Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales).

PREMISE: Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome. METHODS: We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω). RESULTS: Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK. CONCLUSIONS: We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.

The effect of likelihood and impact information on public response to severe weather warnings.

Meteorological services are increasingly moving away from issuing weather warnings based on the exceedance of meteorological thresholds (e.g., windspeed), toward risk-based (or "impact-based") approaches. The UK Met Office's National Severe Weather Warning Service has been a pioneer of this approach, issuing yellow, amber, and red warnings based on an integrated evaluation of information about the likelihood of occurrence and potential impact severity. However, although this approach is inherently probabilistic, probabilistic information does not currently accompany public weather warning communications. In this study, we explored whether providing information about the likelihood and impact severity of forecast weather affected subjective judgments of likelihood, severity, concern, trust in forecast, and intention to take protective action. In a mixed-factorial online experiment, 550 UK residents from 2 regions with different weather profiles were randomly assigned to 1 of 3 Warning Format conditions (Color-only, Text, Risk Matrix) and presented with 3 warnings: high-probability/moderate-impact (amber HPMI); low-probability/high-impact (amber); high-probability/high-impact (red). Amongst those presented with information about probability and impact severity, red high-likelihood/high-impact warnings elicited the strongest ratings on all dependent variables, followed by amber HPMI warnings. Amber low-likelihood/high-impact warnings elicited the lowest perceived likelihood, severity, concern, trust, and intention to take protective responses. Taken together, this indicates that UK residents are sensitive to probabilistic information for amber warnings, and that communicating that severe events are unlikely to occur reduces perceived risk, trust in the warning, and behavioral intention, even though potential impacts could be severe. We discuss the practical implications of this for weather warning communication.

Molecular diffusion in the human nail measured by stimulated Raman scattering microscopy.

The effective treatment of diseases of the nail remains an important unmet medical need, primarily because of poor drug delivery. To address this challenge, the diffusion, in real time, of topically applied chemicals into the human nail has been visualized and characterized using stimulated Raman scattering (SRS) microscopy. Deuterated water (D2O), propylene glycol (PG-d8), and dimethyl sulphoxide (DMSO-d6) were separately applied to the dorsal surface of human nail samples. SRS microscopy was used to image D2O, PG-d8/DMSO-d6, and the nail through the O-D, -CD2, and -CH2 bond stretching Raman signals, respectively. Signal intensities obtained were measured as functions of time and of depth into the nail. It was observed that the diffusion of D2O was more than an order of magnitude faster than that of PG-d8 and DMSO-d6. Normalization of the Raman signals, to correct in part for scattering and absorption, permitted semiquantitative analysis of the permeation profiles and strongly suggested that solvent diffusion diverged from classical behavior and that derived diffusivities may be concentration dependent. It appeared that the uptake of solvent progressively undermined the integrity of the nail. This previously unreported application of SRS has permitted, therefore, direct visualization and semiquantitation of solvent penetration into the human nail. The kinetics of uptake of the three chemicals studied demonstrated that each altered its own diffusion in the nail in an apparently concentration-dependent fashion. The scale of the unexpected behavior observed may prove beneficial in the design and optimization of drug formulations to treat recalcitrant nail disease.

Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels - A Strategy for Brain Cancer Treatments.

PURPOSE: The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression. METHODS: Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg(-1)) or ethanolic lomustine (6.5 mg kg(-1)) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg(-1)) or ethanolic lomustine (daily 1.2 mg kg(-1) - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated. RESULTS: The MET formulation resulted in modest brain targeting (brain/ bone AUC0-4h ratios for MET and ethanolic lomustine = 0.90 and 0.53 respectively and brain/ liver AUC0-4h ratios for MET and ethanolic lomustine = 0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes. CONCLUSIONS: Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity.

Oral particle uptake and organ targeting drives the activity of amphotericin B nanoparticles.

There are very few drug delivery systems that target key organs via the oral route, as oral delivery advances normally address gastrointestinal drug dissolution, permeation, and stability. Here we introduce a nanomedicine in which nanoparticles, while also protecting the drug from gastric degradation, are taken up by the gastrointestinal epithelia and transported to the lung, liver, and spleen, thus selectively enhancing drug bioavailability in these target organs and diminishing kidney exposure (relevant to nephrotoxic drugs). Our work demonstrates, for the first time, that oral particle uptake and translocation to specific organs may be used to achieve a beneficial therapeutic response. We have illustrated this using amphotericin B, a nephrotoxic drug encapsulated within N-palmitoyl-N-methyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycol chitosan (GCPQ) nanoparticles, and have evidenced our approach in three separate disease states (visceral leishmaniasis, candidiasis, and aspergillosis) using industry standard models of the disease in small animals. The oral bioavailability of AmB-GCPQ nanoparticles is 24%. In all disease models, AmB-GCPQ nanoparticles show comparable efficacy to parenteral liposomal AmB (AmBisome). Our work thus paves the way for others to use nanoparticles to achieve a specific targeted delivery of drug to key organs via the oral route. This is especially important for drugs with a narrow therapeutic index.

