267 research outputs found
Reexamination: A Viable Alternative to Patent Litigation?
No full textRecent concern over the state of patent law doctrine has led Congress to pass legislation reforming patent reexamination procedures. The effects of the new procedures will remain uncertain for several years. However, Dale Carlson, Co-chair of the Patent Practice Group at Wiggin & Dana, and Jason Crain, a Yale Law School graduate, discuss the results of a preliminary study of the likely impact of the new inter partes reexamination procedure. In this presentation, Carlson and Crain examine some of the driving forces behind the reform initiative and compare ex parte reexamination procedures with inter partes reexamination procedures. In particular, they address concerns of biases inherent in the new procedure. Ultimately, Carlson and Crain suggest that the new procedure will provide a viable alternative to patent litigation, particularly for small inventors
Reexamination: A Viable Alternative to Patent Litigation?
No full textRecent concern over the state of patent law doctrine has led Congress to pass legislation reforming patent reexamination procedures. The effects of the new procedures will remain uncertain for several years. However, Dale Carlson, Co-chair of the Patent Practice Group at Wiggin \u26 Dana, and Jason Crain, a Yale Law School graduate, discuss the results of a preliminary study of the likely impact of the new inter partes reexamination procedure. In this presentation, Carlson and Crain examine some of the driving forces behind the reform initiative and compare ex parte reexamination procedures with inter partes reexamination procedures. In particular, they address concerns of biases inherent in the new procedure. Ultimately, Carlson and Crain suggest that the new procedure will provide a viable alternative to patent litigation, particularly for small inventors
Quantum drude oscillators for accurate many-body intermolecular forces
No full textOne of the important early applications of Quantum Mechanics was to explain the
Van-der-Waal’s 1/R6 potential that is observed experimentally between two neutral
species, such as noble gas atoms, in terms of correlated uncertainty between interacting
dipoles, an effect that does not occur in the classical limit [London-Eisenschitz,1930].
When many-body correlations and higher-multipole interactions are taken into account
they yield additional many-body and higher-multipole dispersion terms.
Dispersion energies are closely related to electrostatic interactions and polarisation
[Hirschfelder-Curtiss-Bird,1954]. Hydrogen bonding, the dominant force in water, is an
example of an electrostatic effect, which is also strongly modified by polarisation effects.
The behaviour of ions is also strongly influenced by polarisation. Where hydrogen
bonding is disrupted, dispersion tends to act as a more constant cohesive force. It
is the only attractive force that exists between hydrophobes, for example. Thus all
three are important for understanding the detailed behaviour of water, and effects
that happen in water, such as the solvation of ions, hydrophobic de-wetting, and thus
biological nano-structures.
Current molecular simulation methods rarely go beyond pair-wise potentials, and
so lose the rich detail of many-body polarisation and dispersion that would permit
a force field to be transferable between different environments. Empirical force-fields
fitted in the gas phase, which is dominated by two-body interactions, generally do not
perform well in the condensed (many-body) phases. The leading omitted dispersion
term is the Axilrod-Teller-Muto 3-body potential, which does not feature in standard
biophysical force-fields. Polarization is also usually ommitted, but it is sometimes
included in next-generation force-fields following seminal work by Cochran [1971]. In
practice, many-body forces are approximated using two-body potentials fitted to reflect
bulk behaviour, but these are not transferable because they do not reproduce detailed
behaviour well, resulting in spurious results near inhomogeneities, such as solvated
hydrophobes and ions, surfaces and interfaces.
The Quantum Drude Oscillator model (QDO) unifies many-body, multipole
polarisation and dispersion, intrinsically treating them on an equal footing, potentially leading to simpler, more accurate, and more transferable force fields when it is applied
in molecular simulations. The Drude Oscillator is simply a model atom wherein a
single pseudoelectron is bound harmonically to a single pseudonucleus, that interacts
via damped coulomb interactions [Drude,1900].
Path Integral [Feynman-Hibbs,1965] Molecular Dynamics (PIMD) can, in principle,
provide an exact treatment for moving molecules at finite temperature on the Born-
Oppenheimer surface due to their pseudo-electrons. PIMD can be applied to large
systems, as it scales like N log(N), with multiplicative prefactor P that can be
effectively parallelized away on modern supercomputers. There are other ways to
treat dispersion, but all are computationally intensive and cannot be applied to large
systems. These include, for example, Density Functional Theory provides an existence
proof that a functional exists to include dispersion, but we dont know the functional.
We outline the existing methods, and then present new density matrices to improve
the discretisation of the path integral.
