Quantitative analysis of H5N1 DNA hybridization on nanowell array electrode.

A nanowell array electrode-based electrochemical quantitative system without amplification was developed and applied for the detection of H5N1 target DNA. An 18-mer probe was immobilized on a nanowell array electrode with a diameter of 500 nm, which was coated with streptavidin and a self-assembly monolayer (SAM). The surface properties of probe DNA hybridization with complementary target DNA were characterized using atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The AFM image shows that the depth of nanowell was reduced from 200 nm to 15 nm due to the formation of a DNA hybridization complex on the streptavidin/SAM structure. Differences in charge transfer resistance (deltaR(ct)) in EIS upon hybridization of the probe DNA with complementary target DNA were analyzed and used for the quantitation of H5N1 DNA. This approach shows that the quantitative analysis of H5N1 DNA ranging from 1 pM to 1 microM DNA is possible on a nanowell array electrode.

Specific nucleic acid detection using photophysical properties of quantum dot probes.

In the present study, we report a novel separation-free method to detect and quantify avian influenza virus A (H5N1) nucleic acid without amplification, based on the alteration of photophysical parameters of quantum dot (QD) probes after hybridization with specific complementary target DNA. The target DNA was quantified in a custom-made portable device by simultaneously measuring lifetime and quenching of the QD probes. QD probes (25-mer) showed a 30% lifetime reduction and 40% fluorescence quenching when hybridized with complementary 25-mer target DNA. In comparison with a conventional QD-based assay, this assay provides a simple quantitation of nucleic acids with a single labeling step.

Impact of Supraaortic Intimal Tears on Aortic Diameter Changes After Nontotal Arch Replacement.

BACKGROUND: This study evaluated the impact of the intimal tear location on aortic dilation and reintervention after nontotal arch replacement (non-TAR) for acute type I aortic dissection. METHODS: Between 2009 and 2017, 92 patients who underwent non-TAR for acute type I aortic dissection were enrolled. Intimal tears were analyzed at the supraaortic (SA) segment; segment 1, proximal descending thoracic aorta (DTA); segment 2, distal DTA; and segment 3, abdominal aorta. Aortic diameter was measured at the pulmonary artery bifurcation, celiac axis, maximal abdominal aorta, and maximal thoracoabdominal aorta using serial follow-up computed tomographic scans. The Fisher exact or χ2 test, independent t or Mann-Whitney U test, and log-rank test were used in the statistical analyses. RESULTS: The significant factors for increasing aortic diameter were the first location of intimal tear in the SA segment and segments 1 and 2. In the adjusted analysis, the first location of intimal tear in the SA segment and segment 1 was statistically significant. In the additional adjusted analysis, a segment 1 tear without SA tear was the only significant factor for increasing aortic diameter. The 5-year freedom from reintervention rate was significantly higher in patients with no intimal tear than in those with a segment 1 intimal tear with/without SA tear. CONCLUSIONS: We confirmed that SA and proximal DTA intimal tears are associated with subsequent aortic dilation and reintervention. These proximal aortic intimal tears might warrant aggressive surgical treatment at the initial operation or close postoperative follow-up.

Quantitative analysis of nucleic Acid hybridization on magnetic particles and quantum dot-based probes.

In the present study we describe sandwich design hybridization probes consisting of magnetic particles (MP) and quantum dots (QD) with target DNA, and their application in the detection of avian influenza virus (H5N1) sequences. Hybridization of 25-, 40-, and 100-mer target DNA with both probes was analyzed and quantified by flow cytometry and fluorescence microscopy on the scale of single particles. The following steps were used in the assay: (i) target selection by MP probes and (ii) target detection by QD probes. Hybridization efficiency between MP conjugated probes and target DNA hybrids was controlled by a fluorescent dye specific for nucleic acids. Fluorescence was detected by flow cytometry to distinguish differences in oligo sequences as short as 25-mer capturing in target DNA and by gel-electrophoresis in the case of QD probes. This report shows that effective manipulation and control of micro- and nanoparticles in hybridization assays is possible.

