Evaluation of a sequential global test of improved recovery following stroke as applied to the ICTUS trial of citicoline

The International Citicoline Trial in acUte Stroke is a sequential phase III study of the use of the drug citicoline in the treatment of acute ischaemic stroke, which was initiated in 2006 in 56 treatment centres. The primary objective of the trial is to demonstrate improved recovery of patients randomized to citicoline relative to those randomized to placebo after 12 weeks of follow-up. The primary analysis will take the form of a global test combining the dichotomized results of assessments on three well-established scales: the Barthel Index, the modified Rankin scale and the National Institutes of Health Stroke Scale. This approach was previously used in the analysis of the influential National Institute of Neurological Disorders and Stroke trial of recombinant tissue plasminogen activator in stroke. The purpose of this paper is to describe how this trial was designed, and in particular how the simultaneous objectives of taking into account three assessment scales, performing a series of interim analyses and conducting treatment allocation and adjusting the analyses to account for prognostic factors, including more than 50 treatment centres, were addressed. Copyright (C) 2008 John Wiley & Sons, Ltd.

Reproduction recovery of the crustacean Daphnia magna after chronic exposure to ibuprofen

In mammals, the pharmaceutical ibuprofen (IB), a non-steroidal anti-inflammatory drug, primarily functions by reversibly inhibiting the cyclooxygenase (COX) pathway in the synthesis of eicosanoids (e.g. prostaglandins). Previous studies suggest that IB may act in a similar manner to interrupt production of eicosanoids reducing reproduction in the model crustacean Daphnia magna. On this basis withdrawal of IB should lead to the recovery of D. magna reproduction. Here we test whether the effect of IB is reversible in D. magna, as it is in mammals, by observing reproduction recovery following chronic exposure. D. magna (5-days old) were exposed to a range of IB concentrations (0, 20, 40 and 80 mg l(-1)) for 10 days followed by a 10 day recovery period in uncontaminated water. During the exposure period, individuals exposed to higher concentrations produced significantly fewer offspring. Thereafter, IB-stressed individuals produced offspring faster during recovery, having similar average population growth rates (PGR) (1.15-1.28) to controls by the end of the test. It appears that maternal daphnids are susceptible to IB during egg maturation. This is the first recorded recovery of reproduction in aquatic invertebrates that suffered reproductive inhibition during chronic exposure to a chemical stressor. Our results suggest a possible theory behind the compensatory fecundity that we referred to as 'catch-up reproduction'.

Excision of anabaena PCC 7120 nifD element in escherichia coli: growth kinetics and RecA regulated xisA expression and DNA rearrangement

Anabaena PCC 7120 nifHDK operon is interrupted by an 11 kb DNA element which is excised during the development of heterocysts by Excisase A, encoded by the xisA gene residing on the element. The excision is a site-specific recombination event that occurs at the I I base pair direct repeats flanking the element. Earlier work showed the excision of the I I kb element in Escherichia coli at a frequency 0.3%. We report here the excision of this element at 1.1% and 1.98% in E. coli DH5 alpha, and 1.9% and 10.9% in E. coli JM 101 when grown on Luria broth and minimal media, respectively. Excision of nifD element in isogenic recA(-) (RK1) and recA(+) (RK2) E. coli JM101 P1 transductants, showed similar results to that of E. coli JM101 and DH5 alpha, respectively. A plasmid pMX32, carrying a xisA defective 11 kb element, showed no excision in E. coli RK2 strain. In contrast to Anabaena PCC 7120, excision of nifD element did not increase in E. call DH5 alpha grown in iron-deficient conditions. A PxisA::lacZ transcriptional fusion, used to detect the expression of elusive xisA gene, showed maximal beta-galactosidase activity in the stationary phase. The results suggest that the excision event in E. coli may involve additional factors, such as RecA and that the physiological status can influence the excision of nifD element. (C) 2007 Elsevier Ltd. All rights reserved.

Nucleic acid recovery from complex environmental samples

Effective extraction of nucleic acid from environmental samples is an essential starting point in the molecular analysis of microbial communities in the environment. However, there are many different extraction methods in the literature and deciding which one is best suited to a particular sample is very difficult. This article details the important steps and choices in deciding how to extract nucleic acids from environmental samples and gives specific details of one method that has proven very successful at extracting DNA and RNA from a range of different samples.

