Development of a novel experimental model to investigate radiobiological implications of respiratory motion in advanced radiotherapy

Respiratory motion introduces complex spatio-temporal variations in the dosimetry of radiotherapy. There is a paucity of literature investigating the radiobiological consequences of intrafraction motion and concerns regarding the impact of movement when applied to cancer cell lines in vitro exist. We have addressed this by developing a novel model which accurately replicates respiratory motion under experimental conditions to allow clinically relevant irradiation of cell lines. A bespoke phantom and motor driven moving platform was adapted to accommodate flasks containing medium and cells in order to replicate respiratory motion using varying frequencies and amplitude settings. To study this effect on cell survival in vitro, dose response curves were determined for human lung cancer cell lines H1299 and H460 exposed to a uniform 6 MV radiation field under moving or stationary conditions. Cell survival curves showed no significant difference between irradiation at different dose points for these cell lines in the presence or absence of motion. These data indicate that motion of unshielded cells in vitro does not affect cell survival in the presence of uniform irradiation. This model provides a novel research platform to investigate the radiobiological consequences of respiratory motion in radiotherapy.

Chromobox protein homolog 3 is essential for stem cell differentiation to smooth muscles in vitro and in embryonic arteriogenesis

Smooth muscle cell (SMC) differentiation is a critical process during cardiovascular formation and development, but the underlying molecular mechanism remains unclear.

Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow

X-box binding protein 1 (XBP1) is a key signal transducer in endoplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on maintaining endothelial integrity related to atherosclerosis and to delineate the underlying mechanism. We found that XBP1 was highly expressed at branch points and areas of atherosclerotic lesions in the arteries of ApoE(-/-) mice, which was related to the severity of lesion development. In vitro study using human umbilical vein endothelial cells (HUVECs) indicated that disturbed flow increased the activation of XBP1 expression and splicing. Overexpression of spliced XBP1 induced apoptosis of HUVECs and endothelial loss from blood vessels during ex vivo cultures because of caspase activation and down-regulation of VE-cadherin resulting from transcriptional suppression and matrix metalloproteinase-mediated degradation. Reconstitution of VE-cadherin by Ad-VEcad significantly increased Ad-XBP1s-infected HUVEC survival. Importantly, Ad-XBP1s gene transfer to the vessel wall of ApoE(-/-) mice resulted in development of atherosclerotic lesions after aorta isografting. These results indicate that XBP1 plays an important role in maintaining endothelial integrity and atherosclerosis development, which provides a potential therapeutic target to intervene in atherosclerosis.

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.

Pre-clinical development of a combination microbicide vaginal ring containing dapivirine and darunavir

Objectives: Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here, we report the preclinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir.

Methods: Macaque rings containing 25 mg dapivirine, 300 mg darunavir and 100 mg dapivirine, and 300 mg darunavir were manufactured and characterised by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28-day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values calculated.

Results: Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present, and the release medium. In macaques, serum concentrations of both microbicides were maintained between 101–102 pg/mL. Vaginal fluid levels ranged between 103–104 ng/g and 104–105 ng/g for dapivirine and darunavir, respectively. Tissue concentrations ranges for each drug were: vagina (1.8×103–3.8×103 ng/g) > cervix (9.4×101–3.9×102 ng/g) > uterus (0–108 ng/g) > rectum (0–40 ng/g). Measured IC50 values were > 2 ng/mL for both compounds.

Conclusions: Based on these results, and in light of recent clinical progress of the 25mg dapivirine ring, a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate.

Effects of Undaria pinnatifida, Himanthalia elongata and Porphyra umbilicalis extracts on in vitro α-glucosidase activity and glucose diffusion

Background: Seaweeds are good sources of dietary fibre, which can influence glucose uptake and glycemic control.Objective: To investigate and compare the in vitro inhibitory activity of different extracts from Undaria pinnatifida (Wakame), Himanthalia elongata (Sea spaghetti) and Porphyra umbilicalis (Nori) on α-glucosidase activity and glucose diffusion.Methods: The in vitro effects chloroform-, ethanol- and water-soluble extracts of the three algae were assayed on α- glucosidase activity and glucose diffusion through membrane. Principal Components Analysis (PCA) was applied to identify patterns in the data and to discriminate which extract will show the most proper effect.Results: Only water extracts of Sea spaghetti possessed significant in vitro inhibitory effects on α-glucosidase activity (26.2% less mmol/L glucose production than control, p < 0.05) at 75 min. PCA distinguished Sea spaghetti effects, supporting that soluble fibre and polyphenols were involved. After 6 h, Ethanol-Sea spaghetti and water-Wakame extracts exerted the highest inhibitory effects on glucose diffusion (65.0% and 60.2% vs control, respectively). This extracts displayed the lowest slopes for glucose diffusion-time lineal adjustments (68.2% and 62.8% vs control, respectively).Conclusions: The seaweed hypoglycemic effects appear multi-faceted and not necessarily concatenated. According to present results, ethanol and water extracts of Sea spaghetti, and water extracts of Wakame could be useful for the development of functional foods with specific hypoglycemic properties.

