Influence of a charged graphene surface on the orientation and conformation of covalently attached oligonucleotides: a molecular dynamics study.

Molecular dynamics (MD) simulations of single-stranded (ss) and double-stranded (ds) oligonucleotides anchored via an aliphatic linker to a graphene surface were performed in order to investigate the role of the surface charge density in the structure and orientation of attached DNA. Two types of interactions of DNA with the surface are crucial for the stabilisation of the DNA-surface system. Whereas for a surface with a zero or low positive charge density the dispersion forces between the base(s) and the surface dominate, the higher charge densities applied on the surface lead to a strong electrostatic interaction between the phosphate groups of DNA, the surface and the ions. At high-charge densities, the interaction of the DNA with the surface is strongly affected by the formation of a low-mobility layer of counterions compensating for the charge of the surface. A considerable difference in the behaviour of the ds-DNA and ss-DNA anchored to the layer was observed. The ds-DNA interacts with the surface at low- and zero-charge densities exclusively by the nearest base pair. It keeps its geometry close to the canonical B-DNA form, even at surfaces with high-charge densities. The ss-DNA, owing to its much higher flexibility, has a tendency to maximise the attraction to the surface exploiting more bases for the interaction. The interaction of the polar amino group(s) of the base(s) of ss-DNA with a negatively charged surface also contributes significantly to the system stability.

"Extracorporeal Membrane Oxygenation for Greater Poland" Program: How to Save Lives and Develop Organ Donation?

The "ECMO for Greater Poland" program takes full advantage of the extracorporeal membrane oxygenation (ECMO) perfusion therapy opportunities to promote the health of the 3.5 million inhabitants in the region. The main implementation areas are treatment of patients with hypothermia; severe reversible respiratory failure (RRF); critical states resulting in heart failure, that is, cardiac arrest, cardiogenic shock, or acute intoxication; and promotion of the donor after circulatory death (DCD) strategy in selected organ donor cases, after unsuccessful life-saving treatment, to achieve organ recovery. This organizational model is complex and expensive, so we used advanced high-fidelity medical simulation tests to prepare for real-life experience. Over the course of 4 months we performed scenarios including "ECMO for DCD," "ECMO for extended cardiopulmonary resuscitation," "ECMO for RRF," and "ECMO in hypothermia." Soon after these simulations, Maastricht category II DCD procedures were performed involving real patients and resulting in 2 successful double kidney transplantations for the first time in Poland. One month later we treated 2 hypothermia patients (7 adult patients with heart failure and 5 patients with reversible respiratory failure) with ECMO for the first time in the region. Fortunately, we have discovered an important new role of medical simulation. It can be used not only for skills testing but also as a tool to create non-existing procedures and unavailable algorithms. The result of these program activities will promote the care and treatment of patients in critical condition with ECMO therapy as well as increase the potential organ pool from DCDs in the Greater Poland region of Poland.

Oligonucleotide-based microarray: a new improvement in microarray detection of plant viruses.

Microarrays are one of the new emerging methods in plant virology currently being developed by various laboratories. In this study, a new approach is described on the detection of plant viruses using short synthetic single-stranded oligomers (40 nt) instead of PCR products as capture probes. A microchip detecting potato viruses, PVA, PVS, PVM, PVX, PVY and PLRV, in both single and mixed infections was developed and tested. The chip was also designed to distinguish between the main strains of PVY and PVS. Results of initial tests with PVY(NTN) and PVY(O) strains using several different probes for one virus are presented. Possibilities and advantages of the new oligonucleotide-based microarray approach for plant viral diagnosis are discussed.

Influence of hyperbaric oxygenation on bilirubin and ditaurobilirubin auto-oxidation and porphyrin-sensitized photo-oxidation.

Oxygen concentration controls the reaction rate of porphyrin-sensitized photo-oxidation under constant experimental conditions. The rate of bilirubin and ditaurobilirubin auto-oxidation and meso-tetra-(4-sulphonatophenyl) porphine-sensitized photo-oxidation in aqueous solution increases by about 3.5 times in 0.5 MPa of oxygen. Use of hyperbaric oxygenation in the photodynamic therapy of tumours and in the phototherapy of jaundice is proposed.

Transmembrane potential measurement with carbocyanine dye diS-C3-(5): fast fluorescence decay studies.

