Bystander effects induced by diffusing mediators after photodynamic stress.

The bystander effect, whereby cells that are not traversed by ionizing radiation exhibit various responses when in proximity to irradiated cells, is well documented in the field of radiation biology, Here we demonstrate that considerable bystander responses are also observed after photodynamic stress using the membrane-localizing dye deuteroporphyrin (DP). Using cells of a WTK1 human lymphoblastoid cell line in suspension and a transwell insert system that precludes contact between targeted and bystander cells, we have shown that the bystander signaling is mediated by diffusing species. The extranuclear localization of the photosensitizer used suggests that primary DNA damage is not the trigger for initiating these bystander responses, which include elevated oxidative stress, DNA damage (micronucleus formation), mutagenesis and decreased clonogenic survival. In addition, oxidative stress in the bystander population was reduced by the presence of the membrane antioxidant vitamin E in the targeted cells, suggesting that lipid peroxidation may play a key role in mediating these bystander effects. The fluence responses for these bystander effects are non-linear, with larger effects seen at lower fluences and toxicity to the target cell population. Hence, when considering outcomes of photodynamic action in cells and tissue, bystander effects may be significant, especially at sublethal fluences.

Pre-treatment with Depo-Provera modifies the pharmacokinetics of CMPD167 in rhesus macaques following vaginal ring administration

BACKGROUND: Vaginal ring devices are being developed to provide sustained release of HIV microbicides. To date, only limited pharmacokinetic data is available from animal or human studies. Here we report the effect of Depo-Provera (DP) pre- treatment, commonly used to thin the vaginal epithelium in challenge experiments, on the pharmacokinetic profile of CMPD167 (a small molecule CCR5 co-receptor antagonist) in rhesus macaques following vaginal ring administration.

METHODS: A single 400mg CMPD167 silicone elastomer vaginal ring was inserted into each of twelve female rhesus macaques. Six macaques were treated with (DP) 30 days before ring placement; the other six macaques were untreated. Blood, vaginal fluid and vaginal biopsies were collected prior to and at various times during 28 days of ring placement and assayed for CMPD167 levels by HPLC. Rings were assayed for residual CMPD167 at the end of the study and the calculated amount of CMPD167 released in vivo compared with in vitro release data.

RESULTS: Vaginal fluid, plasma and tissue levels of CMPD167 were detectable throughout ring placement. Significant differences were observed in mean daily vaginal fluid levels between the DP-treated (16–56 mcg/mL) and untreated groups (48–181 mcg/mL). Plasma CMPD167 levels were significantly higher peaking at 4 ng/mL and maintaining levels of 1–2 nM throughout the 14 days of testing in animals pre-treated with DP compared to non DP-treated macaques (<1 ng/mL maintained). Tissue levels were varied between 2–10 g/mL CMPD167 with no significant difference between the DP-treated and untreated macaques.

CONCLUSIONS: The study demonstrates that clinically relevant, and possibly protective doses of CMPD167 are released in the vaginal vault of rhesus macaques from vaginal rings through 28 days duration. DP is known to induce vaginal epithelial thinning and lower vaginal fluid levels, which accounts for the increased plasma levels of CMPD167. In contrast, macaques not treated with DP had minimal absorption into plasma compartments and significantly higher levels of CMPD167 in the vagina, similar to those previously shown to be protective against vaginal challenge.

Electroconvection in a homeotropic bent-rod nematic liquid crystal beyond the dielectric inversion frequency

We characterize the structural transitions in an initially homeotropic bent-rod nematic liquid crystal excited by ac fields of frequency f well above the dielectric inversion point f(i). From the measured principal dielectric constants and electrical conductivities of the compound, the Carr-Helfrich conduction regime is anticipated to extend into the sub-megahertz region. Periodic patterned states occur through secondary bifurcations from the Freedericksz distorted state. An anchoring transition between the bend Freedericksz (1317) and degenerate planar (DP) states is detected. The BF state is metastable well above the Freedericksz threshold and gives way to the DP state, which persists in the field-off condition for several hours. Numerous +1 and -1 umbilics form at the onset of BF distortion, the former being largely of the chiral type. They survive in the DP configuration as linear defects, nonsingular in the core. In the BF regime, not far from fi, periodic Williams-like domains form around the umbilics; they drift along the director easy axis right from their onset. With increasing f, the wave vector of the periodic domains switches from parallel to normal disposition with respect to the c vector. Well above fi, a broadband instability is found.

