The application of multi-parameter flow cytometry to the study of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> batch fermentation processes

Multi-parameter flow cytometric techniques coupled with dual colour fluorescent staining were used to study the physical and metabolic consequences of inclusion body formation in batch cultures of the recombinant Escherichia coli strain MSD3735. This strain contains a plasmid coding for the isopropylthiogalactopyranoside-inducible model eukaryotic protein AP50. It is known that the synthesis of foreign proteins at high concentrations can exert a severe metabolic stress on the host cell and that morphological changes can occur. In this work, using various points of induction, it was shown that inclusion body formation is followed immediately by measurable changes in the characteristic intrinsic light scatter patterns for the individual cell (forward scatter, 90° side scatter) and a concomitant progressive change in the individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability. This work establishes flow cytometry as a potentially valuable tool for monitoring recombinant fermentation processes, providing important information for scale-up. Further, we discuss the possibility of optimising inclusion body formation by manipulating the fermentation conditions based on these rapid real-time measurements.     

Statistical inference and model selection for the 1861 Hagelloch measles epidemic

A stochastic epidemic model is proposed which incorporates heterogeneity in the spread of a disease through a population. In particular, three factors are considered: the spatial location of an individual's home and the household and school class to which the individual belongs. The model is applied to an extremely informative measles data set and the model is compared with nested models, which incorporate some, but not all, of the aforementioned factors. A reversible jump Markov chain Monte Carlo algorithm is then introduced which assists in selecting the most appropriate model to fit the data.     

Solvent removal during synthetic and Nephila fiber spinning

The process by which spiders make their mechanically superior fiber involves removal of solvent (water) from a concentrated protein solution while the solution flows through a progressively narrowing spinning canal. Our aim was to determine a possible mechanism of spider water removal by using a computational model. To develop appropriate computational techniques for modeling of solvent removal during fiber spinning, a study was first performed using a synthetic solution. In particular, the effect of solvent removal during elongational flow (also exhibited in the spinning canal of the spider) on fiber mechanical properties was examined. The study establishes a model for solvent removal during dry spinning of synthetic fibers, assuming that internal diffusion governs solvent removal and that convective resistance is small. A variable internal solvent diffusion coefficient, dependent on solvent concentration, is also taken into account in the model. An experimental setup for dry (air) spinning was used to make fibers whose diameter was on the order of those made by spiders (approximately 1 microm). Two fibers of different thickness, corresponding to different spinning conditions, were numerically modeled for solvent removal and then mechanically tested. These tests showed that the thinner fiber, which lost more solvent under elongational flow, had 5-fold better mechanical properties (elastic modulus of 100 MPa and toughness of 15 MJ/m3) than the thicker fiber. Even though the mechanical properties were far from those of dragline spider silk (modulus of 10 GPa and toughness of 150 MJ/m3), the experimental methodology and numerical principles developed for the synthetic case proved to be valuable when establishing a model for the Nephila spinning process. In this model, an assumption of rapid convective water removal at the spinning canal wall was made, with internal diffusion of water through the fiber as the governing process. Then the diffusion coefficient of water through the initial spinning solution, obtained ex vivo from the Nephila clavipes major ampullate gland, was determined and incorporated into the numerical procedure, along with the wall boundary conditions and canal geometry. Also, a typical fiber reeling speed during web making, as well as the assumption of a dry exiting fiber, were included in the model. The results show that a cross-section of spinning solution (dope), which is initially 70% water, spends 19 s in the spinning canal in order to emerge dry. While the dope cross-section traverses the canal, its velocity increases from 0.37 mm/s at the entrance to 12.5 mm/s at the canal exit. The obtained results thus indicate that simple diffusion, along with the dry wall boundary condition, is a viable mechanism for water removal during typical Nephila fiber spinning.     