The mechanism of carbocyanine dye diS-C3-(5) fluorescence intensity variations with transmembrane potential changes has been studied using time-resolved fluorescence spectroscopy. Clear evidence is given of the transmembrane-potential-dependent partition of the dye among various sites with different fluorescence lifetimes. It was found that fluorescence decay profiles reflect the transmembrane potential changes.

Nitrogen utilization by ruminants during restricted intake of high-concentrate diets.

A series of experiments was conducted to determine the effects of limit-feeding high-concentrate (LFHC) diets on dietary CP requirements of steer calves. When steer calves were fed 80% concentrate diets at 78 g/kg of BW.75, increasing dietary CP resulted in increased ADG (P less than .001). Average daily gain was increased in steers as daily monensin dosage increased from 120 to 180 mg (P less than .05). Increasing the daily monensin dosage to 240 mg did not increase ADG further. There were no (P greater than .10) CP X monensin interactions, suggesting that the monensin response was caused by improved energy utilization and not be the possible protein-sparing effects of ionophores. Steer calves in the second feedyard experiment expressed similar ADG when provided equal NEg as limit-fed, high-moisture ear corn (HMEC) or when given ad libitum access to corn silage. The basal diet did not affect the steers' daily N requirement for growth. Gain per unit of protein intake declined quadratically (P less than .05) with increasing CP intake, indicating that CP requirements were near NRC estimates on both diets. The corn silage-based diet was less digestible (70.3 vs 77.4%; P less than .01) than the HMEC diet when fed to lambs. Fecal output differed (P less than .10) substantially (342 g/d of corn silage vs 205 g/d of HMEC), whereas fecal N output was only slightly higher (6.97 vs 6.34 g/d, respectively; P less than .10). Limited feeding of higher-concentrate diets to steer calves seemed to be an effective management procedure and did not cause acute digestion upset.(ABSTRACT TRUNCATED AT 250 WORDS)

Variability in Coat Protein Sequence Homology Among American and European Sources of Strawberry Vein Banding Virus.

Strawberry vein banding virus (SVBV) isolates from North America, Czech Republic, Norway, and Germany were collected and their variability was determined by dot blot hybridization and confirmed by sequencing of a 431-nucleotide fragment from the middle part of the coat protein gene. Two different substitutions were found between the American and two Czech SVBV isolates, but the other isolates were identical in the compared region to the American isolate. Digoxigenin-labeled probes were prepared from these isolates and used for hybridization with polymerase chain reaction-amplified fragments of 26 Czech SVBV field isolates. No significant differences in the hybridization signal were found with any combination of samples and probes. These results show that the European isolates probably originate from a common ancestor and may have been introduced to Europe from America with planting or breeding material.

Diagnosis of strawberry vein banding virus by a non-radioactive probe.

A non-radioactive digoxigenin-labelled cDNA probe was prepared from genomic DNA of American isolate No. 45058 of strawberry vein banding virus (SVBV). Five different air-dried SVBV-containing strawberry leaf samples originating from National Clonal Germplasm Repository, Corvallis, USA, reacted positively in dot blot hybridization with this probe. Six of twelve strawberry samples from the Czech Republic exhibiting symptoms of SVBV-like infection gave positive reaction with this probe. Our results confirm the spread of SVBV in Central Europe and introduce the first reliable screening method for this virus.

Detection of strawberry vein banding virus by polymerase chain reaction and dot blot hybridization.

Strawberry vein banding virus (SVBV) is one of seventeen members of the family Caulimoviridae. Natural infection with the virus is known in Fragaria species only. Infections caused by SVBV are often symptomless (1), but their significance increases in mixed infections with strawberry crinkle or strawberry latent C viruses (2,3). This virus has been originally found on strawberries in USA and firstly described by Frazier (4), but it is probably world-wide distributed by planting or breeding materials. SVBV has been observed on cultivated strawberries in North America, Australia, Brazil, Japan (5) and recently in Europe (6,7). The concentration of SVBV in infected plants is usually very low. Its detection by ELISA is impossible because of lack of specific antibodies. Evidence of the caulimovirus nature of SVBV has been confirmed by its circular dsDNA genome, shape and size of viral particles (8), presence of cytoplasmic inclusion bodies typical for caulimoviruses, and distant serological relationship with cauliflower mosaic virus (CaMV, 9). In this paper we present detection of SVBV by combination of two detection methods--polymerase chain reaction (PCR) and dot blot hybridization with a non-radioactive probe.

Preferential banding of secondary veins in strawberry is caused by mixed virus infection.