Drifting periodic structures in a degenerate-planar bent-rod nematic liquid crystal beyond the dielectric inversion frequency

We report on the electric-field-generated effects in the nematic phase of a twin mesogen formed of bent-core and calamitic units, aligned homeotropically in the initial ground state and examined beyond the dielectric inversion point. The bend-Freedericksz (BF) state occurring at the primary bifurcation and containing a network of umbilics is metastable; we focus here on the degenerate planar (DP) configuration that establishes itself at the expense of the BF state in the course of an anchoring transition. In the DP regime, normal rolls, broad domains, and chevrons (both defect-mediated and defect-free types) form at various linear defect-sites, in different regions of the frequency-voltage plane. A significant novel aspect common to all these patterned states is the sustained propagative instability, which does not seem explicable on the basis of known driving mechanisms.

Nonlinear theory of solitary waves associated with longitudinal particle motion in lattices - Application to longitudinal grain oscillations in a dust crystal

The nonlinear aspects of longitudinal motion of interacting point masses in a lattice are revisited, with emphasis on the paradigm of charged dust grains in a dusty plasma (DP) crystal. Different types of localized excitations, predicted by nonlinear wave theories, are reviewed and conditions for their occurrence (and characteristics) in DP crystals are discussed. Making use of a general formulation, allowing for an arbitrary (e.g. the Debye electrostatic or else) analytic potential form phi(r) and arbitrarily long site-to-site range of interactions, it is shown that dust-crystals support nonlinear kink-shaped localized excitations propagating at velocities above the characteristic DP lattice sound speed v(0). Both compressive and rarefactive kink-type excitations are predicted, depending on the physical parameter values, which represent pulse- (shock-)like coherent structures for the dust grain relative displacement. Furthermore, the existence of breather-type localized oscillations, envelope-modulated wavepackets and shocks is established. The relation to previous results on atomic chains as well as to experimental results on strongly-coupled dust layers in gas discharge plasmas is discussed.

Weakly nonlinear effects associated with transverse oscillations in dusty plasma crystals

We study the amplitude modulation of transverse dust lattice waves (TDLW) propagating in a single- and double-layer dusty plasma (DP) crystal. It is shown that a modulational instability mechanism, which is related to an intrinsic nonlinearity of the sheath electric field, may occur under certain conditions. Possibility of the formation of localized excitations (envelope solitons) in the dusty plasma crystal is discussed.

Localized excitations of charged dust grains in dusty plasma lattices

The nonlinear aspects of charged dust grain motion in a one-dimensional dusty plasma (DP) monolayer are discussed. Both horizontal (longitudinal, acoustic mode) and vertical (transverse, optic mode) displacements are considered, and various types of localized excitations are reviewed, in a continuum approximation. Dust crystals are shown to support nonlinear kink-shaped supersonic longitudinal solitary excitations, as well as modulated envelope (either longitudinal or transverse) localized modes. The possibility for Discrete Breather (DB-) type excitations (Intrinsic Localized Modes, ILMs) to occur is investigated, from first principles. These highly localized excitations owe their existence to lattice discreteness, in combination with the interaction and/or
substrate (sheath) potential nonlinearity. This possibility may open new directions in DP- related research. The relation to previous results on atomic chains as well as to experimental results on strongly-coupled dust layers in gas discharge plasmas is discussed.