Activation of fractalkine/CX3CR1 by vascular endothelial cells induces angiogenesis through VEGF-A/KDR and reverses hindlimb ischaemia.

AIMS: The present study investigated the detailed mechanism by which fractalkine (Fkn), a CX3C chemokine, induces angiogenesis and its functional implication in alleviating ischaemia in vivo. METHODS AND RESULTS: Fkn induced new vessel formation on the excised rat aorta and chick chorioallantoic membrane (CAM) through CX3CR1 activation. Immunoblotting analysis, promoter assay and electrophoretic mobility shift assay showed that Fkn upregulated hypoxia-inducible factor-1 alpha (HIF-1alpha) by cultured human aortic endothelial cells (ECs), which in turn induced mRNA and protein levels of vascular endothelial growth factor (VEGF)-A through a p42/44 mitogen-activated protein kinase pathway. In vivo Fkn-induced angiogenesis on CAM was completely blocked by functional inhibition of VEGF receptor 2 kinase insert domain-containing receptor (KDR) and Rho GTPase. C57/BL6 mice with CX3CR1(-/-) bone marrow-derived cells developed angiogenesis in the implanted Fkn-mixed Matrigel plug, suggesting CX3CR1 activation in vascular ECs is sufficient for Fkn-induced angiogenesis in vivo. The condition of rat hindlimb ischaemia, which rapidly stimulated mRNA expression of both Fkn and VEGF-A, was significantly alleviated by the injection of whole-length Fkn protein. CONCLUSION: Fkn-induced activation of CX3CR1 by ECs leads to in vivo angiogenesis through two sequential steps: the induction of HIF-1alpha and VEGF-A gene expression by CX3CR1 activation and the subsequent VEGF-A/KDR-induced angiogenesis. The potent induction of angiogenesis by Fkn can be used as a therapeutic strategy for alleviating peripheral ischaemia.

Selection and Characterization of Monoclonal Antibodies Targeting Middle East Respiratory Syndrome Coronavirus through a Human Synthetic Fab Phage Display Library Panning.

Since its first report in the Middle East in 2012, the Middle East respiratory syndrome-coronavirus (MERS-CoV) has become a global concern due to the high morbidity and mortality of individuals infected with the virus. Although the majority of MERS-CoV cases have been reported in Saudi Arabia, the overall risk in areas outside the Middle East remains significant as inside Saudi Arabia. Additional pandemics of MERS-CoV are expected, and thus novel tools and reagents for therapy and diagnosis are urgently needed. Here, we used phage display to develop novel monoclonal antibodies (mAbs) that target MERS-CoV. A human Fab phage display library was panned against the S2 subunit of the MERS-CoV spike protein (MERS-S2P), yielding three unique Fabs (S2A3, S2A6, and S2D5). The Fabs had moderate apparent affinities (Half maximal effective concentration (EC50 = 123-421 nM) for MERS-S2P, showed no cross-reactivity to spike proteins from other CoVs, and were non-aggregating and thermostable (Tm = 61.5-80.4 °C). Reformatting the Fabs into IgGs (Immunoglobulin Gs) greatly increased their apparent affinities (KD = 0.17-1.2 nM), presumably due to the effects of avidity. These apparent affinities were notably higher than that of a previously reported anti-MERS-CoV S2 reference mAb (KD = 8.7 nM). Furthermore, two of the three mAbs (S2A3 and S2D5) bound only MERS-CoV (Erasmus Medical Center (EMC)) and not other CoVs, reflecting their high binding specificity. However, the mAbs lacked MERS-CoV neutralizing activity. Given their high affinity, specificity, and desirable stabilities, we anticipate that these anti-MERS-CoV mAbs would be suitable reagents for developing antibody-based diagnostics in laboratory or hospital settings for point-of-care testing.

Dietary protein and fat intake in relation to risk of colorectal adenoma in Korean.

