Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo).

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON® -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions.

[Determinants of low birth weight among newborns to HIV-infected mothers not eligible for antiretroviral treatment in Africa]. / Déterminants du faible poids de naissance chez des enfants nés de mères séropositives pour le VIH, non éligibles au traitement antirétroviral, en Afrique.

BACKGROUND: Low birth weight (LBW) increases the risk of infant death, but little is known about its rate and determinants among babies born to HIV-infected mothers in sub-Saharan Africa. METHODS: This study was conducted in South Africa, Burkina Faso, Uganda and Zambia, during the recruitment process of the PROMISE-PEP (ANRS 12174) clinical trial. The study sample included 1196 subjects screened between August 2009 and December 2011, respectively 254 in South Africa, 221 in Burkina Faso, 197 in Uganda and 524 in Zambia, all ineligible for antiretroviral therapy. Data were collected during ANRS12174 clinical trial antenatal and postnatal screening visits, and during an inclusion visit for completion of an electronic case report form (eCRF). RESULTS: The mean (±SD) age of mothers was 27±5years and their mean CD4 count was 576±195cells/µL. Most mothers lived in a couple (78.7%), had no employment (72.3%) and had a good level of education (74% had gone to school). Male newborns predominated (51.7%). The mean birth weight was 3043g±435g, and 7.8% ([95%CI: 6.3%-9.3%]) of newborns weighed less than 2500g. In univariate analyses, being married or cohabiting, body mass index, WHO HIV disease stage II, female newborn and low gestational age were associated with risk of LBW. In multivariate regression model, low gestational age (aOR=3.74, P<0.0001) and female newborn (aOR=1.63, P=0.04) were significantly associated with LBW. CONCLUSION: The risk factors for LBW found in HIV-infected women ineligible for antiretroviral therapy were the same as in the general population. There was no evidence of additional risk factors associated with HIV infection.

Caesarean section wound infiltration with ropivacaine versus placebo: Survey of chronic pelvic pain after 4 years' follow-up

Objective.To test the hypothesis that optimal management of postoperative pain may reduce the risk of developing chronic pelvic pain in women who undergo caesarean section.Methods. In a randomised trial in 2006/2007; ropivacaine was infiltrated through all the layers of the anterior abdominal wound in patients undergoing caesarean section. The outcome was a reduction in severe pain or the need for rescue narcotic analgesia within 1 hour after the operation in the ropivacaine group compared with a placebo group (relative risk 0.51; 95 confidence interval 0.38 - 0.69). A follow-up study 4 years later was designed to assess the prevalence of chronic pelvic pain by carrying out telephonic interviews with these women; of whom 77 were contactable and 75 were analysed. Data and statistical analysis were done using Microsoft Excel (2007); Epi Info (version 343) statistical and Review Manager 5 software.Results. Three out of 40 women in the ropivacaine group and 3/35 in the placebo group had persistent pelvic pain (total rate of chronic pelvic pain 8.1).Conclusion. There was no significant difference in the prevalence of chronic pelvic pain between the ropivacaine wound infiltration group (7.5) and the placebo group (8.6) after 4 years' follow-up

Intrapartum amnioinfusion for meconium-stained amniotic fluid: a systematic review of randomised controlled trials.

