The downregulation of microRNA let-7a contributes to the excessive expression of type I collagen in systemic and localized scleroderma.

Systemic and localized scleroderma (SSc and LSc) is characterized by excessive deposition of collagen and tissue fibrosis in the skin. Although they have fundamental common characteristics including autoimmunity, little is known about the exact mechanism that mediates the excessive collagen expression in these disorders. In the current study, we tried to evaluate the possibility that microRNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed using miRNA inhibitors, precursors, or protectors. In the mouse model of bleomycin-induced dermal sclerosis, the overexpression of miRNAs was performed by i.p. miRNA injection. We demonstrated let-7a expression was downregulated in SSc and LSc skin both in vivo and in vitro, compared with normal or keloid skin. The inhibition or overexpression of let-7a in human or mouse skin fibroblasts affected the protein expression of type I collagen or luciferase activity of collagen 3'-untranslated region. Also, we found let-7a was detectable and quantitative in the serum and investigated serum let-7a levels in patients with SSc or LSc. let-7a concentration was significantly decreased in these patients, especially in LSc patients. Moreover, we revealed that the intermittent overexpression of let-7a in the skin by i.p. miRNA injection improved the skin fibrosis induced by bleomycin in mice. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approaches against SSc and LSc.

Photophysical and electrochemical properties of thienylnaphthalimide dyes with excellent photostability.

The development of robust dyes is a highly important theme for any applications of dyes. Here we present photophysical and electrochemical characterization of a set of robust dyes based on the thienylnaphthalimide unit. The set is comprised of the thienylnaphthalimide derivatives with phenyl- (Ph-), 4-nitrophenyl- (NO2Ph-), and 4-(diphenylamino)phenyl (Ph2NPh-) substituents as exemplars covering electron-withdrawing to electron-donating groups. The fluorescence quantum yields of the Ph-TNI increases as the solvent polarity increases, while that of Ph2NPh-TNI showed the opposite trend. Changes in the rates of nonradiative decay were found to be a major factor for these contrasting behaviors. Cyclic voltammetry showed that the substituent effects were more apparent for the HOMO energies rather than the LUMO energies. Density functional theory calculations showed that the first singlet excited state of these compounds is a (1)π,π* state with a significant charge transfer character. Ph-TNI and Ph2NPh-TNI are much more stable against photodegradation than coumarin and fluorescein dyes.

Development of new in vivo imaging methodology and system for the rapid and quantitative evaluation of the visual appearance of facial skin firmness.

BACKGROUND/AIMS: Lack of firmness is a key sign of skin aging, but there are no methods for quantifying the firmness of facial skin as perceived visually. The objective of this study was to develop a convenient and accurate method for this purpose. METHODS: A compact imaging system (Magic Ring) was developed to capture images of facial skin. By using an image-analysis algorithm, the number of facial lines and their direction were analyzed to give an index termed the 'Ageless Vector'. Correlations between the Ageless Vector and visually perceived facial skin firmness, mechanical skin firmness (R5), and actual age were examined for 108 Asian females. The technique was also used to assess the effects of a 14-day skin-moisturization regimen in 47 Asian female volunteers. RESULTS: The Ageless Vector showed highly significant correlation with visually perceived skin firmness (r = 0.816) and significant correlation with mechanical skin firmness (R5) (r = -0.775). Skin moisturization significantly improved both the Ageless Vector (P < 0.05) and the visual grading of apparent skin firmness. CONCLUSIONS: We confirmed by two clinical studies that the imaging methodology using the Ageless Vector and the Magic Ring facial-imaging system was sufficiently sensitive to permit the measurement of apparent visible skin firmness and that it is an excellent method suitable for practical in vivo applications.

Notch signaling generates the "cut here line" on the cuticle of the puparium in Drosophila melanogaster.

During a molt or eclosion, insects shed their cuticle, an extracellular matrix made by underlying epidermal cells, by cleavage along a defined line. This means that the "cut here line" is pre-formed on the cuticle, and its formation is indispensable for insect life. Here, we show that the proper formation of the operculum ridge (OR), which is the "cut here line" on the puparium (pupal case) of Drosophila melanogaster, involves Notch signaling activation in the epidermal cells just beneath the future OR region (OR-forming cells). The inhibition of Notch signaling causes defects in eclosion due to failure in OR cleavage, the chitin organization and several cuticular proteins localization, glucose dehydrogenase (Gld) activity, and OR-forming cell shape. Our findings provide the first insight into the molecular basis of the structure and formation of the "cut here line" on the cuticle.

