Nanozyme-based pollutant sensing and environmental treatment: Trends, challenges, and perspectives.

Nanozymes are defined as nanomaterials exhibiting enzyme-like properties, and they possess both catalytic functions and nanomaterial's unique physicochemical characteristics. Due to the excellent stability and improved catalytic activity in comparison to natural enzymes, nanozymes have established a wide base for applications in environmental pollutants monitoring and remediation. Nanozymes have been applied in the detection of heavy metal ions, molecules, and organic compounds, both quantitatively and qualitatively. Additionally, within the natural environment, nanozymes can be employed for the degradation of organic and persistent pollutants such as antibiotics, phenols, and textile dyes. Further, the potential sphere of applications for nanozymes traverses from indoor air purification to anti-biofouling agents, and even they show promise in combatting pathogenic bacteria. However, nanozymes may have inherent toxicity, which can restrict their widespread utility. Thus, it is important to evaluate and monitor the interaction and transformation of nanozymes towards biosphere damage when employed within the natural environment in a cradle-to-grave manner, to assure their utmost safety. In this context, various studies have concluded that the green synthesis of nanozymes can efficiently overcome the toxicity limitations in real life applications, and nanozymes can be well utilized in the sensing and degradation of several toxic pollutants including metal ions, pesticides, and chemical warfare agents. In this seminal review, we have explored the great potential of nanozymes, whilst addressing a range of concerns, which have often been overlooked and currently restrict widespread applications and commercialization of nanozymes.

ANGPTL7, a therapeutic target for increased intraocular pressure and glaucoma.

Glaucoma is a leading cause of blindness. Current glaucoma medications work by lowering intraocular pressure (IOP), a risk factor for glaucoma, but most treatments do not directly target the pathological changes leading to increased IOP, which can manifest as medication resistance as disease progresses. To identify physiological modulators of IOP, we performed genome- and exome-wide association analysis in >129,000 individuals with IOP measurements and extended these findings to an analysis of glaucoma risk. We report the identification and functional characterization of rare coding variants (including loss-of-function variants) in ANGPTL7 associated with reduction in IOP and glaucoma protection. We validated the human genetics findings in mice by establishing that Angptl7 knockout mice have lower (~2 mmHg) basal IOP compared to wild-type, with a trend towards lower IOP also in heterozygotes. Conversely, increasing murine Angptl7 levels via injection into mouse eyes increases the IOP. We also show that acute Angptl7 silencing in adult mice lowers the IOP (~2-4 mmHg), reproducing the observations in knockout mice. Collectively, our data suggest that ANGPTL7 is important for IOP homeostasis and is amenable to therapeutic modulation to help maintain a healthy IOP that can prevent onset or slow the progression of glaucoma.

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Healing and early stent coverage after ultrathin strut biodegradable polymer-coated sirolimus-eluting stent implantation: SiBi optical coherence tomography study

ABSTRACT AIMS: The aim of SiBi study was to evaluate the early vascular healing and neointimal coverage after implantation of ultrathin (60 µm) biodegradable polymer-coated Tetriflex (Sahajanand Medical Technologies Pvt. Ltd., Surat, India) sirolimus-eluting stent (SES) using optical coherence tomography (OCT) at 4 to 6 weeks after implantation. METHODS: SiBi was a single-center, observational, investigator-initiated study. From January 15, 2018 to April 15, 2018, total 29 consecutive patients who had consented and underwent OCT examination at 4­6 weeks after Tetriflex SES implantation were enrolled. All OCT images were analyzed at an independent core laboratory by analysts who were blinded to patient and procedural information. RESULTS: Of 29 patients, four patients were excluded, as those OCT images were technically inadequate for analysis. Therefore, 25 patients were included in final OCT analysis. Average OCT analysis was performed after 35.3 ± 5 days of Tetriflex implantation. Total 14,024 stent struts in 1,520 cross sections were analyzed. Strut tissue coverage was observed in 91.26 ± 5.53% of struts and malapposed struts were seen in 0.89 ± 1.67%. The mean neointimal hyperplasia (NIH) thickness on the covered struts was 50 ± 30 µm. CONCLUSION: A large percentage of struts were found to be covered with thin layer of NIH evenly distributed along the stent length at around 1 month from stent implantation. The results of this pilot study serve as ethical and scientific backbone to conduct an adequately powered clinical trial to evaluate outcomes of short dual-antiplatelet therapy in context of ultrathin strut stent.

