Improved thrombin binding aptamer analogues containing inversion of polarity sites: structural effects of extra-residues at the ends.

In this paper, we report the investigations, based on NMR, molecular modelling, CD measurements and electrophoresis, of thrombin binding aptamer (TBA) analogues containing an extra-residue at the 3'-end or at both the ends of the original TBA sequence, linked through 3'-3' or 5'-5' phosphodiester bonds. The data indicate that most of the modified aptamers investigated adopt chair-like G-quadruplex structures very similar to that of the TBA and that stacking interactions occur between the 3'-3' or 5'-5' extra residues and the deoxyguanosines of the upper G-tetrad. A comparison of the thermodynamic data of TBA-A and TBA-T containing a 3'-3' extra residue and their canonical versions clearly indicates that the 3'-3' phosphodiester bond is fundamental in endowing the modified aptamers with remarkably higher thermal stabilities than the original TBA.

A novel pyrimidine tetrad contributing to stabilize tetramolecular G-quadruplex structures.

G-quadruplex structures formed by oligodeoxyribonucleotides TGGU(NH2)GGT (AM, U(NH2) = 5-amino-2'-deoxyuridine), TGGU(Br)GGT (BR, U(Br) = 5-bromo-2'-deoxyuridine) and TGGTGGT (TH) have been investigated through circular dichroism, nuclear magnetic resonance, gel electrophoresis and molecular modeling techniques. Collected data indicate that all 7-mer oligonucleotides form tetramolecular parallel G-quadruplex structures with all residues adopting anti glycosidic bonds. In the case of AM, data suggest the occurrence of a novel U(NH2)-tetrad characterized by eight hydrogen bonds that stabilizes the G-quadruplex structure more efficiently than U(Br)- and T-tetrads.

Outstanding effects on antithrombin activity of modified TBA diastereomers containing an optically pure acyclic nucleotide analogue.

Herein, we report optically pure modified acyclic nucleosides as ideal probes for aptamer modification. These new monomers offer unique advantages in exploring the role played in thrombin inhibition by a single residue modification at key positions of the TBA structure.

Effect of hyaluronic acid amide derivative on equine synovial fluid viscoelasticity.

An amphiphilic hyaluronic acid (HA) derivative has been obtained by the amidation of the carboxylic group of the glucuronic acid. This derivative, HYADD4-G (HY4), is the hexadecylamide of 500-730 kDa hyaluronic acid, derived from Streptococcus equi at about 2% degree of substitution (2 mol hexadecylamine per 100 mol hexuronic acid). Its viscoelastic properties, at a concentration of 5 mg/mL in phosphate buffer saline, have been compared with those solutions of native HA, having the same molecular weight. Changes in the viscoelastic properties of equine synovial fluid (SF) when mixed with HY4 over a series of volume ratios-viz 1:2, 1:1, 3:1, and 7:1-have been evaluated. HY4 is able to associate into aqueous solution, and its rheological behavior is typical of a weak gel. Throughout the frequency range investigated (0.1-10 Hz), the elastic modulus G' is higher than the viscous modulus G'', and both moduli are frequency independent, and G' value is about two orders of magnitude higher than that of a comparable solution of native HA. The addition of HY4 to equine synovial fluid (SF) increased its viscoelasticity at all the SF:HY4 ratios tested. These results demonstrate that HY4 is able to integrate with SF, increasing the synovial fluid rheology, and could be an interesting new option in viscosupplement therapy of osteoarthritis, particularly considering its low degree of chemical modification from native HA.

A mini-library of TBA analogues containing 3'-3' and 5'-5' inversion of polarity sites.

Several researches have been devoted to structure-activity relationship and to post-SELEX modifications of the thrombin binding aptamer (TBA), one of the first aptamers discovered by the SELEX methodology. However, no studies on TBA dealing with the effects of introduction of inversion of polarity sites have been reported yet. In this frame, we have undertaken the synthesis and the study of a mini-library composed of several TBA analogues containing a 3'-3' or a 5'-5' inversion of polarity site at different positions into the sequence. Particularly, in this article, we present preliminary results about their structural and biological properties.

