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1483 results in Biotechnology

Page 8 of 75

  • Growth performance and bone characteristics of broiler chickens fed corn-soy diet supplemented with different levels of vitamin premix and sources of mineral premix

  • T. Ao, M.A. Paul, A.J. Pescatore, L.M. Macalintal, M.J. Ford, K.A. Dawson
  • Journal:
    Journal of Applied Animal Nutrition / Volume 7 / 2019
    Published online by Cambridge University Press:
    27 June 2019, e6
    Print publication:
    2019

  • Inorganic trace mineral salts in the premix have a detrimental effect on the stability of vitamins due to redox reactions. A study was conducted to investigate the effect of dietary supplementation of different levels of vitamin premix with different mineral premixes on the performance and bone characteristics of broilers. A 2 x 2 factorial dietary treatment was used with two levels of vitamins and two types of minerals in the premix. A total of 1056, one-day old chicks were randomly assigned to four dietary treatments with 12 replicate pens of 22 chicks for 28 d. An interactive effect between vitamin levels and mineral sources on weight gain and feed intake of chickens was detected. Chickens fed the diet containing 100% vitamin premix with either source of mineral premix had higher (P < 0.01) weight gain and feed intake than those fed the diet containing 30% vitamin premix with either source of mineral premix. However, the chickens fed the diet containing the 30% vitamin premix with the organic minerals had higher (P < 0.01) weight gain and feed intake than those fed the diet containing 30% vitamin premix with inorganic minerals. Chickens fed the diet containing organic mineral premix had lower (P < 0.05) mortality and feed to gain ratio and higher (P < 0.01) bone breaking strength and ash content of tibia than those fed the inorganic mineral premix treatment. Chickens fed the diet containing 100% vitamin premix had higher (P < 0.01) breaking strength of femur and tibia ash than those fed the diet containing 30% vitamin premix. The results from this trial indicated that total replacement of inorganic trace minerals with organic minerals can increase the storage stability of vitamins in feed premixes containing both vitamins and trace minerals, which is reflected in better growth performance in poultry.

  • Mannan rich fraction from yeast modulates inflammatory responses in intestinal cells (HT-29) exposed to Escherichia coli

  • Niall Browne, Aimee Traynor, Karina A. Horgan
  • Journal:
    Journal of Applied Animal Nutrition / Volume 7 / 2019
    Published online by Cambridge University Press:
    18 June 2019, e5
    Print publication:
    2019

  • Mannan from yeast has been demonstrated to limit infection in animals susceptible to gastrointestinal infection, including pigs, poultry and cows, by blocking the mechanism by which gram-negative bacteria adhere to and invade the intestines. Enterotoxigenic Escherichia coli (ETEC) cause post weaning diarrhoea (PWD) which results in poor weight gain and potential death at great economic cost to the farmer. A mannan rich fraction (MRF) was assessed in vitro for its impact on ETEC infection of HT-29 intestinal cell line. Gene expression markers for inflammation (TNFα and IL-1β) and TLR4 (TICAM-1 and LY96) associated recognition of bacteria were significantly elevated following exposure to E. coli alone, but not in combination with MRF compared to the control. HT-29 cells exposed to MRF alone demonstrated significantly reduced expression of immune signalling genes IRAK1, IRF7 and JUN when compared to the control. HT-29 cell protein abundance for TNFα and TLR4 associated proteins were significantly increased in response to E. coli exposure alone while no significant change was observed for MRF treatment with E. coli infection. E. coli adhesion to HT-29 cells was significantly decreased with addition of MRF compared to E. coli infection alone. The action of MRF demonstrated its potential capacity to limit infection on an in vitro level through blocking bacterial interaction with the intestines that leads to infection as marked by a reduction in proinflammatory responses. MRF on its own demonstrated potential anti-inflammatory effects on intestinal cells with the reduction of proinflammatory responses observed.

  • Influence of feeding whole maize, differing in endosperm hardness, on the performance, nutrient utilisation and digestive tract development of broiler starters

  • Y. Singh, V. Ravindran
  • Journal:
    Journal of Applied Animal Nutrition / Volume 7 / 2019
    Published online by Cambridge University Press:
    30 January 2019, e1
    Print publication:
    2019

