In-vitro investigation of out-of-field cell survival following the delivery of conformal, intensity-modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT) plans

The aim of this work is to determine the out-of-field survival of cells irradiated with either the primary field or scattered radiation in the presence and absence of intercellular communication following delivery of conformal, IMRT and VMAT treatment plans. Single beam, conformal, IMRT and VMAT plans were created to deliver 3 Gy to half the area of a T80 flask containing either DU-145 or AGO-1522 cells allowing intercellular communication between the in-and out-of-field cell populations. The same plans were delivered to a similar custom made phantom used to hold two T25 culture flasks, one flask in-field and one out-of-field to allow comparison of cell survival responses when intercellular communication is physically inhibited. Plans were created for the delivery of 8 Gy to the more radio-resistant DU-145 cells only in the presence and absence of intercellular communication. Cell survival was determined by clonogenic assay. In both cell lines, the out-of-field survival was not statistically different between delivery techniques for either cell line or dose. There was however, a statistically significant difference between survival out-of-field when intercellular communication was intact (single T80 culture flask) or inhibited (multiple T25 culture flasks) to in-field for all plans. No statistically significant difference was observed in-field with or without cellular communication to out-of-field for all plans. These data demonstrate out-of-field effects as important determinants of cell survival following exposure to modulated irradiation fields when cellular communication between differentially irradiated cell populations is present. This data is further evidence that refinement of existing radiobiological models to include indirect cell killing effects is required.

DNA damage responses following exposure to modulated radiation fields

During the delivery of advanced radiotherapy treatment techniques modulated beams are utilised to increase dose conformity across the target volume. Recent investigations have highlighted differential cellular responses to modulated radiation fields particularly in areas outside the primary treatment field that cannot be accounted for by scattered dose alone. In the present study, we determined the DNA damage response within the normal human fibroblast AG0-1522B and the prostate cancer cell line DU-145 utilising the DNA damage assay. Cells plated in slide flasks were exposed to 1 Gy uniform or modulated radiation fields. Modulated fields were delivered by shielding 25%, 50% or 75% of the flask during irradiation. The average number of 53BP1 or ?H2AX foci was measured in 2 mm intervals across the slide area. Following 30 minutes after modulated radiation field exposure an increase in the average number of foci out-of-field was observed when compared to non-irradiated controls. In-field, a non-uniform response was observed with a significant decrease in the average number of foci compared to uniformly irradiated cells. Following 24 hrs after exposure there is evidence for two populations of responding cells to bystander signals in-and out-of-field. There was no significant difference in DNA damage response between 25%, 50% or 75% modulated fields. The response was dependent on cellular secreted intercellular signalling as physical inhibition of intercellular communication abrogated the observed response. Elevated residual DNA damage observed within out-of-field regions decreased following addition of an inducible nitric oxide synthase inhibitor (Aminoguanidine). These data show, for the first time, differential DNA damage responses in-and out-of-field following modulated radiation field delivery. This study provides further evidence for a role of intercellular communication in mediating cellular radiobiological response to modulated radiation fields and may inform the refinement of existing radiobiological models for the optimization of advanced radiotherapy treatment plans. © 2012 Trainor et al.

Fasciola hepatica: A light and electron microscope study of sustentacular tissue and heterophagy in the testis

In order to investigate cytolytic activity in the testis of Fasciola hepatica, flukes belonging to several different triclabendazole (TCBZ)-sensitive and TCBZ-resistant isolates, and wildtype flukes from field infections, were studied by light and electron microscopy with a view to identifying sites of heterophagy and macromolecular hydrolysis. At the periphery of the testis tubules in all the flukes examined, large euchromatic nuclei, each bearing a prominent nucleolus, were seen. These were invested with a thin cytoplasmic layer, extensions of which partially enveloped and probably supported the neighbouring spermatogonia. No lateral cell boundaries were identified in this tissue, possibly indicating syncytial organisation. The tissue, considered to be analogous to Sertoli cells in vertebrate testis, was identified as sustentacular tissue. It displayed cytoplasmic features consistent with protein/glycoprotein synthesis (through a granular endoplasmic reticulum-Golgi mediated mechanism) and intracellular digestion/heterophagy (through a lysosomal system). The intracytoplasmic hydrolytic activity of the sustentacular tissue probably serves to scavenge effete cells and cytoplasmic debris, to recycle useful molecules, to promote spermatozoon maturation and possibly to aid osmoregulation within the tubules. Certain protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, or have pheromonal activity. The presence of numerous mitochondria and abundant smooth endoplasmic reticulum is consistent with facilitation of inward and outward movement of micromolecular nutrients, metabolites including excretory products and water. In the sustentacular tissue of certain flukes with dysfunctional spermiogenesis, there was increased heterophagic and cytolytic scavenging activity. The cytoplasmic residual vacuoles remaining after the release of spermatids were also identified as possible sites for lysosome-mediated intracellular digestion and osmoregulation in the testis tubules of F. hepatica. (C) 2012 Elsevier B.V. All rights reserved.

