Optimal protection of stabilised dry live bacteria from bile toxicity in oral dosage forms by bile acid adsorbent resins

We previously found that dried live bacteria of a vaccine strain can be temporarily sensitive to bile acids and suggested that Bile Adsorbing Resins (BAR) can be used in oral vaccine tablets to protect dried bacteria from intestinal bile. Here, we report a quantitative analysis of the ability of BAR to exclude the dye bromophenol blue from penetrating into matrix tablets and also sections of hard capsule shells. Based on this quantitative analysis, we made a fully optimised formulation, comprising 25% w/w of cholestyramine in Vcaps™ HPMC capsules. This gave effectively 100% protection of viability from 4% bile, with 4200-fold more live bacteria recovered from this formulation compared to unprotected dry bacteria. From the image analysis, we found that the filler material or compaction force used had no measurable effect on dye exclusion but did affect the rate of tablet hydration. Increasing the mass fraction of BAR gave more exclusion of dye up to 25% w/w, after which a plateau was reached and no further dye exclusion was seen. More effective dye exclusion was seen with smaller particle sizes (i.e. cholestyramine) and when the BAR was thoroughly dried and disaggregated. Similar results were found when imaging dye penetration into capsule sections or tablets. The predictions of the dye penetration study were tested using capsules filled with dried attenuated Salmonella vaccine plus different BAR types, and the expected protection from bile was found, validating the imaging study. Surprisingly, depending on the capsule shell material, some protection was given by the capsule alone without adding BAR, with Vcaps™ HPMC capsules providing up to 174-fold protection against 1% bile; faster releasing Vcaps Plus™ HPMC capsules and Coni Snap™ gelatin capsules gave less protection.

Influence of temperature on the motility of Pseudomonas oryzihabitans and control of Globodera rostochiensis

The motility and efficacy of Pseudomonas oryzihabitans as a biocontrol agent against the potato cyst nematode Globodera rostochiensis were studied with respect to temperature. The influence of soil moisture on bacterial movement was also tested. In a closed container trial, P. oryzihabitans significantly reduced invasion of second stage juveniles (J2) of G. rostochiensis in potato roots, its effect being more marked at 25 and 21 degreesC than at 17 degreesC. P. oryzihabitans motility in vitro was optimal at 26 degreesC and inhibited at temperatures below 18 degreesC. In soil, both temperature and matric potential affected bacterial movement. At 16 degreesC its movement and survival were suppressed, but they were unaffected at 25 degreesC. At both temperatures the biocontrol agent moved faster in the wetter (- 0.03 MPa) than in the drier soil (- 0.1 MPa). These results suggest that temperature is a key factor in determining the potential of P. or.yzihabitans as a biocontrol agent. (C) 2003 Elsevier Science Ltd. All rights reserved.

Seed dormancy and germination of Ficus lundellii and tropical forest restoration

We investigated seed dormancy and germination in Ficus lundellii Standl. (Moraceae), a native species of Mexico's Los Tuxtlas tropical rain forest. In an 8-h photoperiod at an alternating diurnal (16/8 h) temperature of 20/30 degrees C, germination was essentially complete (96%) within 28 days, whereas in darkness, all seeds remained dormant. Neither potassium nitrate (0.05-0.2%) applied continuously nor gibberellic acid applied either continuously (10-200 ppm) or as a 24 hour pretreatment (2000 ppm) induced germination in the dark. Germination in the light was not reduced by a 24-h hydrochloric acid (0.1-1%) pretreatment, but it was reduced both by a 24-h pretreatment with either H2O2 (0. 1-5 M) or 5% HCl, or by more than 5 days of storage at 40 degrees C (4.5% seed water content). In a study with a 2-dimensional temperature gradient plate, seeds germinated fully and rapidly in the light at a constant temperature of 30 degrees C, and fully but less rapidly in the light at alternating temperatures with low amplitudes (< 12 degrees C) about the optimal constant temperature. The base, optimal and ceiling temperatures for rate of germination were estimated as 13.8, 30.1 and 41.1 degrees C, respectively. In all temperature regimes, light was essential for the germination of F lundellii seeds.

An assessment of the benefits and limitations of the shamba agroforestry system in Kenya and of management and policy requirements for its successful and sustainable reintroduction

The shamba system involves farmers tending tree saplings on state-owned forest land in return for being permitted to intercrop perennial food crops until canopy closure. At one time the system was used throughout all state-owned forest lands in Kenya, accounting for a large proportion of some 160,000 ha. The system should theoretically be mutually beneficial to both local people and the government. However the system has had a chequered past in Kenya due to widespread malpractice and associated environmental degradation. It was last banned in 2003 but in early 2008 field trials were initiated for its reintroduction. This study aimed to: assess the benefits and limitations of the shamba system in Kenya; assess the main influences on the extent to which the limitations and benefits are realised and; consider the management and policy requirements for the system's successful and sustainable operation. Information was obtained from 133 questionnaires using mainly open ended questions and six participatory workshops carried out in forest-adjacent communities on the western slopes of Mount Kenya in Nyeri district. In addition interviews were conducted with key informants from communities and organisations. There was strong desire amongst local people for the system's reintroduction given that it had provided significant food, income and employment. Local perceptions of the failings of the system included firstly mismanagement by government or forest authorities and secondly abuse of the system by shamba farmers and outsiders. Improvements local people considered necessary for the shamba system to work included more accountability and transparency in administration and better rules with respect to plot allocation and stewardship. Ninety-seven percent of respondents said they would like to be more involved in management of the forest and 80% that they were willing to pay for the use of a plot. The study concludes that the structural framework laid down by the 2005 Forests Act, which includes provision for the reimplementation of the shamba system under the new plantation establishment and livelihood improvement scheme (PELIS) [It should be noted that whilst the shamba system was re-branded in 2008 under the acronym PELIS, for the sake of simplicity the authors continue to refer to the 'shamba system' and 'shamba farmers' throughout this paper.], is weakened because insufficient power is likely to be devolved to local people, casting them merely as 'forest users' and the shamba system as a 'forest user right'. In so doing the system's potential to both facilitate and embody the participation of local people in forest management is limited and the long-term sustainability of the new system is questionable. Suggested instruments to address this include some degree of sharing of profits from forest timber, performance related guarantees for farmers to gain a new plot and use of joint committees consisting of local people and the forest authorities for long term management of forests.