Diffusion Monte Carlo (DMC), first proposed by Fermi, allows the fast computation
of high-accuracy energies for static nuclear configurations, making it a useful method for
model development, such as fitting repulsion potentials, but there is no straightforward
way to generate forces. We derived new methods and trial wavefunctions for DMC,
allowing the computation of energies for much larger systems to high accuracy.
A Quantum Drude model of Xenon, fit in the gas-phase, was simulated in the
condensed-phase using both DMC and PIMD. The new DMC methods allowed for
calculation of the bulk modulus and lattice constant of FCC-solid Xenon. Both were
in excellent agreement with experiment even though this model was fitted in the gasphase,
demonstrating the power of Quantum Drudes to build transferable models by
capturing many-body effects. We also used the Xenon model to test the new PIMD
methods.
Finally, we present the outline of a new QDO model of water, including QDO
parameters fitted to the polarisabilities and dispersion coefficients of water
Electrochemical control of reversible DNA hybridisation : for future use in nucleic acid amplification
No full textDenaturation and renaturation is indispensable for the biological function of nucleic
acids in many cellular processes, such as for example transcription for the synthesis
of RNA and DNA replication during cell division. However, the reversible
hybridisation of complementary nucleic acids is equally crucial in nearly all
molecular biology technologies, ranging from nucleic acid amplification
technologies, such as the polymerase chain reaction, and DNA biosensors to next
generation sequencing.
For nucleic acid amplification technologies, controlled DNA denaturation and
renaturation is particularly essential and achieved by cycling elevated temperatures.
Although this is by far the most commonly used method, the management of rapid
temperature changes requires bulky instrumentation and intense power supply.
These factors so far precluded the development of true point-of-care tests for
molecular diagnostics.
This Thesis explored the possibility of using electrochemical means to control
reversible DNA hybridisation by using electroactive intercalators. First,
fluorescence-based melting curve analysis was employed to gain an in depth
understanding of the reversible process of DNA hybridisation. Fundamental
properties, such as stability of the double helix, were investigated by studying the
effect of common denaturing agents, such as formamide and urea, pH and
monovalent salt concentration. Thereafter, four different electroactive intercalators and their effect on the thermodynamic stability of duplex DNA were screened. The
intercalators investigated were methylene blue, thionine, daunomycin and
adriamycin. Absorbance-based melting curve analysis revealed a significant
increase of the melting temperature of duplex DNA in the presence of oxidised
daunomycin. This was not observed in the presence of chemically reduced
daunomycin, which confirmed the hypothesis that switching of the redox-state of
daunomycin altered its properties from DNA binding to non-binding. Accordingly
this altered the thermodynamic stability of duplex DNA. The difference in the
stability of duplex DNA, as a direct result of the redox-state of daunomycin, was
exploited to drive cyclic electrochemically controlled DNA denaturation and
renaturation under isothermal conditions. This proof-of-principle was demonstrated
using complementary synthetic 20mer and 40mer DNA oligonucleotides. Analysis
with in situ UV–vis and circular dichroism spectroelectrochemistry, as two
independent techniques, indicated that up to 80 % of the duplex DNA was
reversibly hybridised. Five cycles of DNA denaturation and renaturation were
achieved and gel electrophoresis as well as NMR showed no degradation of DNA or
daunomycin. As no extreme conditions were implicated, no covalent modification
of DNA was required and isothermal conditions were kept, this finding has great
potential to simplify future developments of miniaturised and portable bioanalytical
systems for nucleic acid-based molecular diagnostics
Study on lipid droplet dynamics in live cells and fluidity changes in model bacterial membranes using optical microscopy techniques
No full textIn this thesis optical microscopy techniques are used to consider aspects of viral and
bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics
in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model
bacterial membranes are studied.
The optical microscopy techniques used to study the effects of murine-cytomegalovirus
(mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are coherent anti-
Stokes Raman scattering (CARS), two-photon fluorescence (TPF) and differential interference
contrast (DIC) microscopies. Using a multimodal CARS and TPF imaging system, the
infection process was monitored by imaging the TPF signal caused by a green fluorescent
protein (GFP)-expressing strain of mCMV, where the amount of TPF detected allowed distinct
stages of infection to be identified. Meanwhile, changes to lipid droplet configuration were
observed using CARS microscopy. Quantitative analysis of lipid droplet numbers and size
distributions were obtained from live cells, which showed significant perturbations as the
infection progressed. The CARS and TPF images were acquired simultaneously and the
experimental design allowed incorporation of an environmental control chamber to maintain
cell viability. Photodamage to the live cell population was also assessed, which indicated that
alternative imaging methods must be adopted to study a single cell over longer periods of
time. To this end, DIC microscopy was used to study the lipid droplet dynamics, allowing
lipid droplet motion to be tracked during infection. In this way, the effects of viral infection
on the mobility and arrangement of the lipid droplets were analysed and quantified. It was
found that the diffusion coefficient of the lipid droplets undergoing diffusive motion increased,
and the droplets undergoing directed motion tended to move at greater speeds as the infection
progressed. In addition, the droplets were found to accumulate and cluster in infected cells.