Consumption of red meat and alcohol are known risk factors for colorectal cancer, but associations for dietary fat remain unclear. We investigated the associations of dietary fat, protein, and energy intake with prevalence of colorectal adenoma.We performed a prospective cross-sectional study on asymptomatic persons who underwent a screening colonoscopy at a single center during a routine health check-up from May to December 2011. Dietary data were obtained via a validated Food Frequency Questionnaire (FFQ), assisted by a registered dietician. We also obtained information on alcohol consumption and smoking status, and measured metabolic syndrome markers including abdominal circumference, blood pressure, fasting glucose, serum triglyceride and high-density lipoprotein cholesterol. We calculated odds ratio (OR) and 95% confidence interval (CI) to evaluate the associations using the polytomous logistic regression models. As a secondary analysis, we also conducted a matched analysis, matched by age and sex (557 cases and 557 non-cases).The study sample included 557 cases (406 males and 151 females) with histopathologically confirmed colorectal adenoma, and 1157 controls (650 males and 507 females). The proportion of advanced adenoma was 28.1% of men and 18.5% of female, respectively. Although vegetable protein intake was inversely associated with the prevalence of colorectal adenoma, further adjustment for potential confounding factors attenuated the association, resulting in no significant associations. There were no significant associations between dietary fat intake and colorectal adenoma in energy-adjusted models. For vegetable protein in women, the OR for the comparison of those in the highest tertile with those in the lowest tertile was 0.47 (95% CI 0.25-0.91, P for trend = 0.07) after adjustment for total energy intake. However, after controlling for metabolic syndrome markers, body mass index, smoking status, alcohol consumption, and family history of colorectal adenoma, which were all significantly high in the colorectal adenoma patients group, the association became attenuated (OR 0.54, 95% CI 0.27-1.11, P for trend = 0.13).In conclusion, we did not observe the significant associations for intakes of total energy, total, animal and vegetable fats, and total, animal and vegetable proteins in relation to colorectal adenoma prevalence.

Comprehensive evaluation of carboxylated nanodiamond as a topical drug delivery system.

The best strategy in the development of topical drug delivery systems may be to facilitate the permeation of drugs without any harmful effects, while staying on the skin surface and maintaining stability of the system. Nanodiamonds (NDs) play a key role with their excellent physicochemical properties, including high biocompatibility, physical adsorption, reactive oxygen species (ROS) scavenging capability, and photostabilizing activity. Z-average sizes of carboxylated ND (ND-COOH) agglutinate decreased significantly as the pH increased. Fluorescein-conjugated ND was observed only on the stratum corneum, and no sample diffused into the dermal layer even after 48 hours. Moreover, ND-COOH and ND-COOH/eugenol complex did not show significant toxic effects on murine macrophage cells. ND improved in vitro skin permeation >50% acting as a "drug reservoir" to maintain a high drug concentration in the donor chamber, which was supported by quartz crystal microbalance results. Moreover, ND-COOH could adsorb a drug amount equivalent to 80% of its own weight. A photostability study showed that ND-COOH increased the photostability ~47% with regard to rate constant of the eugenol itself. A significant decrease in ROS was observed in the ND-COOH and ND-COOH/eugenol complex compared with the negative control during intracellular ROS assay. Moreover, ROS and cupric reducing antioxidant capacity evaluation showed that ND-COOH had synergistic effects of antioxidation with eugenol. Therefore, ND-COOH could be used as an excellent topical drug delivery system with improved permeability, higher stability, and minimized safety issue.

Diffusion-weighted magnetic resonance imaging lesions after repair of acute type A aortic dissection: association with neurocognitive outcomes†‡.

OBJECTIVES: We sought to analyse the preoperative status of arch vessels by postoperative diffusion-weighted magnetic resonance imaging (DWI) as a potential surrogate marker for cerebral thromboembolism and its relationship to neurocognitive outcomes. METHODS: Preoperative computed tomography (CT) and postoperative DWI were available for 50 patients who received surgery for acute type A aortic dissection. Two radiologists evaluated CT and DWI scans. Mini-mental status examinations (MMSE) were performed on the same day with DWI. RESULTS: Mean age of participants was 57 ± 14 years. MMSE and DWI were performed 6 ± 3 days after surgery. New cerebral embolisms were evident in 35 of 50 patients (70%) and often occurred as multiple lesions (28/35, 80%; range 2-21). Among patients with multiple lesions, 23 (66%) were clinically silent. Pathological lesions at the origin of the arch vessels correlated with the number and volume of new DWI lesions (P < 0.05). Degree of neurocognitive dysfunction tested by MMSE was negatively associated with age (r = -0.48, P < 0.0001) and left-sided DWI lesion number and volume (r = -0.74, P < 0.0001; r = -0.707, P < 0.0001). CONCLUSIONS: DWI revealed new cerebral embolisms in 70% of patients following surgery for acute type A aortic dissection. Lesion number and volume significantly correlated with pathological status of arch vessels. MMSE was representative of left-sided lesions.

