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This study aims to evaluate the effect of light-emitting diodes (LEDs) of different wavelengths on the embryonic development, covering behavior, righting behavior, and phototaxis of collector urchins (Tripneustes gratilla). The collector urchins were divided into three groups according to the type of LED illumination they received: full-spectrum (400–750 nm wavelength), red light (630 nm), or blue light (450 nm). The results of the embryonic development experiment indicated that the blue LED group had the highest proportion of embryos reaching the prism stage at the 24th hour and the highest proportion of embryos entering the 4-arm pluteus stage, but it also had the highest death rate at the 48th hour. The full-spectrum and red LED groups exhibited similar speeds of embryonic development. In the experiment on covering behavior performed on adult urchins, our findings indicated that the blue LED group gripped the most acrylic sheets for cover, exhibiting the most covering behavior, followed by the full-spectrum group and then the red LED group. Moreover, behavior varied with coloration, as collector urchins with a lower level of melanin exhibited more covering behavior than those with a higher melanin level. In addition, the righting behavior experiments demonstrated that the blue LED group spent the longest time righting themselves. It is possible that the relatively strong stimulation from the blue LED illumination led to a higher level of stress in the collector urchins and hence slowed their righting. The phototaxis experiment revealed the most significant negative phototactic response in collector urchins when they were under the blue LED light, followed by the full-spectrum light; the red LED light did not induce any positive or negative phototactic response in the collector urchins. This experimental result verified collector urchins’ high sensitivity to and dislike of the blue LED light. The study results confirmed that the blue LED light environment accelerated the embryonic development of collector urchins; however, the relatively strong stimulation from that light also caused them to engage in covering behavior or move away from the light. These results indicate that short-wavelength irradiation significantly affects the embryonic development and behavior pattern of this species.

Fat embolism (FE) is a lethal medical emergency often caused by fracture of long bones and amputation of limbs. Vascular endothelial growth factor (VEGF) promotes angiogenesis and increases vascular permeability. We tested the hypothesis that VEGF plays a critical role in FE-induced acute respiratory distress syndrome (ARDS) and acute lung injury (ALI). Fat tissues were collected from male Sprague-Dawley rats, and animal oil was extracted and mixed with water to form fatty micelles. The micelles were then injected into the tail vein to produce FE and ALI in rats. Lung weight gain was measured as the index of pulmonary edema. The expression of pulmonary VEGF was evaluated by real-time PCR and western blot analysis. Inducible nitric oxide synthase (iNOS) and phosphorylation of mitogen-activated protein kinase (MAPK) were determined by western blot analyses. Interleukin-1β (IL-1β) was quantified by ELISAs. Hematoxylin and eosin staining was used to evaluate the pathological damage of ALI. In this study, we found that animal oil-induced FE significantly increased pulmonary VEGF expression and MAPK phosphorylation. We also evaluated the inflammatory response after FE and found that iNOS and IL-1β significantly increased after FE. Systemic administration of SU-1498, an antagonist of VEGF receptor 2 (VEGFR-2), significantly attenuated the FE-induced inflammatory response and histological damage. This study suggested that VEGF is involved in FE-induced ARDS via the VEGFR-2 and MAPK cascades, which induce IL-1β release and iNOS upregulation. Blockade of could be used to treat FE-induced pulmonary damage.

Significant skin pigmentation changes occur when patients suffer deep burn injuries. These pigmentation disorders may cause not only cosmetic and psychological issues, but more importantly it increases the risk of skin cancer or photoaging. Severe burns significantly effect on the process of repigmentation as the pigmentation is tightly regulated by cell proliferation and differentiation of melanocytes and melanocyte stem cells which are housing in the epidermis and hair follicles of the skin. In the present review, we discuss the possible mechanisms to replenish the melanocytes from the healthy epidermis and hair follicles surrounding burn wounds. The molecular mechanisms of skin repigmentation following healing of burn injuries includes the differentiation of melanoblasts into melanocytes, the distribution and responses of melanocytes and melanocyte stem cells after burn injury, and the regulation of melanin production. We also reviewed advanced therapeutic strategies to treat pigmentation disorders, such as convectional surgery, laser, UV treatment and emerging concepts in skin tissue-engineering.