A Superfamily of Actin-Binding Proteins at the Actin-Membrane Nexus of Higher Plants

Complex animals use a wide variety of adaptor proteins to produce specialized sites of interaction between actin and membranes. Plants do not have these protein families, yet actin-membrane interactions within plant cells are critical for the positioning of subcellular compartments, for coordinating intercellular communication, and for membrane deformation [1]. Novel factors are therefore likely to provide interfaces at actin-membrane contacts in plants, but their identity has remained obscure. Here we identify the plantspecific Networked (NET) superfamily of actin-binding proteins, members of which localize to the actin cytoskeleton and specify different membrane compartments. The founding member of the NET superfamily, NET1A, is anchored at the plasma membrane and predominates at cell junctions, the plasmodesmata. NET1A binds directly to actin filaments via a novel actin-binding domain that defines a superfamily of thirteen Arabidopsis proteins divided into four distinct phylogenetic clades. Members of other clades identify interactions at the tonoplast, nuclear membrane, and pollen tube plasma membrane, emphasizing the role of this superfamily in mediating actin-membrane interactions.

Therapeutic effects of PPARα agonists on diabetic retinopathy in type 1 diabetes models

Retinal vascular leakage, inflammation, and neovascularization (NV) are features of diabetic retinopathy (DR). Fenofibrate, a peroxisome proliferator-activated receptor a (PPARa) agonist, has shown robust protective effects against DR in type 2 diabetic patients, but its effects on DR in type 1 diabetes have not been reported. This study evaluated the efficacy of fenofibrate on DR in type 1 diabetes models and determined if the effect is PPARa dependent. Oral administration of fenofibrate significantly ameliorated retinal vascular leakage and leukostasis in streptozotocin-induced diabetic rats and in Akita mice. Favorable effects on DR were also achieved by intravitreal injection of fenofibrate or another specific PPARa agonist. Fenofibrate also ameliorated retinal NV in the oxygen-induced retinopathy (OIR) model and inhibited tube formation and migration in cultured endothelial cells. Fenofibrate also attenuated overexpression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and vascular endothelial growth factor (VEGF) and blocked activation of hypoxia-inducible factor-1 and nuclear factor-?B in the retinas of OIR and diabetic models. Fenofibrate's beneficial effects were blocked by a specific PPARa antagonist. Furthermore, Ppara knockout abolished the fenofibrate-induced downregulation of VEGF and reduction of retinal vascular leakage in DR models. These results demonstrate therapeutic effects of fenofibrate on DR in type 1 diabetes and support the existence of the drug target in ocular tissues and via a PPARa-dependent mechanism.

Green tea minimally affects biomarkers of inflammation in obese subjects with metabolic syndrome

Green tea (Camellia sinensis) has shown to exert cardioprotective benefits in observational studies. The objective of this clinical trial was to assess the effects of green tea on features of metabolic syndrome and inflammation in obese subjects.

Endocytosis in the retinal and choroidal microcirculation

The endocytosis of horseradish peroxidase (HRP) by the vascular cells of retinal and choroidal blood vessels was compared in immersion and perfusion fixed eyes from individual rats. The mechanisms of endocytosis of HRP appeared identical in both retinal and choroidal vessels. The bulk of internalised tracer occurred in macropinosomes 300-400 nm in diameter. Tracer was localised to a 20-30 nm layer on the internal aspect of the limiting membrane. This layer was coincident with the glycocalyx of the luminal plasma membrane as revealed by ruthenium redosmium tetroxide staining. Horseradish peroxidase was also internalised by a small scattered population of vesicles (100-130 nm in diameter). The size of these vesicles suggested that they may have arisen from clathrin coated regions of the plasma membrane. It is suggested that the endocytosis of HRP in retinal and choroidal vascular endothelium occurs as a function of plasma membrane recycling. Horseradish peroxidase may also be internalised as a 'contaminant' of the glycocalyx in coated pits involved in receptor mediated endocytosis. The smooth 80 nm plasmalemmal caveolae of the retinal and choroidal vascular endothelial cells did not appear to participate either in absorptive endocytosis or vesicular transport.

Does unidirectional vesicular transport occur in retinal vessels?

This paper challenges the hypothesis that the smooth 80 nm plasmalemmal caveolae found in abundance at the abluminal aspect of the endothelium in retinal blood vessels participate in a unidirectional vesicular transport mechanism. Evidence is presented which indicates that horseradish peroxidase, when introduced to the extracellular space of the retina via the vitreous body, may enter the intravascular compartment through junctional incompetence which occurs at or after enucleation of the eye. It is proposed that the plasmalemmal caveolae at the abluminal plasma membrane of endothelial cells in retinal blood vessels are static structures which facilitate the transport of small solutes and ions across the blood retinal barrier.