Leaves of Fragaria ananassa Duch. cv. Redgauntlet with mottle and mild dwarf symptoms were grafted onto F. vesca indicator clones. The youngest leaves developed specific vein banding pattern located preferentially on secondary veins near the edge of the leaves. Electron microscopy of ultrathin sections and negatively stained purified virus preparations from symptom-bearing strawberry leaves revealed presence of different-sized isometric virions. Particles of about 50 nm and 23 nm in diameter were identified as strawberry vein banding virus (SVBV) and tobacco necrosis virus (TNV) D strain. Based on results of electron microscopy, DNA hybridization, enzyme-linked immunosorbent assay (ELISA), and DNA sequencing we propose that the anomalous "leaf edge vein banding" symptoms are caused by a mixed virus infection with SVBV and other viruses such as TNV.

DNA microarray: parallel detection of potato viruses.

DNA microarray assay has become a useful tool for gene expression studies. Less frequent is its application to detection of viruses or diagnostics of virus diseases. Here we show design of a microscope slide-based microarray assay for simultaneous identification of several potato viruses. Different primer pairs were designed or adopted to obtain specific amplicons from six potato viruses: Potato virus A (PVA), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), Potato mop-top virus (PMTV) and Potato leaf-roll virus (PLRV). Purified viral DNA probes were spotted on a microscope slide coated with poly-L-lysine. The same primers were used for preparation of fluorochrome-labeled targets. The latter were denatured and hybridized on the microarray slide (chip). An example of simultaneous assay of two pathogens is given and possibilities of practical application of this type of assay are discussed.

DNA conformational change produced by the site-specific interstrand cross-link of trans-diamminedichloroplatinum(II).

The DNA distortion produced by the interstrand cross-link of trans-diamminedichloroplatinum-(II) has been described by means of gel electrophoresis, chemical probes, and molecular mechanics modeling. Synthetic double-stranded oligodeoxyribonucleotides of varying lengths (19-22 base pairs) were synthesized that contained a unique site-specific interstrand cross-link within their central sequence d(TGCT)/d(AGCT) between complementary guanine and cytosine residues. We find that the platinated deoxyriboguanosine residue adopts syn conformation. The duplex is distorted on both sides of the cross-link, but the bases are still paired. The distortion introduces some flexibility into the helix. In addition, the double helix is unwound and bent toward the major groove.

Quantitative and sequence-specific analysis of DNA-ligand interaction by means of fluorescent intercalator probes.

A novel method of analysis of double-stranded DNA-ligand interaction is presented. The interaction is monitored by the fluorescence of a DNA bis-intercalator oxazole homodimer YoYo-3. The fluorescence intensity or its decay time reflects the modification of the DNA double helix. The DNA sequence is scanned by hybridization with short oligomers having consecutively overlapping complementary sequences to analyse the sequence specificity of binding. In our experiments we used as ligands the minor groove binders netropsin, SN6999 (both with AT-preference), the GC-specific ligand chromomycin A3 as well as the derivative SN6113 (non-specific interaction), which displace the bis-intercalator YoYo-3 or influence the duplex structure in such away that the fluorescence intensity and lifetime decrease in comparison to a ligand-free screening. The changes of fluorescence emission clearly define the binding motif and indicate minor groove interactions with a reduced DNA binding site. Titration of the ligand quantitatively characterizes its binding by determining the dependence of the binding constant on the oligonucleotide sequence.

Mitochondrial DNA D-loop hypervariable regions: Czech population data.

In order to identify polymorphic sites and to find out their frequencies and the frequency of haplotypes, the complete D-loop of mitochondrial DNA (mtDNA) from 93 unrelated Czech Caucasians was sequenced. Sequence comparison showed that 85 haplotypes were found and of these 78 were unique, 6 were observed twice and 1 was observed three times. Genetic diversity (GD) was estimated at 0.999 and the probability of two randomly selected sequences matching (random match probability, RMP) at 1.2%. Additionally these calculations were carried out for hypervariable regions 1, 2 (HV1, HV2), for the area between HV1 and HV2 and for the area of the hypervariable region HV3. The average number of nucleotide differences (ANND) was established to be 10.2 for the complete D-loop. The majority of sequence variations were substitutions, particularly transitions. Deletions were found only in the region where HV3 is situated and insertions in the same place and in poly-C tracts between positions 303 and 315 in HV2. A high degree of length heteroplasmy was found especially in the regions of poly-C tracts between positions 16184 and 16193 in HV1 and between positions 303 and 315 in HV2. Position heteroplasmies were found in two cases.