Na(+) -K(+) -2Cl(-) co-transporter (NKCC1) plays an important role in traumatic brain injury (TBI)-induced brain edema via the MAPK cascade. The transient receptor potential vanilloid type 4 (TRPV4) channel participates in neurogenic inflammation, pain transmission, and edema. In this study, we investigated the relationship between NKCC1 and TRPV4 and the related signaling pathways in TBI-induced brain edema and neuronal damage. TBI was induced by the calibrated weight-drop device. Adult male Wistar rats were randomly assigned into sham and experimental groups for time-course studies of TRPV4 expression after TBI. Hippocampal TRPV4, NKCC1, MAPK, and PI-3K cascades were analyzed by western blot, and brain edema was also evaluated among the different groups. Expression of hippocampal TRPV4 peaked at 8 h after TBI, and phosphorylation of the MAPK cascade and Akt was significantly elevated. Administration of either the TRPV4 antagonist, RN1734, or NKCC1 antagonist, bumetanide, significantly attenuated TBI-induced brain edema through decreasing the phosphorylation of MEK, ERK, and Akt proteins. Bumetanide injection inhibited TRPV4 expression, which suggests NKCC1 activation is critical to TRPV4 activation. Our results showed that hippocampal NKCC1 activation increased TRPV4 expression after TBI and then induced severe brain edema and neuronal damage through activation of the MAPK cascade and Akt-related signaling pathway.

The pigment melanin is produced by melanocytes, is primarily responsible for skin color, and protects it against ultraviolet rays that can cause the destruction of genetic material within the keratinocytes. To elucidate the mechanisms of many diseases associated with melanocytes, such as melanoma and albinism, or burns with uneven pigment distribution, the disease model needs to be established first. In this study, we aimed to construct the melanocyte model from patients in a short period.Sandai virus vector containing 4 stemness genes (Oct4, Sox2, Klf4, c-Myc) was transfected into human adipose-derived stem cells to produce induced pluripotent stem cells (iPSCs). Immunofluorescence staining was used to confirm the expression of specific proteins for iPSCs, including Tra-1-60, Tra-1-81, Oct-4, Sox-2, and Nango. polymerase chain reaction results also showed that specific genes of iPSCs with the ability to cause the differentiation of cells into the 3 germ layers were expressed. In our in vivo experiments, iPSCs were subcutaneously injected into nude mice to induce teratoma formation for 2 months. The morphology of the 3 germ layers was confirmed by hematoxylin and eosin staining. Furthermore, melanocytes were purified by serial induction medium, and their presence was confirmed by flow cytometry and the expression of different markers for melanocytes.

Skin substitutes with existing vascularization are in great demand for the repair of full-thickness skin defects. In the present study, we hypothesized that a pre-vascularized skin substitute can potentially promote wound healing. Novel three-dimensional (3D) skin substitutes were prepared by seeding a mixture of human endothelial progenitor cells (EPCs) and fibroblasts into a human plasma/calcium chloride formed gel scaffold, and seeding keratinocytes onto the surface of the plasma gel. The capacity of the EPCs to differentiate into a vascular-like tubular structure was evaluated using immunohistochemistry analysis and WST-8 assay. Experimental studies in mouse full-thickness skin wound models showed that the pre-vascularized gel scaffold significantly accelerated wound healing 7 days after surgery, and resembled normal skin structures after 14 days post-surgery. Histological analysis revealed that pre-vascularized gel scaffolds were well integrated in the host skin, resulting in the vascularization of both the epidermis and dermis in the wound area. Moreover, mechanical strength analysis demonstrated that the healed wound following the implantation of the pre-vascularized gel scaffolds exhibited good tensile strength. Taken together, this novel pre-vascularized human plasma gel scaffold has great potential in skin tissue engineering.