The Roles of HIF-1α in Radiosensitivity and Radiation-Induced Bystander Effects Under Hypoxia.

Radiation-induced bystander effects (RIBE) may have potential implications for radiotherapy, yet the radiobiological impact and underlying mechanisms in hypoxic tumor cells remain to be determined. Using two human tumor cell lines, hepatoma HepG2 cells and glioblastoma T98G cells, the present study found that under both normoxic and hypoxic conditions, increased micronucleus formation and decreased cell survival were observed in non-irradiated bystander cells which had been co-cultured with X-irradiated cells or treated with conditioned-medium harvested from X-irradiated cells. Although the radiosensitivity of hypoxic tumor cells was lower than that of aerobic cells, the yield of micronucleus induced in bystander cells under hypoxia was similar to that measured under normoxia indicating that RIBE is a more significant factor in overall radiation damage of hypoxic cells. When hypoxic cells were treated with dimethyl sulfoxide (DMSO), a scavenger of reactive oxygen species (ROS), or aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS), before and during irradiation, the bystander response was partly diminished. Furthermore, when only hypoxic bystander cells were pretreated with siRNA hypoxia-inducible factor-1α (HIF-1α), RIBE were decreased slightly but if irradiated cells were treated with siRNA HIF-1α, hypoxic RIBE decreased significantly. In addition, the expression of HIF-1α could be increased in association with other downstream effector molecules such as glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF), and carbonic anhydrase (CA9) in irradiated hypoxic cells. However, the expression of HIF-1α expression in bystander cells was decreased by a conditioned medium from isogenic irradiated cells. The current results showed that under hypoxic conditions, irradiated HepG2 and T98G cells showed reduced radiosensitivity by increasing the expression of HIF-1α and induced a syngeneic bystander effect by decreasing the expression of HIF-1α and regulating its downstream target genes in both the irradiated or bystander cells.

Healing and early stent coverage after ultrathin strut biodegradable polymer-coated sirolimus-eluting stent implantation: SiBi optical coherence tomography study.

AIMS: The aim of SiBi study was to evaluate the early vascular healing and neointimal coverage after implantation of ultrathin (60 µm) biodegradable polymer-coated Tetriflex (Sahajanand Medical Technologies Pvt. Ltd., Surat, India) sirolimus-eluting stent (SES) using optical coherence tomography (OCT) at 4 to 6 weeks after implantation. METHODS: SiBi was a single-center, observational, investigator-initiated study. From January 15, 2018 to April 15, 2018, total 29 consecutive patients who had consented and underwent OCT examination at 4-6 weeks after Tetriflex SES implantation were enrolled. All OCT images were analyzed at an independent core laboratory by analysts who were blinded to patient and procedural information. RESULTS: Of 29 patients, four patients were excluded, as those OCT images were technically inadequate for analysis. Therefore, 25 patients were included in final OCT analysis. Average OCT analysis was performed after 35.3 ± 5 days of Tetriflex implantation. Total 14,024 stent struts in 1,520 cross sections were analyzed. Strut tissue coverage was observed in 91.26 ± 5.53% of struts and malapposed struts were seen in 0.89 ± 1.67%. The mean neointimal hyperplasia (NIH) thickness on the covered struts was 50 ± 30 µm. CONCLUSION: A large percentage of struts were found to be covered with thin layer of NIH evenly distributed along the stent length at around 1 month from stent implantation. The results of this pilot study serve as ethical and scientific backbone to conduct an adequately powered clinical trial to evaluate outcomes of short dual-antiplatelet therapy in context of ultrathin strut stent.

Molecular taxonomy of human ocular outflow tissues defined by single-cell transcriptomics.

