads of these products were determined. DNA was extracted from the model foods and human buccal samples by GIDAGEN Multi-fast DNA isolation kit. A primer specific region of human mitochondrial D-Loop was designed. The level of human DNA contamination in the model foods was determined by real-time PCR. The sensitivity of the technique developed here was 0.00001 ng DNA/PCR. In addition, the applicability of the traceable molecular hygiene control method (TMHCM) was tested in 60 food samples from the market. The results of this study demonstrate that DNA based TMHCM can be used to predict to what extent foods meet the human oriented hygienic conditions. [ABSTRACT FROM AUTHOR]

Experimental studies on single molecules of DNA have reported a rich variety of structural transitions, including coexistence of three phases, in a torsionally constrained molecule. A comprehensive knowledge of these structural transitions is useful for unraveling the in vivo and in vitro behavior of DNA. Our objective is to understand the structural transitions in a torsionally constrained DNA molecule when it is pulled using optical or magnetic tweezers. We use foundational concepts from the Zimm-Bragg helix-coil transition theory and merge them with ideas from the theory of fluctuating elastic rods to model the mechanics of DNA. We also account for the electrostatic interactions between the ions and the negatively charged phosphate backbone of DNA. Using our model, we calculate the force and torque corresponding to the overstretching transition characterized by a 70% jump in the contour length of the molecule and examine the effect of salt concentration on this transition. We also deduce conditions under which the co-existence of B-, S- and P-DNA is possible. We examine how the cooperativity parameter for each transition affects the force-extension curve or torque-rotation curve. We attempt to rationalize the non-monotonic dependence of external work done on the ion concentration by connecting it to the electrostatic dependence of the interfacial energy between two phases of DNA. Our theoretical results are in agreement with multiple experiments documented in the literature and they generate falsifiable predictions that can be tested in new experiments. Statement of significance The overarching objective of this paper is to explore the implications of variation in ion concentration on the structural transitions driven by external forces in a torsionally constrained DNA molecule. A comprehensive understanding of the phase behavior of torsionally constrained DNA is useful because DNA in cells is tightly packaged and is acted upon by molecular machines in different ionic environments. We examine the mechanics of the overstretching transition, characterized by a 70% jump in contour length, wherein a mixture of B- and S-DNA converts into a mixture of S- and P-DNA through a triple point in the phase diagram. Our results are corroborated by experimental data at every step and we make predictions that are experimentally verifiable. [ABSTRACT FROM AUTHOR]

Circularized oligonucleotides, or coligos, were previously found to serve as RNA polymerase III (Pol III) templates in vitro and in human tissue culture cells. Here we randomized the 12-nucleotide larger loop (L-loop) of a well characterized coligo and found unexpectedly that in vitro transcription by FLAG-Pol III was not significantly affected. This observation allowed us to test the variable of coligo L-loop size separately from the variable of its sequence. Transcription efficiency increased with L-loop size from 3 to 12 nucleotides of randomized sequence, and the smallest loop forced initiation to move into the stem region. To test further the need for any specific sequence we compared seven nucleotide L-loops composed of random, abasic and abasic-acyclic nucleotides, and all supported transcription by Pol III. Transcription of a series of coligos containing twelve contiguous randomized nucleotides placed at different locations within the coligo structure provided further evidence that the stem-loop junction structure is important for precise initiation. Nearly the same transcript pattern was formed in vitro by Pol III from yeast and human cells. Overall, these experiments support structure, rather than L-loop sequence, as the major determinant of coligo transcription initiation by Pol III. [ABSTRACT FROM AUTHOR]

The A- and B-form are two of the most common structural conformations of double strand DNA present in nature and they can interchange on the basis of the helices hydration [1,2]. Herein we demonstrate that the use of non-destructive techniques such as Raman spectroscopy coupled with the use of a super-hydrophobic device, allows the clear identification of the DNA hydration state, of the backbone (phosphate + deoxyribose sugar) conformation and of the nucleotides. There is a wide prospect for an increase of knowledge in biomolecules using this combined approach resulting in a significant impact in the study of more complex supramolecular assemblies and of fine chemical variation along the genomic loci undergoing to epigenetic variations. [ABSTRACT FROM AUTHOR]

