Dual-targeted delivery system using hollow silica nanoparticles with H+-triggered bubble generating characteristic coated with hyaluronic acid and AS1411 for cancer therapy.

OBJECTIVE: Herein, a dual-targeting delivery system using mesoporous silica nanoparticles with hollow structures (HMSNs) was developed for the specific delivery of epirubicin (EPI) to cancer cells and introducing a H+-triggered bubble generating nanosystem (BGNS). HMSNs containing EPI are covered by hyaluronic acid (HA) shell and AS1411 aptamer to create the BGNS-EPI-HA-Apt complex, which is highly selective against CD44 marker and nucleolin overexpressed on the surface of tumor cells. METHODS: MTT assay compared the cytotoxicity of different treatments in CHO (Chinese hamster ovary) cells as well as 4T1 (murine mammary carcinoma) and MCF-7 (human breast adenocarcinoma) cells. The internalization of Epi was assessed by flow cytometry along with fluorescence imaging. In vivo studies were conducted on BALB/c mice bearing a tumor from 4T1 cell line where monitoring included measuring tumor volume, mouse weight changes over time alongside mortality rate; accumulation levels for Epi within organs were also measured during this process. RESULTS: The collected data illustrated that BGNS-EPI-HA-Apt complex controlled the release of EPI in a sustained method. Afterward, receptor-mediated internalization via nucleolin and CD44 was verified in 4T1 and MCF-7 cells using fluorescence microscopy assay and flow cytometry analysis. The results of tumor inhibitory effect study exhibited that BGNS-EPI-HA-Apt complex decreased off-target effect and improved on-target effects because of its targeting ability. CONCLUSION: The data acquired substantiates that HA-surface modified HMSNs functionalized with aptamers possess significant potential as a focused platform for efficient transportation of anticancer agents to neoplastic tissues.

Hepatocyte Nuclear Factor 4α (HNF4α) Plays a Controlling Role in Expression of the Retinoic Acid Receptor ß (RARß) Gene in Hepatocytes.

HNF4α, a member of the nuclear receptor superfamily, regulates the genes involved in lipid and glucose metabolism. The expression of the RARß gene in the liver of HNF4α knock-out mice was higher versus wildtype controls, whereas oppositely, RARß promoter activity was 50% reduced by the overexpression of HNF4α in HepG2 cells, and treatment with retinoic acid (RA), a major metabolite of vitamin A, increased RARß promoter activity 15-fold. The human RARß2 promoter contains two DR5 and one DR8 binding motifs, as RA response elements (RARE) proximal to the transcription start site. While DR5 RARE1 was previously reported to be responsive to RARs but not to other nuclear receptors, we show here that mutation in DR5 RARE2 suppresses the promoter response to HNF4α and RARα/RXRα. Mutational analysis of ligand-binding pocket amino acids shown to be critical for fatty acid (FA) binding indicated that RA may interfere with interactions of FA carboxylic acid headgroups with side chains of S190 and R235, and the aliphatic group with I355. These results could explain the partial suppression of HNF4α transcriptional activation toward gene promoters that lack RARE, including APOC3 and CYP2C9, while conversely, HNF4α may bind to RARE sequences in the promoter of the genes such as CYP26A1 and RARß, activating these genes in the presence of RA. Thus, RA could act as either an antagonist towards HNF4α in genes lacking RAREs, or as an agonist for RARE-containing genes. Overall, RA may interfere with the function of HNF4α and deregulate HNF4α targets genes, including the genes important for lipid and glucose metabolism.

CYP26A1 gene promoter is a useful tool for reporting RAR-mediated retinoid activity.

Of numerous genes regulated by retinoic acid (RA), CYP26A1 is the most inducible gene by RA. In this study, we have used a shortened construct form, E4, of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase or red fluorescent protein (RFP) as the reporter gene and have tested its responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of RA in cells cotransfected with retinoic acid receptors. It also responded quantitatively to retinol and other retinoids. An isolated clonal line of HEK293T cells permanently transfected with the promoter driving the expression of RFP responded to both RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was used to assess the retinoid activity of 3 novel synthetic retinoid analogues, as well as of the intact serum samples of rats. Among the synthetic retinoid analogues tested, EC23 is more potent than RA at lower concentrations and was more stable than RA. The retinoid activities could be measured in control rat serum samples and were increased in the serum of RA-treated rats. This system offers a biologically-based alternative to mass-based retinoid analysis.

