A new archosauriform species from the Panchet Formation of India and the diversification of Proterosuchidae after the end-Permian mass extinction.

Proterosuchidae represents the oldest substantial diversification of Archosauromorpha and plays a key role in understanding the biotic recovery after the end-Permian mass extinction. Proterosuchidae was long treated as a wastebasket taxon, but recent revisions have reduced its taxonomic content to five valid species from the latest Permian of Russia and the earliest Triassic (Induan) of South Africa and China. In addition to these occurrences, several isolated proterosuchid bones have been reported from the Induan Panchet Formation of India for over 150 years. Following the re-study of historical specimens and newly collected material from this unit, we erect the new proterosuchid species Samsarasuchus pamelae, which is represented by most of the presacral vertebral column. We also describe cf. proterosuchid and proterosuchid cranial, girdle and limb bones that are not referred to Samsarasuchus pamelae. Phylogenetic analyses recovered Samsarasuchus pamelae within the new proterosuchid clade Chasmatosuchinae. The taxonomic diversity of Proterosuchidae is substantially expanded here, with at least 11 nominal species and several currently unnamed specimens, and a biogeographical range encompassing present-day South Africa, China, Russia, India, Brazil, Uruguay and Australia. This indicates a broader taxonomic, phylogenetic and biogeographic diversification of Proterosuchidae than previously thought in the aftermath of the end-Permian mass extinction.

Climatic influence on the growth pattern of Panthasaurus maleriensis from the Late Triassic of India deduced from paleohistology.

Metoposaurids are representatives of the extinct amphibian clade Temnospondyli, found on almost every continent exclusively in the Late Triassic deposits. Osteohistologically, it is one of the best-known temnospondyl groups, analyzed with a wide spectrum of methods, such as morphology, morphometry, bone histology or computed modelling. The least known member of Metoposauridae is Panthasaurus maleriensis from the Pranhita-Godavari basin in Central India, being geographically the most southern record of this family. For the first time the bone histology of this taxon was studied with a focus on the intraspecific variability of the histological framework and the relationship between the observed growth pattern and climatic and/or environmental conditions. The studied material includes thin-sections of five long bones, a rib, an ilium and an intercentrum belonging most likely to eight individuals ranging from different ontogenetic stages. All bones have a large medullary region with progressively increasing remodeling, surrounded by a lamellar-zonal tissue type. The primary cortex consists of parallel-fibered matrix showing various degrees of organization, less organized collagen fibers in the zones and higher organized in the annuli. Growth marks occur in the form of alternating zones and annuli in every bone except the ilium and the intercentrum. The vascularity becomes less dense towards the outermost cortex in all sampled limb bones. Towards the outermost cortex the zone thickness is decreasing, in contrast to the avascular annuli, that become thicker or are of the same thickness. The growth pattern of P. maleriensis is uniform and represents changes in ontogenetic development. Multiple resting lines are prominent in the outer annuli of the limb bones and the rib and they presumably indicate climatic and environmental influence on the growth pattern. Therefore, a prolonged phase of slowed-down growth occurred during the unfavorable phase, but a complete cessation of growth indicated by Lines of Arrested Growth (LAGs) is not recorded in the studied samples. Based on the histological framework we conclude that the climate had an impact on the growth pattern. As we do not see any LAGs in the Indian metoposaurid, we assume that the local climate was relatively mild in India during the Late Triassic. A similar prolonged phase of slowed down growth without the occurrence of LAGs was observed in Metoposaurus krasiejowensis from the Late Triassic of Krasiejów (Poland). This is in contrast to Moroccan metoposaurid Dutuitosaurus ouazzoui from the Late Triassic of Argana Basin, where LAGs are regularly deposited throughout ontogeny indicating most likely harsher climatic conditions.

Mathematical modeling of the effects of CK2.3 on mineralization in osteoporotic bone.

Osteoporosis is caused by decreased bone mineral density (BMD) and new treatments for this disease are desperately needed. Bone morphogenetic protein 2 (BMP2) is crucial for bone formation. The mimetic peptide CK2.3 acts downstream of BMP2 and increases BMD when injected systemically into the tail vein of mice. However, the most effective dosage needed to induce BMD in humans is unknown. We developed a mathematical model for CK2.3-dependent bone mineralization. We used a physiologically based pharmacokinetic (PBPK) model to derive the CK2.3 concentration needed to increase BMD. Based on our results, the ideal dose of CK2.3 for a healthy individual to achieve the maximum increase of mineralization was about 409 µM injected in 500 µL volume, while dosage for osteoporosis patients was about 990 µM. This model showed that CK2.3 could increase the average area of bone mineralization in patients and in healthy adults.

