Modeling product formation in anaerobic mixed culture fermentations

The anaerobic conversion of organic matter to fermentation products is an important biotechnological process. The prediction of the fermentation products is until now a complicated issue for mixed cultures. A modeling approach is presented here as an effort to develop a methodology for modeling fermentative mixed culture systems. To illustrate this methodology, a steady-state metabolic model was developed for prediction of product formation in mixed culture fermentations as a function of the environmental conditions. The model predicts product formation from glucose as a function of the hydrogen partial pressure (P(H2)), reactor pH, and substrate concentration. The model treats the mixed culture as a single virtual microorganism catalyzing the most common fermentative pathways, producing ethanol, acetate, propionate, butyrate, lactate, hydrogen, carbon dioxide, and biomass. The product spectrum is obtained by maximizing the biomass growth yield which is limited by catabolic energy production. The optimization is constrained by mass balances and thermodynamics of the bioreactions involved. Energetic implications of concentration gradients across the cytoplasmic membrane are considered and transport processes are associated with metabolic energy exchange to model the pH effect. Preliminary results confirmed qualitatively the anticipated behavior of the system at variable pH and P(H2) values. A shift from acetate to butyrate as main product when either P(H2) increases and/or pH decreases is predicted as well as ethanol formation at lower pH values. Future work aims at extension of the model and structural validation with experimental data.     

Fracture formation in vitrified thin films of cryoprotectants

As a part of an ongoing effort to study the continuum mechanics effects associated with cryopreservation, the current report focuses on fracture formation in vitrified thin films of cryoprotective agents. The current study combines experimental observations with continuum mechanics analysis. Experimental results have been developed using a new imaging device, termed a "cryomacroscope", which has been recently presented by the current research team. A newly developed liquid nitrogen-based cooling stage is presented in this paper. The samples under investigation are 0.5 ml droplets of cryoprotective agents, having a characteristic diameter of 20 mm and a characteristic thickness of 1.5 mm. Tested samples included dimethyl sulfoxide (DMSO) in a concentration range from 6 to 8.4M, and the cryoprotectant cocktails VS55 and DP6. Some samples contained small bovine muscle segments, having a characteristic dimension of 1mm, in order to study stress concentration effects. Experimental results show that the onset of fracturing in vitrified films of cryoprotectants is very consistent, occurring over a small temperature range. Fracture pattern, however, was affected by the cooling rate. The presence of tissue segments did not affect the onset temperature of fracture, but affected the fracture pattern. The continuum mechanics analysis solidified the hypothesis that fracture is driven by thermal stress, not by temperature per se, and allowed fracture strain to be inferred from observed fracture temperature. In conjunction with the current report, additional photos of fracture formation in thin films are available at .     

Spatial and temporal dynamics of oviposition behavior of bollworm and three of its predators in Bt and non-Bt cotton fields

Host plants exhibiting insect resistance traits have long been known to influence within‐plant distributions of pests and their natural enemies. Sites and timing of egg deposition are particularly important for synchrony of predators and their prey in the field. Temporal and spatial distribution of eggs of the cotton bollworms [Heliothis virescens (F.) and Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)] and that of the predators Geocoris punctipes (Say) (Heteroptera: Geocoridae), Chrysoperla rufilabris (Burmeister) (Neuroptera: Chrysopidae), and Micromus spec. (Neuroptera: Hemerobiidae) were determined during three cotton seasons, from 2002 to 2004, by collecting and examining plants throughout each season. Comparisons also were made between Bt and non‐Bt cotton to investigate possible changes in oviposition behavior on Bt cotton. The study was conducted in commercial fields with insecticide use to manage pests when economic thresholds were exceeded in both cotton types. Egg densities for predators and bollworms varied among years, but were similar on Bt and non‐Bt cottons. Oviposition of bollworms and G. punctipes correlated spatially within plants, with most eggs laid on structures in the top five nodes of cotton plants and on the three outermost leaves on lateral branches regardless of cotton type. Bollworm oviposition dynamics exhibited two peaks within the season (early July and early August). Eggs of all predators and bollworms collected from the field and incubated in the laboratory had high hatching rates throughout each season (74–100%). Temporal association of predator with bollworm oviposition showed a significant correlation with green lacewings, a delay of 10 days for big‐eyed bugs, and no correlation with brown lacewings. Furthermore, Bt cotton plants exerted no significant effect on temporal or spatial patterns of oviposition of bollworms or the predators, indicating no change in oviposition behavior of bollworm females within plant structures after almost one decade of widespread planting of Bt cotton.     

Chromosomes of three freshwater stingrays (Rajiformes Potamotrygonidae) from the Rio Negro basin, Amazon, Brazil

Potamotrygonidae is the representative family of South American freshwater elasmobranchs. It is a monophyletic group containing 20 species grouped into three genera. Three species belonging to two genera of this family were collected from the middle Negro River, Amazonas, Brazil, and studied cytogenetically: Paratrygon aiereba, Potamotrygon motoro and Potamotrygon orbignyi. Paratrygon aiereba presented 2n = 90 chromosomes and 4M+2SM+10ST+74A. Both species of Potamotrygon presented 2n = 66 chromosomes and differed in their chromosomal formulas: P. motoro had 18M+12SM+10ST+26A and P. orbignyi had 22M+10SM+8ST+26A. No sex heteromorphism was detected. The Fundamental Number (FN) was 106 for the three species. A system of multiple NORs was found in the three species, but with interspecific differences in terms of location and position of the active Ag-NORs sites. Paratrygon aiereba presented only four sites on the short arms of two chromosomal pairs, both in terminal regions. Potamotrygon motoro presented seven sites, on the long and short arms, all in terminal regions of non-homologous chromosomes; P. orbignyi presented eight sites on the long arms, all in terminal regions, of non-homologous chromosomes. The constitutive heterochromatin was in pericentromeric regions of all chromosomes, and no significant interspecific difference was found in relation to this marker.     