Background The most common genetic cause of neurological disorders ALS and FTD is a largely increased number of GGGGCC repeats in C9orf72 gene. Non-canonical structures including G-quadruplexes adopted by expanded repeats are hypothesized to be crucial in pathogenesis. Recently, we have shown that structural polymorphism of oligonucleotide d(G 4 C 2 ) 3 G 4 is reduced by dG to 8Br-dG substitution. High-resolution structure of one of the two major G-quadruplexes adopts antiparallel topology comprising of four G-quartets. Herein, we describe the topology of the second major G-quadruplex structure and influence of folding conditions on relative populations of the two folds. Methods Influence of folding conditions was explored by 1H 1 D NMR. Determination of topology was achieved by 2D NMR complemented with PAGE and CD. UV melting experiment was used to explore thermal stability of structures. Results Two structures adopted by oligonucleotide d(G 4 C 2 ) 3 GG Br GG denoted AQU and NAN coexist in solution and ratio of their populations is determined by pH and rate of cooling when folding from thermally denatured state in the presence of K + ions. Conclusions AQU is kinetically favored and forms by folding at low pH, while NAN is favored thermodynamically and at neutral pH. AQU and NAN share similar antiparallel topology with four G-quartets and three edgewise loops, however they exhibit distinct structural and dynamic properties. General significance Novel G-quadruplex topology adds insight into diverse polymorphism of DNA sequences comprising potentially pathological GGGGCC repeat. Relative populations of the two structures and their dependence on folding conditions contribute to understanding of factors that govern G-quadruplex folding. This article is part of a Special Issue entitled "Gquadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. [ABSTRACT FROM AUTHOR]

Biomedical application of selenium nanoparticles (SeNPs) demands the eco-friendly composite for synthesis of SeNPs. The present study reports an aqueous extract of Allium sativum ( AqEAS ) plug-up the current need. Modern spectroscopic, microscopic and gravimetric techniques were employed to characterize the synthesized nanoparticles. Characterization studies revealed the formation of crystalline spherical shaped SeNPs. FTIR spectrum brings out the presence of different functional groups in AqEAS , which influence the SeNPs formation and stabilization. Furthermore the different aspects of the interaction between SeNPs and CT-DNA were scrutinized by various spectroscopic and cyclic voltametric studies. The results reveals the intercalation and groove binding mode of interaction of SeNPs with stacked base pair of CT-DNA. The Stern–Volmer quenching constant ( K SV ) were found to be 7.02 × 10 6 Mˉ 1 (ethidium bromide), 4.22 × 10 6 Mˉ 1 (acridine orange) and 7.6 × 10 6 Mˉ 1 (Hoechst) indicating strong binding of SeNPs with CT–DNA. The SeNPs - CT-DNA interactions were directly visualized by atomic force microscopy. The present study unveils the cost effective, innocuous, highly stable SeNPs intricate mechanism of DNA interaction, which will be a milestone in DNA targeted chemotherapy. [ABSTRACT FROM AUTHOR]

Oxidative stress is considered as one of the significant causes of DNA damage which in turn contributes to cell death through a series of intermediate processes such as cancer formation, mutation, and aging. Natural sources such as plant and fruit products have provided us with interesting substances of antioxidant activity that could be recruited in protecting the genetic materials of the cells. This study is an effort to discover some of those antioxidants effects in their standard and natural forms by performing an ultrasensitive determination of the products of DNA oxidation using GC–MS/MS. Experiments were used to determine the direct antioxidant activity of the substances contained in the tendrils of Vitis vinifera (var. alphonse) by extracting them and achieving Folin-Ciocalteau and CHROMAC analyses to determine the total phenolic content (TPC) and the antioxidant capacity of the extract, respectively; results revealed a phenolic content of 11.39 ± 0.30 mg Gallic Acid Equivalent (GAE)/g of the plant’s fresh weight (FW) by Folin-Ciocalteau and 8.17 ± 0.49 mg Trolox Equivalent (TE)/g FW by CHROMAC assays. The qualitative analysis of the plant extract by HPLC–DAD technique revealed that two flavonoid glycosides namely rutin and isoquercitrin in addition to chlorogenic acid were contained in the extract. The determination of the DNA oxidation products was performed after putting DNA, rutin and isoquercitrin standard samples with different concentration, and the extract's sample under oxidative stress. Eighteen DNA oxidation products were traced using GC–MS/MS with ultra-sensitivity and the experiments proved a significant decrease in the concentration of the DNA oxidation products when the extract was used as a protectant against the oxidative stress. It is believed by conclusion that the extract of V. vinifera ’s (var. alphonse) tendrils has a good antioxidant activity; hence it is recommended to be used as a part of the daily healthy food list if possible. [ABSTRACT FROM AUTHOR]

