Autoradiographic and scintillation analysis of 5-aminolevulinic acid permeation through epithelialised tissue: Implications for topical photodynamic therapy of superficial gynaecological neoplasias

Purpose. Aminolevulinic acid (5-ALA) diffusion through both keratinised and non-keratinised tissue, used as a model tissue substrates, was evaluated, together with the depth of permeation and the concentration achieved following delivery from bioadhesive patch and proprietary cream formulations. Materials and Methods. Moisture-activated, bioadhesive patches loaded with 5-ALA at concentrations of 19.0, 38.0 and 50.0 mg cm(-2) and an o/w cream (20% w/w 5-ALA) were radiolabelled with C14 5-ALA and applied to excised human vaginal tissue and porcine skin. After 1, 2 and 4 h, tissue was sectioned in two orientations and the 5-ALA concentration at specific depths determined using autoradiography and liquid scintillation counting (LSC). Results. The stratum corneum was a significant barrier to 5-ALA permeation, with concentrations in tissue dependent on application time and drug loading. 5-ALA was detected at 6 mm using autoradiography after 2 h, with LSC showing phototoxic concentrations at 2.375 mm after 4 h of application. Inclusion of oleic acid and dimethyl sulphoxide in bioadhesive patches increased 5-ALA significantly in neonate porcine tissue, but only for patches cast from blends containing 5% w/w oleic acid. Conclusions. The bioadhesive patch described delivered 5-ALA to depths of at least 2.5 mm in tissue types indicative of vulval skin, suggesting that photodynamic therapy of deep vulval intraepithelial neoplasia is feasible using this means of bioadhesive 5-ALA delivery.

Development of a Diagnostic Genetic Test for Simplex and Autosomal Recessive Retinitis Pigmentosa

PURPOSE: Retinitis pigmentosa (RP) causes hereditary blindness in adults (prevalence, approximately 1 in 4000). Each of the more than 30 causative genes identified to date are responsible for only a small percentage of cases. Genetic diagnosis via traditional methods is problematic, and a single test with a higher probability of detecting the causative mutation would be very beneficial for the clinician. The goal of this study therefore was to develop a high-throughput screen capable of detecting both known mutations and novel mutations within all genes implicated in autosomal recessive or simplex RP. DESIGN: Evaluation of diagnostic technology. PARTICIPANTS AND CONTROLS: Participants were 56 simplex and autosomal recessive RP patients, with 360 population controls unscreened for ophthalmic disease. METHODS: A custom genechip capable of resequencing all exons containing known mutations in 19 disease-associated genes was developed (RP genechip). A second, commercially available arrayed primer extension (APEX) system was used to screen 501 individual previously reported variants. The ability of these high-throughput approaches to identify pathogenic variants was assessed in a cohort of simplex and autosomal recessive RP patients. MAIN OUTCOME MEASURES: Number of mutations and potentially pathogenic variants identified. RESULTS: The RP genechip identified 44 sequence variants: 5 previously reported mutations; 22 known single nucleotide polymorphisms (SNPs); 11 novel, potentially pathogenic variants; and 6 novel SNPs. There was strong concordance with the APEX array, but only the RP genechip detected novel variants. For example, identification of a novel mutation in CRB1 revealed a patient, who also had a single previously known CRB1 mutation, to be a compound heterozygote. In some individuals, potentially pathogenic variants were discovered in more than one gene, consistent with the existence of disease modifier effects resulting from mutations at a second locus. CONCLUSIONS: The RP genechip provides the significant advantage of detecting novel variants and could be expected to detect at least one pathogenic variant in more than 50% of patients. The APEX array provides a reliable method to detect known pathogenic variants in autosomal recessive RP and simplex RP patients and is commercially available. High-throughput genotyping for RP is evolving into a clinically useful genetic diagnostic tool.

Effect of growth-promoting 17ß estradiol, 19 nortestosterone and dexamethasone on circulating levels of nine potential biomarker candidates in veal calves

The use of screening methods based on the detection of biological effects of growth promoters is a promising approach to assist residue monitoring. To reveal useful effects on protein metabolism, male and female veal calves at 10 weeks of age were treated thrice with a combination of 25 mg 17ß-estradiol 3-benzoate and 150 mg 19-nortestosterone decanoate with 2 weeks intervals and finally once with 4 mg dexamethasone. Hormone-treated calves showed a significant accelerated growth rate over 6 weeks. Plasma samples of treated and control calves were analysed for immunoreactive inhibin (ir-inhibin), osteocalcin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor-binding protein 2 (IGFBP-2), IGFBP-3, luteinzing hormone (LH), follicle-stimulating hormone (FSH) and prolactin using immunoaffinity assays. Hormone treatment did not affect levels of IGF-1, IGFBP-2, IGFBP-3, LH, FSH and prolactin. The concentration of circulating ir-inhibin decreased, however, significantly (P < 0.05) in bull calves upon administration of the sex steroids, whereas it remained unchanged in the female animals. Dexamethasone treatment decreased significantly (P < 0.05) circulating levels of osteocalcin in both female and male animals. Ir-inhibin and osteocalcin were, therefore, considered as candidates for a protein biomarker-based screening assay for detection of abuse of estrogens, androgens and/or glucocorticoids in cattle fattening, which is being developed in the framework of EU research project BioCop

Antibody Targeting of Camptothecin-Loaded PLGA Nanoparticles to Tumor Cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immunonanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 mu g coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT 116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC50 of 21.8 ng mL(-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC50 of 0.37 ng mL(-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.