Instability of the monofunctional adducts in cis-[Pt(NH3)2(N7-N-methyl-2-diazapyrenium)Cl](2+)-modified DNA: rates of cross-linking reactions in cis-platinum-modified DNA.

Single- and double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adduct have been studied at two NaCl concentrations. In 50 mM and 1 M NaCl, the adducts within the single-stranded oligonucleotides are stable. In contrast, they are unstable within the corresponding double-stranded oligonucleotides. In 50 mM NaCl, the bonds between platinum and guanine or N-methyl-2,7-diazapyrenium residues are cleaved and subsequently, intra- or interstrand cross-links are formed as in the reaction between DNA and cis-DDP. In 1 M NaCl, the main reaction is the replacement of N-methyl-2,7-diazapyrenium residues by chloride which generates double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)Cl]+ adduct. The rates of closure of these monofunctional adducts to bifunctional cross-links have been studied in 60 mM NaClO4. Within d(TG.CT/AGCA), d(CG.CT/AGCG) and d(AG.CT/AGCT) (the symbol.indicates the location of the adducts in the central sequences of oligonucleotides), the half-lifes (t1/2) of the cis-[Pt(NH3)2(dG)Cl]+ adducts are respectively 12, 6 and 2.8 hr and the cross-linking reactions occur between guanine residues on the opposite strands. Within d(AG.TC/GACT), d(CG.AT/ATCG) and d(TGTG./CACA) or d(TG.TG/CACA) t1/2 are respectively 1.6, 8 and larger than 20 hr and the intrastrand cross-links are formed at the d(AG), d(GA) and d(GTG) sites, respectively. The conclusion is that the rates of conversion of cis-platinum-DNA monofunctional adducts to minor bifunctional cross-links are dependent on base sequence. The potential use of the instability of cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adducts is discussed in the context of the antisense strategy.

Distortions induced in DNA by cis-platinum interstrand adducts.

A 22 base pair double-stranded oligonucleotide containing a unique interstrand adduct resulting from chelation of the two guanine residues within the central sequence d(TGCT/AGCA) by a cis-platinum residue has been studied by means of gel electrophoresis, chemical probes, and molecular mechanics. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers suggests that the platinated oligonucleotide is bent. The two cytosine residues (complementary to the platinated guanines) are hyperreactive to hydroxylamine, indicating a large exposure of the two bases to the solvent. The adduct does not induce a local denaturation within the flanking sequences since the adenine residues are not reactive with diethyl pyrocarbonate. This is confirmed by the nonreactivity of the complementary T residues with osmium tetraoxide. These results and the molecular mechanics modeling suggest that the interstrand adduct bends the double helix by approximately 55 degrees toward the major groove, that the double helix conserves its average twist angle, and that the distortion induced by the adduct is localized at the platinated sequence d(GC/CG).

Formation of a DNA monofunctional cis-platinum adduct cross-linking the intercalating drug N-methyl-2,7-diazapyrenium.

Our purpose was to better understand the mutual influence of cis-diamminedichloroplatinum (II) (cis-DDP) and intercalating drugs in their interactions with DNA. The present study deals with the intercalating drug N-methyl-2,7-diazapyrenium (MDAP). Two sets of experiments have been performed. In one set, the reaction between cis-DDP and nucleic acid was carried out in the presence of MDAP. The main adduct is a guanine residue chelated by platinum to a MDAP residue. It has the same spectroscopic properties as the synthesized compound cis-[Pt (NH3)2 (N7-d-guanosine) (N7-MDAP)] , the structure of which has been determined by 1H NMR. This adduct was only formed with double-stranded nucleic acids which reveals the importance of DNA matrix in orienting favorably the reactants. In the second set of experiments, the triamine complex cis-[Pt(NH3)2 (MDAP)CI]++ was reacted with the nucleic acids. At molar ratios drug over nucleotide residue equal or less than 0.10, all the added triamine complexes bind by covalent coordination to double-stranded nucleic acids. With natural DNA, the major adduct is cis-[Pt(NH3)2(d-guanosine) (MDAP)] . Thus the same adduct is formed on one hand in the reaction between DNA, MDAP and cis-DDP and on the other hand in the reaction between the triamine complex and DNA. The triamine complex offers the possibility to study the biological role of the new adduct.