Experimental evaluation of flow and dialysis performance of hollow-fiber dialyzers with different packing densities.

The dialyzer housing structure should be designed in such a way that high dialysis performance is achieved. To achieve high dialysis performance, the flow of the dialysis fluid and blood should be uniform, without channeling and dead spaces. The objective of this study was to evaluate the effect of fiber packing density on the flow of dialysis fluid and blood, and on the dialysis performance of a hollow-fiber dialyzer at defined flow rates for blood (Q (B) = 200 mL/min), dialysis fluid (Q (D) = 500 mL/min), and filtrate (Q (F) = 0 mL/min). We measured Q (D), Q (B), and solute clearance for 3 test dialyzers with dialyzer housing different diameters. To evaluate the flow of dialysis fluid and blood, we measured the residence time of the dialysis fluid and blood in the test dialyzers by use of the pulse-response method. We also measured the clearances of urea, creatinine, vitamin B(12), and lysozyme to evaluate the dialysis performance of the test dialyzers. At packing densities ranging from 48 to 67%, higher packing densities and lower housing diameters of the dialyzer resulted in higher dialysis performance because the dialysis fluid and blood entered the hollow-fiber bundle smoothly and, hence, increased contact area between the dialysis fluid and the blood led to better dialysis performance.

Computational evaluation of dialysis fluid flow in dialyzers with variously designed jackets.

Dialyzer performance strongly depends on the flow of blood and dialysis fluid as well as membrane performance. It is necessary, particularly to optimize dialysis fluid flow, to develop a highly efficient dialyzer. The objective of the present study is to evaluate by computational analysis the effects of dialyzer jacket baffle structure, taper angle, and taper length on dialysis fluid flow. We modeled 10 dialyzers of varying baffle angles (0, 30, 120, 240, and 360 degrees ) with and without tapers. We also modeled 30 dialyzers of varying taper lengths (0, 12.5, 25.0, and 50.0 mm) and angles (0, 2, 4, and 6 degrees ) based on technical data of APS-SA dialyzers having varying surface areas of 0.8, 1.5, and 2.5 m(2) (Rexeed). Dialysis fluid flow velocity was calculated by the finite element method. The taper part was divided into 10 sections of varying fluid resistances. A pressure of 0 Pa was set at the dialysis fluid outlet, and a dialysis fluid flow rate of 500 mL/min at the dialysis fluid inlet. Water was used as the dialysis fluid in the computational analysis. Results for dialysis fluid flow velocity of the modeled dialyzers indicate that taper design and a fully surrounded baffle are important in making the dialysis fluid flow into a hollow-fiber bundle easily and uniformly. However, dialysis fluid flow channeling occurred particularly at the outflowing part with dialyzers having larger taper lengths and angles. Optimum design of dialysis jacket structure is essential to optimizing dialysis fluid flow and to increasing dialyzer performance.

The expression of miR-124 increases in aged skin to cause cell senescence and it decreases in squamous cell carcinoma.

Skin senescence is induced by various factors including intrinsic aging and extrinsic aging. The current study compared the expression of microRNAs in young facial skin and senescent facial skin, and this study identified skin aging-related microRNAs. According to the results from a microRNA PCR Array, miR-124 was the microRNA that increased the most in senescent skin compared to young skin. Real-time PCR with a greater number of samples indicated that the increase in miR-124 levels in senescent facial skin was statistically significant. In situ hybridization was performed, and results indicated that the signal for miR-124 was evident in keratinocytes of senescent skin but not in those of young skin. The morphology of cultured normal human epidermal keratinocytes (NHEKs) transfected with a miR-124 mimic changed to an enlarged and irregular shape. In addition, the number of NHEKs positive for senescence-associated ß-galactosidase (SA-ß-gal) increased significantly as a result of the overexpression of the miR-124 mimic. The expression of miR-124 increased in UVB-irradiated NHEKs compared to controls in a dose-dependent manner. Expression of miR-124 in A431, a human cutaneous squamous cell carcinoma (SCC) cell line, decreased significantly compared to that in NHEKs. Forced overexpression of miR-124 as a result of the transfection of a miR-124 mimic in A431 resulted in the significant suppression of the proportion of cancer cells. The current results indicated that miR-124 increases as a result of cell senescence and that it decreases during tumorigenesis. The effect of supplementation of miR-124 in an SCC cell line suggests that senescence induction therapy with microRNA may be a new therapeutic approach for treatment of SCC.