The conventional outflow pathway is a complex tissue responsible for maintaining intraocular pressure (IOP) homeostasis. The coordinated effort of multiple cells with differing responsibilities ensures healthy outflow function and IOP maintenance. Dysfunction of one or more resident cell types results in ocular hypertension and risk for glaucoma, a leading cause of blindness. In this study, single-cell RNA sequencing was performed to generate a comprehensive cell atlas of human conventional outflow tissues. We obtained expression profiles of 17,757 genes from 8,758 cells from eight eyes of human donors representing the outflow cell transcriptome. Upon clustering analysis, 12 distinct cell types were identified, and region-specific expression of candidate genes was mapped in human tissues. Significantly, we identified two distinct expression patterns (myofibroblast- and fibroblast-like) from cells located in the trabecular meshwork (TM), the primary structural component of the conventional outflow pathway. We also located Schwann cell and macrophage signatures in the TM. The second primary component structure, Schlemm's canal, displayed a unique combination of lymphatic/blood vascular gene expression. Other expression clusters corresponded to cells from neighboring tissues, predominantly in the ciliary muscle/scleral spur, which together correspond to the uveoscleral outflow pathway. Importantly, the utility of our atlas was demonstrated by mapping glaucoma-relevant genes to outflow cell clusters. Our study provides a comprehensive molecular and cellular classification of conventional and unconventional outflow pathway structures responsible for IOP homeostasis.

Glucocorticoid-Induced Ocular Hypertension: Origins and New Approaches to Minimize.

Introduction: Glucocorticoids (GCs) have unique actions in their combined anti-inflammatory and immunosuppressive activities and are among the most commonly-prescribed drugs, particularly for inflammatory conditions. They are often used clinically to treat inflammatory eye diseases like uveitis, optic neuritis, conjunctivitis, keratitis and others, but are often accompanied by side effects, like ocular hypertension that can be vision threatening. Areas covered: The review will focus on the complex molecular mechanism of action of GCs that involve both transactivation and transrepression and their use therapeutically that can cause significant systemic side effects, particularly ocular hypertension that can lead to glaucoma. Expert Opinion: While we are still unclear as to all the mechanisms responsible for GC-induced ocular hypertension, however, there are potential novel therapies that are in development that can separate some of the anti-inflammatory therapeutic efficacy from their ocular hypertension side effect. This review provides some insight into these approaches.

Glucocorticoid Receptor Transactivation Is Required for Glucocorticoid-Induced Ocular Hypertension and Glaucoma.

Purpose: Glucocorticoid (GC)-induced ocular hypertension (GC-OHT) is a serious side effect of prolonged GC therapy that can lead to glaucoma and permanent vision loss. GCs cause a plethora of changes in the trabecular meshwork (TM), an ocular tissue that regulates intraocular pressure (IOP). GCs act through the glucocorticoid receptor (GR), and the GR regulates transcription both through transactivation and transrepression. Many of the anti-inflammatory properties of GCs are mediated by GR transrepression, while GR transactivation largely accounts for GC metabolic effects and side effects of GC therapy. There is no evidence showing which of the two mechanisms plays a role in GC-OHT. Methods: GRdim transgenic mice (which have active transrepression and impaired transactivation) and wild-type (WT) C57BL/6J mice received weekly periocular dexamethasone acetate (DEX-Ac) injections. IOP, outflow facilities, and biochemical changes to the TM were determined. Results: GRdim mice did not develop GC-OHT after continued DEX treatment, while WT mice had significantly increased IOP and decreased outflow facilities. Both TM tissue in eyes of DEX-treated GRdim mice and cultured TM cells isolated from GRdim mice had reduced or no change in the expression of fibronectin, myocilin, collagen type I, and α-smooth muscle actin (α-SMA). GRdim mouse TM (MTM) cells also had a significant reduction in DEX-induced cytoskeletal changes, which was clearly seen in WT MTM cells. Conclusions: We provide the first evidence for the role of GR transactivation in regulating GC-mediated gene expression in the TM and in the development of GC-OHT. This discovery suggests a novel therapeutic approach for treating ocular inflammation without causing GC-OHT and glaucoma.