Endothelial cell confluence regulates Weibel-Palade body formation

Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (VWF) are characteristic of the mammalian endothelium. We hypothesized that vascular-specific antigens such as VWF are linked to endothelial identity and proliferation in vitro. To test this idea, the cellular accumulation of VWF in WPBs was monitored as a function of cell proliferation, confluence and passage number in human umbilical vein endothelial cells (HUVECs). We found that as passage number increased the percentage of cells containing VWF in WPBs was reduced significantly, whilst the protein was still detected within the secretory pathway at all times. However, the endothelial-specific marker protein, PECAM-1, is present on all cells even when WPBs are absent, indicating partial maintenance of endothelial identity. Biochemical studies show that a significant pool of immature pro-VWF can be detected in sub-confluent HUVECs; however, a larger pool of mature, processed VWF is detected in confluent cells. Newly synthesized VWF must thus be differentially sorted and packaged along the secretory pathway in semi-confluent versus confluent endothelial cells. Our studies thus show that WPB formation is linked to the formation of a confluent endothelial monolayer.     

Calibration of galliform molecular clocks using multiple fossils and genetic partitions

For more than a century, members of the traditional avian order Galliformes (i.e., pheasants, partridges, junglefowl, and relatives) have been among the most intensively studied birds, but still a comprehensive timeframe for their evolutionary history is lacking. Thanks to a number of recent cladistic interpretations for several galliform fossils, candidates now exist that can potentially be used as accurate internal calibrations for molecular clocks. Here, we describe a molecular timescale for Galliformes based on cytochrome b and ND2 using nine mostly internal fossil-based anchorpoints. Beyond application of calibrations spanning the entire evolutionary history of Galliformes, care was taken to investigate the effects of calibration choice, substitution saturation, and rate heterogeneity among lineages on divergence time estimation. Results show broad consistency in time estimation with five out of the nine total calibrations. Our divergence time estimates, based on these anchorpoints, indicate that the early history of Galliformes took place in the Cretaceous, including the origin of the basal-most megapode and perhaps cracid lineages, but that the remaining morphological diversification likely started in the earliest Tertiary. The multi-calibration/multi-genetic partition approach used here highlights the importance of understanding the genetic saturation, variation, and rate constancy spectra for the accurate calculation of divergence times by use of molecular clocks.     

Protein kinase A-anchoring protein AKAP95 interacts with MCM2, a regulator of DNA replication

Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix. A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex. AKAP95-MCM2 interaction was mapped to residues 1-195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin. Disruption of AKAP95-MCM2 interaction with an AKAP95-(1-195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import. Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation. Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication. Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner. Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2.     

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's beta-secretase

Cleavage of amyloid precursor protein (APP) by the Alzheimer's beta-secretase (BACE1) is a key step in generating amyloid beta-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with beta-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by alpha-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.     

Predictors of the risk of mortality in neurofibromatosis 2

To evaluate clinical and molecular predictors of the risk of mortality in people with neurofibromatosis 2 (NF2), we analyzed the mortality experience of 368 patients from 261 families in the United Kingdom NF2 registry, using the Cox proportional-hazards model and the jackknife method. Age at diagnosis, intracranial meningiomas, and type of treatment center were informative predictors of the risk of mortality. In Cox models, the relative risk of mortality increased 1.13-fold per year decrease in age at diagnosis (95% confidence interval [CI] 1.08-1.18) and was 2.51-fold greater in people with meningiomas compared with those without meningiomas (95% CI 1.38-4.57). The relative risk of mortality in patients treated at specialty centers was 0.34 compared with those treated at nonspecialty centers (95% CI 0.12-0.98). In a separate model, the relative risk of mortality in people with constitutional NF2 missense mutations was very low compared with those with other types of mutations (nonsense or frameshift mutations, splice-site mutations, and large deletions), but the CI could not be well quantified because there was only one death among people with missense mutations. We conclude that age at diagnosis, the strongest single predictor of the risk of mortality, is a useful index for patient counseling and clinical management (as are intracranial meningiomas). To ensure optimal care, we recommend that people with NF2 be referred to specialty treatment centers.