BACKGROUND: Amnioinfusion (AI) is thought to dilute meconium when present in the amniotic fluid and so reduces the risk of meconium aspiration. OBJECTIVES: To evaluate if AI reduces meconium aspiration syndrome (MAS) and other indicators of morbidity in babies born to women with meconium-stained amniotic fluid (MSAF). SEARCH STRATEGY: PubMed, Medline, EMBASE, and the Cochrane Controlled Trials Register from January 1980 to May 30, 2005, using the keywords 'amnioinfusion' and 'meconium'. SELECTION CRITERIA: Randomised trials comparing AI with no AI for women in labour with MSAF. Trial quality was evaluated using pre-established criteria. DATA COLLECTION AND ANALYSIS: The following morbidity indicators were assessed: MAS, 5-minute Apgar score < 7, arterial cord pH < 7.2, and caesarean section. Studies were stratified according to the level of peripartum surveillance (standard versus limited). Typical relative risks (RRs) with their 95% confidence intervals were calculated for each outcome using a random effects model. MAIN RESULTS: In clinical settings with standard peripartum surveillance, we found no evidence that AI reduced the risk of MAS (RR 0.59, 95% CI 0.28-1.25), 5-minute Apgar score < 7 (RR 0.90, 95% CI 0.58-1.41), or caesarean delivery (RR 0.89, 95% CI 0.73-1.10). In clinical settings with limited peripartum surveillance, AI appeared to reduce the risk of MAS (RR 0.25, 95% CI 0.13-0.47). CONCLUSION: In clinical settings with standard peripartum surveillance, the evidence does not support the use of AI for MSAF. In settings with limited peripartum surveillance, where complications of MSAF are common, AI appears to reduce the risk of MAS. However, this finding requires confirmation by further studies.

Summation theorems for flux and concentration control coefficients of dynamic systems.

Metabolic control analysis (MCA) was developed to quantify how system variables are affected by parameter variations in a system. In addition, MCA can express the global properties of a system in terms of the individual catalytic steps, using connectivity and summation theorems to link the control coefficients to the elasticity coefficients. MCA was originally developed for steady-state analysis and not all summation theorems have been derived for dynamic systems. A method to determine time-dependent flux and concentration control coefficients for dynamic systems by expressing the time domain as a function of percentage progression through any arbitrary fixed interval of time is reported. Time-dependent flux and concentration control coefficients of dynamic systems, provided that they are evaluated in this novel way, obey the same summation theorems as steady-state flux and concentration control coefficients, respectively.

Conditions for effective allosteric feedforward and feedback in metabolic pathways.

Whether an allosteric feedback or feedforward modifier actually has an effect on the steady-state properties of a metabolic pathway depends not only on the allosteric modifier effect itself, but also on the control properties of the affected allosteric enzyme in the pathway of which it is part. Different modification mechanisms are analysed: mixed inhibition, allosteric inhibition and activation of the reversible Monod-Wyman-Changeux and reversible Hill models. In conclusion, it is shown that, whereas a modifier effect on substrate and product binding (specific effects) can be an effective negative feedback mechanism, it is much less effective as a positive feedforward mechanism. The prediction is that catalytic effects that change the apparent limiting velocity would be more effective in feedforward activation.

Comparing the regulatory behaviour of two cooperative, reversible enzyme mechanisms.

It is shown that both the reversible Hill equation and a generalised, reversible Monod-Wyman-Changeux equation can give analogous regulatory behaviour when embedded in a model metabolic pathway.

Experimental evidence for allosteric modifier saturation as predicted by the bi-substrate Hill equation.

The cooperative enzyme reaction rates predicted by the bi-substrate Hill equation and the bi-substrate Monod-Wyman-Changeux (MWC) equation when allosterically inhibited are compared in silico. Theoretically, the Hill equation predicts that when the maximum inhibitory effect at a certain substrate condition has been reached, an increase in allosteric inhibitor concentration will have no effect on reaction rate, that is the Hill equation shows allosteric inhibitor saturation. This saturating inhibitory effect is not present in the MWC equation. Experimental in vitro data for pyruvate kinase, a bi-substrate cooperative enzyme that is allosterically inhibited, are presented. This enzyme also shows inhibitor saturation, and therefore serves as experimental evidence that the bi-substrate Hill equation predicts more realistic allosteric inhibitor behaviour than the bi-substrate MWC equation.

Evaluation of a simplified generic bi-substrate rate equation for computational systems biology.