Establishment of a conditionally immortalized mouse optic nerve astrocyte line.

Optic nerve astrocytes play a major role in axonal degeneration and regeneration. Astrocyte lines are an important tool to elucidate the responsible cellular mechanisms. In this study, we established a conditionally immortalized mouse optic nerve astrocyte line. Astrocytes were cultured from explants derived from postnatal day 4-5 H-2kb-tsA58 transgenic mouse optic nerves. Cells were cultured in defined astrocyte culture medium under permissive (33 °C) or non-permissive (38.5 °C) temperatures with or without interferon-ɤ (IFN-ɤ). Astrocytes were characterized by immunocytochemistry staining using antibodies against glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (NCAM). Cell proliferation rates were determined by cell growth curves and percentage of Ki67 positive cells. Karyotyping was performed to validate the mouse origin of established cell line. Conditional immortalization was assessed by western blot-determined expression levels of SV40 large T antigen (TAg), p53, GFAP and NCAM in non-permissive culture conditions. In addition, phagocytic activity of immortalized cells was determined by flow cytometry-based pHrodo fluorescence analysis. After 5 days in culture, cells migrated out from optic nerve explants. Immunocytochemistry staining showed that migrating cells expressed astrocyte makers, GFAP and NCAM. In permissive conditions, astrocytes had increased expression levels of TAg and p53, exhibited a greater cell proliferation rate as well as a higher percentage of Ki67 positive cells (n = 3, p < 0.05) compared to cells cultured in non-permissive conditions. One cell line (ImB1ON) was further maintained through 60 generations. Karyotyping showed that ImB1ON was of mouse origin. Flow cytometry-based pHrodo fluorescence analysis demonstrated phagocytic activity of ImB1ON cells. Quantitative PCR showed mRNA expression of trophic factors. Non-permissive culture conditions decreased expression of TAg and p53 in ImB1ON, and increased the expression of NCAM. A conditionally immortalized mouse optic nerve astrocyte line was established. This cell line provides an important tool to study astrocyte biological processes.

Glucocorticoid receptor GRß regulates glucocorticoid-induced ocular hypertension in mice.

Prolonged glucocorticoid (GC) therapy can cause GC-induced ocular hypertension (OHT), which if left untreated progresses to iatrogenic glaucoma and permanent vision loss. The alternatively spliced isoform of glucocorticoid receptor GRß acts as dominant negative regulator of GR activity, and it has been shown that overexpressing GRß in trabecular meshwork (TM) cells inhibits GC-induced glaucomatous damage in TM cells. The purpose of this study was to use viral vectors to selectively overexpress the GRß isoform in the TM of mouse eyes treated with GCs, to precisely dissect the role of GRß in regulating steroid responsiveness. We show that overexpression of GRß inhibits GC effects on MTM cells in vitro and GC-induced OHT in mouse eyes in vivo. Ad5 mediated GRß overexpression reduced the GC induction of fibronectin, collagen 1, and myocilin in TM of mouse eyes both in vitro and in vivo. GRß also reversed DEX-Ac induced IOP elevation, which correlated with increased conventional aqueous humor outflow facility. Thus, GRß overexpression reduces effects caused by GCs and makes cells more resistant to GC treatment. In conclusion, our current work provides the first evidence of the in vivo physiological role of GRß in regulating GC-OHT and GC-mediated gene expression in the TM.

Anterior chamber perfusion versus posterior chamber perfusion does not influence measurement of aqueous outflow facility in living mice by constant flow infusion.