Chiral resolution of the epoxyeicosatrienoic acids, arachidonic acid epoxygenase metabolites

An HPLC method for the chiral analysis of the four regioisomeric epoxyeicosatrienoic acids (EETs) is described. The cytochrome P450 arachidonic acid epoxygenase metabolites are resolved, without the need for derivatization, by chiral-phase HPLC on a Chiralcel OJ column. Application of this methodology to the analysis of the liver endogenous EETs demonstrates stereospecific biosynthesis and corroborates the role of cytochrome P450 as the endogenous arachidonic acid epoxygenase.     

Transgenic models to study disorders of respiratory control in newborn mice

Recent studies described the in vivo respiratory phenotype of mutant newborn mice with targeted deletions of genes involved in respiratory control development. Whole-body flow barometric plethysmography is the noninvasive method of choice for studying unrestrained newborn mice. The main characteristics of the early postnatal development of respiratory control in mice are reviewed, including available data on breathing patterns and on hypoxic and hypercapnic ventilatory responses. Mice are very immature at birth, and their instable breathing is similar to that of preterm infants. Breathing pattern abnormalities with prolonged apneas occur in newborn mice that lack genes involved in the development of rhythmogenesis. Some mutant newborn mice have blunted hypoxic and hypercapnic ventilatory responses whereas others exhibit impairments in responses to hypoxia or hypercapnia. Furthermore, combined studies in mutant newborn mice and in humans have helped to provide pathogenic information on genetically determined developmental disorders of respiratory control in humans.     

Utilization of substrate-induced quenching for screening targets promoting NADH and NADPH consumption

Oxidation of reduced nicotinamide adenine dinucleotides is a common event for many biochemical reactions. However, its exploitation for ultrahigh-throughput screening purposes is not an easy task and is affected by various drawbacks. It is known that such nucleotides induce quenching on the fluorescence of several dyes and that this quenching disappears with oxidation of the nucleotide. We have made use of this property to develop an assay for high-throughput screening with NADH and NADPH-dependent reductases. Full screening campaigns have been run with excellent assay quality parameters, and interesting hits have been identified. The method is amenable to miniaturization and allows easy identification of false positives without needing extra secondary assays. Although it is based on monitoring substrate consumption, it is demonstrated that the effect of fractional conversion on assay sensitivity is negligible.     

Reciprocal upregulation of urokinase plasminogen activator and its inhibitor, PAI-2, by Borrelia burgdorferi affects bacterial penetration and host-inflammatory response

The mammalian plasminogen activation system (PAS) is a complex system involved in multiple physiological and pathological processes. Borrelia burgdorferi interacts with certain components of the PAS. Here we further investigate this interaction to determine its effect on bacterial dissemination and host cell migration in vitro. We show that stimulation of monocytic cells with B. burgdorferi induces the transient production and secretion of urokinase plasminogen activator (uPA), shortly followed by its physiological inhibitor, plasminogen activator inhibitor-2 (PAI-2). Mono Mac 6 (MM6) cells as well as peripheral blood monocytes enhanced transmigration of B. burgdorferi across a barrier coated with fibronectin mediated by uPA. Moreover, the induction of PAI-2 or the addition of recombinant PAI-2 did not have a significant effect on the uPA-potentiated transmigration of B. burgdorferi. In contrast, the induction of PAI-2 by B. burgdorferi resulted in significantly diminished invasion by monocytic cells across a reconstituted basement membrane (matrigel), which could be partially restored by treatment with purified uPA. These results show that the PAS plays a twofold role in the pathogenesis of B. burgdorferi infection, both by enhancing bacterial dissemination and by diminishing host-cell inflammatory migration.     

Microtubule target for new antileishmanial drugs based on ethyl 3-haloacetamidobenzoates

A new family of antimicrotubule drugs named (3-haloacetamidobenzoyl) ureas and ethyl 3-haloacetamidobenzoates were found to be cytotoxic to the Leishmania parasite protozoa. While the benzoylureas were shown to strongly inhibit in vitro mammalian brain microtubule assembly, the ethyl ester derivatives were characterized as very poor inhibitors of this process. Ethyl 3-chloroacetamidobenzoate, MF29, was found to be the most efficient drug on the promastigote stage of three Leishmania species (IC50: 0.3-1.8 microM). MF29 maintained its activity against the clinical relevant intracellular stage of L. mexicana with IC50 value of 0.33 microM. It was the only compound that exhibits a high activity on all the Leishmania species tested. This compound appeared to alter parasite microtubule organisation as demonstrated by using antibodies directed against microtubule components and more precisely the class of microtubule decorated by the MAP2-like protein. It is interesting to notice that this MAP2-like protein was identified for the first time in a Leishmania parasite