Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.

A Multiplex Real-Time PCR with High Resolution Melting Analysis for the Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detecting AMR determinants could provide valuable tools for surveillance, epidemiological studies and to inform individual case management. We developed a fast (<1.5 hrs) SYBR-green based real-time PCR method with high resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully-characterized N. gonorrhoeae strains, 19 commensal Neisseria spp., and an additional panel of 193 gonococcal isolates. Results were compared with culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with non-gonococcal Neisseria species and the detection limit was 10(3)-10(4) gDNA copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity 100%, specificity 90%), cefixime (sensitivity 92%, specificity 94%), azithromycin and spectinomycin (both sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations generating resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens but this method can be used to screen collections of gonococcal isolates for AMR more quickly than with current culture-based AMR testing.

Distribution of deep-sea meiobenthos in surface sediments off North Carolina

Observations on the ecology and distribution of meiofauna occurring on the outer continental shelf and continental slope at depths from 50 to 2500 m in the region where the Blake Plateau cuts across the North Carolina slope are reported. Total numbers of meiofauna ranged from 151/100 cm**3 of sediment at 400 m to 1196/100 cm**3 of sediment at 250 m. Sediments of the upper region (50-500 m) consisted of medium-sized calcareous sands with relatively low organic carbon contents, while the deeper sediments (600-2500 m) consisted of sandy silts and silts with organic carbon contents 6-10 times that of the shallower sediments. Two basic faunas appear to be present in the areas investigated; a shallow-water fauna extending from 50 to 500 m and a deep-water fauna from 800 to 2500 m. The shallow-water fauna consists of nematodes (the dominant taxon) and relatively large numbers of harpactacoid copepods, ostracods, benthic foraminifera, polychaetes, gastrotrichs and several other groups, while below 500 m only nematodes and foraminifera are present in large numbers, the latter being especially abundant between 800 and 2000 m. A major change in the meiofauna occurs on the Blake Plateau between the depths of approximately 400-500 m and 600-750 m where the composition of the sediment changes from sand to silty sand. From 50 m to 400-500 m gastrotrichs, turbellaria, tardigrades, kinorhynchs, halicarids, hydrozoans, gnathostomulids, lamellibranchs and cumaceans are commonly encountered; these groups are absent below 500 m. In addition, there are significant reductions in the numbers of harpactacoids, ostracods, nemerteans and polychaetes below 500 m. Examination of the nematode population also show faunal differences between the shallower sediments (50-500 m) and the deeper sediments (600-2500 m). High indices of affinity exist among the faunas between 50 and 500 m and among the faunas between 800 and 2500 m; the fauna at 600-750 m represents a transition between these two regions, but it is more closely related to the deep-water fauna. Changes in the distribution of both the total meiofuna and also the nematodes are highly correlated with changes in sediments composition and bottom water temperatures. It is suggested that changes in grain size and accompanying changes in sources of nutrition, which are the results of Gulf Stream and other current activity, are the dominant environmental factors influencing the meiofauna of the area.

F. Luysii Legionensis Augustiniani Theologiae Doctoris & Diuinorum librorum primi apud salmanticensis interpretis explanationum in eosdem : tomus primus.

Contiene: In Canticum canticorum triplex explanatio ; In psalmum XXVI ; In Abdiam ; In epistol. ad Galatas

Virginibus puerisque, and other papers,

Virginibus puerisque.--Crabbed age and youth.--An apology of idlers.--Ordered south.--Æs triplex.--El Dorado.--The English admirals.--Some portraits by Raeburn.--Child's play.--Walking tours.--Pan's pipes.--A plea for gas lamps.

Virginibus puerisque, and other papers /

Virginibus puerisque -- Crabbed age and youth -- An apology of idlers -- Ordered south -- Aes triplex -- El Dorado -- The English admirals -- Some portraits by Raeburn -- Child's play -- Walking tours -- Pan's pipes -- A plea for gas lamps.

Essays :

Ordered south (1874) -- On the enjoyment of unpleasant places (1874) -- Walking tours (1876) -- Virginibus puerisque. -- An apology for idlers (1877) -- Æs triplex (1878) -- Walt Whitman (1878) -- Crabbed age and youth (1878) -- Henry David Thoreau (1880) -- Samuel Pepys (1881) -- Talk and talkers (1882) -- A gossip on romance (1882) -- The character of dogs (1883) -- A note on realism (1883) -- A humble remonstrance (1884) -- Old mortality (1884) -- The manse (1887) -- A college magazine (1887) -- Books which have influenced me (1887) -- The lantern bearers (1888) -- Pulvis et umbra (1888)

Opera omnia theologica, exegetica, didactica, polemica, philologica ...

Mode of access: Internet.

Essays of Robert Louis Stevenson;

"The text of the following essays is taken from the Thistle ed. of Stevenson's works, published by Charles Scribner's Sons, in New York."--Pref.

LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged Lacl protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Base modified peptidic nucleic acids: synthesis and crystallographic analysis of intermediates

Peptidic Nucleic Acids (PNAs) are achiral, uncharged nucleic add mimetics, with a novel backbone composed of N-(2-aminoethyl)glycine units attached to the DNA bases through carboxymethylene linkers. With the aim of extending and improving upon the molecular recognition properties of PNAs, the aim of this work was to synthesjse PNA building block intermediates containing a series of substituted purine bases for subsequent use in automated PNA synthesis. Four purine bases: 2,6~diaminopurine (D), isoGuanine (isoG), xanthine (X) and hypoxanthine (H) were identified for incorporation into PNAs targeted to DNA, with the promise of increased hybrid stability over extended pH ranges together with improvements over the use of adenine (A) in duplex formation, and cytosine (C) in triplex formation. A reliable, high-yielding synthesis of the PNA backbone component N -('2- butyloxycarbonyl-aminoethyl)glycinate ethyl ester was establishecl. The precursor N~(2-butyloxycarbonyl)amino acetonitrile was crystallised and analysed by X-ray crystallography for the first time. An excellent refinement (R = 0.0276) was attained for this structure, allowing comparisons with known analogues. Although chemical synthesis of pure, fully-characterised PNA monomers was not achieved, chemical synthesis of PNA building blocks composed of diaminopurine, xanthine and hypoxanthine was completely successful. In parallel, a second objective of this work was to characterise and evaluate novel crystalline intermediates, which formed a new series of substituted purine bases, generated by attaching alkyl substituents at the N9 or N7 sites of purine bases. Crystallographic analysis was undertaken to probe the regiochemistry of isomers, and to reveal interesting structural features of the new series of similarly-substituted purine bases. The attainment of the versatile synthetic intermediate 2,6-dichloro~9- (carboxymethyl)purine ethyl ester, and its homologous regioisomers 6-chloro~9- (carboxymethyl)purine ethyl ester and 6-chloro-7-(carboxymethyl)purine ethyl ester, necessitated the use of X-ray crystallographic analysis for unambiguous structural assignment. Successful refinement of the disordered 2,6-diamino-9-(carboxymethyl) purine ethyl ester allowed comparison with the reported structure of the adenine analogue, ethyl adenin-9-yl acetate. Replacement of the chloro moieties with amino, azido and methoxy groups expanded the internal angles at their point of attachment to the purine ring. Crystallographic analysis played a pivotal role towards confirming the identity of the peralkylated hypoxanthine derivative diethyl 6-oxo-6,7-dihydro-3H-purlne~3,7~djacetate, where two ethyl side chains were found to attach at N3 and N7,

Design, synthesis and biological evaluation of potential antibacterial oligonucleotides

Purine and pyrimidine triplex-forming oligonucleotides (TFOs), as potential antibacterial agents, were designed to bind by Hoogsteen and reverse Hoogsteen hydrogen bonds in a sequence specific manner in the major groove of genomic DNA at specific polypurine sites within the gyrA gene of E. coli and S. pneumoniae. Sequences were prepared by automated synthesis, with purification and characterisation determined by high performance liquid chromatograpy, capillary electrophoresis and mass spectrometry. Triplex stability was assessed using melting curves where the binding of the third strand to the duplex target, was assessed over a temperature range of 0-80°C, and at pH 6.4 and 7.2. The most successful of the unmodified TFOs (6) showed a Tm value of 26 °C at both pH values with binding via reverse Hoogsteen bonds. Binding to genomic DNA was also demonstrated by spectrofluorimetry, using fluorescein-labelled TFOs, from which dissociation constants were determined. Modifications in the form of 5mC, 5' acridine attachment, phosphorothioation, 2'-0-methylation and phosphoramidation, were made in order to. increase Tm values. Phosphoramidate modification was the most with increased Tm values of 42°C. However, the final purity of these sequences was poor due to their difficult syntheses. FACS (fluorescent activated cell sorting) analysis was used to determine the potential uptake of a fluorescently labelled analogue of 6 via passive, coJd shock mediated, and anionic liposome aided, uptake. This was established at 20°C and 37°C. At both temperatures anionic lipid-mediated uptake produced unrivalled fluorescence, equivalent to 20 and 43% at 20 and 37°C respectively. Antibacterial activity of each oligonucleotide was assessed by viable count anaJysis relying on passive uptake, cold shocking techniques, chlorpromazine-mediated uptake, and, cationic and anionic lipid-aided uptake. All oligonucleotides were assessed for their ability to enhance uptake, which is a major barrier to the effectiveness of these agents. Compound 6 under cold shocking conditions produced the greatest consistent decline in colony forming units per ml. Results for this compound were sometimes variable indicating inconsistent uptake by this particular assay method.

O gênero geastrum pers. (phallomycetidae, basidiomycota) em algumas áreas de Mata Atlântica e Caatinga no Rio Grande do Norte, Brasil

A taxonomic survey of representatives of the genus Geastrum took place during the rainy season from 2006 to 2009 in four districts in the state of Rio Grande do Norte: Parque Estadual Dunas do Natal (Atlantic Forest), Mata do Jiqui EMPARN (Atlantic Forest) and Estação Ecológica do Seridó (Caatinga). Fourteen species were recorded: G. entomophilum, G. fimbriatum, G. hirsutum, G. javanicum, G. lageniforme, G. lloydianum, G. minimum, G. morganii, G. ovalisporum, G. pectinatum, G. saccatum, G. schweinitzii, G. setiferum and G. triplex. Of these species, eleven occurred in the Atlantic Forest and six in the Caatinga. A new species has been recorded to science, G. entomophilum, other as a first record for Brazil, G. morganii, six new records for the Northeast, and ten new records for Rio Grande do Norte. The material was tumbled in the Herbarium UFRN. Additionally, a survey of the species of the genus deposited in the Herbarium UFRN was accomplished, resulting in 244 herbarium specimens belonging to thirty-three species. Of these ones, twenty-three were collected in Brazil and ten are from Czech Republic, Europe, as donation from the VZ Herbarium