BRCA1, FANCD2 and Chk1 are potential molecular targets for the modulation of a radiation-induced DNA damage response in bystander cells

Radiotherapy is an important treatment option for many human cancers. Current research is investigating the use of molecular targeted drugs in order to improve responses to radiotherapy in various cancers. The cellular response to irradiation is driven by both direct DNA damage in the targeted cell and intercellular signalling leading to a broad range of bystander effects. This study aims to elucidate radiation-induced DNA damage response signalling in bystander cells and to identify potential molecular targets to modulate the radiation induced bystander response in a therapeutic setting. Stalled replication forks in T98G bystander cells were visualised via bromodeoxyuridine (BrdU) nuclear foci detection at sites of single stranded DNA. γH2AX co-localised with these BrdU foci. BRCA1 and FANCD2 foci formed in T98G bystander cells. Using ATR mutant F02-98 hTERT and ATM deficient GM05849 fibroblasts it could be shown that ATR but not ATM was required for the recruitment of FANCD2 to sites of replication associated DNA damage in bystander cells whereas BRCA1 bystander foci were ATM-dependent. Phospho-Chk1 foci formation was observed in T98G bystander cells. Clonogenic survival assays showed moderate radiosensitisation of directly irradiated cells by the Chk1 inhibitor UCN-01 but increased radioresistance of bystander cells. This study identifies BRCA1, FANCD2 and Chk1 as potential targets for the modulation of radiation response in bystander cells. It adds to our understanding of the key molecular events propagating out-of-field effects of radiation and provides a rationale for the development of novel molecular targeted drugs for radiotherapy optimisation.

DNA methylation subgroups and the CpG island methylator phenotype in gastric cancer: a comprehensive profiling approach

BACKGROUND: Methylation-induced silencing of promoter CpG islands in tumor suppressor genes plays an important role in human carcinogenesis. In colorectal cancer, the CpG island methylator phenotype (CIMP) is defined as widespread and elevated levels of DNA methylation and CIMP+ tumors have distinctive clinicopathological and molecular features. In contrast, the existence of a comparable CIMP subtype in gastric cancer (GC) has not been clearly established. To further investigate this issue, in the present study we performed comprehensive DNA methylation profiling of a well-characterised series of primary GC.

METHODS: The methylation status of 1,421 autosomal CpG sites located within 768 cancer-related genes was investigated using the Illumina GoldenGate Methylation Panel I assay on DNA extracted from 60 gastric tumors and matched tumor-adjacent gastric tissue pairs. Methylation data was analysed using a recursively partitioned mixture model and investigated for associations with clinicopathological and molecular features including age, Helicobacter pylori status, tumor site, patient survival, microsatellite instability and BRAF and KRAS mutations.

RESULTS: A total of 147 genes were differentially methylated between tumor and matched tumor-adjacent gastric tissue, with HOXA5 and hedgehog signalling being the top-ranked gene and signalling pathway, respectively. Unsupervised clustering of methylation data revealed the existence of 6 subgroups under two main clusters, referred to as L (low methylation; 28% of cases) and H (high methylation; 72%). Female patients were over-represented in the H tumor group compared to L group (36% vs 6%; P = 0.024), however no other significant differences in clinicopathological or molecular features were apparent. CpG sites that were hypermethylated in group H were more frequently located in CpG islands and marked for polycomb occupancy.

CONCLUSIONS: High-throughput methylation analysis implicates genes involved in embryonic development and hedgehog signaling in gastric tumorigenesis. GC is comprised of two major methylation subtypes, with the highly methylated group showing some features consistent with a CpG island methylator phenotype.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

Bladder interstitial cells:an updated review of current knowledge

The field of bladder research has been energized by the study of novel interstitial cells (IC) over the last decade. Several subgroups of IC are located within the bladder wall and make structural interactions with nerves and smooth muscle, indicating integration with intercellular communication and key physiological functions. Significant progress has been made in the study of bladder ICs' cellular markers, ion channels and receptor expression, electrical and calcium signalling, yet their specific functions in normal bladder filling and emptying remain elusive. There is increasing evidence that the distribution of IC is altered in bladder pathophysiologies suggesting that changes in IC may be linked with the development of bladder dysfunction. This article summarizes the current state of the art of our knowledge of IC in normal bladder and reviews the literature on IC in dysfunctional bladder.