  • Use of whole wheat, along with compounded feeds, for poultry feeding is a common practice in many parts of the world. However, studies investigating the use of other grains are limited. In the present study, the influence of including whole maize, with differing hardness, in broiler diets on the performance, nutrient utilisation and digestive tract development was examined. The experimental design was a 3 × 2 factorial arrangement of treatments, evaluating maize hardness (hard, semi-hard or soft) with diets based on ground maize or 115 g/kg whole maize replacing ground maize. The three maize cultivars were ground in a hammer mill to pass through a 4 mm sieve and six diets were developed based on one of the three cultivars. Following mixing, all diets were cold pelleted through a 3 mm die. Each of the six diets was fed to six replicate cages (eight birds per cage) from day 1 to 21 post-hatch. Maize hardness and whole maize inclusion had no effect (P>0.05) on weight gain. Maize hardness influenced (P<0.05) feed intake and feed per gain, while these two parameters were unaffected (P>0.05) by the inclusion of whole maize. The apparent metabolisable energy (AME) was unaffected (P>0.05) by maize hardness and whole maize inclusion. Maize hardness increased the ileal digestibility of nitrogen (P<0.05) and starch (P=0.06). The relative weight of the gizzard was greater (P<0.05) in birds fed hard and semi-hard maize compared to soft maize, and was greater (P<0.05) when whole maize was included. These results indicated that 115 g/kg of ground maize can be replaced by whole maize in broiler starter diets with no adverse effects on growth performance and thus can reduce the cost of feed manufacture.

  • Effect of different phytases derived from E. coli AppA gene on the performance, bone mineralisation and nutrient digestibility of broiler chicken

  • K. Kozlowski, L. Nollet, A. Lanckriet, E. Vanderbeke, P. Mielnik, N. Outchkourov, S. Petkov
  • Journal:
    Journal of Applied Animal Nutrition / Volume 7 / 2019
    Published online by Cambridge University Press:
    07 August 2019, e7
    Print publication:
    2019

  • This study evaluated the effects of three different thermostable phytase variants, based on the AppA gene from E. coli (AppAT1, AppAT2 and AppAT3) on growth performance, nutrient digestibility and bone mineralisation in broiler chickens at inclusion levels of 250 and 500 FTU/kg. The eight treatment groups included a positive control (PC) which was sufficient in Ca and P, a negative control (NC, the same basal formulation as the PC, but reduced in Ca and P), and NC supplemented with AppAT1 at 250 and 500 FTU/kg (AppAT1-250 and AppAT1-500), AppAT2 at 250 and 500 FTU/kg (AppAT2-250 and AppAT2-500) and with AppAT3 at 250 and 500 FTU/kg (AppAT3-250 and AppAT3-500). Over the entire feeding period, body weight (BW) and average daily gain (ADG) were significantly higher in the PC group, with all phytase supplemented groups being statistically the same, compared to the NC group. Feed conversion (FCR) for the PC-fed birds (1.479) was significantly (P<0.05) better compared to the NC birds (1.582) and those fed the AppAT3-250 diet (1.523). Reduced levels of Ca and P in the NC group led to significantly (P<0.05) lower tibia ash (40.9%) compared to the PC group (47.4%). Birds fed the phytase diets had significantly higher tibia ash compared to the NC birds, with those from the AppAT2-500 and AppAT3-500 groups being statistically the same as the PC group. Diets AppAT1-500, AppAT2-250, AppAT2-500 and AppAT3-500 significantly increased Ca digestibility compared to the NC. Apparent total track digestibility (ATTD) of P was improved for AppAT1-500 and AppAT2-250. The ATTD of Ca and P for all of the phytase supplemented groups reached the same level of the PC and AppAT1-500 group. It was concluded that adding any of the phytases tested, especially when included at 500 FTU/kg to a feed reduced in Ca and P, led to improved performance and bone mineralisation back to the same levels as seen for the Ca and P sufficient diet.

  • Effect of Calsporin® (Bacillus subtilis C-3102) addition to the diet on faecal quality and nutrient digestibility in healthy adult dogs

  • S. Schauf, N. Nakamura, C. Castrillo
  • Journal:
    Journal of Applied Animal Nutrition / Volume 7 / 2019
    Published online by Cambridge University Press:
    08 April 2019, e3
    Print publication:
    2019

