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Diabetic retinopathy (DR) is a pathophysiologic vasculopathic process with obscure mechanisms and limited effective therapeutic strategies. Aryl hydrocarbon receptor (AhR) is an important regulator of xenobiotic metabolism and an environmental sensor. The aim of the present study was to investigate the role of AhR in the development of DR and elucidate the molecular mechanism of its downregulation. DR was evaluated in diabetes-induced retinal injury in wild type and AhR knockout (AhR-/-) mice. Retinal expression of AhR was determined in human donor and mice eyes by immunofluorescence since AhR activity was examined in diabetes. AhR knockout (AhRKO) mice were used to induce diabetes with streptozotocin, high-fat diet, or genetic double knockout with diabetes spontaneous mutation (Leprdb) (DKO; AhR-/-×Leprdb/db) for investigating structural, functional, and metabolic abnormalities in vascular and epithelial retina. Structural molecular docking simulation was used to survey the pharmacologic AhR agonists targeting phosphorylated AhR (Tyr245). Compared to diabetic control mice, diabetic AhRKO mice had aggravated alterations in retinal vasculature that amplified hallmark features of DR like vasopermeability, vascular leakage, inflammation, blood-retinal barrier breakdown, capillary degeneration, and neovascularization. AhR agonists effectively inhibited inflammasome formation and promoted AhR activity in human retinal microvascular endothelial cells and pigment epithelial cells. AhR activity and protein expression was downregulated, resulting in a decrease in DNA promoter binding site of pigment epithelium-derived factor (PEDF) by gene regulation in transcriptional cascade. This was reversed by AhR agonists. Our study identified a novel of DR model that target the protective AhR/PEDF axis can potentially maintain retinal vascular homeostasis, providing opportunities to delay the development of DR.

Background Amphiprion ocellaris is one of the rare reef fish species that can be reared in aquaria. It is increasingly used as a model species for Eco‐Evo‐Devo. Therefore, it is important to have an embryonic development table based on high quality images that will allow for standardized sampling by the scientific community. Results Here we provide high‐resolution time‐lapse videos to accompany a detailed description of embryonic development in A ocellaris. We describe a series of developmental stages and we define six broad periods of embryogenesis: zygote, cleavage, blastula, gastrula, segmentation, and organogenesis that we further subdivide into 32 stages. These periods highlight the changing spectrum of major developmental processes that occur during embryonic development. Conclusions We provide an easy system for the determination of embryonic stages, enabling the development of A ocellaris as a coral reef fish model species. This work will facilitate evolutionary development studies, in particular studies of the relationship between climate change and developmental trajectories in the context of coral reefs. Thanks to its lifestyle, complex behavior, and ecology, A ocellaris will undoubtedly become a very attractive model in a wide range of biological fields., Key Findings Six periods have been identified to describe the embryonic development of the false clown‐fish Amphiprion ocellaris.We provide a staging series organized similarly to other models allowing comparative stud‐ies of embryonic development.We provide high‐resolution time‐lapse videos to accompany a detailed description of embry‐onic development in Amphiprion ocellaris.These data provide a general framework that can be used as a tool to standardize studies and experimental procedures throughout false clownfish development.