Systemic oxidative-nitrosative-inflammatory stress during acute exercise in hypoxia; implications for microvascular oxygenation and aerobic capacity

New Findings

What is the central question of this study?Exercise performance is limited during hypoxia by a critical reduction in cerebral and skeletal tissue oxygenation. To what extent an elevation in systemic free radical accumulation contributes to microvascular deoxygenation and the corresponding reduction in maximal aerobic capacity remains unknown.What is the main finding and its importance?We show that altered free radical metabolism is not a limiting factor for exercise performance in hypoxia, providing important insight into the fundamental mechanisms involved in the control of vascular oxygen transport.

Exercise performance in hypoxia may be limited by a critical reduction in cerebral and skeletal tissue oxygenation, although the underlying mechanisms remain unclear. We examined whether increased systemic free radical accumulation during hypoxia would be associated with elevated microvascular deoxygenation and reduced maximal aerobic capacity (). Eleven healthy men were randomly assigned single-blind to an incremental semi-recumbent cycling test to determine  in both normoxia (21% O_{2) and hypoxia (12% O2) separated by a week. Continuous-wave near-infrared spectroscopy was employed to monitor concentration changes in oxy- and deoxyhaemoglobin in the left vastus lateralis muscle and frontal cerebral cortex. Antecubital venous blood samples were obtained at rest and at  to determine oxidative (ascorbate radical by electron paramagnetic resonance spectroscopy), nitrosative (nitric oxide metabolites by ozone-based chemiluminescence and 3-nitrotyrosine by enzyme-linked immunosorbent assay) and inflammatory stress biomarkers (soluble intercellular/vascular cell adhesion 1 molecules by enzyme-linked immunosorbent assay). Hypoxia was associated with increased cerebral and muscle tissue deoxygenation and lower  (P < 0.05 versus normoxia). Despite an exercise-induced increase in oxidative–nitrosative–inflammatory stress, hypoxia per se did not have an additive effect (P > 0.05 versus normoxia). Consequently, we failed to observe correlations between any metabolic, haemodynamic and cardiorespiratory parameters (P > 0.05). Collectively, these findings suggest that altered free radical metabolism cannot explain the elevated microvascular deoxygenation and corresponding lower  in hypoxia. Further research is required to determine whether free radicals when present in excess do indeed contribute to the premature termination of exercise in hypoxia.}

Cellular signalling effects in high precision radiotherapy

Radiotherapy is commonly planned on the basis of physical dose received by the tumour and surrounding normal tissue, with margins added to address the possibility of geometric miss. However, recent experimental evidence suggests that intercellular signalling results in a given cell's survival also depending on the dose received by neighbouring cells. A model of radiation-induced cell killing and signalling was used to analyse how this effect depends on dose and margin choices. Effective Uniform Doses were calculated for model tumours in both idealised cases with no delivery uncertainty and more realistic cases incorporating geometric uncertainty. In highly conformal irradiation, a lack of signalling from outside the target leads to reduced target cell killing, equivalent to under-dosing by up to 10% compared to large uniform fields. This effect is significantly reduced when higher doses per fraction are considered, both increasing the level of cell killing and reducing margin sensitivity. These effects may limit the achievable biological precision of techniques such as stereotactic radiotherapy even in the absence of geometric uncertainties, although it is predicted that larger fraction sizes reduce the relative contribution of cell signalling driven effects. These observations may contribute to understanding the efficacy of hypo-fractionated radiotherapy.

Cable correction of membrane currents recorded from root hairs of Arabidopsis thaliana L.

Membrane currents were recorded under voltage clamp from root hairs of Arabidopsis thaliana L. using the two-electrode method. Concurrent measurements of membrane voltage distal to the point of current injection were also carried out to assess the extent of current dissipation along the root hair axis. Estimates of the characteristic cable length, λ, showed this parameter to be a function both of membrane voltage and of substrate concentration for transport. The mean value for λ at 0 mV was 103 ± 20 μm (n=17), but ranged by as much as 6-fold in any one cell for membrane voltages from -300 to +40 mV and was affected by 0.25 to 3-fold at any one voltage on raising [K⁺]₀ from 0.1 to 10 mol m^-3. Current dissipation along the length of the cells lead to serious distortions of the current-voltage [I-V) characteristic, including consistent underestimates of membrane current as well as a general linearization of the I-V curve and a masking of conductance changes in the presence of transported substrates. In some experiments, microelectrodes were also placed in neighbouring epidermal cells to record the extent of intercellular coupling. Even with current-passing microelectrodes placed at the base of root hairs, coupling was ≤5% (voltage deflection of the epidermal cell ≤5% that recorded at the site of current injection), indicating an appreciable resistance to current passage between cells. These results demonstrate the feasibility of using root hairs as a 'single-cell model' in electrophysiological analyses of transport across the higher-plant plasma membrane; they also confirmed the need to correct for the cable properties of these cells on a cell-by-cell basis. © 1994 Oxford University Press.