  • This study evaluated the effect of Bacillus subtilis C-3102 (Calsporin®) addition to the diet on faecal characteristics and nutrient digestibility in healthy adult dogs. Sixteen Beagles received either a low-energy control diet (CON; 3.35 Mcal metabolisable energy (ME)/kg with 21.8, 27.9, and 50.3% ME as protein, fat, and nitrogen-free extractives (NFE), respectively) or the same diet supplemented with Bacillus subtilis at 1 × 109 CFU/kg diet as probiotic (PRO) for four weeks in a parallel design (eight dogs per diet). In the prior two weeks, all dogs received a high-energy diet (Advance Medium Adult, Affinity Petcare®, 3.81 Mcal ME/kg ME with 24.8, 41.2, and 34% ME protein, fat, and NFE, respectively). Faecal consistency, dry matter (DM), pH, and NH3 were analysed on fresh samples collected at the start and weekly throughout the study. Additional samples were collected for the determination of lactate and short-chain fatty acids (SCFA) on days 0 and 21. In week four, a five–day total faecal collection was conducted in six dogs from each diet for the determination of nutrient apparent digestibility. Dogs fed the PRO diet had more firm faeces (P = 0.011) than control dogs and a higher faecal DM content in the first two weeks (P < 0.05). Feeding the PRO diet resulted in a decline in NH3 over four weeks (P = 0.05) and in faecal pH in the first two weeks (P < 0.05) alongside an increase in SCFA content (P = 0.044), mainly acetate (P = 0.024). Faecal lactate did not differ between diets (P > 0.10). Dogs fed the PRO diet showed a higher apparent digestibility of fat (P = 0.031) and NFE (P = 0.038) compared to control dogs. Dog food supplementation with Calsporin® at 1 × 109 CFU/kg improved faecal quality, enhanced fat and carbohydrate digestibility, and contributed to the gut health of dogs by reducing gut ammonia and increasing SCFA content.

Dynamics of Engineered Artificial Membranes and Biosensors
  • William Hoiles, Vikram Krishnamurthy, Bruce Cornell
  • Published online:
    25 May 2018
    Print publication:
    03 May 2018
  • Learn about the state of the art in building artificial membranes and synthetic biological devices, and in constructing mathematical models for their dynamics at multiple time and spatial scales with this comprehensive book. Drawing on recent advances in bioengineering and biochemistry, it describes how to engineer tethered bilayer lipid membranes, bioelectronic interfaces, high-resolution biosensors, and diagnostic devices for non-invasive cellular measurements and electroporation. Multi-physics models combining atomistic (molecular dynamics and coarse-grained molecular dynamics), mesoscopic (Poisson–Nernst–Planck), and macroscopic (reaction-rate theory) dynamics provide a complete structure-to-function description of these devices. Experiments and dynamic models explain how anti-microbial peptides penetrate membranes, how molecular machine biosensors built out of artificial membranes can detect femtomolar concentrations, and how electroporation can be controlled. Supported by atomistic simulation code online, this is essential reading for researchers, students and professionals in bioengineering, chemical engineering, biophysics, applied mathematics, and electrical engineering.

  • Appendix A - Elementary Primer on Partial Differential Equations (PDEs)

  • from Appendices
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 395-403

  • Summary

    Chapters 10–12 described continuum models of engineered membranes that involved partial differential equations (PDEs). This appendix gives a brief introduction to the classification of PDEs, linear PDEs, nondimensionalization of PDEs, and numerical methods for solving PDEs. Uniqueness and existence results are not discussed here since they involve advanced results in functional analysis that are outside the scope of this book. For a comprehensive advanced treatment of PDEs at a graduate mathematics level see [115].

    Linear, Semilinear, and Nonlinear Partial Differential Equations

    PDEs are typically classified into linear, semilinear, and nonlinear. Linear PDEs are important as several methods exist to obtain closed-form solutions of these PDEs (under simple boundary conditions), including separation of variables, superposition, Fourier series, Laplace transform, and Fourier transform. The solution to some semilinear PDEs can be obtained using the symmetry method or method of characteristics. When a linear or semilinear PDE has more sophisticated boundary conditions (as is the case with engineered membranes), the PDE needs to be solved numerically. Similarly, nonlinear PDEs typically do not have an exact solution and must also be solved numerically.

    Consider a generic PDE of two scalar variables (space x and time t), where u(x, t) is a function of the variables x and t. A linear PDE has the form

    where a(x, t), b(x, t), c(x, t), and d(x, t) are generic functions of x and t but not u. A PDE is semilinear if the coefficient function a(x, t) of the highest partial derivative is dependent on x and t but not u. Therefore, (A.1) is a semilinear PDE if the coefficient functions can be expressed as a(x, t), b(x, t, u), c(x, t, u), and d(x, t, u). If the PDE is neither linear nor semilinear, it is nonlinear. Linear, semilinear, and nonlinear PDEs are all used to model the dynamics of engineered membranes. For example:

    Linear. Poisson's equation ((10.3) on page 180), the Nernst–Planck equation ((10.9) on page 182), the Poisson–Fermi equation ((11.23) on page 233), and the Fokker– Planck equation ((14.28) on page 318).

  • 11 - Electroporation Models in Engineered Artificial Membranes

  • from Part III - Dynamic Models for Artificial Membranes: From Atoms to Device
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 212-249

  • Summary

    Introduction

    A lipid membrane that contains negligible defects and no ion channels will not allow ions to pass through the membrane. Indeed a membrane can be viewed as an electric capacitor. However, if an excitation is applied to the membrane this will cause the temporary breakdown of the membrane, causing water-filled pores to form that are large enough to allow the passage of ions and molecules through the membrane – this process is called electroporation.