Physiologically Based Pharmacokinetic Modeling of Fluorescently Labeled Block Copolymer Nanoparticles for Controlled Drug Delivery in Leukemia Therapy.

A physiologically based pharmacokinetic (PBPK) model was developed that describes the concentration and biodistribution of fluorescently labeled nanoparticles in mice used for the controlled delivery of dexamethasone in acute lymphoblastic leukemia (ALL) therapy. The simulated data showed initial spikes in nanoparticle concentration in the liver, spleen, and kidneys, whereas concentration in plasma decreased rapidly. These simulation results were consistent with previously published in vivo data. At shorter time scales, the simulated data predicted decrease of nanoparticles from plasma with concomitant increase in the liver, spleen, and kidneys before decaying at longer timepoints. Interestingly, the simulated data predicted an unaccounted accumulation of about 50% of the injected dose of nanoparticles. Incorporation of an additional compartment into the model justified the presence of unaccounted nanoparticles in this compartment. Our results suggest that the proposed PBPK model can be an excellent tool for prediction of optimal dose of nanoparticle-encapsulated drugs for cancer treatment.

Significance of an index of insulin resistance in non-diabetic patients with impaired fasting glucose with acute myocardial infarction and its correlation to short term outcome.

AIMS AND OBJECTIVES: To investigate the potential role of a simple insulin resistance index i.e. Homeostatic Model Assessment-Insulin Resistance (HOMA-IR) in identifying insulin resistance in patients admitted with an acute coronary syndrome without diabetes mellitus and its effect on short term outcome on cardiovascular morbidity and mortality. METHODS: Seventy non-diabetic patients with impaired fasting glucose admitted with a diagnosis of acute myocardial infarction and followed prospectively for a median of six months for outcomes. Admission fasting glucose and insulin concentrations were measured and from these values an Insulin Resistance index was calculated. RESULTS: During hospital course a high incidence of heart failure (11/;29 vs5/;41; p<0.05) and mortality (4/29 vs 1/41; p=0.152) was seen in patients with Index > 2 as compared to patients with Index <2, but mortality data failed to show any statistical significance because of small sample size. At six months patients with Index >2 had high incidence of heart failure (9/25 vs 4/40; p<0.020), reinfarct (2/25 vs. 0; p=0.141), repeated admissions (7/25 vs 3/40; p=0.092) and mortality (1/25 vs 1/40; p=0.181). Six out of twenty-five (p<0.001) patients with Index > 2 required antidiabetic treatment to achieve glycemic control. CONCLUSIONS: A simple index of insulin resistance (Homeostatic Model Assessment-Insulin Resistance) measured on patients admitted with myocardial infarction provides an important predictive measure of poor outcome and is superior to admission glucose measurement. It may be useful in identifying patients admitted with myocardial infarction who could benefit from alternative early invasive strategies.

Modeling and observer design for recombinant Escherichia coli strain.

A mathematical model for recombinant bacteria which includes foreign protein production is developed. The experimental system consists of an Escherichia Coli strain and plasmid pIT34 containing genes for bioluminescence and production of a protein, beta-galactosidase. This recombinant strain is constructed to facilitate on-line estimation and control in a complex bioprocess. Several batch experiments are designed and performed to validate the developed model. The design of a model structure, the identification of the model parameters and the estimation problem are three parts of a joint design problem. A nonlinear observer is designed and an experimental evaluation is performed on a batch fermentation process to estimate the substrate consumption.

Bilaterally symmetric Fourier approximations of the skull outlines of temnospondyl amphibians and their bearing on shape comparison.

