high-throughput-fluorometric-measurement-potential-soil-extracellular

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be ...

Colorado State University

Three distinct, yet interrelated, terms-wellness, recovery, and self-management-have received increasing attention in the research, consumer, and provider communities. This article traces the origins of these terms, seeking to understand how they apply, individually and in conjunction with one another to mental health consumers. Each shares a common perspective that is health-centered rather than disease-centered and that emphasizes the role of consumers as opposed to professional providers as the central determinants of health and well-being. Developing approaches combining elements of each construct may hold promise for improving the overall health and well-being of persons with serious mental disorders.

Integrating wellness, recovery, and self-management for mental health consumers.

We previously synthesized a series of potent and selective A(3) adenosine receptor (AR) agonists (North-methanocarba nucleoside 5'-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed "click" chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A(3)AR activation was preserved in these multivalent conjugates, which bound with apparent K(i) of 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A(3)AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A(3) and P2Y(14) receptors (via amide-linked uridine-5'-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.

Polyamidoamine (PAMAM) dendrimer conjugates of "clickable" agonists of the A3 adenosine receptor and coactivation of the P2Y14 receptor by a tethered nucleotide.

Persons with serious mental illnesses (SMI) have elevated rates of comorbid medical conditions, but may also face challenges in effectively managing those conditions.

The Health and Recovery Peer (HARP) Program: a peer-led intervention to improve medical self-management for persons with serious mental illness.

The P2Y(14) receptor is a relatively broadly expressed G protein-coupled receptor that is prominently associated with immune and inflammatory cells as well as with many epithelia. This receptor historically was thought to be activated selectively by UDP-glucose and other UDP-sugars. However, UDP is also a very potent agonist of this receptor, and may prove to be one of its most important cognate activators.

Signalling and pharmacological properties of the P2Y receptor.

We introduce a nearly automatic procedure to locate and count the quantum dots in images of kinesin motor assays. Our procedure employs an approximate likelihood estimator based on a two-component mixture model for the image data; the first component has a normal distribution, and the other component is distributed as a normal random variable plus an exponential random variable. The normal component has an unknown variance, which we model as a function of the mean. We use B-splines to estimate the variance function during a training run on a suitable image, and the estimate is used to process subsequent images. Parameter estimates are generated for each image along with estimates of standard errors, and the number of dots in the image is determined using an information criterion and likelihood ratio tests. Realistic simulations show that our procedure is robust and that it leads to accurate estimates, both of parameters and of standard errors.

A mixture model for quantum dot images of kinesin motor assays.

Kinesin stepping requires both tethered diffusion of the free head and conformational changes driven by the chemical state of the motor. We present a numerical method using matrix representations of approximating Markov chains and renewal theory to compute important experimental quantities for models that include both tethered diffusion and chemical transitions. Explicitly modeling the tethered diffusion allows for exploration of the model under perturbation of the neck linker; comparisons are made between the computed models and in vitro assays.

A matrix computational approach to kinesin neck linker extension.

Kinesin stepping is thought to involve both concerted conformational changes and diffusive movement, but the relative roles played by these two processes are not clear. The neck linker docking model is widely accepted in the field, but the remainder of the step--diffusion of the tethered head to the next binding site--is often assumed to occur rapidly with little mechanical resistance. Here, we investigate the effect of tethering by the neck linker on the diffusive movement of the kinesin head, and focus on the predicted behavior of motors with naturally or artificially extended neck linker domains. The kinesin chemomechanical cycle was modeled using a discrete-state Markov chain to describe chemical transitions. Brownian dynamics were used to model the tethered diffusion of the free head, incorporating resistive forces from the neck linker and a position-dependent microtubule binding rate. The Brownian dynamics and chemomechanical cycle were coupled to model processive runs consisting of many 8 nm steps. Three mechanical models of the neck linker were investigated: Constant Stiffness (a simple spring), Increasing Stiffness (analogous to a Worm-Like Chain), and Reflecting (negligible stiffness up to a limiting contour length). Motor velocities and run lengths from simulated paths were compared to experimental results from Kinesin-1 and a mutant containing an extended neck linker domain. When tethered by an increasingly stiff spring, the head is predicted to spend an unrealistically short amount of time within the binding zone, and extending the neck is predicted to increase both the velocity and processivity, contrary to experiments. These results suggest that the Worm-Like Chain is not an adequate model for the flexible neck linker domain. The model can be reconciled with experimental data if the neck linker is either much more compliant or much stiffer than generally assumed, or if weak kinesin-microtubule interactions stabilize the diffusing head near its binding site.