    Electroporation is the process of aqueous pore formation in biological membranes when a voltage potential (e.g., ion gradient) is applied across the membrane. Several microbiology techniques are based on the use of electroporation to increase the permeability of the cell membrane to allow the passage of macromolecules, drugs, DNA, and antimicrobial peptides into the cell. These microbiology techniques are focused on how to design the excitation potential to allow the passage of these molecules through the membrane while ensuring the membrane is not irreversibly damaged. In this chapter we construct mesoscopic diffusion models of electroporation in engineered tethered membranes. The formulation in this chapter relies heavily on the Smoluchowski–Einstein equation, which is a probabilistic model for the number of aqueous pores formed. Additionally, we use statistical mechanics and electrodiffusive dynamic models to estimate lumped circuit parameters such as membrane conductance, membrane capacitance, and double-layer capacitance in the presence of electroporation. Recall that Chapters 4 and 6 dealt with the construction and experimental measurement methods for the electroporation measurement platform. Put simply, this chapter gives mathematical models and associated insight at the mesoscopic level to explain the experimental results of Chapter 6.

    Applications of Electroporation

    Electroporation has been used in a variety of in vitro and in vivo biotechnical applications for antitumor treatment, protein insertion, cell fusion, and gene and drug delivery.

    The process of electroporation results in the formation of pores in the membrane that allow molecules and ions to pass through the hydrophobic bilayer membrane. During electroporation the lipid molecules remain intact; they merely undergo a collective configurational change to form nanometer-sized aqueous pores. These aqueous pores allow ions and molecules to pass through the membrane. There are two types of electroporation:

    Reversible electroporation. Reversible electroporation occurs if, after the excitation potential is removed, the lipids can rearrange to form an impermeable membrane.

  • 12 - Electroporation Measurements in Engineered Membranes

  • from Part III - Dynamic Models for Artificial Membranes: From Atoms to Device
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 250-281

  • Summary

    Introduction

    The electroporation measurement platform (EMP) discussed in Chapters 4 and 6 is a synthetic biological device built out of artificial membranes to study electroporation in a controlled environment. In this chapter we apply the continuum models for electroporation developed in Chapter 11 to predict and interpret the response of the EMP device. Specifically we evaluate how accurately the continuum models predict the response of the EMP to membranes containing different tether densities, lipid types, and sterols and excited using different waveforms. Thus, this chapter (which validates electroporation models with experimental data in precisely controlled environments) together with the previous chapter (which formulates continuum models) gives a complete treatment of electroporation at the mesoscopic level.

    Before proceeding, the reader should recall that electroporation is the process of aqueous pore formation resulting from changes in the transmembrane potential. A schematic of the electroporation process is given in Figure 12.1. To ensure only the process of electroporation is present, the following test is performed for all experiments involving the electroporation measurement platform (see Chapter 6 for details of such experiments). An excitation potential Vs is applied and the resulting current is recorded; then the negative potential −Vs is applied and the resulting current recorded. If the current response resulting from Vs is related to the current response of −Vs by a sign change then we can conclude that only the process of electroporation is present. This conclusion follows from the dynamic model of engineered membranes given by Figure 11.4 and (11.17). Recall that the process of electroporation is transmembrane potential symmetric; that is, the polarity or sign of the applied transmembrane potential does not change the dynamics the electroporation process. All experimental measurements reported below satisfied this test.

    Organization of Chapter

    Given the mesoscopic models in Chapter 11, in this chapter we evaluate important parameters of the engineered membrane using both in silico and in vitro techniques. More importantly, we give significant insight into the process of electroporation by discussing several aspects.

    Figure 12.2 illustrates the aspects of electroporation that we discuss, which comprises three topics. The first is the aqueous pore conductance and dynamics (number and size of aqueous pores).

  • Part II - Building Engineered Membranes, Devices, and Experimental Results

  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 59-60

  • Summary

    Part II of this book deals with the construction, formation, and operation of engineered tethered membrane devices. Detailed descriptions are provided on the molecular components of engineered membranes, methods for inserting peptides and proteins, and how to measure the structure and dynamics of these biomimetic devices.

    Four biomimetic devices built out of engineered tethered membranes are discussed: the ion-channel switch (ICS) biosensor, the electroporation measurement platform (EMP), the electrophysiological response platform (ERP), and the pore formation measurement platform (PFMP). Several real-world examples are provided such as how the ICS biosensor can be used for the rapid detection of influenza A, how the PFMP can be used to infer the pore-formation dynamics of the antimicrobial peptide PGLa (peptidylglycylleucine- carboxyamide), how the EMP can be used to study the membrane conductance dynamics, and how the ERP can be used as a noninvasive method for measuring the response of ion channels and cells.

