15 research outputs found
An RNA interference (RNAi) toolkit and its utility for functional genetic analysis of Leishmania (Viannia)
RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of th
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Multivalent ions induce lateral structural inhomogeneities in polyelectrolyte brushes
Subtle details about a polyelectrolyte's surrounding environment can dictate its structural features and potential applications. Atomic force microscopy (AFM), surface forces apparatus (SFA) measurements, and coarse-grained molecular dynamics simulations are combined to study the structure of planar polyelectrolyte brushes [poly(styrenesulfonate), PSS] in a variety of solvent conditions. More specifically, AFM images provide a first direct visualization of lateral inhomogeneities on the surface of polyelectrolyte brushes collapsed in solutions containing trivalent counterions. These images are interpreted in the context of a coarse-grained molecular model and are corroborated by accompanying interaction force measurements with the SFA. Our findings indicate that lateral inhomogeneities are absent from PSS brush layers collapsed in a poor solvent without multivalent ions. Together, AFM, SFA, and our molecular model present a detailed picture in which solvophobic and multivalent ion-induced effects work in concert to drive strong phase separation, with electrostatic bridging of polyelectrolyte chains playing an essential role in the collapsed structure formation
Amorphous formulations of indomethacin and griseofulvin prepared by electrospinning
Following an array of optimization
experiments, two series of electrospun
polyvinylpyrrolidone (PVP) fibers were prepared. One set of fibers
contained various loadings of indomethacin, known to form stable glasses,
and the other griseofulvin (a poor glass former). Drug loadings of
up to 33% w/w were achieved. Electron microscopy data showed the fibers
largely to comprise smooth and uniform cylinders, with evidence for
solvent droplets in some samples. In all cases, the drug was found
to exist in the amorphous physical state in the fibers on the basis
of X-ray diffraction and differential scanning calorimetry (DSC) measurements.
Modulated temperature DSC showed that the relationship between a formulationâs
glass transition temperature (<i>T</i><sub>g</sub>) and
the drug loading follows the GordonâTaylor equation, but not
the Fox equation. The results of GordonâTaylor analysis indicated
that the drug/polymer interactions were stronger with indomethacin.
The interactions between drug and polymer were explored in more detail
using molecular modeling simulations and again found to be stronger
with indomethacin; the presence of significant intermolecular forces
was further confirmed using IR spectroscopy. The amorphous form of
both drugs was found to be stable after storage of the fibers for
8 months in a desiccator (relative humidity <25%). Finally, the
functional performance of the fibers was studied; in all cases, the
drug-loaded fibers released their drug cargo very rapidly, offering
accelerated dissolution over the pure drug
Multivalent ions induce lateral structural inhomogeneities in polyelectrolyte brushes
Subtle details about a polyelectrolyteâs surrounding environment can dictate its structural features and potential applications. Atomic force microscopy (AFM), surface forces apparatus (SFA) measurements, and coarse-grained molecular dynamics simulations are combined to study the structure of planar polyelectrolyte brushes [poly(styrenesulfonate), PSS] in a variety of solvent conditions. More specifically, AFM images provide a first direct visualization of lateral inhomogeneities on the surface of polyelectrolyte brushes collapsed in solutions containing trivalent counterions. These images are interpreted in the context of a coarse-grained molecular model and are corroborated by accompanying interaction force measurements with the SFA. Our findings indicate that lateral inhomogeneities are absent from PSS brush layers collapsed in a poor solvent without multivalent ions. Together, AFM, SFA, and our molecular model present a detailed picture in which solvophobic and multivalent ionâinduced effects work in concert to drive strong phase separation, with electrostatic bridging of polyelectrolyte chains playing an essential role in the collapsed structure formation.Published versio
An RNA Interference (RNAi) Toolkit and Its Utility for Functional Genetic Analysis of <i>Leishmania</i> (<i>Viannia</i>)
RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia, and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis (Lbr). We developed vectors facilitating generation of long-hairpin or âstem-loopâ (StL) RNAi knockdown constructs, using GatewayTM site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as quinonoid dihydropteridine reductase (QDPR), a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation (A600), and essential genes of the intraflagellar transport (IFT) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and reporter firefly luciferase (LUC) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful âhypomorphicâ approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a finding that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages
Comparing Solvophobic and Multivalent Induced Collapse in Polyelectrolyte Brushes
Coarse-grained
molecular dynamics enhanced by free-energy sampling
methods is used to examine the roles of solvophobicity and multivalent
salts on polyelectrolyte brush collapse. Specifically, we demonstrate
that while ostensibly similar, solvophobic collapsed brushes and multivalent-ion
collapsed brushes exhibit distinct mechanistic and structural features.
Notably, multivalent-induced heterogeneous brush collapse is observed
under good solvent polymer backbone conditions, demonstrating that
the mechanism of multivalent collapse is not contingent upon a solvophobic
backbone. Umbrella sampling of the potential of mean-force (PMF) between
two individual brush strands confirms this analysis, revealing starkly
different PMFs under solvophobic and multivalent conditions, suggesting
the role of multivalent âbridgingâ as the discriminating
feature in trivalent collapse. Structurally, multivalent ions show
a propensity for nucleating order within collapsed brushes, whereas
poor-solvent collapsed brushes are more disordered; this difference
is traced to the existence of a metastable PMF minimum for poor solvent
conditions, and a global PMF minimum for trivalent systems, under
experimentally relevant conditions