Mice are now routinely utilized in studies of aqueous humor outflow dynamics. In particular, conventional aqueous outflow facility (C) is routinely measured via perfusion of the aqueous chamber by a number of laboratories. However, in mouse eyes perfused ex-vivo, values for C are variable depending upon whether the perfusate is introduced into the posterior chamber (PC) versus the anterior chamber (AC). Perfusion via the AC leads to posterior bowing of the iris, and traction on the iris root/scleral spur, which may increase C. Perfusion via the PC does not yield this effect. But the equivalent situation in living mice has not been investigated. We sought to determine whether AC versus PC perfusion of the living mouse eye may lead to different values for C. All experiments were conducted in C57BL/6J mice (all ♀) between the ages of 20 and 30 weeks. Mice were divided into groups of 3-4 animals each. In all groups, both eyes were perfused. C was measured in groups 1 and 2 by constant flow infusion (from a 50 µL microsyringe) via needle placement in the AC, and in the PC, respectively. To investigate the effect of ciliary muscle (CM) tone on C, groups 3 and 4 were perfused live via the AC or PC with tropicamide (muscarinic receptor antagonist) added to the perfusate at a concentration of 100 µM. To investigate immediate effect of euthanasia, groups 5 and 6 were perfused 15-30 min after death via the AC or PC. To investigate the effect of CM tone on C immediately following euthanasia, groups 7 and 8 were perfused 15-30 min after death via the AC or PC with tropicamide added to the perfusate at a concentration of 100 µM. C in Groups 1 (AC perfusion) and 2 (PC perfusion) was computed to be 19.5 ± 0.8 versus 21.0 ± 2.1 nL/min/mmHg, respectively (mean ± SEM, p > 0.4, not significantly different). In live animals in which tropicamide was present in the perfusate, C in Group 3 (AC perfusion) was significantly greater than C in Group 4 (PC perfusion) (22.0 ± 4.0 versus 14.0 ± 2.0 nL/min/mmHg, respectively, p = 0.0021). In animals immediately following death, C in groups 5 (AC perfusion) and 6 (PC perfusion) was computed to be 21.2 ± 2.0 versus 22.8 ± 1.4 nL/min/mmHg, respectively (mean ± SEM, p = 0.1196, not significantly different). In dead animals in which tropicamide was present in the perfusate, C in group 7 (AC perfusion) was greater than C in group 8 (PC perfusion) (20.6 ± 1.4 versus 14.2 ± 2.6 nL/min/mmHg, respectively, p < 0.0001). C in eyes in situ in living mice or euthanized animals within 15-30 min post mortem is not significantly different when measured via AC perfusion or PC perfusion. In eyes of live or freshly euthanized mice, C is greater when measured via AC versus PC perfusion when tropicamide (a mydriatic and cycloplegic agent) is present in the perfusate.

TGFß2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways.

Purpose: Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor ß2 (TGFß2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFß2 signaling pathways in CLAN formation. Methods: Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFß2, with or without the inhibitors of TGFß receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFß2 plus inhibitors for 10 days or pretreated with TGFß2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4',6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. Results: TGFß2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFß receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFß receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. Conclusions: TGFß2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.

Dexamethasone-Induced Ocular Hypertension in Mice: Effects of Myocilin and Route of Administration.

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious adverse effect of prolonged GC therapy that can lead to iatrogenic glaucoma and permanent vision loss. An appropriate mouse model can help us understand precise molecular mechanisms and etiology of GC-induced OHT. We therefore developed a novel, simple, and reproducible mouse model of GC-induced OHT. GC-induced myocilin expression in the trabecular meshwork (TM) has been suggested to play an important role in GC-induced OHT. We further determined whether myocilin contributes to GC-OHT. C57BL/6J mice received weekly periocular conjunctival fornix injections of a dexamethasone-21-acetate (DEX-Ac) formulation. Intraocular pressure (IOP) elevation was relatively rapid and significant, and correlated with reduced conventional outflow facility. Nighttime IOPs were higher in ocular hypertensive eyes compared to daytime IOPs. DEX-Ac treatment led to increased expression of fibronectin, collagen I, and α-smooth muscle actin in the TM in mouse eyes. No changes in body weight indicated no systemic toxicity associated with DEX-Ac treatment. Wild-type mice showed increased myocilin expression in the TM on DEX-Ac treatment. Both wild-type and Myoc-/- mice had equivalent and significantly elevated IOP with DEX-Ac treatment every week. In conclusion, our mouse model mimics many aspects of GC-induced OHT in humans, and we further demonstrate that myocilin does not play a major role in DEX-induced OHT in mice.