Disruption of vitellogenesis and spermatogenesis by triclabendazole (TCBZ) in a TCBZ-resistant isolate of Fasciola hepatica following incubation in vitro with a P-glycoprotein inhibitor

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2 h in R(+)-VPL (100 μ m), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL-1, or 133·1 μ m) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Interferon regulatory factor (IRF) 3 is critical for the development of experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is an animal model of autoimmune inflammatory demyelination that is mediated by Th1 and Th17 cells. The transcription factor interferon regulatory factor 3 (IRF3) is activated by pathogen recognition receptors and induces interferon-beta production.

METHODS: To determine the role of IRF3 in autoimmune inflammation, we immunised wild-type (WT) and irf3-/- mice to induce EAE. Splenocytes from WT and irf3-/- mice were also activated in vitro in Th17-polarising conditions.

RESULTS: Clinical signs of disease were significantly lower in mice lacking IRF3, with reduced Th1 and Th17 cells in the central nervous system. Peripheral T-cell responses were also diminished, including impaired proliferation and Th17 development in irf3-/- mice. Myelin-reactive CD4+ cells lacking IRF3 completely failed to transfer EAE in Th17-polarised models as did WT cells transferred into irf3-/- recipients. Furthermore, IRF3 deficiency in non-CD4+ cells conferred impairment of Th17 development in antigen-activated cultures.

CONCLUSION: These data show that IRF3 plays a crucial role in development of Th17 responses and EAE and warrants investigation in human multiple sclerosis.

Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery

The design of a non-viral gene delivery vehicle capable of delivering and releasing a functional nucleic acid cargo intracellularly remains a formidable challenge. For systemic gene therapy to be successful a delivery vehicle is required that protects the nucleic acid cargo from enzymatic degradation, extravasates from the vasculature, traverses the cell membrane, disrupts the endosomal vesicles and unloads the cargo at its destination site, namely the nucleus for the purposes of gene delivery. This manuscript reports the extensive investigation of a novel amphipathic peptide composed of repeating RALA units capable of overcoming the biological barriers to gene delivery both in vitro and in vivo. Our data demonstrates the spontaneous self-assembly of cationic DNA-loaded nanoparticles when the peptide is complexed with pDNA. Nanoparticles were < 100 nm, were stable in the presence of serum and were fusogenic in nature, with increased peptide α-helicity at a lower pH. Nanoparticles proved to be non-cytotoxic, readily traversed the plasma membrane of both cancer and fibroblast cell lines and elicited reporter-gene expression following intravenous delivery in vivo. The results of this study indicate that RALA presents an exciting delivery platform for the systemic delivery of nucleic acid therapeutics.

RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro

Background: Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).

Methodology/Principal Findings: Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.

Conclusions/Significance: In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

Lack of in vitro-in vivo correlation for a UC781-releasing vaginal ring in macaques

This study describes the preclinical development of a matrix-type silicone elastomer vaginal ring device designed to provide controlled release of UC781, a non-nucleoside re- verse transcriptase inhibitor. Testing of both human- and macaque-sized rings in a sink condition in vitro release model demonstrated continuous UC781 release in quantities consid- ered sufficient to maintain vaginal fluid concentrations at levels 82–860-fold higher than the in vitro IC50 (2.0 to 10.4 nM) and therefore potentially protect against mucosal trans- mission of HIV. The 100-mg UC781 rings were well tolerated in pig-tailed macaques, did not induce local inflammation as determined by cytokine analysis and maintained median con- centrations in vaginal fluids of UC781 in the range of 0.27 to 5.18 mM during the course of the 28-day study. Analysis of residual UC781 content in rings after completion of both the in vitro release and macaque pharmacokinetic studies revealed that 57 and 5 mg of UC781 was released, respectively. The pharmacokinetic analysis of a 100-mg UC781 vaginal ring in pig-tailed macaques showed poor in vivo–in vitro correlation, attributed to the very poor solubility of UC781 in vaginal fluid and resulting in a dissolution-controlled drug release mecha- nism rather than the expected diffusion-controlled mechanism.

Small GTPase RhoA and its effector rho kinase mediate oxygen glucose deprivation-evoked in vitro cerebral barrier dysfunction

BACKGROUND AND PURPOSE: Enhanced vascular permeability attributable to disruption of blood-brain barrier results in the development of cerebral edema after stroke. Using an in vitro model of the brain barrier composed of human brain microvascular endothelial cells and human astrocytes, this study explored whether small GTPase RhoA and its effector protein Rho kinase were involved in permeability changes mediated by oxygen-glucose deprivation (OGD), key pathological phenomena during ischemic stroke.