Self-Expandable Stent for Repairing Coarctation of the Left-Circumferential Aortic Arch with Right-sided Descending Aorta and Aberrant Right Subclavian Artery with Kommerell's Aneurysm.

Endovascular treatment offers a great advantage in the management of main arteries stenoses. However, simultaneous presence of a group of anomalies may complicate the situation. Here we present a case of 21-year-old man with aortic coarctation. Radiographic imaging and angiography demonstrated aortic coarctation of the left-circumferential aortic arch, right-sided descending aorta, and Kommerell's diverticulum at the origin of right subclavian artery. These anomalies have rarely been reported to concurrently exist in the same case and the treatment is challenging. Percutaneous treatment for repair of aortic coarctation was successfully performed with deployment of self-expanding nitinol stents. Follow-up demonstrated the correction of blood pressure and improvement of the symptoms. It appears that deployment of self-expandable nitinol stents present a viable option for the management of coarcted aorta in patients having all or some of these anomalies together.

Mitral valve resistance determines hemodynamic consequences of severe rheumatic mitral stenosis and immediate outcomes of percutaneous valvuloplasty.

INTRODUCTION: The mitral valve area (MVA) poorly reflects the hemodynamic status of (MS). In this study, we compared the MVA with mitral valve resistance (MVR) with regard to the determination of hemodynamic consequences of MS and the immediate outcomes of percutaneous balloon mitral valvuloplasty (PBMV). METHODS: In a prospective study, 36 patients with severe rheumatic MS with left ventricular ejection fraction (LVEF) >50% were evaluated. They underwent transthoracic echocardiography (TTE) and catheterization. The MVA was measured by two-dimensional planimetry and pressure half-time (PHT), and the MVR was calculated using the equation: 1333 × transmitral pressure gradient mean transmitral diastolic flow rate. RESULTS: The patients' mean age was 47.8±10.5 years. MVR ≥140.6 dynes·s/cm5 detected systolic pulmonary arterial pressure (sPAP) >55 mm Hg with a sensitivity of 100% and a specificity of 74%. The sensitivity and specificity of MVA<0.75 cm2 to discriminate elevated sPAP were 81% and 89%, respectively. PHT ≥323.5 mseconds had a sensitivity of 78% and a specificity of 96% to detect an elevated sPAP. To predict a successful PBMV, preprocedural MVR ≥106.1 dynes·s/cm5 had a sensitivity of 100% and a specificity of 67% (area under the curve [AUC]=0.763; 95% confidence interval [CI]=0.520-1.006; P=.034); preprocedural MVA <0.95 cm2 had a sensitivity of 78% and a specificity of 73% (AUC=0.730; 95% CI=0.503-0.956; P=.065); and preprocedural PHT ≥210.5 mseconds had a sensitivity of 73% and a specificity of 78% (AUC=0.707; 95% CI=0.474-0.941; P=.095). CONCLUSIONS: MVR seems to be more accurate than MVA in determining the hemodynamic consequences of severe MS as determined by sPAP. In addition, preprocedural MVR detected successful PBMVs.

Hepatocyte nuclear factor 4α (HNF4α) in coordination with retinoic acid receptors increases all-trans-retinoic acid-dependent CYP26A1 gene expression in HepG2 human hepatocytes.