Aorta Remodeling after Endovascular Treatment of a Chronic DeBakey IIIb Aneurysm and Simultaneous Palliation of a Renal Cell Carcinoma.

We report the case of a patient with a chronic DeBakey type IIIb aneurysm who underwent thoracic endovascular aortic repair to seal the primary entry tear and stent-graft insertion to cover the re-entry tear at the renal artery. The procedure was performed in order to achieve complete thrombosis in the entire thoracoabdominal false lumen, leading to favorable aortic remodeling. Simultaneously, ethanol ablation and renal artery embolization were performed to treat a renal tumor suspicious of renal cell carcinoma. Radical nephrectomy then confirmed clear cell carcinoma. To the best of our knowledge, no other cases of this type have been reported in the Korean literature.

Prognostic factors for aorta remodeling after thoracic endovascular aortic repair of complicated chronic DeBakey IIIb aneurysms.

OBJECTIVES: The use of thoracic endovascular aortic repair (TEVAR) for chronic DeBakey III type b (CDIIIb) aneurysms is controversial. We analyzed the potential prognostic factors affecting aorta remodeling after this procedure. METHODS: A total of 20 patients with CDIIIb aneurysms underwent TEVAR, with full coverage of reentry tears at the descending thoracic aorta. The potential factors affecting false lumen (FL) remodeling were analyzed, including reentry tears (communicating channels visible on the computed tomography angiogram), large intimal tears below the stent graft (≥ 2 consecutive axial cuts on the computed tomography angiogram), visceral branches arising from the FL, and intercostal arteries (ICAs) arising from the FL. RESULTS: All the patients had uneventful in-hospital courses; 2 patients (10%) required reintervention during the follow-up period. Thirteen patients (65%) had complete thrombosis of the FL at stent graft segment. Compared with the complete thrombosis group, the partial thrombosis group had more reentry tears (1.8 vs 2.3, P = .48), large intimal tears (0.8 vs 1.7, P < .05), visceral branches arising from the FL (1.2 vs 2.3, P < .05), and ICAs arising from the FL (3.8 vs 5.1, P = .35). Reentry tears, visceral branches, and ICAs from the FL were significant negative prognostic factors for FL shrinkage (P < .05). CONCLUSIONS: Although reentry tears above the celiac trunk were fully covered, the visceral branches and ICAs from the FL and all communicating channels below the celiac trunk kept the FL pressurized and were unfavorable prognostic factors for aorta remodeling after TEVAR for CDIIIb aneurysms.

Total Arch Replacement with Stented Elephant Trunk in DeBakey Type I Acute Aortic Dissection.

A 67-year-old female patient was treated with conventional total arch replacement and insertion of a stented elephant trunk (SET) graft into the descending thoracic aorta for acute DeBakey type I aortic dissection at one time. She had been treated with right coronary artery stent insertion for acute myocardial infarct 4 days earlier, and at that time, she was diagnosed with acute DeBakey type I aortic dissection from the ascending aorta to the suprarenal artery based on trans-esophageal echocardiography and aorta computed tomography. Through a median sternotomy, we inserted the SET graft through the opened aorta to the descending aorta. We also performed anastomosis between the proximal stented graft and the distal aortic arch, and then performed total arch replacement. For acute DeBakey type I aortic dissection, we report total arch replacement with insertion of a SET graft as a combination of conventional surgery and the interventional technique.

Effects of intermittent lower body perfusion on end-organ function during repair of acute DeBakey type I aortic dissection under moderate hypothermic circulatory arrest.