HDAC Inhibitor-Mediated Epigenetic Regulation of Glaucoma-Associated TGFß2 in the Trabecular Meshwork.

PURPOSE: Elevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG) results from glaucomatous damage to the trabecular meshwork (TM). The glaucoma-associated factor TGFß2 is increased in aqueous humor and TM of POAG patients. We hypothesize that histone acetylation has a role in dysregulated TGFß2 expression. METHODS: Protein acetylation was compared between nonglaucomatous TM (NTM) and glaucomatous TM (GTM) cells using Western immunoblotting (WB). Nonglaucomatous TM cells were treated with 10 nM thailandepsin-A (TDP-A), a potent histone deacetylase inhibitor for 4 days. Total and nuclear proteins, RNA, and nuclear protein-DNA complexes were harvested for WB, quantitative PCR (qPCR), and chromatin immunoprecipitation (ChIP) assays, respectively. Paired bovine eyes were perfused with TDP-A versus DMSO, or TDP-A versus TDP-A plus the TGFß pathway inhibitor LY364947 for 5 to 9 days. Intraocular pressure, TM, and perfusate proteins were compared. RESULTS: We found increased acetylated histone 3 and total protein acetylation in the GTM cells and TDP-A treated NTM cells. Chromatin immunoprecipitation assays showed that TDP-A induced histone hyperacetylation associated with the TGFß2 promoter. This change of acetylation significantly increased TGFß2 mRNA and protein expression in NTM cells. In perfusion-cultured bovine eyes, TDP-A increased TGFß2 in the perfusate as well as elevated IOP. Histologic and immunofluorescent analyses showed increased extracellular matrix and cytoskeletal proteins in the TM of TDP-A treated bovine eyes. Cotreatment with the TGFß pathway inhibitor LY364947 blocked TDP-A-induced ocular hypertension. CONCLUSIONS: Our results suggest that histone acetylation has an important role in increased expression of the glaucoma-associated factor TGFß2. Histone hyperacetylation may be the initiator of glaucomatous damage to the TM.

Time and Cell Type Dependency of Survival Responses in Co-cultured Tumor and Fibroblast Cells after Exposure to Modulated Radiation Fields.

Advanced radiotherapy techniques such as intensity-modulated radiation therapy (IMRT) achieve high levels of conformity to the target volume through the sequential delivery of highly spatially and temporally modulated radiation fields, which have been shown to impact radiobiological response. This study aimed to characterize the time and cell type dependency of survival responses to modulated fields using single cell type (SCT) and mixed cell type (MCT) co-culture models of transformed fibroblast (AG0-1522b) cells, prostate (DU-145) and lung (H460) cancer cells. In SCT cultures, in-field responses showed no significant time dependency while out-of-field responses occurred early, and plateaued 6 h after irradiation in both DU-145 and H460 cells. Under modulated beam configurations MCT co-cultures showed cell-specific, differential out-of-field responses depending on the irradiated in-field and responding out-of-field cell type. The observed differential out-of-field responses may be due to the genetic background of the cells, in particular p53 status, which has been shown to mediate radiation-induced bystander effects (RIBEs). These data provide further insight into the radiobiological parameters that influence out-of-field responses, which have potential implications for advanced radiotherapy modalities and may provide opportunities for biophysical optimization in radiotherapy treatment planning.

Tissue specific up regulation of ACE2 in rabbit model of atherosclerosis by atorvastatin: role of epigenetic histone modifications.