CYP26A1 expression is very highly induced by retinoic acid (RA) in the liver, compared to most other tissues, suggesting that a liver-enriched factor may be required for its physiological transcriptional response. HNF4α is a highly conserved liver-specific/enriched member of nuclear receptor superfamily. In this study, we hypothesized that HNF4α and RARs may cooperate in an RA-dependent manner to induce a high level of CYP26A1 expression in liver cells. Partial inhibition of endogenous HNF4α by siRNA reduced the level of RA-induced CYP26A1 mRNA in HepG2 cells. Cotransfection of HNF4α, with or without RARs, demonstrated RA-dependent activation of a human CYP26A1 promoter-luciferase construct. Analysis of a 2.5-kbp putative CYP26A1 promoter sequence identified five potential HNF4α DNA response elements: H1 located in a proximal region overlapping with an RAR element-1 (RARE1 or R1); H2 and H3 in the distal region, close to RARE2 (R2) and RARE3 (R3); and H4 and H5 in intermediary regions. In EMSA and ChIP analyses HNF4α and RARs binding in the proximal and distal CYP26A1 promoter regions was significantly higher in RA-treated cells. Mutational analysis of the individual HNF4α DNA-response elements identified H1 as the major site for HNF4α binding because mutation of H1 inhibited the promoter activity by ~90%, followed by H2 mutation with less than 40% inhibition. Our results indicate that HNF4α coordinates with RARs in an RA-dependent manner to strongly induce CYP26A1 gene expression in the liver, which may explain the high level of response to RA observed in vivo.

Lymphangiogenesis in Odontogenic Keratocysts Compared with Dentigerous Cysts.

Statment of the Problem: Podoplanin can indicate the lymphangiogenesis. On the other hand, lymphangiogenesis affects the biological behavior of lesions. The clinical behavior of odontogenic keratocysts (OKC) and dentigerous cysts (DC) is different. Purpose: This study aimed to evaluate the immunohistochemical expression of podoplanin and to investigate lymphangiogenesis in OKCs as compared to DCs. Materials and Method: In this experimental laboratory study, sixty paraffined blocks, including 30 OKC and 30 DC samples, were examined in this study, all of which were histopathologically non-inflamed. To evaluate lymphangiogenesis, the immunohistochemical reaction of D2-40 was evaluated via cytoplasmic and membrane staining of lymphatic endothelial cells. The expression of podoplanin in the epithelial cells of two cyst groups was also examined. To analyze the collected data and compare the results between the two groups of cysts, independent samples t-test, Mann-Whitney U test, and Chi-square test were performed in SPSS version 22. The significance level was set at 0.05. Results: The mean lymph node count and podoplanin expression were significantly higher in the OKC epithelium as compared to DC (p< 0.001). Based on the results, 90% of OKC samples and 43.3% of DC samples showed grade 3 staining. Conclusion: The rate of lymphangiogenesis and podoplanin expression in the epithelium were higher in OKCs compared to DCs. According to the results, the expression of podoplanin may be a useful marker for determining the invasiveness and proliferation of OKC.

Outcomes of chronic total occlusion percutaneous coronary intervention from the RAIAN (RAjaie - Iran) registry.

OBJECTIVE: While most of the evidence in CTO interventions emerge from Western and Japanese studies, few data have been published up today from the Middle East. Objective of this study was to evaluate technical success rates and clinical outcomes of an Iranian population undergoing CTO PCI in a tertiary referral hospital. Moreover, we sought to evaluate the efficacy of our CTO teaching program. METHODS: This is a retrospective single-center cohort study including 790 patients who underwent CTO PCI performed by operators with different volumes of CTOs PCI performed per year. According to PCI result, all patients have been divided into successful (n = 555, 70.3 %) and unsuccessful (n = 235, 29.7 %) groups. Study endpoints were Major Adverse Cardiovascular Events and Health Status Improvement evaluated using the Seattle Angina Questionnaire at one year. RESULTS: A global success rate of 70 % for antegrade and 80 % for retrograde approach was shown despite the lack of some CTO-dedicated devices. During the enrollment period, the success rate increased significantly among operators with a lower number of CTO procedures per year. One-year MACE rate was similar in both successful and unsuccessful groups (13.5 % in successful and 10.6 % in unsuccessful group, p = 0.173). One year patients' health status improved significantly only in successful group. CONCLUSIONS: No significant differences of in-hospital and one-year MACE were found between the successful and unsuccessful groups. Angina symptoms and quality of life significantly improved after successful CTO PCI. The RAIAN registry confirmed the importance of operator expertise for CTO PCI success.

DHRS3, a retinal reductase, is differentially regulated by retinoic acid and lipopolysaccharide-induced inflammation in THP-1 cells and rat liver.