The second part of this thesis presents a study on the effects of an antimicrobial peptide
on model bacterial membranes. Giant unilamellar vesicles (GUVs) were produced as a simple
model of E. Coli membrane using a 3:1 mixture of DPPC and POPG lipids. Incorporating
Laurdan fluorescent dye into the lipid membrane of the GUVs allowed the membrane fluidity
to be probed and visualised using TPF microscopy, whereby the fluidity was quantified by
determining the general polarization (GP) values. Studying GUVs comprising single lipid and
mixed lipid compositions over a temperature range from 25 C to 55 C enabled the lipid phase
bands to be identified on the basis of GP value as gel phase and liquid crystalline phase. As
such, the changes in lipid phase as a result of interaction with AMP were quantified, and phase
domains were identified. It was found that the amount of liquid crystalline phase domains
increased significantly as a result of AMP interaction
Mobile Press-Register sleeve MP0117594
No full textFaulkner State Community College baseball / Timmer Hale, Corey Swindle, Jason Crain, Les Hall / Bay Minette / FSCC batters get ready for playoff game against Middle Georgia / [Work order included
Advancing our understanding of lipid bilayer interactions: a molecular dynamics study
No full textIn recent years, advances in computer architecture and lipid force field parameters
have made Molecular Dynamics (MD) a powerful tool for gaining atomistic
resolution of biological membranes on timescales that other tools simply cannot
explore. With many key biological processes involving membranes occurring on
the nanosecond timescale, MD allows us to probe the dynamics and energetics
of these interactions in molecular detail. Specifically, we can observe the
interactions taking place as a peptide or protein comes into contact with a lipid
bilayer, and how this may shape or alter the bilayer either locally (changes
in headgroup orientation, lipid fluidity) or in bulk (lipid demixing, membrane
curvature). The resolution achieved through atomistic MD can be directly
compared with other tools such as NMR and EPR to gain a full perspective of
how these biological systems behave over different timescales. As my background
is in computational physics, this thesis not only looks into broadening our
understanding of various interactions with biological membranes, but also into
the development of construction and analytical software to assist in my research
and benefit others in the field.
One aspect of biological membranes that could vastly benefit from MD simulations
is that of antimicrobial peptides (AMPs). These peptides primarily target
and destroy microbes by permeabilising the cell membrane through a variety of
proposed mechanisms, where each mechanism relies on the AMP to adopt specific
conformations upon contact with bacterial membranes. In this thesis, I present
an investigation into the interactions between a synthetic AMP and an inhibitor
peptide designed to regulate antimicrobial activity through the formation of a
coiled coil structure, which restricts the AMP from adopting new conformations.
Simulations captured the spontaneous formation of coiled coils between these
peptides, and specific residues in their sequences were identified that promote
unfolding. This knowledge may lead to better design of coiled coil forming peptides.
Another aspect of biological membranes that can be explored with MD is the
interactions between model bacterial membranes and amphipathic helices, such
as the MinD membrane targeting sequence (MinD-MTS). This 11-residue helix
is responsible for anchoring the MinD protein to the inner membrane of Bacillus
subtilis and plays a crucial role in bacterial cell division. MinD is known to exhibit
sensitivity to transmembrane potentials (TMVs), whereby its localisation and
binding affinity to bacterial membranes are disrupted upon removal of the TMV.
Simulations revealed rapid insertions of MinD-MTS peptides into the headgroup
region of a model bacterial membrane. Analytical software was constructed
to measure the membrane properties of the lipids surrounding inserted MinDMTS
peptides, which revealed splayed lipid tails and suggests the MinD-MTS
may be capable of inducing membrane curvature. Additional simulations were
conducted to investigate the influence of a TMV on model bacterial membranes,
where software was constructed to measure changes in membrane properties. An
analysis of these simulations suggests that a TMV is capable of lowering the
transition temperature of a model bacterial membrane by a few degrees, yielding
increased fluidity in the lipids and increased perturbations on the membrane
surface.
