DNA Phosphorus Research Articles

DNA mapping in the capsid of giant bacteriophage phiEL (Caudovirales: Myoviridae: Elvirus) by analytical electron microscopy

Giant phiKZ-like bacteriophages have a unique protein formation inside the capsid, an inner body (IB) with supercoiled DNA molecule wrapped around it. Standard cryo-electron microscopy (cryo-EM) approaches do not allow to distinguish this structure from the surrounding nucleic acid of the phage. We previously developed an analytical approach to visualize protein-DNA complexes on Escherichia coli bacterial cell slices using the chemical element phosphorus as a marker. In the study presented, we adapted this technique for much smaller objects, namely the capsids of phiKZ-like bacteriophages. Following electron microscopy techniques were used in the study: analytical (AEM) (electron energy loss spectroscopy, EELS), and cryo-EM (images of samples subjected to low and high dose of electron irradiation were compared). We studied DNA packaging inside the capsids of giant bacteriophages phiEL from the Myoviridae family that infect Pseudomonas aeruginosa. Phosphorus distribution maps were obtained, showing an asymmetrical arrangement of DNA inside the capsid. We developed and applied an IB imaging technique using a high angle dark-field detector (HAADF) and the STEM-EELS analytical approach. Phosphorus mapping by EELS and cryo-electron microscopy revealed a protein formation as IB within the phage phiEL capsid. The size of IB was estimated using theoretical calculations. The developed technique can be applied to study the distribution of phosphorus in other DNA- or RNA-containing viruses at relatively low concentrations of the element sought.

Vitamin D3 Supplementation Effects on Spermatogram and Oxidative Stress Biomarkers in Asthenozoospermia Infertile Men: a Randomized, Triple-Blind, Placebo-Controlled Clinical Trial.

The objective is to evaluate the effects of vitamin D3 (VD3) on sperm parameters and seminal and serum oxidative stress (OS) biomarkers in asthenozoospermia infertile men. This randomized, triple-masking, placebo-controlled clinical trial conducted on 86 asthenozoospermia infertile men with serum 25 hydroxy vitamin D3 (25-OH-D3) < 30ng/ml in the infertility clinic of Ahvaz Jahad Daneshgahi, Iran. Patients were randomly allocated to groups A and B, who received daily 4000IU vitamin D3 (VD3) and matching placebo respectively for 3months. Demographic data, dietary intake, physical activity, sun exposure, anthropometric indices, serum and seminal levels of MDA (Malondialdehyde), 8-hydroxy-2- Dioxy Guanosine (8-OHDG), total antioxidant capacity (TAC) and calcium, sperm DNA fragmentation index (DFI), serum 25-OH-D3, parathyroid hormone (PTH), phosphorus, and sperm parameters were assessed. VD3 supplementation had no significant effects on body weight, body mass index (BMI), waist circumference (WC), body fat (BF), 8-OHDG, DFI, semen volume, sperm count, and normal sperm morphology, but increased post-intervention mean and mean change of serum 25-OH-D3 (P < 0.001, P < 0.001), PTH (P < 0.001, P < 0.001) and phosphorus (P = 0.009, P = 0.049) and seminal calcium (P = 0.035, P = 0.038) and serum calcium (P = 0.008, P = 0.009), seminal TAC (P < 0.001, P < 0.001), and serum TAC (P = 0.007, P = 005), total sperm motility (P < 0.001, P < 0.001) and progressive sperm motility (P < 0.001, P < 0.001) and decreased seminal MDA (P = 0.017, P = 0.004) and serum MDA (P = 006, P = 0.005) significantly compared to the baseline and placebo group respectively. VD3 supplementation may modulate OS and affect sperm motility in men with asthenozoospermia and serum 25-OH-D3 < 30ng/ml. Iran Clinical Trials Registry, ID: IRCT20151128025274N4, registered on 28 March 2018, URL of trial registry record: https://www.irct.ir/trial/29983.