Present work illustrates a scheme of quantitative description of the shape of the skull outlines of temnospondyl amphibians using bilaterally symmetric closed Fourier curves. Some special points have been identified on the Fourier fits of the skull outlines, which are the local maxima, or minima of the distances from the centroid of the points at the skull outline. These points denotes break in curvature of the outline and their positions can be compared to differentiate the skull shapes. The ratios of arc-lengths of the posterior and lateral outline of 58 temnospondyl skulls have been plotted to generate a triaguarity series of the skulls. This series grades different families, some of their genera and species as well as some individuals according to their posterior and lateral skull length ratios. This model while comparing different taxa, takes into account the entire arc-length of the outline of the temnospondyl skulls, and does not depend on few geometric or biological points used by earlier workers for comparing skull shapes.

Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process.

On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.

Quaternary ammonium functionalized poly(propylene imine) dendrimers as effective antimicrobials: structure-activity studies.

Quaternary ammonium functionalized poly(propyleneimine) dendrimers were synthesized and their antibacterial properties were evaluated using a bioluminescence method. These quaternary ammonium dendrimers are very potent biocides. The antibacterial properties depend on the size of the dendrimer, the length of hydrophobic chains in the quaternary ammonium groups, and the counteranion. Since these dendrimers are well characterized and monodisperse, they also serve as an effective system to study the structure-activity relationship. The antimicrobial properties of these dendrimer biocides have a parabolic dependence on molecular weight, which is different from the bell-shaped molecular weight dependence of conventional polymer biocides. The dependence on the hydrophobic chain of the quaternary ammonium structure is similar to conventional polymer biocides, and shows a parabolic relationship with dendrimer biocides carrying C10 hydrophobes the most potent. The antimicrobial properties of these novel biocides with bromide anions are more potent than those with chloride anions. Biocides derived from hyperbranched polymers were also synthesized and found to possess somewhat lower effectiveness.

Cecropins induce the hyperosmotic stress response in Escherichia coli.

Cecropin A and B, below or near their minimum inhibitory concentrations in viable Escherichia coli, interfered with the rapid NaCl-induced hyperosmotic shrinkage of the cytoplasmic volume (plasmolysis), and also activated the promoter of the hyperosmotic stress gene osmY. The same promoter was also expressed by hyperosmolar NaCl or sucrose, two of the most commonly used antimicrobial food preservatives. Stress responses were monitored during the logarithmic growth phase of E. coli strains that contain specific promoters fused to a luxCDABE operon on a plasmid. The luminescence assay, developed to monitor the transcriptional response to stresses, is based on the premise that organisms often respond and adapt to sublethal environmental adversities by increased expression of stress proteins to restore homeostasis. The luminescence response from these fusion strains to a specific stress occurs as the transcription at the promoter site is activated. Cecropins induced luminescence response only from the osmY-luxCDABE fusion, but not the corresponding stress promoter activation associated with macromolecular or oxidative damage, or leakage of the cytoplasmic content including the proton gradient. The inhibitory effect of cecropins on plasmolysis is interpreted to suggest that the primary locus of action of these antimicrobial peptides in the periplasmic space is on the coupling between the inner and outer membrane.

Osmotic stress in viable Escherichia coli as the basis for the antibiotic response by polymyxin B.

Cationic antimicrobial peptides, such as polymyxin B (PxB), below growth inhibitory concentration induce expression of osmY gene in viable E. coli without leakage of solutes and protons. osmY expression is also a locus of hyperosmotic stress response induced by common food preservatives, such as hypertonic NaCl or sucrose. High selectivity of PxB against Gram-negative organisms and the basis for the hyperosmotic stress response at sublethal PxB concentrations is attributed to PxB-induced mixing of anionic phospholipid between the outer layer of the cytoplasmic membrane with phospholipids in the inner layer of the outer membrane. This explanation is supported by PxB-mediated rapid and direct exchange of anionic phospholipid between vesicles. This mechanism is consistent with the observation that genetically stable resistance against PxB could not be induced by mutagenesis.

A miniature bioreactor for sensing toxicity using recombinant bioluminescent Escherichia coli cells.

A miniature bioreactor was fabricated as a contactor between biosensing cells and toxic materials. This miniature bioreactor (58 mL working volume) showed performance similar to that of a conventional bioreactor, as well as the advantages of easy installation, facile operation, and small medium requirements during long-term continuous operation. A performance evaluation measured the response to ethanol in continuous operation by using a recombinant bioluminescent Escherichia coli strain. Continuous cultures were repeatedly induced by the ethanol challenge. Steady-state cell concentrations (OD) were found to be decreased, the induced specific bioluminescence (SBL) peak value was found to be increased, and the peak response time, which is the time constant of this continuous monitoring system, was found to be decreased with increasing dilution rate. Finally on- and off-line bioluminescence monitoring was shown to be reliable, suggesting that this system is suitable for applications such as monitoring the influent and effluent streams of waste water biotreatment plants.