    We also discuss how to perform experimental measurements using engineered tethered membranes to determine the structure and dynamics of the membrane and macromolecules in the membrane. The experimental techniques discussed include electrical measurements, and spectroscopy and imaging techniques (e.g., X-radiation refractometry, neutron reflectometry, fluorescence recovery after photobleaching, and nuclear magnetic resonance). These measurement methods not only yield important biological details about the membrane but also verify its structure. Since engineered tethered membranes mimic real biological membranes, the experimental studies reported involving antimicrobial peptides, electroporation, growth of cells, and other properties of the membrane give significant insight into how biological membranes function.

    Parts II and III of the book together give a complete account of how to engineer artificial membranes: building them, mathematically modeling their dynamics, and interpreting and refining their design. The reader interested in mathematical modeling of engineered membranes can read §4.1, §§5.1–5.4, and parts of Chapter 6 before proceeding to Part III. For a laboratory exercise on building engineered tethered membranes, refer to §4.4.

  • 1 - Motivation and Outline

  • from Part I - Introduction and Background
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 3-8

  • Summary

    In simple terms a biological membrane consists of two layers of fat that slide on each other – hence membranes are called lipid bilayers. Biological membranes include more than just the cell membrane. Within the cell, there are several structures that are bound by membranes; these structures are called organelles and include the nucleus, mitochondria, endoplasmic reticulum, Gogli apparatus, and lysomes.

    Why Membranes?

    Why biological membranes? Membranes and the macromolecules embedded in them perform extremely important biological functions. They form selectively permeable barriers that enclose cells and organelles within the cell. The cell membrane is the target of physical, chemical, and biological agents such as thermal and mechanical stress, toxins, hormones, viruses, and microbes.

    Biological membranes exhibit remarkable properties. Embedded in the membrane are protein macromolecules that perform crucial functions for a living cell. For example, ion channels are subnanosized pores formed out of proteins in the membrane that selectively open and close and allow ions to flow into the cell. It is known that almost 25 percent of genes code for membrane proteins. Also, more than 50 percent of available drugs target membrane proteins [369]. Cytoskeletal filaments and sterols, such as cholesterol, give structural stability to the membrane. Antimicrobial drugs bind to specific sites in the membrane of bacterial cells and induce pores in the membrane that compromise the integrity of the membrane and lead to bacterial cell death. Applying a voltage across a membrane causes the membrane to spontaneously form pores – this process is called electroporation and is crucial for drug delivery mechanisms. A membrane has several moving parts that comprise lipids and macromolecules that perform a variety of biological tasks. For example, the assembly of new cellular membranes commonly results from old membranes in which membrane-bound enzymes construct new lipid molecules. These new lipid molecules then either diffuse into the old membrane or form vesicles which can merge with other membranes via vesicle fusion. Vesicle fusion requires the coordination of several proteins and macromolecules as biological membranes do not spontaneously fuse. The process of vesicle fusion and cellular membrane assembly is still an active area of research.

  • 14 - Coarse-Grained Molecular Dynamics

  • from Part III - Dynamic Models for Artificial Membranes: From Atoms to Device
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 295-352

  • Summary

    Introduction

    We now move on to a lower level of model abstraction compared to the continuum models of the previous chapters. This chapter describes how coarse-grained molecular dynamics (CGMD) can be used to study important properties of engineered artificial membranes (see Figure 14.1 for perspective on the levels of modeling abstraction). CGMD provides a dynamic simulation model of the engineered tethered membrane that is close to atomic resolution. As explained below, CGMD simulations can be used to estimate important parameters such as the diffusion coefficient of lipids and thereby facilitate building and designing membrane devices such as biosensors (discussed in Part II).

    Why use CGMD simulations when one can use continuum models (at a lower spatial resolution) or all-atom molecular dynamics (MD) simulations (at a higher spatial resolution)? In simple terms, CGMD strikes a compromise between full-atom MD (which is often computationally intractable) and continuum theory (which can be inaccurate at a molecular spatial scale). All-atom MD (described in Chapter 15) deals with the trajectory of individual atoms, whereas in continuum theory the probability distribution of the atoms is propagated over time. Since an atom's trajectory is dependent on the trajectory of all other atoms due to intermolecular forces, the computational cost of evaluating all the atom trajectories in an MD simulation is restricted to systems on the nanometer length scale and nanosecond time scale. The main idea of CGMD is to construct “pseudoatoms” (also known as beads) that represent groups of atoms. CGMD then involves evaluating the trajectory of these interacting pseudoatoms instead of specific atom trajectories. An example is the atomistic representation of the DphPC lipid in an MD simulation compared with the coarse-grained representation of the DphPC lipid. In the MD simulation, the DphPC lipid is represented by 154 atoms, while a coarse-grained representation of the lipid ranges from 1 to 11 beads (or pseudoatoms) depending on the coarse-grained force field used. There are 23,716 atom-to-atom interactions between two DphPC lipids in theMDsimulation, while in the CGMD simulation there are between 1 and 169 bead interactions – this significantly reduces the computational cost of CGMD simulations compared with MD simulations. Note that since CGMD only models the trajectory of the beads and not specific atoms, the CGMD model should not be used when atom trajectories are required such as in the estimation of the charge density across an engineered membrane.