Chronic sun exposure-related fusion oncogenes EGFR-PPARGC1A in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) differs from SCC of other organs in its strong association with chronic sun exposure. However, the specific driver mutations in cSCC remain unknown. Fusion genes in established cSCC cell lines (A431 and DJM-1) were predicted by transcriptome sequence, and validated by Sanger sequence, fluorescence in situ hybridization and G-banding. By transcriptome sequencing, we identified fusion gene EGFR-PPARGC1A in A431, which were expressed in 31 of 102 cSCCs. The lesions harboring the fusion gene tended to be located in sun-exposed areas. In vivo cutaneous implantation of EGFR-PPARGC1A-expressing NIH3T3 induced tumors resembling human cSCC, indicating its potent tumorigenicity. NIH3T3 transfected with EGFR-PPARGC1A as well as A431 showed increased cell proliferation activity. With regard to underlying mechanism, EGFR-PPARGC1A protein causes constitutive tyrosine phosphorylation, and induces the phosphorylation of wild-type full-length epidermal growth factor receptor (EGFR) by dimerization. Conversely, the RNAi-mediated attenuation of EGFR or CRISPR/Cas9-mediated knockdown of the fusion gene in A431 led to a decrease in the cell number, and may have therapeutic value. Our findings advance the knowledge concerning genetic causes of cSCC and the function of EGFR, with potential implications for new diagnostic and therapeutic approaches.

Effect of a low dose of midazolam on high blood pressure in dental patients: a randomised, double-blind, placebo-controlled, two-centre study.

Some patients have transient hypertension before dental treatment as a result of anxiety and stress. Midazolam is an anxiolytic, and thought to be effective for the management of this sort of transient hypertension. We have evaluated in a randomised, controlled trial whether a low dose of midazolam can lower blood pressure in dental patients to an acceptable level without excessive sedation. Suitable patients were randomised to be given midazolam (trial group) or physiological saline (control group) intravenously. Blood pressure, heart rate, degree of anxiety, and amount of sedation were measured before and after injection. After injection, blood pressure in the trial group significantly decreased to clinically acceptable levels compared with controls. The degree of anxiety in the trial group was also significantly less than that in the control group, but there were no significant differences in sedation. These results suggest that injection of a low dose of midazolam stabilises the blood pressure of dental patients with transient hypertension.

NUP160-SLC43A3 is a novel recurrent fusion oncogene in angiosarcoma.

Angiosarcoma is a malignant vascular tumor originating from endothelial cells of blood vessels or lymphatic vessels. The specific driver mutations in angiosarcoma remain unknown. In this study, we investigated this issue by transcriptome sequencing of patient-derived angiosarcoma cells (ISO-HAS), identifying a novel fusion gene NUP160-SLC43A3 found to be expressed in 9 of 25 human angiosarcoma specimens that were examined. In tumors harboring the fusion gene, the duration between the onset of symptoms and the first hospital visit was significantly shorter, suggesting more rapid tumor progression. Stable expression of the fusion gene in nontransformed human dermal microvascular endothelial cells elicited a gene-expression pattern mimicking ISO-HAS cells and increased cell proliferation, an effect traced in part to NUP160 truncation. Conversely, RNAi-mediated attenuation of NUP160 in ISO-HAS cells decreased cell number. Confirming the oncogenic effects of the fusion protein, subcutaneous implantation of NUP160-SLC43A3-expressing fibroblasts induced tumors resembling human angiosarcoma. Collectively, our findings advance knowledge concerning the genetic causes of angiosarcoma, with potential implications for new diagnostic and therapeutic approaches.