Вплив Дерозалу на обмін азотистих та фосфорних сполук у печінці і м’язовій тканині курей за умов хронічного експерименту

Pesticides are toxic chemicals which are widely used as an effective means against pests and plant diseases as well as for animals’ protection against ectoparasites. They are also used to control rodents which transmit infectious diseases to people and animals (encephalitis, malaria, leptospirosis, rabies, listeriosis, etc.). Biosynthesis of protein in tissues depends on many circumstances and, first of all, on the complete set of amino acids, the state of enzymes’ system and on the presence of stimulators and inhibitors of synthesis reaction. Xenobiotics including benzimidazole derivatives are a part of these compositions. In Ukraine there are more than 20 medicines based on carbendazim. Thus, the analysis of the its influence on protein metabolism is relevant. In the research the hens breed Rhode Island of the 38th line for egg and meat of 30 days of age, with the mass 1000-1200 gr. was used. They were divided into 2 groups - test (n=36) and 1 control (n=18). Нens were equally cared and fed with two food allowances: the first part up to 40 days of age and the second part from 40 days of age up to the end of the experiment (60 days). Every day throughout 60 days hens of the first and second testing groups received feed with the medicine Derosal (BAYER, Germany) in the form of 50 % aqueous suspension in dose of 90 and 900 mg. per 1 kg of body mass (0.01 and 0.1 LD50 for hens) accordingly. The control group of hens was fed without addition of the medicine. The research was carried out on the 30th and 60th day. During the experiment it was found that addition of carbendazim to fodder slowed down the use of its nutrients. This situation was characterized by the fact that on the 30th and 60th day of the research the decrease of body mass gaining up to 10 % occurred, the decrease of protein and non-protein nitrogen content in liver and muscles in all the hens’ groups occurred. The increase of concentration of DNA and RNA phosphorus, the content of acid-soluble phosphate, phosphorus of phospholipid as well as lipoid phosphorus that indicates the slight deceleration of protein synthesis were determined, DNA and RNA in liver and hens’ muscles, also indicated the stimulation of synthesis of phosphorus acid ether and the reaction of glycolysis under the conditions of pesticide adding.&nbsp

Bioavailability of organic phosphorus in an eutrophic lake: Insights from an in-situ experiment

Abstract The bioavailability of organic phosphorus (Porg) in surface sediments of an eutrophic lake was studied in an in-situ experiment. On the temporal scale, soluble reactive phosphorus (SRP) in the surface water showed an initial decrease and then increased in western and middle parts of the lake. In contrast, the SRP remained about 0.22 mg L−1 throughout the year in the eastern area of the lake. Four Porg compounds, phosphonate, orthophosphate monoesters, phospholipids and deoxyribonucleic acid phosphorus, were detected using solution 31P nuclear magnetic resonance spectroscopy. Orthophosphate monoesters were the dominant Porg in the sediment extracts, higher than the orthophosphate diesters deoxyribonucleic acid phosphorus and phospholipids. The SRP and Porg showed obvious seasonal variations in different lake regions. The surface water SRP had a negative relationship with sedimentary Porg in the western and middle lake regions. From spring to autumn, SRP in the water column decreased while Porg in the surface sediment increased; from autumn to winter, SRP in the water column increased and Porg in the surface sediment decreased. According to aquatic metabolism, we inferred that excessive external phosphorus input was the “trigger” and the aquatic plants were the “pump” for Porg biogeochemical cycling in the shallow lake. Therefore, appropriate human intervention, such as the harvest of aquatic plants, is vital for eutrophication control in freshwater ecosystems.

Ultrasound-enhanced gene delivery to alfalfa cells by hPAMAM dendrimer nanoparticles.