  • 13 - Electrophysiological Response of Ion Channels and Cells

  • from Part III - Dynamic Models for Artificial Membranes: From Atoms to Device
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 282-294

  • Summary

    Introduction

    In this chapter we continue our study of continuum models for engineered membranes. We construct mesoscopic models for the electrophysiological response platform (ERP). The ERP is a synthetic biological device built out of artificial membranes for measuring ion-channel dynamics and the electrophysiological response of cells grown on the surface of the engineered membrane. Recall that the ERP was synthesized in Chapter 3; our aim now is to model how the ERP works mathematically. The ERP can be used for drug screening and diagnosing diseases in which ion-channel functionality is disrupted (i.e., channelopathies). To estimate the electrophysiological response of cells and embedded ion channels requires that the electroporation dynamics of the tethered membrane be accounted for in the mesoscopic model of the ERP.

    Motivation: Ion-Channel and Cellular Measurements

    For measuring the electrophysiological response of individual ions or groups of ions, patch clamping is the gold standard. Patch clamping involves electrical measurements of the current-voltage response at different electrolyte concentrations. Patch clamping produces information-rich data which are widely used to study and validate ion-channel gating models. However, patch clamping is a labor-intensive process requiring a highly skilled experimenter to micromanipulate a glass pipette under a microscope to record data from one cell or membrane segment at a time.

    Regarding measuring the electrophysiological response of cells, there are two classical methods. The first is to use substrate-integrated microelectrode arrays, and the second is to use sharp or patch microelectrodes that puncture the cell membrane [400]. A limitation of these invasive cell-measurement techniques is that, because they use sharp and patch microelectrodes, only a limited number of cells can be measured. Moreover, these techniques can cause the interior of the cell to leak into the electrolyte. Hence, these techniques can only measure the response of cells for short periods of time before they destroy the cell. Substrate-integrated microelectrode arrays provide a noninvasive method for measuring the electrophysiological response of cells; however, a major challenge when using these sensors is to ensure sufficient cell adhesion and coverage [46, 400]. An emerging technology to ensure cell adhesion is to use a metal electrode coated with a polycationic film onto which an adhesion protein, such as Glycocalyx or Fibronectin, is used to bind with the cell membrane [400].

  • 4 - Formation of Engineered Tethered Membranes

  • from Part II - Building Engineered Membranes, Devices, and Experimental Results
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 61-82

  • Summary

    Introduction

    Engineered membranes are synthetic lipid bilayers that are used to mimic biological cell membranes in biophysical and physiological applications. Recall from Chapter 3 that engineered membranes are a fundamental component in building high-resolution sensing devices (such as the ion-channel switch [ICS] biosensor), building stable platforms for measuring the electrophysiological response of cells, and also for using diagnostic tools and implantable devices. From a more fundamental point of view, engineered membranes allow us to study the dynamics of lipids, proteins, peptides, and cells in a controlled environment.

    Recalling the various types of artificial membranes summarized in Figure 3.2 on page 32, this book focuses exclusively on artificial tethered bilayer lipid membranes (tBLMs). The atomic structure of these engineered tethered membranes has been determined using several experimental techniques such as electrical measurements, X-ray reflectometry, and neutron reflectometry as will be discussed in Chapter 7. The unique feature of such membranes is that they are tethered to a conductive solid surface, namely, the bioelectronic interface. Tethering is used to improve the mechanical stability of the engineered membrane. The conductive gold surface to which the membrane is tethered also provides a convenient electrode to generate localized ion fluxes.

    This chapter discusses two important synthesis aspects of engineered tethered membranes:

  • (i) How does one build a tethered membrane?

  • (ii) How does one insert specific lipids, proteins, and peptides into the membrane?