Effect of the introduction of an A-residue into a quadruplex forming oligonucleotide containing a 5'-5' polarity of inversion site.

Preliminary NMR studies on structure formed by sequence 3'-TGA-5'-5'-GGT-3' are described. We proposed the formation of a tetramolecular quadruplex in which strands are equivalent to each other and three G-tetrads are present. The possibility of the occurrence of an A-tetrad also is discussed.

A topological classification of G-quadruplex structures.

A topological classification of most quadruplex structures is proposed, based on two main characteristics: 1) the relative orientation of the strands and 2) the nature of the loops connecting the strands.

Effects of fibronectin and laminin on structural, mechanical and transport properties of 3D collageneous network.

Recent studies, on cells cultured in 3D collagen gels, have shown that, beside from their well known biochemical role, fibronectin (FN) and laminin (LM) affect cell functions via a modification of mechanical and structural properties of matrix due to interaction with collagen molecules. Though biochemical properties of FN and LM have been widely studied, little is known about their role in collagen matrix assembly. The aim of this work was to characterize FN- and LM-based collagen semi-interpenetrating polymer networks (semi-IPNs), in order to understand how these biomacromolecular species can affect collagen network assembly and properties. Morphology, viscoelasticity and diffusivity of collagen gels and FN- and LM-based collagen semi-IPNs were analysed by Confocal Laser Scanning microscopy (CLSM), Environmental Scanning Electron microscopy (ESEM), Transmission Electron microscopy (TEM), Rheometry and Fluorescence Recovery After Photobleaching (FRAP) techniques. It was found that FN and LM were organized in aggregates, interspersed in collagen gel, and in thin fibrils, distributed along collagen fibres. In addition, high FN and LM concentrations affected collagen fibre assembly and structure and induced drastic effects on rheological and transport properties.

[Nonvaccine Streptococcus pneumoniae serotypes causing acute bacterial meningitis]. / Streptococcus pneumoniae serotipo no vacunal como agente causal de meningitis bacteriana aguda.

The pneumococcal heptavalent conjugate vaccine protects children aged less than 2 years old from invasive pneumococcal disease (IPD). Efficacy is 89-93% in the US population and 71-86% in European studies. The vaccine confers active immunization against the main serotypes causing IPD (4, 6B, 9V, 14, 18C, 19F y 23F). We describe 2 children who presented with pneumococcal meningitis caused by nonvaccine serotypes. As a result of the widespread use of the heptavalent vaccine, there may be a shift in the serotypes causing IPD.

Streptococcus pneumoniae serotipo no vacunal como agente causal de meningitis bacteriana aguda / Nonvaccine Streptococcus pneumoniae serotypes causing acute bacterial meningitis

La vacuna conjugada heptavalente protege a niños de menos de 2 años de la enfermedad neumocócica invasiva (ENI) en el 89-93 % en población estadounidense y en el 71-86 % en población europea. Confiere una inmunización activa frente a los serotipos mayoritariamente causantes de ENI (4, 6B, 9V, 14, 18C, 19F y 23F). Presentamos 2 casos de meningitis neumocócica por serotipos no vacunales. Es posible que haya un desplazamiento de serotipos causantes de ENI, como consecuencia de la generalización de la vacuna heptavalente

The pneumococcal heptavalent conjugate vaccine protects children aged less than 2 years old from invasive pneumococcal disease (IPD). Efficacy is 89-93 % in the US population and 71-86 % in European studies. The vaccine confers active immunization against the main serotypes causing IPD (4, 6B, 9V, 14, 18C, 19F y 23F). We describe 2 children who presented with pneumococcal meningitis caused by nonvaccine serotypes. As a result of the widespread use of the heptavalent vaccine, there may be a shift in the serotypes causing IPD

Factors associated with poor outcomes in patients with lupus nephritis.