Supplement_S1_The_immunofluorescent_staining_of_EPCs_of_CD31 for Development of a Novel Pre-Vascularized Three-Dimensional Skin Substitute Using Blood Plasma Gel by Niann-Tzyy Dai, Wen-Shyan Huang, Fang-Wei Chang, Lin-Gwei Wei, Tai-Chun Huang, Jhen-Kai Li, Keng-Yen Fu, Lien-Guo Dai, Pai-Shan Hsieh, Nien-Chi Huang, Yi-Wen Wang, Hsin-I Chang, Roxanne Parungao, and Yiwei Wang in Cell Transplantation

Traumatic brain injury (TBI) is one of the most prevalent causes of worldwide mortality and morbidity. We previously had evidenced that TBI induced Na-K-2Cl co-transporter (NKCC1) upregulation in hippocampus. Here, we aim to investigate the role of NKCC1 in TBI-induced neurogenesis and the detailed mechanisms. The TBI-associated alternations in the expression of NKCC1, HIF-1α, VEGF, MAPK cascade, and CREB phosphorylation were analyzed by Western blot. TBI-induced neurogenesis was determined by immuno-fluorescence labeling. Chromatin immunoprecipitation was used to elucidate whether HIF-1α would activate VEGF gene after TBI. We found that the level of hippocampal NKCC1 and VEGF began to rise 8 h after TBI, and both of them reached maxima at day 7. Along with the upregulation of NKCC1 and VEGF, MAPK cascade was activated and hippocampal neurogenesis was promoted. Administration of CREB antisense oligonucleotide significantly attenuated the expression of HIF-1α, while HIF-1α antisense oligonucleotide exhibited little effect on the expression of CREB. However, HIF-1α antisense oligonucleotide administration did effectively suppress the expression of VEGF. Our results of the chromosome immunoprecipitation also indicated that HIF-1α could directly act on the VEGF promoter and presumably would elevate the VEGF expression after TBI. All these results have illustrated the correlation between NKCC1 upregulation and TBI-associated neurogenesis. The pathway involves the activation of Raf/MEK/ERK cascade, CREB phosphorylation, and HIF-1α upregulation, and finally leads to the stimulation of VEGF expression and the induction of neurogenesis.

Background Nogo-A is a member of the reticulon family of membrane-associated proteins and plays an important role in axonal remodeling. The present study aimed to investigate alterations in Nogo-A expression following traumatic brain injury (TBI)-induced inflammation and neuronal damage. Methods A weight-drop device was used to deliver a standard traumatic impact to rats. Western blot, RT-PCR and ELISA were used to analyze the expression of Nogo-A and IL-1β. Nogo-A antisense, and an irrelevant control oligonucleotide was intracerebroventricularly infused. We also performed H & E staining and luxol fast blue staining to evaluate the neuronal damage and demyelination resulting from TBI and various treatments. Results Based on RT-PCR and western blot analyses, the expression of Nogo-A was found to be significantly upregulated in the hippocampus beginning eight hours after TBI. In addition, TBI caused an apparent elevation in IL-1β levels and severe neuronal damage and demyelination in the tested animals. All of the TBI-associated molecular and cellular consequences could be effectively reversed by treating the animals with the anti-inflammatory drug indomethacin. More importantly, the TBI-associated stimulation in the levels of both Nogo-A and IL-1β could be effectively inhibited by a specific Nogo-A antisense oligonucleotide. Conclusions Our findings suggest that the suppression of Nogo-A expression appears to be an early response conferred by indomethacin, which then leads to decreases in the levels of IL-1β and TBI-induced neuron damage.

The die edge-seals and circuit under fad (CUF) are structurally optimized through 3D finite element analysis (FEA). Die edge-seals having the mechanically reinforced structure units are demonstrated essential. It is also shown that FEA help extract useful structural design concepts for CUP. The Poisson effects leading to a dual-ring-shaped high stress distribution area are confirmed to be detrimental for wire bonding and wire-pull tests. Excellent agreements are obtained as compared with the experimental data

The process development, characterization and performance evaluation of low-k dielectrics to form multi-level Cu interconnects for the 65 nm CMOS technology node are presented. Significant modifications and improvements over 90nm node have been implemented to overcome those challenges as design rules shrink, which include top via corner rounding control for the robust EM/SM reliability, and inline e-beam inspection for via/trench processes optimization. An in-house developed ECP additive "Trameca" for good Cu gap filling and a controllable hump height for good CMP performance are adopted to achieve tight Rs distributions. The facts that 100% yields of 2.1 millions via chain structure and open/short free on 5m long comb/meander structures along with SM/EM meeting the spec all demonstrated the technology to be a highly manufacturable BEOL process for 65 nm technology node.