Impact of polyplex micelles installed with cyclic RGD peptide as ligand on gene delivery to vascular lesions

Gene therapy is expected to open a new strategy for the treatment of refractory vascular diseases, so the development of appropriate gene vectors for vascular lesions is needed. To realize this requirement with a non-viral approach, cyclo(RGDfK) peptide (cRGD) was introduced to block copolymer, poly(ethylene glycol)-block-polycation carrying ethylenediamine units (PEG-PAsp(DET)). cRGD recognizes αvβ3 and αvβ5 integrins, which are abundantly expressed in vascular lesions. cRGD-conjugated PEG-PAsp(DET) (cRGD-PEG-PAsp(DET)) formed polyplex micelles through complexation with plasmid DNA (pDNA), and the cRGD-PEG-PAsp(DET) micelles achieved significantly more efficient gene expression and cellular uptake as compared with PEG-PAsp(DET) micelles in endothelial cells and vascular smooth muscle cells. Intracellular tracking of pDNA showed that cRGD-PEG-PAsp(DET) micelles were internalized via caveolae-mediated endocytosis, which is associated with a pathway avoiding lysosomal degradation, and that PEG-PAsp(DET) micelles were transported to acidic endosomes and lysosomes via clathrin-mediated endocytosis. Further, in vivo evaluation in rat carotid artery with a neointimal lesion revealed that cRGD-PEG-PAsp(DET) micelles realized sustained gene expression, while PEG-PAsp(DET) micelles facilitated rapid but transient gene expression. These findings suggest that introduction of cRGD to polyplex micelles might create novel and useful functions for gene transfer and contribute to the establishment of efficient gene therapy for vascular diseases

A gene expression test for depression

Purpose: Recently, we could distinguished patients with major depressive disorder (MDD) from nonpsychiatric controls with high accuracy using a panel of five gene expression markers (ARHGAP24, HDAC5, PDGFC, PRNP, and SLC6A4) in leukocyte. In the present study, we examined whether this biological test is able to discriminate patients with MDD from those without MDD, including those with schizophrenia and bipolar disorder. Patients and methods: We measured messenger ribonucleic acid expression levels of the aforementioned five genes in peripheral leukocytes in 17 patients with schizophrenia and 36 patients with bipolar disorder using quantitative real-time polymerase chain reaction (PCR), and we combined these expression data with our previous expression data of 25 patients with MDD and 25 controls. Subsequently, a linear discriminant function was developed for use in discriminating between patients with MDD and without MDD. Results: This expression panel was able to segregate patients with MDD from those without MDD with a sensitivity and specificity of 64% and 67.9%, respectively. Conclusion: Further research to identify MDD-specific markers is needed to improve the performance of this biological test

Test-retest reproducibility of cerebral adenosine A(2A) receptor quantification using [C-11]preladenant

Objective To evaluate the reproducibility of cerebral adenosine A(2A) receptor (A(2A)R) quantification using [C-11]preladenant ([C-11]PLN) and PET in a test-retest study. Methods Eight healthy male volunteers were enrolled. Dynamic 90 min PET scans were performed twice at the same time of the day to avoid the effect of diurnal variation. Subjects refrained from caffeine from 12 h prior to scanning, and serum caffeine was measured before radioligand injection. Arterial blood was sampled repeatedly during scanning and the fraction of the parent compound in plasma was determined. Total distribution volume (V-T) was estimated using 1- and 2-tissue compartment models (1-TCM and 2-TCM, respectively) and Logan graphical analysis (Logan plot) (t* = 30 min). Plasma-free fraction (f(P)) of [C-11]PLN was measured and used for correction of V-T values. Distribution volume ratio (DVR) was calculated from V-T of target and reference regions and obtained by noninvasive Logan graphical reference tissue model (LGAR) (t* = 30 min). Absolute test-retest variability (aTRV), and intra-class correlation coefficient (ICC) of V-T and DVR were calculated as indexes of repeatability. Correlation between DVR and serum concentration of caffeine (a nonselective A(2A)R blocker) was analyzed by Pearson's correlation analysis. Results Regional time-activity curves were well described by 2-TCM models. Estimation of V-T by 2-TCM produced some erroneous values; therefore, the more robust Logan plot was selected as the appropriate model. Global mean aTRV was 20% for V-T and 14% for V-T/f(P) (ICC, 0.72 for V-T and 0.87 for V-T/f(P)). Global mean aTRV of DVR was 13% for Logan plot and 10% for LGAR (ICC, 0.70 for Logan plot and 0.81 for LGAR). DVR estimates using LGAR and Logan plot were in good agreement (r(2) = 0.96). Coefficients of variation for V-T, V-T/f(P), DVR (Logan plot), and DVR (LGAR) were 47%, 47%, 27%, and 18%, respectively. Despite low serum caffeine levels, significant concentration-dependent effects on [C-11]PLN binding to target regions were observed (p < 0.01). Conclusions In this study, moderate test-retest reproducibility and large inter-subject differences were observed with [C-11]PLN PET, possibly attributable to competition by baseline amount of caffeine. Analysis of plasma caffeine concentration is recommended during [C-11]PLN PET studies