The objective of this study was to identify the factors associated with important clinical outcomes in a case-control study of 213 patients with lupus nephritis. Included were 47% Hispanics, 44% African Americans and 9% Caucasians with a mean age of 28 years. Fifty-four (25%) patients reached the primary composite outcome of doubling serum creatinine, end-stage renal disease or death during a mean follow-up of 37 months. Thirty-four percent African Americans, 20% Hispanics and 10% Caucasians reached the primary composite outcome (P < 0.05). Patients reaching the composite outcome had predominantly proliferative lupus nephritis (WHO classes: 30% III, 32% IV, 18% V and 5% II, P < 0.025) with higher activity index score (7 +/- 6 versus 5 +/- 5, P < 0.05), chronicity index (CI) score (4 +/- 3 versus 2 +/- 2 unit, P < 0.025), higher baseline mean arterial pressure (MAP) (111 +/- 21 versus 102 +/- 14 mmHg, P < 0.025) and serum creatinine (1.9 +/- 1.3 versus 1.3 +/- 1.0 mg/dL, P < 0.025), but lower baseline hematocrit (29 +/- 6 versus 31 + 5%, P < 0.025) and complement C3 (54 +/- 26 versus 65 + 33 mg/dL, P < 0.025) compared to controls. More patients reaching the composite outcome had nephrotic range proteinuria compared to controls (74% versus 56%, P < 0.025). By multivariate analysis, CI (hazard ratio [95% CI] 1.18 [1.07-1.30] per point), MAP (HR 1.02 [1.00-1.03] per mmHg), and baseline serum creatinine (HR 1.26 [1.04-1.54] per mg/dL) were independently associated with the composite outcome. We concluded that hypertension and elevated serum creatinine at the time of the kidney biopsy as well as a high CI are associated with an increased the risk for chronic renal failure or death in patients with lupus nephritis.

Evaluation of injection augmentation treatment of hyaluronic acid based materials on rabbit vocal folds viscoelasticity.

The viscoelastic properties of vocal folds after injection of hyaluronic acid (hyaluronan, HA) based materials have been studied in an animal model (rabbit) six months after injection. The results indicate that the viscoelastic properties of the vocal folds injected with the HA based materials are similar to the healthy vocal folds (non-injected samples) used as control. Histological analysis has been also performed to investigate on the fate of the injected materials after six months from the implant. The HA based materials remain up to six months and they recruited fibroblasts that induce the ingrowth of new connective tissue resulting in an endogenous soft tissue augmentation. The HA based compounds are good candidate for further studies aimed at restoring/preserving the vibratory capacity of the vocal folds with injection treatment in glottal insufficiency.

Once-weekly epoetin alfa for treating the anemia of chronic kidney disease.

BACKGROUND AND AIM: Anemia occurs in approximately 47% of patients with chronic kidney disease (CKD) not on dialysis. Recombinant human erythropoietin (r-HuEPO, epoetin alfa) has been proven safe and effective for anemia treatment in patients with CKD using a three times-weekly regimen. The current study was conducted to evaluate the clinical safety and efficacy of a less frequent dosing regimen (once weekly) in this population. METHODS: This prospective, multicenter, open-label, non-randomized study enrolled 1,557 adult anemic (hemoglobin (Hb) < or = 10 g/dl) CKD patients not on dialysis. Epoetin alfa 10,000 U was administered subcutaneously once weekly for 16 weeks. Titration to 20,000 U once weekly at week 5 was permitted if patients had an increase in Hb < 1 g/dl. Safety and efficacy were assessed by changes in health-related quality of life (Linear Analog Scale Assessment (LASA) and Kidney Disease Questionnaire (KDQ)), changes in hematologic parameters and transfusion utilization, and incidence and severity of adverse events. RESULTS: 1,338 patients were evaluable for efficacy. Mean Hb level increased from 9.1 g/dl at baseline to 11.6 g/dl at study completion (last observed value after baseline) (p < 0.0001). Overall, 89.8% of patients responded to once-weekly dosing, exhibiting an increase in Hb level of > or = 1 g/dl from baseline. The percentage of patients that required transfusion decreased from 11.1% (baseline) to 3.7% (during the study) (p < 0.0001). All quality-of-life parameters improved significantly from baseline (p < 0.0001). Mean LASA scores for energy, activity and overall quality of life increased from baseline to study completion by 27.9 mm (70.5%), 24.5 mm (57.0%) and 22.6 mm (47.4%), respectively. All 5 KDQ domains showed statistically significant improvements (p < 0.0001). Hb change was a strong predictor for all 5 KDQ domains and the overall score (p < 0.0001). Treatment with once-weekly epoetin alfa was well tolerated, similar to that reported with three times-weekly dosing. CONCLUSION: Once-weekly epoetin alfa therapy is safe and effective for treating anemia in patients with CKD not on dialysis, and is associated with significant improvements in functional status and quality of life.