Copper is one of the predominant components of pesticides employed in agriculture and known to be highly toxic once it reaches aquatic organisms. The impact of sublethal concentrations of this metal on wild insects is not yet completely understood. Studies addressing alterations in different levels of gene expression are still lacking. We previously demonstrated that in a wild population of Chironomus riparius , HSP and CYP families of genes were up-regulated at the transcriptional level after copper exposure. Here, we analyse the impact of copper at the genomic, translational and protein functional level, obtaining a comprehensive picture of the molecular reply to this metal. We studied genotoxicity in C. riparius larvae by Comet Assay, the translational response by polysomal profiling and the detoxification capacity by the CYP450 enzymes activity. Fourth-instar larvae from a mountain stream polluted by agricultural land run-off (NE-Italy) were exposed for 3 h copper concentrations ≤ LC 50 . We report DNA damage induced by copper, even at sublethal levels, as demonstrated by significant increases in all the comet parameters at concentrations ≥1 mg L −1 . By estimating the transcript-specific translational efficiency, we observe a specific up-regulation of CYP4G . Furthermore, the enzymatic activity of CYP450 enzymes is increased at all sublethal copper concentrations, confirming the role of this protein family in the detoxification processes. Surprisingly, the HSP transcripts are up-regulated at the transcriptional level, but these changes are buffered at the translational level suggesting the existence of still unknown post-transcriptional controls that may be connected to survival processes. [ABSTRACT FROM AUTHOR]

Nanoscale biosensors, owing to their high-sensitivity and extremely low limits-of-detection, have enabled the realization of highly complex and sophisticated miniaturized platforms for several important healthcare applications, the most predominant one being disease diagnosis. In particular, nanomaterial facilitated electrochemical detection of DNA hybridization has had an exceptional impact on fields such as genetics and cancerous mutation detection Here we report an ultrasensitive electrochemical platform using electrospun semi-conducting Manganese (III) Oxide (Mn 2 O 3 ) nanofibers for DNA Hybridization detection. The proposed platform coalesces the inherent advantages of metal-oxide nanofibers and electrochemical transduction techniques, resulting in label-free zeptomolar detection of DNA hybridization. As proof of concept, we demonstrate zeptomolar detection of Dengue consensus primer (limit of detection: 120×10 –21 M) both in control as well as spiked serum samples. Our reported detection limit is superior in comparison with previously reported electrochemical DNA hybridization sensors for Dengue virus detection, spanning both labeled and label-free transductions. This ultra-sensitivity, we believe, is a result of synthesizing a low bandgap electrospun metal-oxide nanomaterial corresponding to a specific oxidation state of Manganese. This methodology can be extended for detection of any hybridization of interest by simply adapting an appropriate functionalization protocol and thus is very generic in nature. [ABSTRACT FROM AUTHOR]

In this study, we tried coupling the small signal molecules that could intercalate into DNA double helix with hybridization chain reaction (HCR) technique to fabricate a multiple immunosensor. Doxorubicin hydrochloride (DXH) and methylene blue (MB) were used as signal molecules and alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) were selected as model biomarkers. The immunosensor mainly consists of three parts as follows: First, two different primary antibodies (Ab 1 ) immobilized on the surface of gold nanoparticles (Au NPs); Second, secondary antibodies (Ab 2 ) conjugated with DNA primer; Third, long DNA concatemers from HCR were used as a carrier to intercalate amounts of signal molecules (DXH or MB). A sandwich immunocomplex was formed among primary antibodies, target biomarkers and secondary antibodies conjugated with DNA primer via specific recognition reaction. Afterwards, DNA concatemers intercalating amounts of DXH or MB were linked to DNA primer via DNA hybridization. Square wave voltammetry (SWV) was employed to record the response signals from electroactive molecules DXH and MB, and two distinguishable signals were obtained, which peak potentials were at about −0.30 V (corresponding to MB) and −0.70 V (corresponding to DXH, both vs SCE), respectively. The signal intensities of MB and DXH were linearly related to the logarithm of biomarkers concentration in the range of 0.05 pg mL −1 –25 ng mL −1 , and the limit of detection were 0.03 pg mL −1 for CEA and 0.02 pg mL −1 for AFP (at S/N=3), respectively. Furthermore, the immunosensor exhibited a sensitive electrochemical response to biomarkers in human serum samples and the results obtained were in accordance with reference method, indicating the immunosensor can be applied to real sample analysis in clinic diagnosis. [ABSTRACT FROM AUTHOR]