Monte Carlo analysis of neck linker extension in kinesin molecular motors.

The overall objective of this study is to evaluate the morphological [scanning electron microscopy (SEM)], physicochemical [differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), chemical composition analysis, Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR)], and biochemical properties of Candida rugosa lipase (CRL) immobilized on a natural biopolymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. CRL was immobilized by physical adsorption with efficiency of 30%. Compared with free CRL enzyme, there were slight changes in immobilized CRL activity as a function of temperature (from 37°C to 45°C), but a similar optimal pH value of 7.0. Inactivation rate constants for immobilized CRL enzyme were 0.009 and 0.334 h⁻¹, and half-lives were 77 and 2 h at 40°C and 60°C, respectively. Kinetic parameters obtained for immobilized CRL include the Michaelis-Menten constant of K(m) = 213.18 mM and maximum reaction velocity of V(max) = 318.62 U/g. The operational stability of immobilized CRL was tested repeatedly, and after 12 cycles of reuse, the enzyme retained 50% activity. Based on our results, we propose that PHBV-immobilized CRL could serve as a promising biocatalyst in several industrial applications.

Immobilization of Candida rugosa lipase on poly(3-hydroxybutyrate-co-hydroxyvalerate): a new eco-friendly support.

The cell response to virus infection and virus perturbation of that response is dynamic and is reflected by changes in cell susceptibility to infection. In this study, we evaluated the response of human epithelial cells to sequential infections with human respiratory syncytial virus strains A2 and B to determine if a primary infection with one strain will impact the ability of cells to be infected with the second as a function of virus strain and time elapsed between the two exposures. Infected cells were visualized with fluorescent markers, and location of all cells in the tissue culture well were identified using imaging software. We employed tools from spatial statistics to investigate the likelihood of a cell being infected given its proximity to a cell infected with either the homologous or heterologous virus. We used point processes, K-functions, and simulation procedures designed to account for specific features of our data when assessing spatial associations. Our results suggest that intrinsic cell properties increase susceptibility of cells to infection, more so for RSV-B than for RSV-A. Further, we provide evidence that the primary infection can decrease susceptibility of cells to the heterologous challenge virus but only at the 16 h time point evaluated in this study. Our research effort highlights the merits of integrating empirical and statistical approaches to gain greater insight on in vitro dynamics of virus-host interactions.

Exploratory Spatial Analysis of in vitro Respiratory Syncytial Virus Co-infections.

We develop a stochastic model for variable-length stepping of kinesins engineered with extended neck linkers. This requires that we consider the separation in microtubule binding sites between the heads of the motor at the beginning of a step. We show that this separation is stationary and can be included in the calculation of standard experimental quantities. We also develop a corresponding matrix computational framework for conducting computer experiments. Our matrix approach is more efficient computationally than large-scale Monte Carlo simulation. This efficiency greatly eases sensitivity analysis, an important feature when there is considerable uncertainty in the physical parameters of the system. We demonstrate the application and effectiveness of our approach by showing that the worm-like chain model for the neck linker can explain recently published experimental data. While we have focused on a particular scenario for kinesins, these methods could also be applied to myosin and other processive motors.

Kinesins with extended neck linkers: a chemomechanical model for variable-length stepping.

This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution.

Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system.

In 2008 all WHO member states endorsed a target of 90% reduction in measles mortality by 2010 over 2000 levels. We developed a model to estimate progress made towards this goal.

Assessment of the 2010 global measles mortality reduction goal: results from a model of surveillance data.

We describe a system of stochastic differential equations (SDEs) which model the interaction between processive molecular motors, such as kinesin and dynein, and the biomolecular cargo they tow as part of microtubule-based intracellular transport. We show that the classical experimental environment fits within a parameter regime which is qualitatively distinct from conditions one expects to find in living cells. Through an asymptotic analysis of our system of SDEs, we develop a means for applying in vitro observations of the nonlinear response by motors to forces induced on the attached cargo to make analytical predictions for two parameter regimes that have thus far eluded direct experimental observation: (1) highly viscous in vivo transport and (2) dynamics when multiple identical motors are attached to the cargo and microtubule.