Structure and function analysis of the poliovirus cis-acting replication element (CRE)

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes

Nucleotides in the terminal loop of the poliovirus 2C cis-acting replication element (2C(CRE)), a 61 nt structured RNA, function as the template for the addition of two uridylate (U) residues to the viral protein VPg. This uridylylation reaction leads to the formation of VPgpUpU, which is used by the viral RNA polymerase as a nucleotide-peptide primer for genome replication. Although VPg primes both positive- and negative-strand replication, the specific requirement for 2C(CRE)-mediated uridylylation for one or both events has not been demonstrated. We have used a cell-free in vitro translation and replication reaction to demonstrate that 2C(CRE) is not required for the initiation of the negative-sense strand, which is synthesized in the absence of 2C(CRE)-mediated VPgpUpU formation. We propose that the 3' poly(A) tail could serve as the template for the formation of a VPg-poly(U) primer that functions in the initiation of negative-sense strands.

First 5f-element complexes with the tetradentate BTBP ligand. Synthesis and crystal structure of uranyl(VI) compounds with CyMe4BTBP

Treatment of [UO2(OTf)(2)] or [UO2I2(thf)(3)] with 1 equiv. of CyMe4BTBP in anhydrous acetonitrile led to the formation of [UO2(CyMe4BTBP)(OTf)(2)] (1) and [UO2(CyMe4BTBP)I-2] (2) which crystallized as the cationic forms [UO2(CyMe4BTBP)(py)][OTf](2) (3) and [UO2I(CyMe4BTBP)][I] (4) in pyridine and acetonitrile, respectively. These compounds are unique examples of structurally characterized actinide complexes with a BTBP molecule; this ligand adopts a planar conformation in the equatorial plane of the {UO2}(2+) ion. In pyridine, 1 is dissociated into [UO2(OTf)(2)(PY)(3)] and free CyMe4BTBP and the thermodynamic parameters (K, Delta H, Delta S) of this equilibrium have been determined by H-1 NMR spectroscopy. The ethoxide derivative [UO2(OEt)(CyMe4BTBP)][OTf] (5) crystallized from a solution of I in a mixture of ethanol and acetone under air, and the dinuclear mu-oxo complex [{UO2(CyMe4BTBP)}(2)(mu-O)][I](2) (6) was obtained from [UO2I(thf)(2.7)] and CyMe4BTBP. The crystal structures of 6 and of the analogous derivatives [{UO2(py)(4)}(2)(mu-O)][I](2)(7) and [{UO2(TPTZ)(py)}(2)(mu-O)][I-3](2)(8) exhibit a flexible [{UO2}-O-{UO2}](2+) moiety.

Designs for an adaptive tuned vibration absorber with variable shape stiffness element

An adaptive tuned vibration absorber (ATVA) with a smart variable stiffness element is capable of retuning itself in response to a time-varying excitation frequency., enabling effective vibration control over a range of frequencies. This paper discusses novel methods of achieving variable stiffness in an ATVA by changing shape, as inspired by biological paradigms. It is shown that considerable variation in the tuned frequency can be achieved by actuating a shape change, provided that this is within the limits of the actuator. A feasible design for such an ATVA is one in which the device offers low resistance to the required shape change actuation while not being restricted to low values of the effective stiffness of the vibration absorber. Three such original designs are identified: (i) A pinned-pinned arch beam with fixed profile of slight curvature and variable preload through an adjustable natural curvature; (ii) a vibration absorber with a stiffness element formed from parallel curved beams of adjustable curvature vibrating longitudinally; (iii) a vibration absorber with a variable geometry linkage as stiffness element. The experimental results from demonstrators based on two of these designs show good correlation with the theory.

Finite element aided design evolution of composite leaf spring

This paper shows the process of the virtual production development of the mechanical connection between the top leaf of a dual composite leaf spring system to a shackle using finite element methods. The commercial FEA package MSC/MARC has been used for the analysis. In the original design the joint was based on a closed eye-end. Full scale testing results showed that this configuration achieved the vertical proof load of 150 kN and 1 million cycles of fatigue load. However, a problem with delamination occurred at the interface between the fibres going around the eye and the main leaf body. To overcome this problem, a second design was tried using transverse bandages of woven glass fibre reinforced tape to wrap the section that is prone to delaminate. In this case, the maximum interlaminar shear stress was reduced by a certain amount but it was still higher than the material’s shear strength. Based on the fact that, even with delamination, the top leaf spring still sustained the maximum static and fatigue loads required, the third design was proposed with an open eye-end, eliminating altogether the interface where the maximum shear stress occurs. The maximum shear stress predicted by FEA is reduced significantly and a safety factor of around 2 has been obtained. Thus, a successful and safe design has been achieved.