Cationic polyamidoamine (PAMAM) dendrimers are highly branched nanoparticles with unique molecular properties, which make them promising nanocarriers for gene delivery into cells. This research evaluated the ability of hyperbranched PAMAM (hPAMAM)-G2 with a diethylenetriamine core to interact with DNA, its protection from ultrasonic damage, and delivery to alfalfa cells. Additionally, the effects of ultrasound on the efficacy of hPAMAM-G2 for the delivery and expression of the gus A gene in the alfalfa cells were investigated. The electrophoresis retardation of plasmid DNA occurred at an N/P ratio (where N is the number of hPAMAM nitrogen atoms and P is the number of DNA phosphorus atoms) of 3 and above, and hPAMAM-G2 dendrimers completely immobilized the DNA at an N/P ratio of 4. The analysis of the DNA dissociated from the dendriplexes revealed a partial protection of the DNA from ultrasound damage at N/P ratios lower than 2, and with increasing N/P ratios, the DNA was better protected. Sonication of the alfalfa cells in the presence of ssDNA-FITC-hPAMAM increased the ssDNA delivery efficiency to 36%, which was significantly higher than that of ssDNA-FITC-hPAMAM without sonication. Additionally, the efficiency of transfection and the expression of the gus A gene were dependent on the N/P ratio and the highest efficiency (1.4%) was achieved at an N/P ratio of 10. The combination of 120 s of ultrasound and hPAMAM-DNA increased the gusA gene transfection and expression to 3.86%.

Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.

Development of a Novel Coarse-Grained Model of the Nucleosome Core Particle for Long-Timescale Simulations

The Nucleosome Core Particle (NCP) is the fundamental unit of DNA packaging in eukaryotic cells. It consists of ∼147 base pairs of DNA that are wrapped around an octameric protein core. Despite extensive experimental and theoretical work, the detailed physical mechanisms of important processes such as nucleosome opening, NCP-NCP interactions, and chromatin folding are not fully understood. Atomistic molecular dynamics (MD) simulations can in theory be a useful method to study these processes, however the timescales accessible to MD simulations are too short to explore these mechanisms. Therefore, we have used all-atom MD simulation data to derive a new Coarse-Grained (CG) model of the NCP. In this model, NCP residues are modeled using spherical beads centered on the DNA phosphorus and protein α-carbon atoms. DNA is represented by two types of beads that distinguish between base pairs (i.e. A-T and C-G), whereas protein residues are divided into two types based on their structure (i.e. α-helices and coils/loops). Force field parameters were derived using a hybrid approach of the iterative Boltzmann inversion method for bonded parameters, and Force Matching for non-bonded parameters. Our CG model significantly speeds up NCP simulations, and is expected to be useful in examining nucleosome opening, NCP-NCP interactions, and chromatin folding. Moreover, it is capable of reproducing the structural properties of the underlying atomistic system.

Beyond the fringe: when science moves from innovative to nonsense

Microbiology has experienced examples of highly productive researchers who have gone beyond just interpreting their experimental results with hypotheses and published nonsense that was readily recognized as such by readers. Although the most discussed cases of this pathology come from physics, studies of single-celled microorganisms, virology, and immunology have provided many examples. Five cases are described here along with some generalizations. These are the Lamarckian inheritance of acquired characteristics reported by distinguished and experienced researchers, vectorless DNA transfer and incorporation of bacterial DNA into chromosomes of plants years before vector construction of genetically modified plants was invented, water with memory of immunoglobulin IgE, a new electromagnetic radiation method for identifying bacterial and viral pathogens by the discoverer of human immunodeficiency virus, and the claim of isolation of a new bacterial isolate with arsenic replacing phosphorus in DNA. These examples represent very dissimilar areas, and the only common factor is hubris on the part of experienced researchers. Secondarily, failure of peer review sometimes happens, and journal editors do not step in, sometimes even when alerted before publication. These failures of the publishing process teach us that unnecessary mistakes occur and should warn us all to watch our own enthusiasms.