    • We stress that engineered tethered membranes have several advantages compared to other types of artificial membranes. One can precisely control the membrane stability via the tether density. Also a tethered membrane can withstand electrochemical excitations in the range of −900 to +900 mV and convection and advection flows, and it has a shelf life on the order of months. This remarkable stability, compared to traditional whole cell voltage clamping experiments, for example, is a result of the disulfide chemistry, the nanometer hydrophilic submembrane space between the bioelectronic gold interface and the membrane, and the flexibility of lipid composition. For example, it is standard to use 100 percent tether density to ensure the membrane is sufficiently stable to allow the cells to grow on the surface of the membrane.

  • Appendix B - Tutorial on Coarse-Grained Molecular Dynamics with Peptides

  • from Appendices
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 404-411

  • Summary

    In this appendix we use GROMACS 5.1, PyMOL 1.7x, and VMD 1.9.3, together with the MARTINI force field to outline how to set up coarse-grained molecular dynamics (CGMD) simulations of a peptide. Our description includes how to construct the all-atom structure of the peptide, how to build all-atom models of bilayer lipid membranes, how to insert the peptides into the membrane, and how to construct publication-quality figures. The material in this appendix is relevant to Chapter 14 for constructing coarse-grained molecular dynamics simulations using the MARTINI force field. Note that performing CGMD simulations and MD simulations are identical except for the definitions of the bead-particle interactions and simulation time steps used. All the files associated with these tutorials are provided at www.cambridge.org/ engineered-artificial-membranes.

    Constructing the All-Atom and Coarse-Grained Structure of a Peptide

    This section illustrates how to construct the all-atom and coarse-grained structure of the antimicrobial PGLa peptide using VMD and PyMOL. Recall that molecular dynamics (MD) and PyMOL were discussed in Chapter 2, and PGLa was discussed in Chapter 10. The goal is to construct the all-atom and coarse-grained structures of PGLa illustrated in Figure B.1.

    To construct the all-atom coordinate structure of the PGLa peptide the following steps are used.

  • (i)Obtain the amino-acid sequence of interest. For PGLa the amino acid sequence is:

    • GMASKAGAIAGKIAKVALKAL-NH2.

  • (ii) Open VMD, and proceed to Extensions → Modeling → Molfacture. Click the “Start Molefacture”, leaving the entry field blank.

  • (iii) In Molefacture, Build→Protein Builder. Enter the amino-acid sequence of PGLa, and select the α-helix secondary structure (this is the expected secondary structure of PGLa in both the surface and transmembrane configuration as discussed in Chapter 14). Now click build.

  • (iv) After pressing build, the all-atom structure of PGLa will appear in the VMD display. In VMD Main, select File→Save Coordinates, and save the all-atom PGLa structure as PGLa.pdb. The file PGLa.pdb contains the all-atom representation of the PGLa peptide in the protein data bank format.

    • Notice that this PGLa.pdb file can be viewed in both VMD and PyMOL.

    Having constructed the PGLa.pdb coordinate file, we now map the all-atom structure into a coarse-grained structure for use with the MARTINI force field. This mapping operation is performed using the martinize.py python script (available from http://md.chem.rug.nl/cgmartini/index.php/home).

  • 6 - Physiochemical Membrane Platforms

  • from Part II - Building Engineered Membranes, Devices, and Experimental Results
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 103-117

  • Summary

    Introduction

    This chapter describes the construction and operation of three important tetheredmembrane- based sensor platforms, namely, the pore formation measurement platform (PFMP), the electroporation measurement platform (EMP), and the electrophysiological response platform (ERP). These three platforms are constructed to mimic real cell membranes and allow experimental measurements of the dynamics and biochemical reaction pathways present in biological membranes (e.g., the physiochemical properties of membranes). As such, the results from the PFMP, EMP, and ERP elucidate the structural and dynamic properties of real biological membranes. An understanding of the structural and dynamic properties of biological membranes’ interaction with macromolecules is also useful for rational drug design and therapeutic protocols as discussed in §3.7. A schematic diagram of these platforms is provided in Figure 6.1; recall that these platform devices were introduced in Chapter 3.

    The PFMP, EMP, and ERP are designed to perform specific tasks. The PFMP is designed to estimate the reaction dynamics of pore-forming peptides and proteins. The dynamics of pore-forming peptides and proteins are crucial to the attack and defense mechanisms of biological organisms. Understanding the chemical kinetics of poreforming peptides and proteins provides vital information of use to pharmacologists to target specific classes of peptides and proteins for in-depth pharmaceutical screening of novel drugs. The principal operation of the PFMP is that changes in the membrane conductance can be related to the number of formed pores in the membrane. These changes in membrane conductance can be measured experimentally using impedance measurements. The EMP is designed to study the dynamics of electroporation in tethered membranes. Electroporation is a process in which a transmembrane potential induces the formation of aqueous pores in the tethered membrane. The formation of aqueous pores is important for the transport of molecules through the membrane. The ERP is designed to measure the conductance dynamics of embedded ion channels and to measure the electrophysiological response of cells grown on the surface of the tethered membrane. These results are important for drug screening and diagnosis of channelopathic diseases.