Asymptotic analysis of microtubule-based transport by multiple identical molecular motors.

A new source of lipase from Bacillus sp. ITP-001 was immobilized by physical adsorption on the polymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. The support and immobilized lipase were characterised, compared to the lyophilised lipase, with regard to the specific surface area, adsorption-desorption isotherms, pore volume (V(p)) and size (dp) by nitrogen adsorption, differential scanning calorimetry, thermogravimetric analysis, chemical composition analysis, Fourier transform infrared spectroscopy and biochemical properties. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0, whereas the optimum pH for the free enzyme was at pH 7.0; the optimum temperature of activity was 80 and 37 °C for the free and immobilized enzyme, respectively. The inactivation rate constant for the immobilized enzyme at 37 °C was 0.0038 h⁻¹ and the half-life was 182.41 h. The kinetic parameters obtained for the immobilized enzyme gave a Michaelis-Menten constant (K(m)) of 49.10 mM and a maximum reaction velocity (V(max)) of 205.03 U/g. Furthermore, the reuse of the lipase immobilized by adsorption allowed us to observe that it could be reused for 10 successive cycles, duration of each cycle (1 h), maintaining 33 % of the initial activity.

Immobilization and characterisation of a lipase from a new source, Bacillus sp. ITP-001.

We show that, for a wide range of models, the empirical velocity of processive motor proteins has a limiting Pearson type VII distribution with finite mean but infinite variance. We develop maximum likelihood inference for this Pearson type VII distribution. In two simulation studies, we compare the performance of our MLE with the performance of standard Student's t-based inference. The studies show that incorrectly assuming normality (1) can lead to imprecise inference regarding motor velocity in the one-sample case, and (2) can significantly reduce power in the two-sample case. These results should be of interest to experimentalists who wish to engineer motors possessing specific functional characteristics.

Article	Year
Integrating wellness, recovery, and self-management for mental health consumers. Community mental health journal\| PubMed ID: 20033488	2010
Polyamidoamine (PAMAM) dendrimer conjugates of "clickable" agonists of the A3 adenosine receptor and coactivation of the P2Y14 receptor by a tethered nucleotide. Bioconjugate chemistry\| PubMed ID: 20121074	2010
The Health and Recovery Peer (HARP) Program: a peer-led intervention to improve medical self-management for persons with serious mental illness. Schizophrenia research\| PubMed ID: 20185272	2010
Signalling and pharmacological properties of the P2Y receptor. Acta physiologica (Oxford, England)\| PubMed ID: 20345417	2010
A mixture model for quantum dot images of kinesin motor assays. Biometrics\| PubMed ID: 20707870	2011
A matrix computational approach to kinesin neck linker extension. Journal of theoretical biology\| PubMed ID: 20951143	2011
Monte Carlo analysis of neck linker extension in kinesin molecular motors. PLoS computational biology\| PubMed ID: 21079666	2010
Immobilization of Candida rugosa lipase on poly(3-hydroxybutyrate-co-hydroxyvalerate): a new eco-friendly support. Journal of industrial microbiology & biotechnology\| PubMed ID: 21870100	2012
Exploratory Spatial Analysis of in vitro Respiratory Syncytial Virus Co-infections. Viruses\| PubMed ID: 21994640	2010
Kinesins with extended neck linkers: a chemomechanical model for variable-length stepping. Bulletin of mathematical biology\| PubMed ID: 21997362	2012
Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences\| PubMed ID: 22100550	2011
Assessment of the 2010 global measles mortality reduction goal: results from a model of surveillance data. Lancet\| PubMed ID: 22534001	2012
Asymptotic analysis of microtubule-based transport by multiple identical molecular motors. Journal of theoretical biology\| PubMed ID: 22575549	2012
Immobilization and characterisation of a lipase from a new source, Bacillus sp. ITP-001. Bioprocess and biosystems engineering\| PubMed ID: 23673896	2013
Estimating Velocity for Processive Motor Proteins with Random Detachment. Journal of agricultural, biological, and environmental statistics\| PubMed ID: 23730145	2013

Barbara E. Fricks

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