Rosie Redfield

Rosie Redfield

Discoverer Asks for Time, Patience Over Arsenic Bacteria Controversy

Three weeks ago, NASA astrobiology fellow Felisa Wolfe-Simon published a paper in Science about bacteria that can use arsenic instead of phosphorus in DNA and other biomolecules. Then came a torrent of criticism that she and her co-authors declined to respond to. Last week, Wolfe-Simon agreed to share some of her thoughts in an interview with Science 's news department.

Quantification of phosphorus in DNA using capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry

We have analyzed phosphorus in an enzymatically digested DNA molecule using capillary electrophoresis (CE) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). The DNA concentration was quantified by the phosphorus value obtained in the CE-ICP-MS analysis. The CE-ICP-MS measurement, for which the interface device AIF-01 equipped three layered nebulizer was adopted, was achieved with limited μL/min nebulizing without loss of sample in the vaporizing chamber. The samples of nucleotides and free phosphate were separated well in the CE-ICP-MS measurement, and the calibration curve (0.1–10 μg/mL) of the phosphorus showed a linear ( R 2 = 0.999) increase in intensity. After digestion of the 100-bp double-strand DNA sample to deoxyribonucleotide-5′-monophosphates (dNMPs) by phosphodiesterase-I, phosphorus was detected by CE-ICP-MS without further purification steps. In this study, we applied two calculation schemes of DNA analysis using a dNMP concentration obtained from CE-ICP-MS. Comparative CE-ICP-MS analysis with DNA digested to dNMPs showed that the assay gave an equal value obtained from the total DNA quantification using fluorescence detection. The detection limits of the DNA sample obtained from these species and phosphorus in nucleotides using CE-ICP-MS were 3.1–26 ng/mL. These LOD values were equal to the conventional fluorescence determination of DNA.

A strategy for establishing accurate quantitation standards of oligonucleotides: quantitation of phosphorus of DNA phosphodiester bonds using inductively coupled plasma–optical emission spectroscopy

A novel approach for quantitation of DNA (oligonucleotides) with an unprecedented accuracy of approximately 1% is reported. Quantitation of DNA is commonly performed by measuring UV absorption or fluorescence from dyes intercalated into DNA. Both methods need accurate quantitation standards to yield more comparable results between laboratories. For establishing technically authentic standards for DNA quantitation, a new measurement approach carrying an inherent capability of absolute quantitation is demanded. The proposed approach is based on the stoichiometric existence of phosphorus (P) in DNA. The quantity of P from the phosphodiester backbone of a purified oligonucleotide was accurately determined using inductively coupled plasma–optical emission spectroscopy (ICP–OES) with yttrium internal standard via acid digestion. The number of moles of oligonucleotides was then calculated from that of P using the stoichiometry. The major issues regarding the validity of the suggested approach were (i) effective removal of extra P sources, (ii) quantitative recovery of P through the digestion process, and (iii) oligomeric purity of the target oligonucleotide. These issues were investigated experimentally using various analytical techniques such as matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI–TOF MS), capillary electrophoresis, electrical conductometry, UV spectrometry, and gravimetry. In conclusion, it is feasible to certify pure oligonucleotide reference materials with uncertainties less than 1% using the proposed approach.

Effects of ecdysteroids on Chlorella vulgaris

The effects of three ecdysteroids, 20‐hydroxyecdysone (20E), 2‐deoxy‐20‐hydroxyecdysone (2d20E) and 20‐hydroxyecdysone 22‐acetate (20E22Ac), on growth and the levels of cellular components in Chlorella vulgaris Beijerinck (Trebouxiophyceae) are reported and compared with data previously reported for ecdysone (E; Bajguz A and Koronka A, Plant Physiol Biochem 39: 707–715, 2001). All three 20‐hydroxyecdysteroids stimulate growth of C. vulgaris cells over a wide concentration range (10−16 to 10−7 M). Optimal stimulation is observed at 10−9 M with each ecdysteroid. High concentrations (&gt;10−6 M) are cytotoxic. The potency ranking of the ecdysteroids is 20E &gt; 20E22Ac &gt; 2d20E &gt; E. Levels per cell of DNA, RNA, protein, sugars, organic and inorganic phosphorus, chlorophylls a and b and phaeophytins a and b are all stimulated by ecdysteroid treatment when compared with the untreated control cells. Possible modes of action of ecdysteroids on C. vulgaris cells are discussed.