Recovery of gallic acid with colloidal gas aphrons (CGA)

In the present paper the potential application of colloidal gas aphrons (CGA) to the recovery of antioxidants from wine-making waste extracts is investigated. CGA were generated by stirring a buffered solution (400 ml) of a cationic surfactant(cetyltrimethylammonium bromide, CTAB) at 8000 rpm for 10 minutes. Trials were carried out on standard solutions (2 ml) of gallic acid (GA) 200 mg/l with varying volumes of colloidal gas aphrons (20-60 ml) generated with varying concentrations of CTAB (2 and 4 mM). Influence of pH, solvent (buffered aqueous solution and ethanol), CTAB to GA molar ratio on recovery were studied. Best recovery (63%) was achieved from an aqueous solution of GA and at a CTAB to GA molar ratio of 16. Separation is mainly driven by electrostatic interactions but pH conditions are to be optimised to preserve the GA antioxidant power.

(-)Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons

Emerging evidence suggests that the cellular actions of flavonoids relate not simply to their antioxidant potential but also to the modulation of protein kinase signalling pathways. We investigated in primary cortical neurons, the ability of the flavan-3-ol, (-)epicatechin, and its human metabolites at physiologically relevant concentrations, to stimulate phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity. (-)Epicatechin at 100-300 nmol/L stimulated a rapid, extracellular signal-regulated kinase (ERK)- and PI3K-dependent, increase in CREB phosphorylation. At micromolar concentrations, stimulation was no longer apparent and at the highest concentration tested (30 mu mol/L) (-)epicatechin was inhibitory. (-)Epicatechin also stimulated ERK and Akt phosphorylation with similar bell-shaped concentration-response characteristics. The human metabolite 3 '-O-methyl-(-)epicatechin was as effective as (-)epicatechin at stimulating ERK phosphorylation, but (-)epicatechin glucuronide was inactive. (-)Epicatechin and 3 '-O-methyl-(-)epicatechin treatments (100 nmol/L) increased CRE-luciferase activity in cortical neurons in a partially ERK-dependent manner, suggesting the potential to increase CREB-mediated gene expression. mRNA levels of the glutamate receptor subunit GluR2 increased by 60%, measured 18 h after a 15 min exposure to (-)epicatechin and this translated into an increase in GluR2 protein. Thus, (-)epicatechin has the potential to increase CREB-regulated gene expression and increase GluR2 levels and thus modulate neurotransmission, plasticity and synaptogenesis.

Effect of dietary phytate and microbial phytase on mineral and trace element bioavailability- a literature review

Phytic acid (PA) is the main phosphorus storage compound in cereals, legumes and oil seeds. In human populations where phytate-rich cereals such as wheat, maize and rice are a staple food, phytate may lead to mineral and trace element deficiency. Zinc appears to be the trace element whose bioavailability is most influenced by PA. Furthermore, several studies in humans as well as in monogastric animals clearly indicate an inhibition of non-haem iron absorption at marginal iron supply due to phytic acid. In fact PA seems to be, at least partly, responsible for the low absorption efficiency and high incidence of iron deficiency anaemia evident in most developing countries, where largely vegetarian diets are consumed Microbial phytases have provided a realistic means of improving mineral availability from traditionally high-phytate diets. In fact it has been consistently shown that Aspergillus phytases significantly enhance the absorption of calcium, magnesium and zinc in pigs and rats. Furthermore there are a few studies in humans indicating an improvement of iron bioavailability due to microbial phytase.

Recovery and purification of surfactin from fermentation broth by a two-step ultrafiltration process

Surfactin is a bacterial lipopeptide produced by Bacillus subtilis and it is a powerful surfactant, having also antiviral, antibacterial and antitumor properties. The recovery and purification of surfactin from complex fermentation broths is a major obstacle to its commercialization; therefore, two-step membrane filtration processes were evaluated using centrifugal and stirred cell devices while the mechanisms of separation were investigated by particle size and surface charge measurements. In a first step of ultrafiltration (UF-1), surfactin was retained effectively by membranes at above its critical micelle concentration (CMC); subsequently in UF-2, the retentate micelles were disrupted by addition of 50% (v/v) methanol solution to allow recovery of surfactin in the permeate. Main protein contaminants were effective]), retained by the membrane in UF-2. Ultrafiltration was carried out either using centrifugal devices with 30 and 10 kDa MWCO regenerated cellulose membranes, or a stirred cell device with 10 kDa MWCO polyethersulfone (PES) and regenerated cellulose (RC) membranes. Total rejection of surfactin was consistently observed in UF-1, while in UF-2 PES membranes had the lowest rejection coefficient of 0.08 +/- 0.04. It was found that disruption of surfactin micelles, aggregation of protein contaminants and electrostatic interactions in UF-2 can further improve the selectivity of the membrane based purification technique. (C) 2007 Elsevier B.V. All rights reserved.