Growing body of evidence points out the crucial role of ACE2 in preventing atherosclerosis. However, data on how atherosclerosis affects ACE2 expression in heart and kidney remains unknown. Atherosclerosis was induced by feeding New Zealand White rabbits with high cholesterol diet (HCD - 2%) for 12 weeks and atorvastatin was administered (5mg/kg/day p.o) in last 3 weeks. ACE2 mRNA and protein expression was assessed by Western blotting and real time PCR. HCD fed rabbits developed atherosclerosis as confirmed by increase in plasma total cholesterol, LDL and triglycerides as well as formation atherosclerotic plaques in arch of aorta. The ACE2 protein but not mRNA expression was reduced in heart and kidney of HCD rabbits. Interestingly, atorvastatin increased the ACE2 protein expression in heart and kidney of HCD rabbits. However, atorvastatin increased ACE2 mRNA in heart but not in kidney of HCD rabbits. Atorvastatin increased the occupancy of histone H3 acetylation (H3-Ac) mark on ACE2 promoter region in heart of HCD rabbits indicating direct or indirect epigenetic up-regulation of ACE2 by atorvastatin. Further, atorvastatin suppressed Ang II-induced contractile responses and enhanced AT2 receptor mediated relaxant responses in atherosclerotic aorta. We propose that atherosclerosis is associated with reduced ACE2 expression in heart and kidney. We also show an unexplored potential of atorvastatin to up-regulate ACE2 via epigenetic histone modifications. Our data suggest a novel way of replenishing ACE2 expression for preventing not only atherosclerosis but also other cardiovascular disorders.

Mechanisms of DNA damage response to targeted irradiation in organotypic 3D skin cultures.

DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2.

Preparation and evaluation of hepatic stellate cell selective, surface conjugated, peroxisome proliferator-activated receptor-gamma ligand loaded liposomes.

Hepatic stellate cells (HSCs) activation leads to major fibrogenic response in liver fibrosis. Selective localization of drug to HSCs can provide effective antifibrotic therapy. Thus, objectives of study were to prepare peroxisome proliferator-activated receptor-γ ligand (rosiglitazone) loaded mannose 6-phosphate modified human serum albumin (M6P-HSA) conjugated liposomes and evaluate pharmacokinetically and pharmacodynamically in rats for application of findings of studies in development of suitable and relevant product for treatment of liver fibrosis. The HSA was derivatized with mannose 6-phosphate and then coupled to optimized liposomes. Drug distribution in liver and other tissues after intravenous administration in carbon tetrachloride-induced liver fibrosis model rats was studied. Histopathological examination, estimation of biochemical markers, and grading of liver fibrosis was performed to evaluate pharmacodynamic efficacy of prepared formulation. The M6P-HSA conjugation to liposomes enhanced rosiglitazone liver uptake significantly (2.61 folds) and disappeared from systemic circulation at double rate. Favorable pharmacokinetics resulted in improved histopathological morphology, biochemical markers level, and decreased fibrosis grade. Hence, critical scrutiny of results suggested preferential and enhanced drug localization in pathogenic cells of liver providing a thinking which may result in development of product that can provide cure or at least prevention to this progressive disease necessitating liver transplant.

Genomic instability after targeted irradiation of human lymphocytes: evidence for inter-individual differences under bystander conditions.

Environmental (222)radon exposure is a human health concern, and many studies demonstrate that very low doses of high LET alpha-particle irradiation initiate deleterious genetic consequences in both irradiated and non-irradiated bystander cells. One consequence, radiation-induced genomic instability (RIGI), is a hallmark of tumorigenesis and is often assessed by measuring delayed chromosomal aberrations. We utilised a technique that facilitates transient immobilization of primary lymphocytes for targeted microbeam irradiation and have reported that environmentally relevant doses, e.g. a single (3)He(2+) particle traversal to a single cell, are sufficient to induce RIGI. Herein we sought to determine differences in radiation response in lymphocytes isolated from five healthy male donors. Primary lymphocytes were irradiated with a single particle per cell nucleus. We found evidence for inter-individual variation in radiation response (RIGI, measured as delayed chromosome aberrations). Although this was not highly significant, it was possibly masked by high levels of intra-individual variation. While there are many studies showing a link between genetic predisposition and RIGI, there are few studies linking genetic background with bystander effects in normal human lymphocytes. In an attempt to investigate inter-individual variation in the induction of bystander effects, primary lymphocytes were irradiated with a single particle under conditions where fractions of the population were traversed. We showed a marked genotype-dependent bystander response in one donor after exposure to 15% of the population. The findings may also be regarded as a radiation-induced genotype-dependent bystander effect triggering an instability phenotype.