Reparability of lethal lesions produced by phosphorus photoabsorption in yeast cells.

The characteristics of DNA lesions produced by the photoabsorption of phosphorus in yeast cells were studied using monochromatized soft X-rays tuned to the absorption peak of the phosphorus K-edge (2153 eV) and below the peak energy (2147 eV). The repaired fractions of DNA double-strand breaks (dsb) were measured relatively by using both a mutant, rad 54-3, which shows the temperature-sensitive dsb repair-deficient phenotype, and a wild-type strain. The repaired fraction of lesion in rad 54-3, which corresponds to the relative yield of dsb reparable by the RAD 54 pathway, was not affected by the phosphorus photoabsorption. Repair of the produced lesions in the wild-type cells was also measured by comparing the surviving fraction of the immediately plated cells to that of those cells plated after holding in a non-nutrient medium for 80 hrs. The recovery of the surviving fraction after the holding treatment was dependent upon the irradiated X-ray energy. These results suggest that irreparable lesions are produced by the inner-shell photoabsorption of phosphorus in DNA, although its yield is small.

Analysis of biosensor chips for identification of nucleic acids.

Two novel DNA-sequencing methods are described that use DNA hybridization biosensor chips. These two techniques involve either labeling the free nucleic acid with enriched stable isotopes or hybridizing DNA without labels to immobilized peptide nucleic acid (PNA) and detecting the phosphorus present in the DNA but not in the PNA. Sputter-initiated resonance ionization microprobe analysis was used to detect the presence of enriched tin isotope-labeled DNA and of phosphorus in natural DNA as a means to identify the presence of DNA after hybridization to oligodeoxynucleotides (ODNs) or PNAs, respectively, immobilized on a biosensor chip. The data clearly demonstrate that excellent discrimination between complementary and noncomplementary sequences can be obtained during hybridization of DNA to either ODNs or PNAs. The capability to detect different enriched stable isotope-labeled DNAs simultaneously allows high degrees of multiplexing which may be very advantageous for hybridization kinetics studies in complex systems, as well as significantly increasing the speed of analysis. Alternatively, by using natural DNA with PNA biosensor chips, discrimination for single-point mutation could be increased because of improved hybridization kinetics and direct analysis of genomic DNA may become possible without amplification. Both methods have the potential to provide a rapid method for DNA/RNA sequencing, diagnostics, and mapping.

Electron spectroscopic imaging of encapsidated DNA in vaccinia virus.

We have used electron spectroscopic imaging to locate the phosphorus in vaccinia DNA in situ in unstained, ultrathin sections of virions. The phosphorus of the DNA backbone appeared to form a halo on the core periphery surrounding a phosphorus-impoverished central element. These results constrain models for how DNA could be packaged into mature vaccinia particles.

11] Solid-state NMR of DNA

Publisher Summary This chapter discusses solid-state nuclear magnetic resonance (NMR) of DNA. Solid-state NMR is one of the most useful techniques, for probing the structure and dynamics of DNA. Since it can measure the orientational as well as distance information, it is a useful complement to liquid-state NMR and the crystallographic methods. The problem, until now, has been resolution. Even highly oriented DNA samples are poorly ordered, by comparison with, for example, single crystals; therefore, if several spin species are present in the sample, it will be difficult to distinguish the contributions from the individual species. For this reason, most solid-state NMR studies of DNA have concentrated on phosphorus, sodium, or deuterium; there is formally only a single phosphorus and sodium species in DNA, whereas deuterium can be selectively attached to the C-8 carbon of purines. Two other variables affect the properties of DNA; excess salt content and humidity. Some excess salt is almost always present in DNA prepared from electrolyte solutions; its quantity can be controlled by modifying the ionic strength of the solution, from which the DNA is precipitated or in which it is bathed after precipitation.