Both retinoid status and inflammation have been shown to control the level of expression of retinoid homeostatic genes. In the present study, DHRS3, previously shown to possess retinal reductase activity, was identified by microarray analysis of THP-1 monocytes as a possible gene target of all-trans-retinoic acid (RA). In these cells, DHRS3 mRNA increased 30- to 40-fold after treatment with ≤20 nM RA for 24 h, while DHRS3 protein also increased. Of several synthetic retinoids tested, only Am580, a RA receptor-α-selective retinoid, increased DHRS3 mRNA expression. The full-length DHRS3 cDNA was cloned from rat liver and subjected to in vitro transcription-translation. Two major ∼30- and 35-kDa proteins were detected. In adult rat tissues, DHRS3 mRNA was most abundant in the adrenal gland, liver, and ovary. In the liver, DHRS3 is expressed in hepatocytes and possibly in all liver cells. To evaluate whether DHRS3 is regulated in the liver by RA and/or inflammatory stimuli, we treated rats for 6 h with RA or LPS or both. DHRS3 mRNA was doubled by RA but reduced by >90% after treatment with LPS in the absence and presence of RA. On the basis of our results, DHRS3 mRNA expression is regulated by RA in a tissue- or cell-type specific manner; the RA-induced increase in DHRS3 may contribute to retinoid storage; and a reduction of DHRS3 expression in the liver during inflammation may contribute to the perturbation of whole body vitamin A metabolism that has previously been shown to occur in conditions of inflammatory stress.

Cytochrome P450s in the regulation of cellular retinoic acid metabolism.

The active metabolite of vitamin A, retinoic acid (RA), is a powerful regulator of gene transcription. RA is also a therapeutic drug. The oxidative metabolism of RA by certain members of the cytochrome P450 (CYP) superfamily helps to maintain tissue RA concentrations within appropriate bounds. The CYP26 family--CYP26A1, CYP26B1, and CYP26C1--is distinguished by being both regulated by and active toward all-trans-RA (at-RA) while being expressed in different tissue-specific patterns. The CYP26A1 gene is regulated by multiple RA response elements. CYP26A1 is essential for embryonic development, whereas CYP26B1 is essential for postnatal survival as well as germ cell development. Enzyme kinetic studies have demonstrated that several CYP proteins are capable of metabolizing at-RA; however, it is likely that CYP26A1 plays a major role in RA clearance. Thus, pharmacological approaches to limiting the activity of CYP26 enzymes may extend the half-life of RA and could be useful clinically in the future.

Relationship Between Preprocedural Lipid Levels and Periprocedural Myocardial Injury in Patients Undergoing Elective Percutaneous Coronary Intervention.

BACKGROUND: Periprocedural myocardial injury is a predictor of cardiovascular morbidity and mortality after percutaneous coronary intervention. METHODS: The authors examined the effects of preprocedural lipid levels (low-density lipoprotein, high-density lipoprotein, and triglycerides) in 977 patients with coronary artery disease who underwent elective percutaneous coronary intervention. RESULTS: Elevated cardiac troponin I level (≥5× the upper limit of normal) was used to indicate periprocedural myocardial injury. Serum lipid samples were collected 12 hours preprocedurally. Cardiac troponin I was collected 1, 6, and 12 hours postprocedurally. Correlations between preprocedural lipid levels and postprocedural cardiac troponin I were studied. Low-density lipoprotein levels were less than 70 mg/dL in 70% of patients and greater than 100 mg/dL in only 7.4% of patients; 13% had triglyceride levels greater than or equal to 150 mg/dL, and 96% had high-density lipoprotein levels less than 40 mg/dL. Patients with elevated cardiac troponin I had significantly lower left ventricular ejection fraction than did those with cardiac troponin I levels less than 5× the upper limit of normal (P = .01). Double-and triple-vessel disease were more common in patients with elevated cardiac troponin I (P < .002). Multivariable logistic and linear regression analyses revealed no statistically significant associations between lipid levels and postprocedural cardiac troponin I elevation, possibly because such large proportions of included patients had low levels of low-density lipoprotein (70%) and a history of statin intake (86%). CONCLUSION: The authors found no association between lipid profile and periprocedural myocardial injury.

Multiple cytochrome P-450 genes are concomitantly regulated by vitamin A under steady-state conditions and by retinoic acid during hepatic first-pass metabolism.

Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improvement of dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate, and supplemented) and acutely after administration of a therapeutic dose of all-trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P-450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P < 0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding retinoic acid receptor (RAR)ß, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log 2(6) in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naive rats, evidenced by increased CYP26A1 gene expression and increased conversion of [(3)H]RA to polar metabolites. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling retinol-binding protein (RBP)4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change.

Design and synthesis of targeted star-shaped micelle for guided delivery of camptothecin: In vitro and in vivo evaluation.

This study aimed to synthesize a star-shaped micelle using 3-azido-2,2-bis(azidomethyl)propan-1-ol (pentaerythritol triazide) core, as an initiator for the synthesis of three-arm polylactic acid (PLA) block. Then, the ends of the PLA arms were converted to PLA triazide followed by conjugation to the three alkyne-PEG-maleamide through click reaction. The maleamide ends were available for coupling with sulfhydryl-modified DNA aptamer against epithelial cell adhesion molecule in order to offer targeted delivery of encapsulated drug, camptothecin to the site of action. The successful synthesis of the star-shaped polymers was confirmed via1HNMR. Hydrophobic anti-cancer drug, camptothecin was encapsulated into the micelles core implementing solvent switching method providing loading content (LC%) and encapsulation efficiency (EE%) of 3.7 ± 0.4 and 73.7 ± 8.2, respectively. The size of both non-targeted and aptamer-targeted micelles was determined to be 154 and 192 nm, respectively with polydispersity index below 0.3. In vitro drug release evaluation at 37 °C, pH 7.4 showed a controlled release pattern for camptothecin during 72 h. In vitro cytotoxicity of the prepared non-targeted and targeted micelles was carried out on human colorectal adenocarcinoma (HT29) and mouse colon carcinoma (C26) as EpCAM positive cell lines and Chinese hamster ovary (CHO) as EpCAM negative cell line. The results verified significantly higher cytotoxicity of the targeted micelles on HT29 and C26 cell lines, while no obvious difference was observed between targeted and non-targeted formulation on CHO cell line. The in vivo therapeutic efficiency investigation on BALB/c C26 tumor-bearing mice showed superior capability of the targeted formulation on tumor suppression and survival rate of the treated mice. The developed platform exhibited excellent characteristics to diminish camptothecin drawbacks and its adverse effects while considerably increasing its therapeutic index.

Liver-specific cytochrome P450 CYP2C22 is a direct target of retinoic acid and a retinoic acid-metabolizing enzyme in rat liver.

Several cytochrome P450 (CYP) enzymes catalyze the C4-hydroxylation of retinoic acid (RA), a potent inducer of cell differentiation and an agent in the treatment of several diseases. Here, we have characterized CYP2C22, a member of the rat CYP2C family with homology to human CYP2C8 and CYP2C9. CYP2C22 was expressed nearly exclusively in hepatocytes, where it was one of the more abundant mRNAs transcripts. In H-4-II-E rat hepatoma cells, CYP2C22 mRNA was upregulated by all-trans (at)-RA, and Am580, a nonmetabolizable analog of at-RA. In comparison, in primary human hepatocytes, at-RA increased CYP2C9 but not CYP2C8 mRNA. Analysis of the CYP2C22 promoter region revealed a RA response element (5'-GGTTCA-(n)5-AGGTCA-3') in the distal flanking region, which bound the nuclear hormone receptors RAR and RXR and which was required for transcriptional activation response of this promoter to RA in CYP2C22-luciferase-transfected RA-treated HepG2 cells. The cDNA-expressed CYP2C22 protein metabolized [3H]at-RA to more polar metabolites. While long-chain polyunsaturated fatty acids competed, 9-cis-RA was a stronger competitor. Our studies demonstrate that CYP2C22 is a high-abundance, retinoid-inducible, hepatic P450 with the potential to metabolize at-RA, providing additional insight into the role of the CYP2C gene family in retinoid homeostasis.

Marriage of phospholipid and block copolymer in lipopolymersome hybrid structure for efficient tumor accumulation.