Synthesis of 5-methylamino-2'-deoxyuridine derivatives.

Reductive amination of 3',5'-O-(tetraisopropyldisilyloxane-1,3-diyl)-2'-deoxy-5-formyluridine with several aliphatic and aromatic amines, in various solvents, is described. In the case of aliphatic amines, the expected C-5 substituted methylamino pyrimidine nucleosides are formed along with by-products deriving from opening of the pyrimidine ring. Relative amounts of the by-products depend upon the polarity of the solvent employed.

Solid-phase synthesis of oligonucleotides containing a bipyridine ligand at the 3'-3' inversion of polarity site.

The preparation of a solid support useful for the synthesis of oligonucleotides with a 3'-3' inversion of polarity, via a linker containing a chelating molecule, namely 2,2'-bipyridine, is described.

Extracción de muestras a través de catéter venoso central para control de tiempo de tromboplastina parcial en pacientes con perfusión de heparina sódica / Sample extraction through central venous catheters to control partial thromboplastin time in patients undergoing heparin sodium perfusion

La necesidad de realizar controles sistemáticos del tiempo de tromboplastina parcial activado (TTPA) en pacientes sometidos a tratamiento con heparina sódica (no fraccionada) en perfusión continua, para mantener niveles terapéuticos de anticoagulación, nos hace plantearnos dos preguntas: ¿Es posible extraer la muestra de sangre del catéter por donde se está perfundiendo la dilución de heparina, sin alterar los valores del TTPA?, ¿Qué volumen de sangre habrá que desechar para que los valores del TTPA no sean alterados? Para la obtención de los valores de TTPA en estos pacientes se extrajeron dos muestras simultáneamente, una a través del catéter venoso central por donde se perfundía heparina, desechando 10 o 20 ml de sangre previamente según el grupo al que se asignaba el paciente, y otra mediante venopunción directa o del catéter periférico localizado en el brazo contralateral al de la perfusión de heparina. Se estudió si había significación estadística entre los valores obtenidos por ambos métodos.Al comparar los resultados del TTPA de las muestras, extrayendo previamente 10 o 20 ml de sangre, con los obtenidos a través de venopunción o catéter venoso periférico, se observan diferencias estadísticamente significativas. Estas diferencias fueron menores cuando se desechaban 20 ml que cuando se desechaban 10 ml.Como conclusión se propone realizar los controles de TTPA obteniendo la muestra mediante venopunción directa o bien a través del catéter periférico localizado en el brazo contralateral al de la perfusión de heparina. (AU)

2'-Deoxy-8-(propyn-1-yl)adenosine-containing oligonucleotides: effects on stability of duplex and quadruplex structures.

2'-Deoxy-8-(propyn-1-yl)adenosine has been incorporated in synthetic oligodeoxyribonucleotides and its influence on thermal stability of duplex and quadruplex structures investigated by UV, CD and 1H NMR. The obtained results seem to indicate that the presence of the modified base negatively affects the stability of double stranded DNA whereas remarkably increases the stability of parallel quadruplex structures.