Non-covalent DNA groove-binding by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

The cooked meat mutagen 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) is metabolized in vivo to electrophilic intermediates that covalently bind to DNA guanines. Here we address the mechanism of PhIP's non-covalent interaction with DNA by using spectroscopic and computational methodologies. NMR methodologies indicated that upon addition of DNA, PhIP aromatic protons underwent a small, 0.11-0.12 p.p.m. upfield shift. DNA phosphorus resonances of non-covalent PhIP-DNA complexes broadened and slightly shifted upfield, while DNA base imino proton resonances shifted slightly downfield relative to DNA alone. UV and fluorescence spectra of PhIP titrated with DNA showed no detectable shifting and hypochromism of absorbance or fluorescence bands. In the presence of DNA, PhIP fluorescence was efficiently quenched by acrylamide, but not by silver ion. Further, the NMR spectra suggest that PhIP is in fast exchange with the DNA, and is slightly specific for adenine-thymine (A-T) sequences. Finally, structural arguments based on quantum chemistry calculations suggested that PhIP and its metabolites are unlikely to intercalate into DNA. These data collectively indicate that PhIP non-covalently binds in a groove of DNA.

The metabolic fate of 14C-parathion by some fresh water phytoplankton and its possible effects on the algal metabolism

Abstract The growth and total carbohydrate contents of Nostoc muscorum and Tolypothrix tenuis were greatly and significantly reduced by the application of parathion. “Chlorophyll a”, carotene biosynthesis and the rate of glucose absorption were enhanced after supplementation of parathion to the culture media of both cyanobacteria. Nitrogen released to the media, total nitrogen content and total nitrogen fixed were increased in both organisms‐ Increase in protein content was accompanied by remarkable drop in amino, peptide and ammonia fractions‐ Phosphorus uptake, RNA, DNA and total phosphorus content were accelerated to reach maximum accumulation at the highest insecticide level. In metabolism study using 14C‐labelled compound, parathion was readily degraded by Nostoc and Tolypothrix. Following ten days incubation, the aqueous fractions contained 21.1% and 18.1% of the initial activity in Nostoc and Tolypothrix respectively. TLC analysis of the hydrolytic products revealed the presence of three metabolite...

Seasonal and Diel Variability in Dissolved DNA and in Microbial Biomass and Activity in a Subtropical Estuary

Dissolved DNA and microbial biomass and activity parameters were measured over a 15-month period at three stations along a salinity gradient in Tampa Bay, Fla. Dissolved DNA showed seasonal variation, with minimal values in December and January and maximal values in summer months (July and August). This pattern of seasonal variation followed that of particulate DNA and water temperature and did not correlate with bacterioplankton (direct counts and [H]thymidine incorporation) or phytoplankton (chlorophyll a and CO(2) fixation) biomass and activity. Microautotrophic populations showed maxima in the spring and fall, whereas microheterotrophic activity was greatest in late summer (September). Both autotrophic and heterotrophic microbial activity was greatest at the high estuarine (low salinity) station and lowest at the mouth of the bay (high salinity station), irrespective of season. Dissolved DNA carbon and phosphorus constituted 0.11 +/- 0.05% of the dissolved organic carbon and 6.6 +/- 6.5% of the dissolved organic phosphorus, respectively. Strong diel periodicity was noted in dissolved DNA and in microbial activity in Bayboro Harbor during the dry season. A noon maximum in primary productivity was followed by an 8 p.m. maximum in heterotrophic activity and a midnight maximum in dissolved DNA. This diel periodicity was less pronounced in the wet season, when microbial parameters were strongly influenced by episodic inputs of freshwater. These results suggest that seasonal and diel production of dissolved DNA is driven by primary production, either through direct DNA release by phytoplankton, or more likely, through growth of bacterioplankton on phytoplankton exudates, followed by excretion and lysis.