Bio-sensing with butterfly wings: naturally occurring nano-structures for SERS-based malaria parasite detection.

Surface enhanced Raman scattering (SERS) is a powerful tool with great potential to provide improved bio-sensing capabilities. The current 'gold-standard' method for diagnosis of malaria involves visual inspection of blood smears using light microscopy, which is time consuming and can prevent early diagnosis of the disease. We present a novel surface-enhanced Raman spectroscopy substrate based on gold-coated butterfly wings, which enabled detection of malarial hemozoin pigment within lysed blood samples containing 0.005% and 0.0005% infected red blood cells.

Imaging the uptake of gold nanoshells in live cells using plasmon resonance enhanced four wave mixing microscopy.

Gold nanoshells (GNS) are novel metal nanoparticles exhibiting attractive optical properties which make them highly suitable for biophotonics applications. We present a novel investigation using plasmon-enhanced four wave mixing microscopy combined with coherent anti-Stokes Raman scattering (CARS) microscopy to visualize the distribution of 75 nm radius GNS within live cells. During a laser tolerance study we found that cells containing nanoshells could be exposed to < 2.5 mJ each with no photo-thermally induced necrosis detected, while cell death was linearly proportional to the power over this threshold. The majority of the GNS signal detected was from plasmon-enhanced four wave mixing (FWM) that we detected in the epi-direction with the incident lasers tuned to the silent region of the Raman spectrum. The cellular GNS distribution was visualized by combining the epi-detected signal with forwards-detected CARS at the CH2 resonance. The applicability of this technique to real-world nanoparticle dosing problems was demonstrated in a study of the effect of H2S on nanoshell uptake using two donor molecules, NaHS and GYY4137. As GYY4137 concentration was increased from 10 µM to 1 mM, the nanoshell pixel percentage as a function of cell volume (PPCV) increased from 2.15% to 3.77%. As NaHS concentration was increased over the same range, the nanoshell PPCV decreased from 12.67% to 11.47%. The most important factor affecting uptake in this study was found to be the rate of H2S release, with rapid-release from NaHS resulting in significantly greater uptake.

Nanoparticulate peptide delivery exclusively to the brain produces tolerance free analgesia.

The delivery of peptide drugs to the brain is challenging, principally due to the blood brain barrier and the low metabolic stability of peptides. Exclusive delivery to the brain with no peripheral exposure has hitherto not been demonstrated with brain quantification data. Here we show that polymer nanoparticles encapsulating leucine5-enkephalin hydrochloride (LENK) are able to transport LENK exclusively to the brain via the intranasal route, with no peripheral exposure and nanoparticle localisation is observed within the brain parenchyma. Animals dosed with LENK nanoparticles (NM0127) showed a strong anti-nociceptive response in multiple assays of evoked and on going pain whereas animals dosed intranasally with LENK alone were unresponsive. Animals did not develop tolerance to the anti-hyperalgesic activity of NM0127 and NM0127 was active in morphine tolerant animals. A microparticulate formulation of clustered nanoparticles was prepared to satisfy regulatory requirements for nasal dosage forms and the polymer nanoparticles alone were found to be biocompatible, via the nasal route, on chronic dosing.

Limiting the level of tertiary amines on polyamines leads to biocompatible nucleic acid vectors.

We have designed an efficient, synthetic nucleic acid vector, which is relatively non-toxic. [N-(2-ethylamino)-6-O-glycolchitosan - EAGC] polymers were 10-50 fold less toxic than Lipofectamine 2000, able to complex DNA, mRNA and siRNA into positively charged (zeta potential=+40 - 50mV), 50-450nm nanoparticles. The level of tertiary amine N-2-ethylamino substitution (DStert) was inversely proportional to the IC50 of the EAGC polymers in the A431 cell line: IC50=6.18DStert-0.9, r2=0.9991. EAGC polyplexes were stable against a heparin challenge, able to protect the nucleic acids from nuclease degradation and achieve levels of transfection comparable to Lipofectamine 2000 formulations. The relative biocompatibility of the vector allowed 10 fold higher doses of DNA (1µg compared to 0.1µg per well with Lipofectamine 2000) and siRNA (10.7µg per well vs 1.3µg with Lipofectamine 2000) to be applied to cells, when compared to Lipofectamine 2000. Finally intranasal application of EAGC - siRNA complexes resulted in siRNA transfer to the neurons of the olfactory bulb.

