Evaluation of LightCycler as a Platform for Nucleic Acid Sequence-Based Amplification (NASBA) in Real-Time Detection of Enteroviruses

The nucleic acid sequence-based amplification (NASBA) assay has been demonstrated to be more sensitive for detection of enteroviruses (EV) than RT-PCR. Many laboratories, however, do not have a dedicated instrument for the NASBA assay. This study aimed to evaluate the use of the Roche LightCycler as a platform for performing the NASBA assay for detection of EV. A diverse subgenera of EV were used to assess the specificity of the NASBA assay, including coxsackie, echovirus, poliovirus, and other enteroviruses together with related and unrelated viruses, including rhinovirus, respiratory syncytial virus, herpes simplex virus, adenovirus, influenza virus A, and cytomegalovirus. All species of EV tested were successfully detected using NASBA and no cross reactivity with other viruses was observed. Using serial dilutions of EV to assess sensitivity, the NASBA assay was compared to an in-house EV RT-PCR assay. The NASBA assay demonstrated a higher level of sensitivity. Fifty-one clinical samples positive for EV by viral culture were also evaluated. All NASBA results obtained were concordant with viral culture results. This study confirmed that the NASBA assay for the detection of EV could be readily performed on the LightCycler and easily incorporated into the workflow of a diagnostic laboratory equipped with a LightCycler, thereby eliminating the need for additional instrumentation.     

Microwave-assisted synthesis of thieno[2,3-c]pyridine derivatives as a new series of allosteric enhancers at the adenosine A(1) receptor

The microwave-assisted aromatization method has been used for the synthesis of a series of novel thieno[2,3-c]pyridines. This rapid method produces compounds in good yield within minutes in comparison with conventional heating method. The synthesized molecules have been evaluated as a potential new series of allosteric enhancers acting at the adenosine A(1) receptor. In a functional assay, one compound (3h) showed activity comparable with that of reference compound PD 81,723.     

New strategies for the synthesis of A3 adenosine receptor antagonists

New A(3) adenosine receptor antagonists were synthesized and tested at human adenosine receptor subtypes. An advanced synthetic strategy permitted us to obtain a large amount of the key intermediate 5 that was then submitted to alkylation procedures in order to obtain the derivatives 6-8. These compounds were then functionalised into ureas at the 5-position (compounds 9-11, 18 and 19) to evaluate their affinity and selectivity versus hA(3) adenosine receptor subtype; in particular, compounds 18 and 19 displayed a value of affinity of 4.9 and 1.3 nM, respectively. Starting from 5, the synthetic methodologies employed permitted us to perform a rapid and a convenient divergent synthesis. A further improvement allowed the regioselective preparation of the N(8)-substituted compound 7. This method could be used as an helpful general procedure for the design of novel A(3) adenosine receptor antagonists without the difficulty of separating the N(8)-substituted pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines from the corresponding N(7)-isomers.     

Different Tolerance to Light Stress in NO3−- and NH4+-Grown Phaseolus vulgaris L.

Abstract: NH₄⁺‐grown plants are more sensitive to light stress than NO₃^?‐grown plants, as indicated by reduced growth and intervenal chlorosis of French bean (Phaseolus vulgaris L.). Measuring the time course of F_v/F_m ratios under photoinhibitory light regimes did not reveal any difference in PS II damage between NO₃^?‐ and NH₄⁺‐grown plants, in spite of some indications of higher energy quenching in NO₃^?‐grown plants. Also, a direct action of NH₄⁺ as an uncoupler at the thylakoid membrane could be excluded. Instead, biochemical analysis revealed enhanced lipid peroxidation and higher activity of scavenging enzymes in NH₄⁺‐grown plants indicating that these plants make use of metabolic pathways with stronger radical formation. Evidence for higher rates of photorespiration in NH₄⁺‐grown plants came from experiments showing that electron flux and O₂ evolution were decreased by SHAM in NH₄⁺‐grown plants, and by antimycin A in NO₃^?‐grown plants. Further, the comparison of electron flux and of photoacoustic measurements of O₂ evolution suggested that in NH₄⁺‐grown plants the Mehler reaction was also increased, at least in the induction phase. However, the major cause of N form‐dependent stress sensitivity is assumed to be in the coupling between photosynthesis and respiration, i.e., NO₃^?‐grown plants can utilize the TCA cycle for the generation of C skeletons for amino acid synthesis, thus improving the ATP: reductant balance, whereas NH₄⁺‐grown plants have enhanced rates of photorespiration.     