Assessment of bio-activities of the crude extract and components of Withania somnifera leaves by bioinformatics

Traditional herbal medicines are now increasingly being appreciated with Western models of integrative health sciences and evidence-based approach both in the basic research and clinic scenario. Ashwagandha is a commonly used plant in Ayurvedic, Indian traditional medicine. Medicinal value of Ashwagandha (WithaniasomniferaDunal) extends from anti-inflammatory, anti-arthritic, anti-rheumatic, rejuvenation and anti-cancer. Based on the belief that holistic multi-site mechanism of action offers greater chance of success, the traditional Ayurvedicmedicine practices the use of whole herb or its crude extract. It opposes with the mainstream of pharmaceutical industry that uses single and purified molecules. In the present study, we used bioinformatics approach to reveal the mechanism of action of (i) crude extract of Ashwagandha leaf extract and its purified components, (ii) Withanone and (iii) Withaferin A. Whereas p53-p21 was identified as a common signaling pathway for the three kinds of reagents, specific signaling pathways for Withaferin-A and Withanone were identified. Whereas the crude extract and Withanone were selectively toxic to human cancer cells, WithaferinA showed cytotoxicity to the normal cells too. The study suggested that the crude extract or a combinational formulamay be a superior and safenatural reagent for cancer treatment

First clinical assessment of [ 18 F]MC225, a novel fluorine-18 labelled PET tracer for measuring functional P-glycoprotein at the blood-brain barrier

Objective: 5-(1-(2-[18F]fluoroethoxy))-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalen ([18F]MC225) is a selective substrate for P-glycoprotein (P-gp), possessing suitable properties for measuring overexpression of P-gp in the brain. This is the first-in-human study to examine safety, radiation dosimetry and P-gp function at the blood-brain barrier (BBB) of [18F]MC225 in healthy subjects. Methods: [18F]MC225 biodistribution and dosimetry were determined in 3 healthy male subjects, using serial 2 h and intermittent 4 and 6 h whole-body PET scans acquired after [18F]MC225 injection. Dynamic [18F]MC225 brain PET (90 min) was obtained in 5 healthy male subjects. Arterial blood was sampled at various time intervals during scanning and the fraction of unchanged [18F]MC225 in plasma was determined. T1-weighted MRI was performed for anatomical coregistration. Total distribution volume (VT) was estimated using 1- and 2-tissue-compartment models (1-TCM and 2-TCM, respectively). VT was also estimated using the Logan graphical method (Logan plot) (t* = 20 min). Surrogate parameters without blood sampling (area-under the curve [AUC] of regional time-activity curves [TACs] and negative slope of calculated TACs) were compared with the VT values. Results: No serious adverse events occurred throughout the study period. Although biodistribution implied hepatobiliary excretion, secretion of radioactivity from liver to small intestine through the gallbladder was very slow. Total renal excreted radioactivity recovered during 6 h after injection was 0.9). AUCs of TACs were positively correlated with VT (2-TCM) values (r2: AUC0-60 min = 0.61, AUC0-30 min = 0.62, AUC30-60 min = 0.59, p < 0.0001). Negative slope of SUV TACs was negatively correlated with VT (2-TCM) values (r2 = 0.53, p < 0.0001). Conclusions: This initial evaluation indicated that [18F]MC225 is a suitable and safe PET tracer for measuring P-gp function at the BBB. Keywords: Blood–Brain barrier; Dosimetry; First-in-human; P-glycoprotein; Positron emission tomography

Current Status of the Development of a Transportable and Compact VLBI System by NICT and GSI