  • 7 - Experimental Measurement Methods for Engineered Membranes

  • from Part II - Building Engineered Membranes, Devices, and Experimental Results
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 118-136

  • Summary

    Introduction

    In previous chapters we described how to construct engineered membranes and devices including the ion-channel switch biosensor, electroporation measurement platform, and electrophysiological response platform. An important question is: How can one verify that the artificial membranes constructed in previous chapters have the precisely engineered structure that we have claimed? This chapter focuses on how experimental measurements can be performed on engineered membranes to estimate important biological parameters such as the membrane conductance and capacitance, and also how such measurements can be used to verify the structure of the membrane. We describe three important measurement methods to determine the properties of engineered membranes:

  • (i) impedance measurements,

  • (ii) time-dependent electrical measurements, and

  • (iii) spectroscopy and imaging techniques.

    • Impedance measurement and time-dependent electrical measurement techniques require minimal experimental setup and low-cost laboratory equipment compared with optical and imaging techniques. For example, a standard computer sound card can be used to perform impedance measurements; in comparison, spectroscopic and imaging techniques require both labor-intensive experimental setup and access to equipment that can perform fluorescence correlation spectroscopy, X-ray and neutron reflectometry, and electron microscopy. In practice this is why either impedance measurements or time-dependent electrical measurements are used for estimating properties of engineered membranes. The measurement methods in this chapter also play an important role in Part III, where we construct dynamic models of engineered membranes which can be used to relate the experimental measurements from tethered membranes to biological parameters of interest (e.g., reaction pathway of pore-forming peptides, the concentration estimation of analyte species, and the population and dynamics of aqueous pores in the membrane).

    Electrical Response of Engineered Membranes

    A very useful yet straightforward procedure for performing measurements of an engineered membrane is to apply an excitation potential across the membrane and then record the current response. Indeed, in Part III, we construct mathematical models for the electrical response of engineered membranes at several levels of abstraction (from atoms to device).

    When the engineered tethered membrane is excited by a low-level (below 50 mV) external alternating voltage, the ions in the electrolyte solution migrate to and from the gold electrode surface and insulating barrier of the membrane. Additionally, ions can also pass through aqueous pores and ion channels in the membrane.

  • 3 - Engineered Artificial Membranes

  • from Part I - Introduction and Background
  • William Hoiles, University of British Columbia, Vancouver, Vikram Krishnamurthy, Cornell University, New York, Bruce Cornell, University of Technology Sydney
  • Book:
    Dynamics of Engineered Artificial Membranes and Biosensors
    Published online:
    25 May 2018
    Print publication:
    03 May 2018, pp 30-58

  • Summary

    Membranes

    Biological membranes are ubiquitous, ranging from the cell membrane which encompasses a cell, to membranes for organelles within the cell such as the nucleus and mitochondria. This book focuses exclusively on the construction and modeling of engineered artificial membranes. We use the term “engineered membranes” to emphasize the fact that we will precisely engineer various components of the artificial membrane to achieve specific structural properties. Additionally, we model the dynamics of these engineered membranes from atoms to device. The results from artificial membranes elucidate the structural and dynamic properties of biological membranes. The interaction of biological membranes with macromolecules is key to the fight against human disease (rational drug design and construction of therapeutic protocols). As such, engineered artificial membranes in combination with accurate mathematical models provide an important tool for innovation in the pharmaceutical industry.

    Since artificial membranes are meant to mimic biological membranes, we start by discussing the key properties of one of the most important biological membranes, namely, the cell membrane. The cell membrane provides an interface between the cell and its environment as illustrated in Figure 3.1. The extracellular fluid (depicted in Figure 3.1) contains all components that are not contained in the cell, and the cytosol is composed of components that are contained in the cell. Cell membranes are dynamic and respond to their environment as the result of an external stimulus. To recap from Chapter 2, the cell membrane is composed of three primary components:

  • (i) Lipids. Lipids are primary components of the cell membrane. All lipids are composed of a hydrophilic head (attracted to water) and hydrophobic tails (water neutral). The lipids separate the extracellular fluid from the cytosol of the cell.

  • (ii) Macromolecules. These are responsible for cellular communication both in cells and between cells. Given the low permeability of biological membranes, the primary task of macromolecules is to transport molecules from the extracellular fluid to the interior of the cell. Additionally, there exist other macromolecules which act as molecular receptors, detect vesicles carrying cellular cargo, and facilitate the transport of other macromolecules across the membrane from the extracellular fluid to the cytosol, or from the cytosol to the extracellular fluid.

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