Synthesis and biological effects of a new series of 2-amino-3-benzoylthiophenes as allosteric enhancers of A1-adenosine receptor

New derivatives of PD 81,723, an allosteric enhancer of agonist binding to the A1-adenosine receptor, have been synthesized and evaluated in an intact cell assay. Compounds 3a, 3o and 3p appeared to be more potent than PD 81,723 and at a concentration of 0.1 microM caused significant reductions of cAMP content of CHO cells expressing the human A1-adenosine receptor. Compounds 4e and 4o appeared to be allosteric enhancers at a low concentration and antagonists at a higher concentration, whereas compounds 3c, 3g, 3s and 4l appeared to be weak antagonists that are also allosteric enhancers at the higher concentration of 10 microM.     

The Effects of Synbiotic Supplementation on Glucose Metabolism and Lipid Profiles in Patients with Diabetes: a Systematic Review and Meta-Analysis of Randomized Controlled Trials

Probiotics and Antimicrobial Proteins - Although several studies have evaluated the effect of synbiotic intake on metabolic profiles in patients with diabetes, findings are inconsistent. This...     

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with K_b values of 21–63 nm at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.     

Prevalent and Incident Bacterial Vaginosis Are Associated with Sexual and Contraceptive Behaviours in Young Australian Women

Background

To determine prevalence and incidence of bacterial vaginosis (BV) and risk factors in young sexually-active Australian women.

Methods

1093 women aged 16–25 years were recruited from primary-care clinics. Participants completed 3-monthly questionnaires and self-collected vaginal smears 6-monthly for 12-months. The primary endpoint was a Nugent Score = 7–10 (BV) and the secondary endpoint was a NS = 4–10 (abnormal flora [AF]). BV and AF prevalence estimates and 95% confidence intervals (95%CI) were derived, and adjusted odds ratios (AOR) calculated to explore epidemiological associations with prevalent BV and AF. Proportional-hazards regression models were used to examine factors associated with incident BV and AF.

Results

At baseline 129 women had BV [11.8% (95%CI: 9.4–14.2)] and 188 AF (17.2%; 15.1–19.5). Prevalent BV was associated with having a recent female partner [AOR = 2.1; 1.0–4.4] and lack of tertiary-education [AOR = 1.9; 1.2–3.0]; use of an oestrogen-containing contraceptive (OCC) was associated with reduced risk [AOR = 0.6; 0.4–0.9]. Prevalent AF was associated with the same factors, and additionally with >5 male partners (MSP) in 12-months [AOR = 1.8; 1.2–2.5)], and detection of C.trachomatis or M.genitalium [AOR = 2.1; 1.0–4.5]. There were 82 cases of incident BV (9.4%;7.7–11.7/100 person-years) and 129 with incident AF (14.8%; 12.5–17.6/100 person-years). Incident BV and AF were associated with a new MSP [adjusted rate ratio (ARR) = 1.5; 1.1–2.2 and ARR = 1.5; 1.1–2.0], respectively. OCC-use was associated with reduced risk of incident AF [ARR = 0.7; 0.5–1.0].

Conclusion

This paper presents BV and AF prevalence and incidence estimates from a large prospective cohort of young Australian women predominantly recruited from primary-care clinics. These data support the concept that sexual activity is strongly associated with the development of BV and AF and that use of an OCC is associated with reduced risk.     

Metabolic characterization of the R6/2 transgenic mouse model of Huntington's disease by high-resolution MAS 1H NMR spectroscopy

The metabolic consequences of Huntington's disease in the R6/2 mouse model were investigated using NMR spectroscopy and pattern recognition to characterize selected brain regions, muscle, blood, and urine. Global increases in relative brain concentrations of osmolytes, creatine, glutamine, and lactate, and decreases in acetate and N-acetylaspartate were found together with striatal-specific lower concentrations of GABA and choline. Clear differentiation of R6/2 and wild-type mice was also obtained for urine and blood metabolite profiles that may have applicability for monitoring HD in human populations.     

Airway pressures in an asymmetrically branched airway model of the dog respiratory system

A computer model of the mechanical properties of the dog respiratory system based on the asymmetrically branching airway model of Horsfield et al. (11) is described. The peripheral ends of this airway model were terminated by a lumped-parameter impedance representing gas compression in the alveoli, and lung and chest wall tissue properties were derived from measurements made in this laboratory. Using this model we predicted the respiratory system impedance and the distribution of pressures along the airways in the dog lung. Predicted total respiratory system impedances for frequencies between 4 and 64 Hz at three lung volumes were found to compare quite closely to measured impedances in dogs. Serial pressure distributions were found to be frequency-dependent and to result in higher pressures in the lung periphery than at the airway opening at some frequencies. The implications of this finding for high-frequency ventilation are discussed.