Drug delivery into microneedle-porated nails from nanoparticle reservoirs.

This study demonstrates the potential of polymeric nanoparticles as drug reservoirs for sustained topical drug delivery into microneedle-treated human nail. Laser scanning confocal microscopy was used to image the delivery of a fluorescent model compound from nanoparticles into the nail. A label-free imaging technique, stimulated Raman scattering microscopy, was applied, in conjunction with two-photon fluorescence imaging, to probe the disposition of nanoparticles and an associated lipophilic 'active' in a microneedle-porated nail. The results provide clear evidence that the nanoparticles function as immobile reservoirs, sequestered on the nail surface and in the microneedle-generated pores, from which the active payload can be released and diffuse laterally into the nail over an extended period of time.

Evaluation of drug delivery to intact and porated skin by coherent Raman scattering and fluorescence microscopies.

Stimulated Raman scattering microscopy was used to assess the permeation of topically applied drugs and formulation excipients into porcine skin. This chemically selective technique generates high-resolution 3D images, from which semi-quantitative information may be elucidated. Ibuprofen, applied as a close-to-saturated solution in propylene glycol, was directly observed to crystallise in/on the skin, as the co-solvent permeated more rapidly, resulting in precipitation of the drug. Coherent Raman scattering microscopy is also an excellent tool, in conjunction with more conventional confocal fluorescence microscopy, with which to image micro/nanoparticle-based formulations. Specifically, the uptake of particles into thermal ablation transport pathways in the skin has been examined.

Nanofiber-based delivery of therapeutic peptides to the brain.

The delivery of therapeutic peptides and proteins to the central nervous system is the biggest challenge when developing effective neuropharmaceuticals. The central issue is that the blood-brain barrier is impermeable to most molecules. Here we demonstrate the concept of employing an amphiphilic derivative of a peptide to deliver the peptide into the brain. The key to success is that the amphiphilic peptide should by design self-assemble into nanofibers wherein the active peptide epitope is tightly wrapped around the nanofiber core. The nanofiber form appears to protect the amphiphilic peptide from degradation while in the plasma, and the amphiphilic nature of the peptide promotes its transport across the blood-brain barrier. Therapeutic brain levels of the amphiphilic peptide are achieved with this strategy, compared with the absence of detectable peptide in the brain and the consequent lack of a therapeutic response when the underivatized peptide is administered.

Exploring uptake mechanisms of oral nanomedicines using multimodal nonlinear optical microscopy.

Advances in pharmaceutical nanotechnology have yielded ever increasingly sophisticated nanoparticles for medicine delivery. When administered via oral, intravenous, ocular and transcutaneous delivery routes, these nanoparticles can elicit enhanced drug performance. In spite of this, little is known about the mechanistic processes underlying interactions between nanoparticles and tissues, or how these correlate with improved pharmaceutical effects. These mechanisms must be fully understood before nanomedicines can be rationally engineered to optimise their performance. Methods to directly visualise these particulates within tissue samples have traditionally involved imaging modalities requiring covalent labelling of fluorescent or radioisotope contrast agents. We present CARS, second harmonic generation and two photon fluorescence microscopy combined as a multi-modal label-free method for pinpointing polymeric nanoparticles within the stomach, intestine, gall bladder and liver. We demonstrate for the first time that orally administered chitosan nanoparticles follow a recirculation pathway from the GI tract via enterocytes, to the liver hepatocytes and intercellular spaces and then to the gall bladder, before being re-released into the gut together with bile.

Spectroscopy on the wing: naturally inspired SERS substrates for biochemical analysis.

We show that naturally occurring chitinous nanostructures found on the wings of the Graphium butterfly can be used as substrates for surface-enhanced Raman scattering when coated with a thin film of gold or silver. The substrates were found to exhibit excellent biocompatibility and sensitivity, making them ideal for protein assaying. An assay using avidin/biotin binding showed that the substrates could be used to quantify protein binding directly from changes in the surface-enhanced Raman scattering (SERS) spectra and were sensitive over a concentration range comparable with a typical enzyme-linked immunosorbent assays (ELISA) assay. A biomimetic version of the wing nanostructures produced using a highly reproducible, large-scale fabrication process, yielded comparable enhancement factors and biocompatibility. The excellent biocompatibility of the wings and biomimetic substrates is unparalleled by other lithographically produced substrates, and this could pave the way for widespread application of ultrasensitive SERS-based bioassays.