METHODS: OGD increased RhoA and Rho kinase protein expressions in human brain microvascular endothelial cells and human astrocytes while increasing or unaffecting that of endothelial nitric oxide synthase in respective cells. Reperfusion attenuated the expression and activity of RhoA and Rho kinase in both cell types compared to their counterparts exposed to equal periods of OGD alone while selectively increasing human brain microvascular endothelial cells endothelial nitric oxide synthase protein levels. OGD compromised the barrier integrity as confirmed by decreases in transendothelial electric resistance and concomitant increases in flux of permeability markers sodium fluorescein and Evan's blue albumin across cocultures. Transfection of cells with constitutively active RhoA also increased flux and reduced transendothelial electric resistance, whereas inactivation of RhoA by anti-RhoA Ig electroporation exerted opposite effects. In vitro cerebral barrier dysfunction was accompanied by myosin light chain overphosphorylation and stress fiber formation. Reperfusion and treatments with a Rho kinase inhibitor Y-27632 significantly attenuated barrier breakdown without profoundly altering actin structure.

CONCLUSIONS: Increased RhoA/Rho kinase/myosin light chain pathway activity coupled with changes in actin cytoskeleton account for OGD-induced endothelial barrier breakdown.

Progress in the development of immunoanalytical methods incorporating recombinant antibodies to small molecular weight biotoxins

Rapid immunoanalytical screening of food and environmental samples for small molecular weight (hapten) biotoxin contaminations requires the production of antibody reagents that possess the requisite sensitivity and specificity. To date animal-derived polyclonal (pAb) and monoclonal (mAb) antibodies have provided the binding element of the majority of these assays but recombinant antibodies (rAb) isolated from in vitro combinatorial phage display libraries are an exciting alternative due to (1) circumventing the need for experimental animals, (2) speed of production in commonly used in vitro expression systems and (3) subsequent molecular enhancement of binder performance. Short chain variable fragments (scFv) have been the most commonly employed rAb reagents for hapten biotoxin detection over the last two decades but antibody binding fragments (Fab) and single domain antibodies (sdAb) are increasing in popularity due to increased expression efficiency of functional binders and superior resistance to solvents. rAb-based immunochromatographic assays and surface plasmon resonance (SPR) biosensors have been reported to detect sub-regulatory levels of fungal (mycotoxins), marine (phycotoxins) and aquatic biotoxins in a wide range of food and environmental matrices, however this technology has yet to surpass the performances of the equivalent mAb- and pAb-based formats. As such the full potential of rAb technology in hapten biotoxin detection has yet to be achieved, but in time the inherent advantages of engineered rAb are set to provide the next generation of ultra-high performing binder reagents for the rapid and specific detection of hapten biotoxins.

Control of biofilm growth through photodynamic treatments combined with chemical inhibitors: in vitro evaluation methods

The rock/atmosphere interface is inhabited by a complex microbial community including bacteria, algae and fungi. These communities are prominent biodeterioration agents and remarkably influence the status of stone monuments and buildings. Deeper comprehension of natural biodeterioration processes on stone surfaces has brought about a concept of complex microbial communities referred to as "subaerial biofilms". The practical implications of biofilm formation are that control strategies must be devised both for testing the susceptibility of the organisms within the biofilm and treating the established biofilm. Model multi-species biofilms associated with mineral surfaces that are frequently refractory to conventional treatment have been used as test targets. A combination of scanning microscopy with image analysis was applied along with traditional cultivation methods and fluorescent activity stains. Such a polyphasic approach allowed a comprehensive quantitative evaluation of the biofilm status and development. Effective treatment strategies incorporating chemical and physical agents have been demonstrated to prevent biofilm growth in vitro. Model biofilm growth on inorganic support was significantly reduced by a combination of PDT and biocides

Prostaglandin E2 as a regulator of germ cells during ovarian development

CONTEXT: The formation of primordial follicles occurs during fetal life yet is critical to the determination of adult female fertility. Prior to this stage, germ cells proliferate, enter meiosis, and associate with somatic cells. Growth and survival factors implicated in these processes include activin A (INHBA), the neurotrophins BDNF and NT4 (NTF5), and MCL1. The prostaglandins have pleiotrophic roles in reproduction, notably in ovulation and implantation, but there are no data regarding roles for prostaglandins in human fetal ovarian development.

OBJECTIVE: The aim of the study was to investigate a possible role for prostaglandin (PG) E(2) in human fetal ovary development.

DESIGN: In vitro analysis of ovarian development between 8 and 20 wk gestation was performed.

MAIN OUTCOME MEASURE(S): The expression patterns of PG synthesis enzymes and the PGE(2) receptors EP2 and EP4 in the ovary were assessed, and downstream effects of PGE(2) on gene expression were analyzed.

RESULTS: Ovarian germ cells express the PG synthetic enzymes COX2 and PTGES as well as the EP2 and EP4 receptors, whereas COX1 is expressed by ovarian somatic cells. Treatment in vitro with PGE(2) increased the expression of BDNF mRNA 1.7 +/- 0.16-fold (P = 0.004); INHBA mRNA, 2.1 +/- 0.51-fold (P = 0.04); and MCL1 mRNA, 1.15 +/- 0.06-fold (P = 0.04), but not that of OCT4, DAZL, VASA, NTF5, or SMAD3.

CONCLUSIONS: These data indicate novel roles for PGE(2) in the regulation of germ cell development in the human ovary and show that these effects may be mediated by the regulation of factors including BDNF, activin A, and MCL1.