The evaluation of a generic simplified bi-substrate enzyme kinetic equation, whose derivation is based on the assumption of equilibrium binding of substrates and products in random order, is described. This equation is much simpler than the mechanistic (ordered and ping-pong) models, in that it contains fewer parameters (that is, no K(i) values for the substrates and products). The generic equation fits data from both the ordered and the ping-pong models well over a wide range of substrate and product concentrations. In the cases where the fit is not perfect, an improved fit can be obtained by considering the rate equation for only a single set of product concentrations. Due to its relative simplicity in comparison to the mechanistic models, this equation will be useful for modelling bi-substrate reactions in computational systems biology.

Software tools that facilitate kinetic modelling with large data sets: an example using growth modelling in sugarcane.

A solution to manage cumbersome data sets associated with large modelling projects is described. A kinetic model of sucrose accumulation in sugarcane is used to predict changes in sucrose metabolism with sugarcane internode maturity. This results in large amounts of output data to be analysed. Growth is simulated by reassigning maximal activity values, specific to each internode of the sugarcane plant, to parameter attributes of a model object. From a programming perspective, only one model definition file is required for the simulation software used; however, the amount of input data increases with each extra interrnode that is modelled, and likewise the amount of output data that is generated also increases. To store, manipulate and analyse these data, the modelling was performed from within a spreadsheet. This was made possible by the scripting language Python and the modelling software PySCeS through an embedded Python interpreter available in the Gnumeric spreadsheet program.

Is there an optimal ribosome concentration for maximal protein production?

A core model is presented for protein production in Escherichia coli to address the question whether there is an optimal ribosomal concentration for non-ribosome protein production. Analysing the steady-state solution of the model over a range of mRNA concentrations, indicates that such an optimum ribosomal content exists, and that the optimum shifts to higher ribosomal contents at higher specific growth rates.

Developing the sterile insect technique for Cryptophlebia leucotreta (Lepidoptera: Tortricidae): influence of radiation dose and release ratio on fruit damage and population growth in field cages.

The effect of radiation dose and different release ratios of treated (T) to untreated (U) Cryptophlebia leucotreta (Meyrick) (Lepidoptera: Tortricidae), on the incidence of fruit damage, the competitiveness of the treated males, and population growth was examined inside field cages. Navel orange trees were individually enclosed in large nylon mesh cages. Newly emerged adult moths treated with either 150 or 200 Gy of gamma radiation were released into the cages at ratios of 5T:1U or 10T:1U. The fruit was collected after 4 wk, and the number of damaged fruit and larval entries per cage were recorded for each treatment. Infested fruit was maintained in the laboratory until all emerging F1 progeny were collected and outcrossed to untreated moths of the opposite sex. Treatment had a significant effect on the mean number of larval entries and on the number of undamaged fruit per cage. The number of larval entries as well as the number of F1 progeny per cage decreased as the overflooding ratio increased. A significant reduction in egg hatch was observed in the progeny of crosses between F1 females or F1 males originating from the treatment cages compared with crosses of F1 moths originating from the control cages. The lowest mean number of fertile F1 adult females and males was obtained from the 150 Gy and 10T:1U ratio treatment. This treatment also showed the lowest per generation rate of increase (< 1 from the parental [P1] to the F1 generation), suggesting that growth in the fertile population would have been prevented if releases of treated moths at this dose and ratio were maintained in the field.

The systems biology markup language (SBML): a medium for representation and exchange of biochemical network models.

MOTIVATION: Molecular biotechnology now makes it possible to build elaborate systems models, but the systems biology community needs information standards if models are to be shared, evaluated and developed cooperatively. RESULTS: We summarize the Systems Biology Markup Language (SBML) Level 1, a free, open, XML-based format for representing biochemical reaction networks. SBML is a software-independent language for describing models common to research in many areas of computational biology, including cell signaling pathways, metabolic pathways, gene regulation, and others. AVAILABILITY: The specification of SBML Level 1 is freely available from http://www.sbml.org/

Radiation biology and inherited sterility in false codling moth (Lepidoptera: Tortricidae).