Regulation of cyclin D1 by the BRCA1-BARD1 complex

Background BRCA1 and cyclin D₁ are both essential for normal breast development and mutation or aberration of their expression is associated with breast cancer [1,2]. Cyclin D₁ is best known as a G₁ cyclin where it regulates the G₁ to S phase transition by acting as a rate-limiting subunit of CDK4/6 kinase activity. More recently, however, Stacey has demonstrated that cyclin D₁ levels in G₂/M determine whether a cell continues to proliferate or exits the cell cycle [3]. The majority of BRCA1 in the cell is bound to BARD1 through their N-terminal RING domains. Heterodimerization is essential for the stability and correct localization of the complex and confers ubiquitin ligase activity to BRCA1. The importance of the ligase activity of BRCA1 to breast cancer development is inferred from the fact that N-terminal diseaseassociated mutations are proposed to reduce ligase activity [4]. Methods Protein–protein interactions were demonstrated using yeast-two-hybrid and coimmunoprecipitation. Protein levels were altered through overexpression, siRNA and antisense technology. The effect of proteasome inhibitors and cycloheximide treatment was also examined. Results We initially identified cyclin D₁ as a binding partner of BARD1 in a yeast-two-hybrid screen and defined the minimal binding region as the N-terminus of BARD1. This interaction was confirmed in vivo by coimmunoprecipitation. The N-terminus of BARD1 also binds BRCA1 and imparts ubiquitin ligase activity to the complex. Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Traditionally polyubiquitin chains linked through lysine 48 target proteins for degradation by the 26 S proteasome. We have demonstrated that cyclin D₁ protein levels are inversely related to BRCA1 and BARD1 levels in several model systems. Furthermore, regulation of cyclin D1 levels occurs through a post-transcriptional mechanism and requires the ligase activity of BRCA1. Interestingly, this phenomenon is cell-cycle regulated, occurring in G₂/M. Conclusion We propose that cyclin D₁ is a potential substrate for BRCA1 ubiquitination and that this targets cyclin D₁ for proteasomal-mediated degradation. Future work will focus on ascertaining the functional consequence of cyclin D₁ regulation by the BRCA1–BARD1 complex; in particular, the impact of BRCA1, mediated through regulation of cyclin D₁, on the proliferation versus differentiation decision.

BRCA1 transcriptionally regulates genes associated with the basal breast cancer phenotype

Background Ten to twenty per cent of breast tumours exhibit a basallike genetic profile and these tumours carry a poor prognosis. Breast tumours which contain germline mutations for BRCA1 commonly exhibit a molecular profile similar to basal breast tumours. BRCA1 is a tumour suppressor gene which is mutated in up to 5–10% of breast cancer cases and is involved in multiple cellular processes including DNA damage control, cell cycle checkpoint control, apoptosis, ubiquitination and transcriptional regulation.

Methods Microarray-based profiling was carried out using the HCC1937EV and HCC1937BR breast cancer cell lines. Basal gene and protein expression levels were analysed by qRT-PCR and western blotting. ChIP analyses were performed and demonstrated that BRCA1 regulates basal gene expression through a transcriptional mechanism involving c-myc.

Results We have previously carried out microarray-based expression profiling to examine differences in gene expression when BRCA1 is reconstituted in BRCA1 mutated HCC1937 breast cancer cells. We observed that p-cadherin and the cytokeratin 5 and cytokeratin 17 genes, which are strongly correlated with the basal phenotype, are differentially expressed when BRCA1 is reconstituted. In addition, qRT-PCR and ChIP analysis of BRCA1 reconstituted cells show that BRCA1 represses the expression of these basal genes by a transcriptional mechanism. Furthermore, abrogation of endogenous BRCA1 protein in the T47D cell line using siRNA results in reexpression of these basal genes, suggesting that BRCA1 expression levels may be important in basal gene expression. We have also demonstrated that BRCA1 is physically associated with the promoter regions of basal genes through an association with c-myc. Consequently, we have confirmed that siRNA inhibition of c-myc in T47D cells results in re-expression of these genes.

Conclusions Our results suggest that BRCA1 is involved in the transcriptional regulation of genes associated with the basal phenotype and that BRCA1 controls basal gene expression through a transcriptional mechanism involving c-myc. Further work is now concentrating on defining the relationship between BRCA1 and basal gene expression and how this may affect clinical responses to breast cancer chemotherapy.