Despite the noticeable advantages of the liposomes and polymersomes, they also revealed some drawbacks that could be minimized by preparing hybrid vesicular systems and integrating the advantage of both vehicles into one system named lipopolymersome. Lipopolymesome incorporates the biodegradability, stability, adjustability and chemical flexibility of polymersomes with the elasticity, soft nature and biocompatibility of liposomes. In the current study, wereported the development of five nanoscale lipopolymersomal hybrid vesicular systems consisting different molar ratios of dipalmitoylphosphatidylcholine (DPPC) and poly (ethylene glycol)-poly (lactic acid) (PEG-PLA) (PEG-PLA: DPPC ratio of 100:0, 50:50: 25:75, 75:25 and 0:100). Rhod-6G-loaded hybrid vesicles were prepared via film rehydration. Then, the efficacy of five formulations were evaluated in terms of loading capacity, release pattern, cellular uptake, andin vivobiodistribution in ectopic tumor model in mice. The obtained results demonstrated that the self-assembly, loading capacity, cargo release and stability of hybrid nanoscale lipopolymersomes can be tuned by incorporation of amphiphilic lipid-polymers at various ratios. In this regard, the prepared hybrid nanovesicles consisting of DPPC-PEG-PLA (25:75) exhibited great potential through superior loading capacity, stability and tumor accumulation compared with other systems. It could be concluded that the prepared lipopolymersome offers important opportunities for the development of novel hybrid carriers for efficient transportation of therapeutics into tumor site.

Using the human CYP26A1 gene promoter as a suitable tool for the determination of RAR-mediated retinoid activity.

We have used a shortened construct form of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase (known as E4) or a red fluorescent protein, RFP (known as E4.2) as the reporter gene and examined their responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of at-RA in cells cotransfected with retinoic acid receptors (RAR). The promoter also responded quantitatively to retinol and various other retinoids. An isolated clonal line of HEK293T cells that was permanently transfected with the promoter driving the expression of RFP responded to both at-RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was also used to assess the retinoid activity of 3 novel synthetic retinoid analogues. Among them, EC23 was shown to be more potent than at-RA at lower concentrations and also more stable than at-RA. The promoter was also used to estimate the retinoid activities of intact rat serum samples as well as extracts of rat liver and lung, using retinol and at-RA as the reference standards. The retinoid activities could be measured in control rat serum samples and were increased in the serum of at-RA-treated rats. The total retinol and at-RA levels in the rat liver and lung samples determined by this promoter-based assay were compared with total retinol levels determined by the UPLC as the conventional methods. This system should offer a biologically-based alternative to mass-based retinoid analysis.

The first prospective implementation of markerless lung target tracking in an experimental quality assurance procedure on a standard linear accelerator.

The ability to track tumour motion without implanted markers on a standard linear accelerator (linac) could enable wide access to real-time adaptive radiotherapy for cancer patients. We previously have retrospectively validated a method for 3D markerless target tracking using intra-fractional kilovoltage (kV) projections acquired on a standard linac. This paper presents the first prospective implementation of markerless lung target tracking on a standard linac and its quality assurance (QA) procedure. The workflow and the algorithm developed to track the 3D target position during volumetric modulated arc therapy treatment delivery were optimised. The linac was operated in clinical QA mode, while kV projections were streamed to a dedicated computer using a frame-grabber software. The markerless target tracking accuracy and precision were measured in a lung phantom experiment under the following conditions: static localisation of seven distinct positions, dynamic localisation of five patient-measured motion traces, and dynamic localisation with treatment interruption. The QA guidelines were developed following the AAPM Task Group 147 report with the requirement that the tracking margin components, the margins required to account for tracking errors, did not exceed 5 mm in any direction. The mean tracking error ranged from 0.0 to 0.9 mm (left-right), -0.6 to -0.1 mm (superior-inferior) and -0.7 to 0.1 mm (anterior-posterior) over the three tests. Larger errors were found in cases with large left-right or anterior-posterior and small superior-inferior motion. The tracking margin components did not exceed 5 mm in any direction and ranged from 0.4 to 3.2 mm (left-right), 0.7 to 1.6 mm (superior-inferior) and 0.8 to 1.5 mm (anterior-posterior). This study presents the first prospective implementation of markerless lung target tracking on a standard linac and provides a QA procedure for its safe clinical implementation, potentially enabling real-time adaptive radiotherapy for a large population of lung cancer patients.