OBJECTIVES: To avoid deep hypothermia-related side effects, moderate hypothermic circulatory arrest (HCA) is commonly employed during aortic arch repair, thereby jeopardizing end-organ protection. We sought to analyse the effect of intermittent lower body perfusion (ILBP) on end-organ function during repair of acute DeBakey type I aortic dissection (AIAD). METHODS: Between May 2008 and May 2011, 107 patients underwent surgical repair for AIAD. All operations were performed with selective cerebral perfusion (SCP) under either moderate HCA only (n = 57) or moderate HCA with ILBP (n = 50). Adverse outcomes, including operative mortality, permanent neurological deficit, temporary neurological deficit, renal failure requiring dialysis and hepatic dysfunction, were compared between the two groups. RESULTS: The mean body temperature at the initiation of SCP was 28.7 ± 1.9 °C. Overall operative mortality occurred in 6 (5.6%) patients. The incidences of permanent neurological deficit and temporary neurological deficit were 1.9 and 4.7%, respectively. None of the 9 (8.4%) patients who suffered postoperative renal failure requiring dialysis received ILBP. The laboratory data showed significantly lower levels of hepatic and kidney enzymes in the ILBP group (P < 0.05). CONCLUSIONS: Significantly lower levels of hepatic and kidney enzymes indicate more effective end-organ protection with the use of ILBP. Our data suggest that ILBP provides more effective end-organ protection during repair of aortic arch under moderate HCA.

The effect of cholesterol in the liposome bilayer on the stabilization of incorporated Retinol.

The effect of cholesterol in the liposome bilayer on the stability of incorporated retinol was studied. Retinol was incorporated into liposomes containing soybean phosphatidylcholine (PC) and cholesterol (CH) at various ratios, and the liposomes were prepared as multilamellar vesicles by the dehydration-rehydration method. Retinol readily incorporated into liposomes at a ratio of 0.01:1 (w/w) retinol:lipid, with over 94.52% being incorporated in all conditions studied. The incorporation efficiency of retinol increased slightly with increasing CH content in the liposome and with increasing pH of the hydration buffer. Average particle size increased as the CH content increased, and mean particle sizes at pH 5, 7, and 9 were 30.27, 89.53, and 41.42 microm, respectively. The time course of retinol degradation in aqueous solution in liposomes with various ratios of PC to CH was determined under a variety of pH conditions (pH 5, 7, and 9), and temperatures (4, 25, 37, and 50 degrees C). The stability of incorporated retinol was enhanced by increasing the CH content. At pH 7.0 and 4 degrees C, for example, 90.17% of the retinol in liposomes containing 50:50 (PC:CH) remained after 10 days of storage, whereas 51.46% remained at 100:0 (PC:CH). These results indicate that CH in liposomes greatly increases the incorporation efficiency of retinol and the stability of incorporated retinol.

Electrotransfection of mammalian cells using microchannel-type electroporation chip.

Transfection of DNA molecules into mammalian cells with electric pulsations, which is so-called electroporation, is a powerful and widely used method that can be directly applied to gene therapy. However, very little is known about the basic mechanisms of DNA transfer and cell response to the electric pulse. We developed a microelectroporation chip with poly(dimethylsiloxane) (PDMS) to investigate the mechanism of electroporation as a first step of DNA transfer and to introduce the benefits of miniaturization into the genetic manipulation. The microelectroporation chip has a microchannel with a height of 20 microm and a length of 2 cm. Owing to the transparency of PDMS, we could in situ observe the uptake process of propidium iodide (PI) into SK-OV-3 cells, which shows promise in visualization of gene delivery in living cells. We also noticed the geometric effect on the degree of electroporation in microchannels with diverse channel width. This experimental result shows that the geometry can be another parameter to be considered for the electroporation when it is performed in microchannels with an exponential decaying pulse generator. Cell culturing is possible within the microelectroporation chip, and we also successfully transfected SK-OV-3 cells with enhanced green fluorescent protein genes, which demonstrates the feasibility of the microelectroporation chip in genetic manipulation.