Sustained Release of the CCR5 Inhibitors CMPD167 and Maraviroc from Vaginal Rings in Rhesus Macaques

Antiretroviral entry inhibitors are now being considered as vaginally administered microbicide candidates for prevention of sexual transmission of human immunodeficiency virus. Previous studies testing the entry inhibitors maraviroc and CMPD167 in aqueous gel formulations showed efficacy in the macaque challenge model, although protection was highly dependent on the time period between initial gel application and subsequent challenge. In this paper, we describe the sustained release of the entry inhibitors maraviroc and CMPD167 from matrix-type silicone elastomer vaginal rings both in vitro and in vivo. Both inhibitors were released continuously over 28 days from rings in vitro, at rates of 100-2500 µg/day. In 28-day pharmacokinetic studies in rhesus macaques, the compounds were measured in the vaginal fluid and vaginal tissue; steady state fluid concentrations were ~106 fold greater than IC50 values for SHIV-162P3 inhibition in macaque lymphocytes in vitro. Plasma concentrations for both compounds were very low. Pretreatment of macaques with Depo-Provera® (DP), as commonly used in macaque challenge studies, was shown to significantly modify the bio-distribution of the inhibitors, but not the overall amount released. Vaginal fluid and tissue concentrations were significantly decreased while plasma levels increased with DP pretreatment. These observations have implications for designing macaque challenge experiments, and also for ring performance during the human female menstrual cycle. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Electrostatic waves in superthermal dusty plasmas: review of recent advancement

Real plasmas are often caracterized by the presence of excess energetic particle populations, resulting in a long-tailed non-Maxwellian distribution. In Space plasma physics, this phenomenon is usually modelled via a kappa-type distribution. This presentation is dedicated to an investigation, from first principles, of the effect of superthermality on the characteristics of dusty plasma modes. We employ a kappa distribution function to model the superthermality of the background components (electrons and/or ions). Background superthermality is shown to modify the charge screening mechanism in dusty plasmas, thus affecting the linear dispersion laws of both low- and higher frequency DP modes substantially. Various experimentally observed effects may thus be interpreted as manifestations of superthermality. Focusing on the features of nonlinear excitations (solitons) as they occur in different dusty plasma modes, we investigate the role of superthermality in their propagation dynamics (existence laws, stability profile) and characteristics (geometry).

Modelling solute and microorganism transport through a peri-alpine gravel aquifer by considering the aquifer as a dual porosity fractured medium.

Approach:
In-situ passive gradient comparative artificial tracer testing, undertaken using solutes (Uranine and Iodide), Bacteria (E.coli and P.putida) and bacteriophage (H40/1), permitted comparison of the mobility of different sized microorganisms relative to solutes in the sand and gravel aquifer underlying Dornach, Germany.
Tracer breakthrough curves reveal that even though uranine initially arrived at observation wells at the same time as microbiological tracers, maximum relative concentrations were sometimes less than those of microbiological tracers, while solute breakthrough curves proved more disperse.
Monitoring uranine breakthrough with depth suggested tracers arrived in observation wells in discrete 0.5m-1m thick intervals, over the aquifer’s 12m saturated thickness. Nearby exposures of aquifer material suggested that the aquifer consisted of sandy gravels enveloping sequences of open framework (OW) gravel up to 1m thick. Detailed examination of OW units revealed that they contained lenses of silty sand up to 1m long x 30cm thick., while granulometric data suggested that the gravel was two to three orders of magnitude more permeable than the enveloping sandy gravel.
Solute and microorganism tracer responses could not be simulated using conventional advective-dispersive equation solutions employing the same velocity and dispersion terms. By contrast solute tracer responses, modelled using a dual porosity approach for fractured media (DP-1D) corresponded well to observed field data. Simulating microorganism responses using the same transport terms, but no dual porosity term, generated good model fits and explained the higher relative concentration of the bacteria, compared to the non-reactive solute, even with first order removal to account for lower RR. Geologically, model results indicate that the silty units within open framework gravels are accessible to solute tracers, but not to microorganisms.
Importance:
Results highlight the benefits of geological observations developing appropriate conceptual models of solute and micro organism transport and in developing suitable numerical approaches to quantifying microorganism mobility at scales appropriate for the development of groundwater supply (wellhead) protection zones.