MARBLE (Multiple Antenna Radio-interferometer for Baseline Length Evaluation) is under development by NICT and GSI. The main part of MARBLE is a transportable VLBI system with a compact antenna. The aim of this system is to provide precise baseline length over about 10 km for calibrating baselines. The calibration baselines are used to check and validate surveying instruments such as GPS receiver and EDM (Electro-optical Distance Meter). It is necessary to examine the calibration baselines regularly to keep the quality of the validation. The VLBI technique can examine and evaluate the calibration baselines. On the other hand, the following roles are expected of a compact VLBI antenna in the VLBI2010 project. In order to achieve the challenging measurement precision of VLBI2010, it is well known that it is necessary to deal with the problem of thermal and gravitational deformation of the antenna. One promising approach may be connected-element interferometry between a compact antenna and a VLBI2010 antenna. By measuring repeatedly the baseline between the small stable antenna and the VLBI2010 antenna, the deformation of the primary antenna can be measured and the thermal and gravitational models of the primary antenna will be able to be constructed. We made two prototypes of a transportable and compact VLBI system from 2007 to 2009. We performed VLBI experiments using theses prototypes and got a baseline length between the two prototypes. The formal error of the measured baseline length was 2.7 mm. We expect that the baseline length error will be reduced by using a high-speed A/D sampler

Trans-complemented hepatitis C virus particles as a versatile tool for study of virus assembly and infection

AbstractIn this study, we compared the entry processes of trans-complemented hepatitis C virus particles (HCVtcp), cell culture-produced HCV (HCVcc) and HCV pseudoparticles (HCVpp). Anti-CD81 antibody reduced the entry of HCVtcp and HCVcc to almost background levels, and that of HCVpp by approximately 50%. Apolipoprotein E-dependent infection was observed with HCVtcp and HCVcc, but not with HCVpp, suggesting that the HCVtcp system is more relevant as a model of HCV infection than HCVpp. We improved the productivity of HCVtcp by introducing adapted mutations and by deleting sequences not required for replication from the subgenomic replicon construct. Furthermore, blind passage of the HCVtcp in packaging cells resulted in a novel mutation in the NS3 region, N1586D, which contributed to assembly of infectious virus. These results demonstrate that our plasmid-based system for efficient production of HCVtcp is beneficial for studying HCV life cycles, particularly in viral assembly and infection

Robotic anal preserving posterior pelvic exenteration combined with the transanal-vaginal approach

Robotic surgery is increasingly being applied for rectal cancer and its feasibility and safety have been reported. However, problems associated with advanced robotic surgery such as pelvic exenteration include lengthy operation time and difficulty in controlling unexpected bleeding. A 47-year-old woman had undergone laparoscopic left hemicolectomy for descending colon cancer three years previously (pT3N0M0 pStageII). And had undergone bilateral oophorectomy for ovarian metastases one year previously. Follow-up CT detected a peritoneal metastasis in the pelvic space. After seven courses of systemic chemotherapy, she received robotic anal preserving posterior pelvic exenteration combined with the transanal-vaginal approach. The postoperative course was uneventful. There is no evidence of recurrent disease 8 months after surgery. In conclusion, robotic anal preserving posterior pelvic exenteration combined with the transanal-vaginal approach is a safe and feasible minimally invasive approach for the treatment of advanced rectal malignancies

Oligomerization of Hepatitis C Virus Core Protein is Crucial for Interaction with the Cytoplasmic Domain of E1 Envelope Protein

Hepatitis C virus (HCV) contains two membrane-associated envelope glycoproteins, E1 and E2, which assemble as a heterodimer in the endoplasmic reticulum (ER). In this study, predictive algorithms and genetic analyses of deletion mutants and glycosylation site variants of the E1 glycoprotein were used to suggest that the glycoprotein can adopt two topologies in the ER membrane: the conventional type I membrane topology and a polytopic topology in which the protein spans the ER membrane twice with an intervening cytoplasmic loop (amino acid residues 288 to 360). We also demonstrate that the E1 glycoprotein is able to associate with the HCV core protein, but only upon oligomerization of the core protein in the presence of tRNA to form capsid-like structures. Yeast two-hybrid and immunoprecipitation analyses reveal that oligomerization of the core protein is promoted by amino acid residues 72 to 91 in the core. Furthermore, the association between the E1 glycoprotein and the assembled core can be recapitulated using a fusion protein containing the putative cytoplasmic loop of the E1 glycoprotein. This fusion protein is also able to compete with the intact E1 glycoprotein for binding to the core. Mutagenesis of the cytoplasmic loop of E1 was used to define a region of four amino acids (residues 312 to 315) that is important for interaction with the assembled HCV core. Taken together, our studies suggest that interaction between the self-oligomerized HCV core and the E1 glycoprotein is mediated through the cytoplasmic loop present in a polytopic form of the E1 glycoprotein