Characterization of the stress response of a bioluminescent biological sensor in batch and continuous cultures.

The effects of temperature, growth stage, and inducer (ethanol) concentration on the kinetics and magnitude of the stress response were investigated by using an Escherichia coli strain with the grpE heat shock promoter fused to the Vibrio fischeri lux genes. When stressed, the cells responded by changing the level of specific light emission, which was measured both on- and off-line. These measurements were used to characterize and optimize the sensitivity of the construct by determining the conditions at which the culture exhibited maximum specific bioluminescence and minimum response time to ethanol induction in batch cultivation. The results of the batch study were then applied to continuous cultivation, and the effect of dilution rate was determined. These results are of considerable interest in the development of an on-line biological sensor system for the detection and toxicity assessment of chemical pollutants.

Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene.

The tendency of recombinant protein in bacteria to partition into soluble and insoluble forms is attributed, in general, to a kinetic competition between protein folding and aggregation. However, little experimental work has actually been performed in vivo on the kinetics and mechanisms of protein folding and aggregation. Results are presented here from radiolabeling experiments which monitored the kinetics of recombinant protein aggregation in actively growing cultures. The strain used was an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene. The rate of CheY aggregation was found to be time dependent in that the tendency of CheY to aggregate was greater for newly translated molecules, i.e., those translated within the previous several minutes, than for molecules translated less recently. CheY protein molecules that were translated less recently continued to aggregate for several hours but at a lower rate. The movement of soluble CheY to the insoluble form was enhanced at elevated growth temperatures and inhibited by the presence of chloramphenicol. The latter observation suggests that ongoing translation facilitates the movement of soluble CheY to the insoluble form. The implications of these results for the mechanism of protein aggregation in vivo, i.e., inclusion body formation, are discussed.

Synthesis of non-translating or translating specialized ribosomes causes feedback regulation of ribosomal RNA synthesis in Escherichia coli.

Specialized ribosomes carry a mutant anti-Shine-Dalgarno region that disrupts the complementary base pairing that stabilizes the translation initiation complex with E. coli mRNAs. It has been reported that production of specialized ribosomes does not cause the inhibition of chromosomal rRNA synthesis that follows production of wild-type ribosomes. We proposed that enabling translation on specialized ribosomes by providing mRNA with a complementary mutation in the Shine-Dalgarno region would restore feedback regulation and inhibit chromosomal rRNA synthesis. With both our system and the system studied previously, we saw feedback regulation regardless of whether the specialized ribosomes were translating. As reported previously, transcription from plasmid-borne promoters decreased as chromosomal rRNA synthesis was repressed, suggesting that the lambda PL and tac promoters may be sensitive to the effector(s) of feedback regulation.

Mathematical model of temperature-sensitive plasmid replication.

The copy number of a series of plasmids constructed at Odense University is regulated by the lambda PR/PRM promoters and the temperature-sensitive cI857 repressor. At low temperatures, these plasmids exhibit the low copy number of the parent plasmid R1 (5-6 per cell). At high temperatures, the plasmids exhibit runaway replication, reaching copy numbers of greater than 1,000 per cell. A detailed mathematical model of the temperature-sensitive replication of these plasmids has been developed incorporating three features: replication of the parent plasmid, regulation of the lambda PR/PRM promoters by the cI repressor, and thermal denaturation of the cI857 repressor. Models of the first two of these features have been described by others. We revised and extended those models, described the thermal denaturation of the cI857 repressor, and integrated these features to give a comprehensive model of temperature-sensitive plasmid replication. Model predictions were compared to experimental measurements of both steady-state copy numbers as a function of temperature and the change in copy number following temperature shifts up and down. The model accurately describes the qualitative behavior of the system and gives reasonable quantitative results. This is particularly significant since all the parameter values used in this model were determined independently: that is, there was no adjustment of parameter values to match our experimental data. The regulatory system that gives rise to the temperature-sensitive replication of these plasmids is widely used in biotechnology applications, so the elements of the model related to this regulation should be applicable to a wide variety of systems.

Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions.

Heat shock gene expression is induced by a variety of environmental stresses, including the presence of many chemicals. To address the utility of this response for pollutant detection, two Escherichia coli heat shock promoters, dnaK and grpE, were fused to the lux genes of Vibrio fischeri. Metals, solvents, crop protection chemicals, and other organic molecules rapidly induced light production from E. coli strains containing these plasmid-borne fusions. Introduction of an outer membrane mutation, tolC, enhanced detection of a hydrophobic molecule, pentachlorophenol. The maximal response to pentachlorophenol in the tolC+ strain was at 38 ppm, while the maximal response in an otherwise isogenic tolC mutant was at 1.2 ppm. Stress responses were observed in both batch and chemostat cultures. It is suggested that biosensors constructed in this manner may have potential for environmental monitoring.

Properties conferred on Clostridium thermocellum endoglucanase CelC by grafting the duplicated segment of endoglucanase CelD.

The DNA sequence encoding the duplicated 22 amino acid segment of Clostridium thermocellum endoglucanase CelD was fused to the 3'-terminus of the celC gene encoding C.thermocellum endoglucanase CelC. The presence of the duplicated segment endowed CelC with the capacity to form cytoplasmic inclusion bodies containing active enzyme when the hybrid gene was expressed in Escherichia coli. Inclusion body formation prevented proteolytic cleavage of the duplicated segment. The intact hybrid protein CelC-Cel'D was purified from inclusion bodies and characterized. In contrast to CelC, CelC-Cel'D was able to bind to CipA, a protein acting as a scaffolding component of the C.thermocellum cellulase complex (cellulosome). However, the catalytic properties of CelC-Cel'D were similar to those of CelC. These results suggest that foreign proteins tagged with the duplicated segment could be incorporated into the cellulosome in order to modify the enzymatic properties of the complex. The formation of inclusion bodies by proteins carrying the duplicated segment may also prove a convenient means of purifying cloned gene products that are sensitive to proteolytic degradation.

Real-time compensation of the inner filter effect in high-density bioluminescent cultures.

Bioluminescence has recently become a popular research tool in several fields, including medicine, pharmacology, biochemistry, bioprocessing, and environmental engineering. Beginning with purely qualitative goals, scientists are now targeting more demanding applications where accurate, quantitative interpretation of bioluminescence is necessary. Using the recent advances in fiber-optic technology, bioluminescence is easily monitored in vivo and in real time. However, the convenience of this measurement is often concealing an unsuspected problem: the bioluminescence signal might be corrupted by a large error caused by the extinction of light by biological cells. Since bioluminescent cultures not only emit light but also absorb and scatter it, the measured signal is related in a complex, nonlinear, and cell-concentration-dependent manner to the "true" bioluminescence. This light extinction effect, known as the "inner filter effect," is significant in high-density cultures. Adequate interpretation of the bioluminescence signal can be difficult without its correction. Here, we propose a real-time algorithm for elimination of the inner filter effect in a bioreactor. The algorithm yields the bioluminescence which would be measured if the glowing culture was completely transparent. This technique has been successfully applied to batch and continuous cultivation of recombinant bioluminescent Escherichia coli.

Specialized ribosomes in Escherichia coli.

Specialized ribosomes, developed by de Boer and co-workers at Genentech, carry a mutation in the anti-Shine-Dalgarno region of 16 S ribosomal RNA, the site of messenger RNA binding. A complementary mutation in the ribosome binding site (the Shine-Dalgarno region) of a particular messenger RNA results in specific and efficient translation of that messenger RNA on the specialized ribosomes. With this system, a fraction of the cell's ribosomes can be dedicated to the translation of a single messenger RNA; the remaining wild-type ribosomes carry out the normal cellular translation processes. Specialized ribosomes have been used for the overproduction of proteins, analysis of the feedback regulation of ribosomal RNA synthesis, and mutational analysis of 16 S ribosomal RNA. Experimental results related to each of these topics are summarized and discussed, and other potential applications are described. In particular, we propose a novel technique for mutational analysis of 23 S ribosomal RNA, the primary RNA constituent of the large ribosomal subunit, using the specialized ribosome approach.