An essential set of basic DNA response elements is required for receptor-dependent transcription of the lecithin:retinol acyltransferase (Lrat) gene.

Lecithin:retinol acyltransferase (LRAT) is essential for vitamin A storage. Nuclear run-on assays demonstrated transcriptional regulation of the Lrat gene in vivo by all-trans-retinoic acid (RA) and other retinoids. Analysis of a 2.5 kb segment of rat genomic DNA revealed that the region approximately 300 bp upstream from the transcription start site (TSS) is necessary for high luciferase (Luc) reporter activity in HEK293T and HepG2 cells. Although this region lacks retinoid receptor binding elements, it responded to the nuclear receptors RARalpha, RARbeta or RARgamma, with RXRalpha, with and without ligand. Removal of -111 bp from the TSS, which is well conserved in human, rat and mouse genomes, completely eliminated activity. This region contains several basic elements (TATA box, SP3 site, AP-1 site, CAAT box), all of which were essential. Nuclear extracts from RA-treated cells exhibited enhanced binding. Therefore, this proximal region together with basal transcription factors may be sufficient to drive Lrat expression.

Ligand activation of peroxisome proliferator-activated receptor-beta/delta inhibits cell proliferation in human HaCaT keratinocytes.

Although there is strong evidence that ligand activation of peroxisome proliferator-activated receptor (PPAR)-beta/delta induces terminal differentiation and attenuates cell growth, some studies suggest that PPARbeta/delta actually enhances cell proliferation. For example, it was suggested recently that retinoic acid (RA) is a ligand for PPARbeta/delta and potentiates cell proliferation by activating PPARbeta/delta. The present study examined the effect of ligand activation of PPARbeta/delta on cell proliferation, cell cycle kinetics, and target gene expression in human HaCaT keratinocytes using two highly specific PPARbeta/delta ligands [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy acetic acid (GW0742) and 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-acetic acid (GW501516)] and RA. Both PPARbeta/delta ligands and RA inhibited cell proliferation of HaCaT keratinocytes. GW0742 and GW501516 increased expression of known PPARbeta/delta target genes, whereas RA did not; RA increased the expression of known retinoic acid receptor/retinoid X receptor target genes, whereas GW0742 did not affect these genes. GW0742, GW501516, and RA did not modulate the expression of 3-phosphoinositide-dependent protein kinase or alter protein kinase B phosphorylation. GW0742 and RA increased annexin V staining as quantitatively determined by flow cytometry. The effects of GW0742 and RA were also examined in wild-type and PPARbeta/delta-null primary mouse keratinocytes to determine the specific role of PPARbeta/delta in modulating cell growth. Although inhibition of keratinocyte proliferation by GW0742 was PPARbeta/delta-dependent, inhibition of cell proliferation by RA occurred in both genotypes. Results from these studies demonstrate that ligand activation of PPARbeta/delta inhibits keratinocyte proliferation through PPARbeta/delta-dependent mechanisms. In contrast, the observed inhibition of cell proliferation in mouse and human keratinocytes by RA is mediated by PPARbeta/delta-independent mechanisms and is inconsistent with the notion that RA potentiates cell proliferation by activating PPARbeta/delta.

A new amplified π-shape electrochemical aptasensor for ultrasensitive detection of aflatoxin B1.

There is a prompt need for determination of aflatoxin B1 (AFB1) in food products to avoid distribution and consumption of contaminated food products. In this study, an accurate electrochemical sensing strategy was presented for detection of AFB1 based on aptamer (Apt)-complementary strands of aptamer (CSs) complex which forms a π-shape structure on the surface of electrode and exonuclease I (Exo I). The presence of π-shape structure as a double-layer physical barrier allowed detection of AFB1 with high sensitivity. In the absence of AFB1, the π-shape structure remained intact, so only a weak peak current was recorded. Upon the addition of AFB1, the π-shape structure was disassembled and a strong current was recorded following the addition of Exo I. Under optimal conditions, the electrochemical signals enhanced as AFB1 concentrations increased with a dynamic range of 7-500pg/mL and a limit of detection (LOD) of 2pg/mL. The developed aptasensor was also used to analyze AFB1 spiked human serum and grape juice samples and the recoveries were 95.4-108.1%.