Finally, another aspect of biological membranes that can be explored through
MD is that of electroporation. This induction of transient water pores in cell
membrane provides an exciting aspect for drug delivery applications into cells,
whereby electric fields are applied to cells to increase the uptake of therapeutic
drugs. Simulations of membranes with high voltage TMVs were conducted that
sought to investigate the implications of electroporation across a variety of bilayer
compositions at different temperatures. Software was constructed to measure
changes in membrane and system properties, which revealed that pore formation
occurred at the same threshold voltage for different bilayer compositions in the fluid phase (~1.9 V) and a higher voltage for DPPC bilayers in the gel phase (~2.4 V). The TMV was found to be highly dependent on the area per lipid (APL),
implying that bilayers with bulkier lipids or those transitioning from gel to fluid
will experience smaller TMVs and fewer pore formations. These simulations
also revealed lipid flip-flopping through pores, where charged lipids tended to
translocate in the direction of the electric field to produce an asymmetrically
charged bilayer. Finally, simulations utilising charged peptides with membranes
yielded electroporation effects, whereby the charged peptides generate an identical
TMV to those produced by an ion imbalance of equal magnitude. This suggests
that charged peptides, such as AMPs, may be capable of permeabilising cell
membranes through electroporation mechanisms
Alumni Visit Lab
No full text"Jason Crain and Apryl Rogers returned to my lab during their 15th reunion in 2010. They are posing in front of my lab bulletin board, that never contained a real photo of them, just the cartoon that Jason had drawn (and it's still there today).
Two Negations for the Price of One
No full textStandard English is typically described as a double negation language. In double negation languages, each negative marker contributes independent semantic force. Two negations in the same clause usually cancel each other out, resulting in an affirmative sentence. Other dialects of English permit negative concord. In negative concord sentences, the two negative markers yield a single semantic negation. This paper explores how English-speaking children interpret sentences with more than one negative element, in order to assess whether their early grammar allows negative concord. According to Zeijlstra’s (2004) typological generalization, if a language has a negative syntactic head, it will be a negative concord language. Since Standard English is often analysed as having a negative head, it represents an apparent exception to Zeijlstra’s generalization. This raises the intriguing possibility that initially, children recognize that English has a negative head (i.e., n’t) and, therefore, assign negative concord interpretations to sentences with two negations, despite the absence of evidence for this interpretation in the adult input. The present study investigated this possibility in a comprehension study with 20 3- to 5-year-old children and a control group of 15 adults. The test sentences were presented in contexts that made them amenable to either a double negation or a negative concord interpretation. As expected, the adult participants assigned the double negation interpretation of the test sentences the majority of the time. In contrast, the child participants assigned the alternative, negative concord interpretation the majority of the time. Children must jettison the negative concord interpretation of sentences with two negative markers, and acquire a double negation interpretation. We propose that the requisite positive evidence is the appearance of negative expressions like nothing in object position. Because such expressions exert semantic force without a second negation, this informs children that they are acquiring a double negation language. © 2016 The Author(s)
Nonlinear laser microscopy for the study of virus–host interactions
No full textBiomedical imaging is a key tool for the study of host-pathogen interactions. New techniques
are enhancing the quality and flexibility of imaging systems, particularly as a result of developments
in laser technologies. This work applies the combination of two advanced laser imaging
methods to study the interactions between a virus and the host cells it infects.
The first part of this work describes the theory and experimental implementation of coherent
anti-Stokes Raman scattering microscopy. This technique—first demonstrated in its current
form in 1999—permits the imaging of microscopic samples without the need for fluorescent
labelling. Chemical contrast in images arises from the excitation of specific vibrations in the
sample molecules themselves. A laser scanning microscope system was set up, based on an
excitation source consisting of two titanium-sapphire lasers synchronized with a commercial
phase-locked loop system. A custom-built microscope was constructed to provide optimal
imaging performance, high detection sensitivity and straightforward adaptation to the specific
requirements of biomedical experiments. The system was fully characterized to determine its
performance.
The second part of this work demonstrates the application of this microscope platform in
virology. The microscope was configured to combine two nonlinear imaging modalities: coherent
anti-Stokes Raman scattering and two-photon excitation. Mouse fibroblast cells were
infected with a genetically modified cytomegalovirus. The modification causes the host cell
to express the green fluorescent protein upon infection. The host cell morphology and lipid
droplet distribution were recorded by imaging with coherent anti-Stokes Raman scattering,
whilst the infection was monitored by imaging the viral protein expression with two-photon
excitation. The cytopathic effects typical of cytomegalovirus infection were observed, including
expansion of the nucleus, rounding of the cell shape, and the appearance of intracellular
viral inclusions. In some cases these effects were accompanied by dense accumulations of lipid
droplets at the nuclear periphery. Imaging was performed both with fixed cells and living. It
was demonstrated that the lipid droplets in a single live cell could be imaged over a period of
7 hours without causing noticeable laser-induced damage. The system is shown to be a flexible
and powerful tool for the investigation of virus replication and its effects on the host cell
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