Discoidin domain receptor 2-microRNA 196a-mediated negative feedback against excess type I collagen expression is impaired in scleroderma dermal fibroblasts.

Systemic sclerosis (SSc) is characterized by excess collagen deposition in the skin, due to intrinsic transforming growth factor-ß (TGF-ß) activation. We tried to determine the expression and the role of discoidin domain receptor 2 (DDR2) in SSc. The expression of DDR2 mRNA and protein was significantly decreased in SSc dermal fibroblasts, which was recovered by knocking down TGF-ß. The knockdown of DDR2 in normal fibroblasts induced microRNA-196a expression, which led to type I collagen downregulation, indicating that DDR2 itself has a negative effect on microRNA-196a expression and inducible effect on collagen expression. In SSc fibroblasts, however, the DDR2 knockdown did not affect TGF-ß signaling and microRNA-196a expression. The microRNA-196a levels were significantly decreased in normal fibroblasts treated with TGF-ß and in SSc fibroblasts. Taken together our data indicate that, in SSc fibroblasts, intrinsic TGF-ß stimulation induces type I collagen expression, and also downregulates DDR2 expression. This probably acts as a negative feedback mechanism against excess collagen expression, as a decreased DDR2 expression is supposed to stimulate the microRNA-196a expression and further change the collagen expression. However, in SSc fibroblasts the microRNA-196a expression was downregulated by TGF-ß signaling. DDR2-microRNA-196a pathway may be a previously unreported negative feedback system, and its impairment may be involved in the pathogenesis of SSc.

Evaluation of dialyzer jacket structure and hollow-fiber dialysis membranes to achieve high dialysis performance.

The objective of this study was to determine the optimum dialyzer jacket structure and hollow-fiber dialysis membrane, both of which are indispensable factors for achieving high dialysis performance, by clarifying the relationship between the dialysis performance and the flow of dialysate and blood in a hollow-fiber dialyzer. We evaluated the clearance, dialysate, and blood flow for four commercially available hollow-fiber dialyzers, namely, the APS-15S, APS-15SA, TS-1.6UL, and CX-1.6U. To evaluate dialysate and blood flow, we measured the residence-time distribution of dialysate and blood flow of these dialyzers by the pulse-response method. We also determined the clearances of urea, creatinine, vitamin B(12), and lysozyme to evaluate the dialysis performance of these dialyzers. While the baffle and taper structures allow effective supply of dialysate into the dialyzer jacket, the hollow-fiber shape, inner diameter, and packing density significantly influence the dialysate flow. In dialyzers with long taper-holding slits, the slit area is a key design parameter for achieving optimum dialysate flow. Similarly, the blood flow is significantly influenced by the structure of the inflowing and outflowing blood ports at the header of a dialyzer, and the shape and inner diameter of the hollow fibers. Hollow fibers with smaller inner diameters cause an increase in blood pressure, which causes blood to enter the hollow fibers more easily. The hollow-fiber shape hardly affects the blood flow. While improved dialysate and blood flow cause higher clearance of low molecular-weight substances, higher membrane area and pure-water permeability accelerate internal filtration, thereby causing an increase in the clearance of large molecular-weight substances.

Flow uniformity in oxygenators with different outlet port design.

This study reports on evaluation of the optimum design of a blood outlet port structure for providing uniform flow by visualizing the blood flow in an extracapillary membrane oxygenator. We tested a cylindrical type extracapillary membrane oxygenator, HPO-20. The HPO-20 has a tangential blood outlet port and is thus referred to as "Tangential HPO-20." We engineered "Vertical HPO-20" with a vertical blood outlet port by modifying the Tangential HPO-20. To visualize the blood-side flow, a total of 120 insulated copper-wire electrodes were placed in the "Tangential" and the Vertical HPO-20s. The test solution flow was visualized by the dimensionless fluid arrival time reaching each electrode. The test solution flow in the Tangential HPO-20 was not uniform, particularly at the outside blood channel. The flow was more uniform in the Vertical HPO-20. The blood flow in an extracapillary membrane oxygenator with a vertical blood outlet port is well distributed so that it produces more uniform blood flow than that with a tangential outlet port because of the small stagnation and reduced channeling.