Bayesian decision procedures for dose-escalation based on evidence of undesirable events and therapeutic benefit.

In this paper, Bayesian decision procedures are developed for dose-escalation studies based on bivariate observations of undesirable events and signs of therapeutic benefit. The methods generalize earlier approaches taking into account only the undesirable outcomes. Logistic regression models are used to model the two responses, which are both assumed to take a binary form. A prior distribution for the unknown model parameters is suggested and an optional safety constraint can be included. Gain functions to be maximized are formulated in terms of accurate estimation of the limits of a therapeutic window or optimal treatment of the next cohort of subjects, although the approach could be applied to achieve any of a wide variety of objectives. The designs introduced are illustrated through simulation and retrospective implementation to a completed dose-escalation study. Copyright © 2006 John Wiley & Sons, Ltd.

The role of voltage gated T-type Ca2+ channel isoforms in mediating "capacitative" Ca2+ entry in cancer cells

The mechanism by which Ca2+ enters electrically non-excitable cells is unclear. The sensitivity of the Ca2+ entry pathway in electrically non-excitable cells to inhibition by extracellular Ni2+ was used to direct the synthesis of a library of simple, novel compounds. These novel compounds inhibit Ca2+ entry into and, consequently, proliferation of several cancer cell lines. They showed stereoselective inhibition of proliferation and Ca2+ influx with identical stereoselective inhibition of heterologously expressed Cav3.2 isoform of T-type Ca2+ channels. Proliferation of human embryonic kidney (HEK)293 cells transfected with the Cav3.2 Ca2+ channel was also blocked. Cancer cell lines sensitive to our compounds express message for the Cav3.2 T-type Ca2+ channel isoform, its delta25B splice variant, or both, while a cell line resistant to our compounds does not. These observations raise the possibility that clinically useful drugs can be designed based upon the ability to block these Ca2+ channels.

Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites

Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics.

Regulation of cell cycle and stress responses to hydrostatic pressure in fission yeast

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress.

Exact error estimates and optimal randomized algorithms for integration

Exact error estimates for evaluating multi-dimensional integrals are considered. An estimate is called exact if the rates of convergence for the low- and upper-bound estimate coincide. The algorithm with such an exact rate is called optimal. Such an algorithm has an unimprovable rate of convergence. The problem of existing exact estimates and optimal algorithms is discussed for some functional spaces that define the regularity of the integrand. Important for practical computations data classes are considered: classes of functions with bounded derivatives and Holder type conditions. The aim of the paper is to analyze the performance of two optimal classes of algorithms: deterministic and randomized for computing multidimensional integrals. It is also shown how the smoothness of the integrand can be exploited to construct better randomized algorithms.

Quasi-optimal assimilation of satellite retrievals for incremental 4D-Var

An interface between satellite retrievals and the incremental version of the four-dimensional variational assimilation scheme is developed, making full use of the information content of satellite measurements. In this paper, expressions for the function that calculates simulated observations from model states (called “observation operator”), together with its tangent linear version and adjoint, are derived. Results from our work can be used for implementing a quasi-optimal assimilation of satellite retrievals (e.g., of atmospheric trace gases) in operational meteorological centres.

Spectral design and verification of HIRDLS filters and antireflection coatings using an integrated system performance approach

The HIRDLS instrument contains 21 spectral channels spanning a wavelength range from 6 to 18mm. For each of these channels the spectral bandwidth and position are isolated by an interference bandpass filter at 301K placed at an intermediate focal plane of the instrument. A second filter cooled to 65K positioned at the same wavelength but designed with a wider bandwidth is placed directly in front of each cooled detector element to reduce stray radiation from internally reflected in-band signals, and to improve the out-of-band blocking. This paper describes the process of determining the spectral requirements for the two bandpass filters and the antireflection coatings used on the lenses and dewar window of the instrument. This process uses a system throughput performance approach taking the instrument spectral specification as a target. It takes into account the spectral characteristics of the transmissive optical materials, the relative spectral response of the detectors, thermal emission from the instrument, and the predicted atmospheric signal to determine the radiance profile for each channel. Using this design approach an optimal design for the filters can be achieved, minimising the number of layers to improve the in-band transmission and to aid manufacture. The use of this design method also permits the instrument spectral performance to be verified using the measured response from manufactured components. The spectral calculations for an example channel are discussed, together with the spreadsheet calculation method. All the contributions made by the spectrally active components to the resulting instrument channel throughput are identified and presented.