The tip-sample water bridge and light emission from scanning tunnelling microscopy

The light emission spectrum from a scanning tunnelling microscope (LESTM) is investigated as a function of relative humidity and shown to provide a novel and sensitive means for probing the growth and properties of a water meniscus on the nanometre scale. An empirical model of the light emission process is formulated and applied successfully to replicate the decay in light intensity and spectral changes observed with increasing relative humidity. The modelling indicates a progressive water filling of the tip-sample junction with increasing humidity or, more pertinently, of the volume of the localized surface plasmons responsible for light emission; it also accounts for the effect of asymmetry in structuring of the water molecules with respect to the polarity of the applied bias. This is juxtaposed with the case of a non-polar liquid in the tip-sample nanocavity where no polarity dependence of the light emission is observed. In contrast to the discrete detection of the presence/absence of a water bridge in other scanning probe experiments through measurement of the feedback parameter for instrument control, LESTM offers a means of continuously monitoring the development of the water bridge with sub-nanometre sensitivity. The results are relevant to applications such as dip-pen nanolithography and electrochemical scanning probe microscopy.

Lack of Association Between the Trp719Arg Polymorphism in Kinesin-Like Protein-6 and Coronary Artery Disease in 19 Case-Control Studies

Objectives: We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455), and clinical coronary artery disease (CAD).
Background: Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with noncarriers.
Methods: The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in 19 case-control studies of nonfatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports.
Results: A total of 17,000 cases and 39,369 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the 19 studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with noncarriers. Regression analyses and fixed-effects meta-analyses ruled out with high degree of confidence an increase of <2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early-onset disease (younger than 50 years of age for men and younger than 60 years of age for women) compared with similarly aged controls as well as all non-European subgroups.
Conclusions: The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study.

Interview with Paul DeMarinis

Since the early 1970s, the American electronic media artist Paul DeMarinis (b. 1948, Cleveland, Ohio, USA) has created works that re-imagine modes of communication and reinvent the technologies that enable communication. His works (see Table 1) have taken shape as recordings, performances, electronic inventions, and site-specific and interactive installations; many are considered landmarks in the histories of electronic music and media art. Paul DeMarinis pioneered live performance with computers, collaborated on landmark works with artists like David Tudor and Robert Ashley, undertook several tours with the Merce Cunningham Dance Company, and brought to life obscure technologies such as the flame loudspeaker (featured in his 2004 sculpture Firebirds). His interactive installation The Music Room (1982), commissioned by Frank Oppenheimer for the Exploratorium in San Francisco, was the first automatic music work to reach a significant audience. His album Music As A Second Language (1991) marks one of the most extensive explorations of the synthesized voice and speech melodies to date. Installations like The Edison Effect (1989-1993), in which lasers scan ancient recordings to produce music, and The Messenger (1998/2005), in which electronic mail messages are displayed on alphabetic telegraph receivers, illustrate a creative process that Douglas Kahn (1994) has called "reinventing invention." [etc]

Metronidazole Population Pharmacokinetics in Preterm Neonates Using Dried Blood-Spot Sampling

OBJECTIVES: To characterize the population pharmacokinetics of metronidazole in preterm neonates.
PATIENTS AND METHODS: Data were collected prospectively from 32 preterm neonates who received intravenous metronidazole for the treatment of or prophylaxis against necrotizing enterocolitis. Dried
blood spots (n 203) on ?lter paper were analyzed by highperformance liquid chromatography, and the data were subjected to pharmacokinetic analysis performed by using nonlinear mixed-effect modeling.
RESULTS: A 1-compartment model best described the data. Signi?cant covariates were weight (WT) and postmenstrual age (PMA). The ?nal population models for metronidazole clearance (CL) and volume of distribution (V) were: CL 0.0247 (WT/1.00)0.75 (1 0.107 [PMA 30]) and V 0.726 WT, where CL is in liters per hour, WT is in kilograms, PMA is in weeks, and V is in liters. This model predicts that the half-life of metronidazole decreases rapidly from 40 hours at 25 weeks’ PMA to 19 hours at 32 weeks’ PMA, after which it starts to plateau. This decrease in half-life is the result of a 5-fold increase in CL compared with only a 2.5-fold increase in V during the same period.
CONCLUSIONS: Currently, there are no speci?c dose recommendations for metronidazole in preterm neonates. However, a dosing scheme for preterm neonates that takes into consideration both the weight and PMA has been suggested and should avoid administration of doses that are excessive or more frequent than necessary.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11-19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.