[Sample taking through central venous catheter for the control of partial thromboplastin time in patients with heparin sodium perfusion]. / Extracción de muestras a través de catéter venoso central para control de tiempo de tromboplastina parcial en pacientes con perfusión de heparina sódica.

The need to systematically monitor activated partial thromboplastin time (APTT) in patients undergoing continuous perfusion of heparin sodium (non-fractionated) in order to maintain therapeutic levels of anticoagulation leads to two questions: 1. can blood be withdrawn from the catheter through which the heparin solution is being perfused without altering APTT values? and 2. how much blood should be discarded so that APTT values remain unchanged? To obtain APTT values in these patients, two samples were extracted simultaneously: one through the central venous catheter through which the heparin was being perfused, after previously discarding 10 or 20 ml of blood according to the group to which the patient was assigned and the other through direct venous puncture or through the peripheral catheter inserted in the arm not being used for heparin perfusion. The values obtained by both methods were analyzed for statistical significance. Comparison of the results of APPT in the samples, having previously extracted 10 or 20 ml of blood, with those obtained through venous puncture or through peripheral venous catheter revealed statistically significant differences. These differences were lower when 20 ml were discarded than when 10 ml were discarded. In conclusion, APPT monitoring should be performed whether the sample is obtained through direct venous puncture or through a peripheral catheter inserted in the arm not being used for heparin perfusion.

NMR-derived solution structure of a 17mer hydroxymethyluracil-containing DNA.

Incorporation of 5-(hydroxymethyl)-2'-deoxyuridine into DNA in place of thymine by SPO1, a Bacillus subtilis bacteriophage, allows the viral DNA to bind selectively to transcription factor 1. We have synthesized a TF1-binding site: d(5'-ACCHACHCHHHGHAGGT-3')-d(5'-ACCHACAAAGAGHAGGT-3') and studied this molecule using NMR spectroscopy. The chemical shifts of exchangeable and non-exchangeable protons were sequentially assigned. Absence of corresponding NOEs in the imino-imino region suggested that the end base pairs did not form Watson-Crick hydrogen bond. Restrained molecular dynamics calculation yielded a family of B-DNA structures whose r.m.s.d. was 0.66 A (all atoms) for the internal 15 bp. The helical twist was 38.5 degrees per step. The base pairs were situated directly on the helix axis (X-displacement = -0.2 A). All sugars exhibited C2'-endo puckering with P = 167.3 degrees and upsilon(max)= 38.2 degrees. The OH groups of all hmU bases resided on the 3' side of the base plane and may affect the base orientation relative to the sugar plane as the average chi value for all hmU was 4 degrees more positive than that of other nucleosides (258 degrees versus 254 degrees ). Positive roll angles (rho) and small flanking twists (omega) at hmU suggested that the two hmU-A base pair steps open toward the minor grooves.

Affinity, stability and polarity of binding of the TATA binding protein governed by flexure at the TATA Box.

The TATA binding protein (TBP), which plays a central role in gene regulation as an essential component of all three nuclear transcription systems, sharply kinks the TATA box at two sites and severely contorts the intervening DNA segment. DNA constructs with precisely localized flexure have been used to investigate the special repertoire of mechanisms and properties that arise from TBP interacting with the TATA box. DNA flexure precisely localized to the sites of TBP-mediated DNA kinking increases the affinity of TBP more than 100-fold; unexpectedly, this increase in affinity is achieved almost exclusively by increasing the stability of the TBP-DNA complex rather than the rate of its formation. In vitro transcription with RNA polymerase III provides a first demonstration that the orientation of TBP on the TATA box is governed by DNA deformability, its C-proximal repeat contacting the more flexible end of the TATA box. Exceptionally stable TBP-DNA complexes reach their orientational equilibrium very slowly; in these circumstances, assembly of stable ("committed") transcription initiation complexes can freeze far-from-equilibrium orientations of TBP on the TATA box, causing transcription polarity to be determined by a kinetic trapping mechanism.