False codling moth, Cryptophlebia leucotreta (Meyrick), male and female mature pupae and newly emerged adults were treated with increasing doses of gamma radiation and either inbred or out-crossed with fertile counterparts. For newly emerged adults, there was no significant relationship between dose of radiation and insect fecundity when untreated females were mated to treated males (N female by T male). However, fecundity of treated females mated to either untreated (T female by N male) or treated males (T female by T male) declined as the dose of radiation increased. A similar trend was observed when mature pupae were treated. The dose at which 100% sterility was achieved in treated females mated to untreated males (T female by N male) for both adults and pupae was 200 Gy. In contrast, newly emerged adult males treated with 350 Gy still had a residual fertility of 5.2% when mated to untreated females, and newly emerged adult males that were treated as pupae had a residual fertility of 3.3%. Inherited effects resulting from irradiation of parental (P1) males with selected doses of radiation were recorded for the F1 generation. Decreased F1 fecundity and fertility, increased F1 mortality during development, and a significant shift in the F1 sex ratio in favor of males was observed when increasing doses of radiation were applied to the P1 males.

Experimental supply-demand analysis of anaerobic yeast energy metabolism.

Experimental supply-demand analysis of yeast fermentative energy metabolism shows that control of the glycolytic flux is shared between supply and demand. In glucose limited chemostat cultures the supply block was modulated in a dilution rate change and demand block via a benzoic acid titration. Under these conditions the supply block had a flux control of 0.90 and the demand block a flux control of 0.10.

Modelling cellular processes with Python and Scipy.

This paper shows how Python and Scipy can be used to simulate the time-dependent and steady-state behaviour of reaction networks, and introduces Pysces, a Python modelling toolkit.

How to distinguish between the vacuum cleaner and flippase mechanisms of the lmrA multi-drug transporter in Lactococcus lactis.

A numerical model of the LmrA multi-drug transport system of Lactococcus lactis is used to explore the possibility of distinguishing experimentally between two putative transport mechanisms, i.e., the vacuum-cleaner and the flippase mechanisms. This comparative model also serves as an example of numerical simulation with the scripting language Python and its scientific add-on Scipy.

Building the cellular puzzle: control in multi-level reaction networks.

Quantitative conceptual tools dealing with control and regulation of cellular processes have been mostly developed for and applied to the pathways of intermediary metabolism. Yet, cellular processes are organized in different levels, metabolism forming the lowest level in a cascade of processes. Well-known examples are the DNA-mRNA-enzyme-metabolism cascade and the signal transduction cascades consisting of covalent modification cycles. The reaction network that constitutes each level can be viewed as a "module" in which reactions are linked by mass transfer. Although in principle all of these cellular modules are ultimately linked by mass transfer, in practice they can often be regarded as "isolated" from each other in terms of mass transfer. Here modules can interact with each other only by means of regulatory or catalytic effects-a chemical species in one module may affect the rate of a reaction in another module by binding to an enzyme or transport system or by acting as a catalyst. This paper seeks to answer two questions about the control and regulation of such multi-level reaction networks: (i) How can the control properties of the system as a whole be expressed in terms of the control properties of individual modules and the effects between modules? (ii) How do the control properties of a module in its isolated state change when it is embedded in the whole system through its connections with the other modules? In order to answer these questions a quantitative theoretical framework is developed and applied to systems containing two, three or four fully interacting modules; it is shown how it can be extended in principle to n modules. This newly developed theory therefore makes it possible to quantitatively dissect intermodular, internal and external regulation in multi-level systems.

Regulating the cellular economy of supply and demand.

Cellular metabolism is a molecular economy that is functionally organised into supply and demand blocks linked by metabolic products and cofactor cycles. Supply-demand analysis allows the behaviour, control and regulation of metabolism as a whole to be understood quantitatively in terms of the elasticities of supply and demand, which are experimentally measurable properties of the individual blocks. The kinetic and thermodynamic aspects of regulation are clearly distinguished. One important result is the demonstration that when flux is controlled by one block, the other block determines to which degree the concentration of the linking metabolite is homeostatically maintained.