Background: Activated microglia-mediated neuro-inflammation plays a vital aspect in regulating the micromilieu of the central nervous system. Neuro-inflammation involves distinct alterations of microglial phenotypes, containing nocuous pro-inflammatory (M1) phenotype and neuroprotective anti-inflammatory (M2) phenotype. Currently, there is no effective treatment for modulating such alterations. Little evidence shows that melatonin prevents the detrimental cascade of activated microglia-mediated neuro-inflammation. Methods: The expression levels of M1/M2 marker of primary microglia influenced by Melatonin were detected via qPCR. Functional activities were explored by western blotting, luciferase activity, EMSA, and ChIP assay. Structure interaction was assessed by molecular docking and LIGPLOT analysis. ER stress detection was examined by ultrastructure TEM, calapin activity, and ERSE assay. The neurobehavioral evaluations and immunofluorescence staining in animals were used for investigation of Melatonin on the neuroinflammation in vivo. Results: Melatonin had targeted on Peroxisome Proliferator Activated Receptor Delta (PPARd) activity, boosted LPS-stimulated alterations in polarization from the M1 to the M2 phenotype, and thereby inhibited NFkB–IKKb activation in primary microglia. The PPARd agonist L-165041 or over-expression of PPARd plasmid (ov-PPARd) showed similar results. Molecular docking screening, dynamic simulation approaches, and biological studies of melatonin showed that the activated site was located at PPARd (phospho-Thr256-PPARd). Furthermore, we found that activated microglia had lowered PPARd activity as well as the downstream SIRT1 formation via enhancing ER stress. Melatonin, PPARd agonist and ov-PPARd all effectively reversed the above-mentioned effects. Melatonin blocked ER stress by regulating calapin activity and expression in LPS-activated microglia. Additionally, melatonin or L-165041 ameliorated the neurobehavioral deficits in LPS-aggravated neuroinflammatory mice through blocking microglia activities, and also promoted phenotype changes to M2-predominant microglia. Conclusions Melatonin suppressed neuro-inflammation in vitro and in vivo by tuning microglial activation through the ER stress-dependent PPARd/SIRT1 signaling cascade. We proposed that this treatment strategy is an encouraging pharmacological approach for the remedy of neuro-inflammation associated disorders.

Background Cisplatin (CP) is a chemotherapeutic drug for treating melanoma that also causes adverse side effects in cancer patients. Purpose This study investigated the bioefficacy of a phytoagent deoxyelephantopin (DET) in inhibiting B16 melanoma cell activity, its synergism with CP against metastatic melanoma, and its capability to attenuate CP side effects in animals. Methods DET and CP bioactivities were assessed by MTT assay, isobologram analysis, time-lapse microscopy, migration and invasion assays, flow cytometry and western blotting. In vivo bioluminescence imaging was used to detect lung metastasis of B16 cells carrying COX-2 reporter gene in syngeneic mice. H&E staining and immunohistochemistry were used to evaluate the compound/drug efficacy and CP side effects. Nephrotoxicity caused by CP treatment in mice was evaluated by UPLC/ESI-QTOF MS − based metabolomics and haematometry. Result DET, alone or in combination with cisplatin, inhibited B16 cell proliferation, migration, and invasion, and induced cell-cycle arrested at the G2/M phase and de-regulated cell-cycle mediators in cancer cells. In a murine B16COX-Luc metastatic allograft model, CP2 (2 mg/kg) treatment inhibited B16 lung metastasis accompanied by severe body weight loss, renal damage and inflammation, and haematological toxicity. DET10 and CP cotreatment (DET10 + CP1) or sequential treatment (CP2→DET10) significantly inhibited formation of pulmonary melanoma foci and reduced renal damage. DET pretreatment (Pre-DET10) or CP2→DET10 treatment had the longest survival (52 vs. 37 days for tumor control mice). CP treatment caused abnormally accumulated urea cycle metabolites and serotonin metabolite hippuric acid in renal tissues that were not seen with DET alone or in combination with CP. Conclusion The CP and DET combination may be an effective intervention for melanoma with reduced side effects.

Background and purpose: Histone deacetylase (HDAC) inhibitors (HDIs) were shown to modulate the epithelial-mesenchymal transition (EMT) progression and inhibit the migration and invasion of cancer cells. Emerging as a novel class of anti-cancer drugs, HDIs have attracted much attention in the field of drug discovery. This study aimed to the underlying mechanisms of Honokiol in preventing the metastatic dissemination of gastric cancer cells by inhibiting HDAC3 activity/expression.Experimental Approach: Clinical pathological analysis was performed to determine the relationship between HDAC3 and tumor progression. The effects of Honokiol on pharmacological characterization, functional, transcriptional activities, organelle structure changes, and molecular signalings were analyzed using binding assays, Differential Scanning Calorimetry, luciferase reporter assay, HDAC3 activity, ER stress response element activity, transmission electron microscopy, immune-blotting and Wnt/b-catenin activity assays. The in vivo effects of Honokiol on peritoneal dissemination were determined by a mouse model and detected by the PET/CT tomography.Key Results: HDAC3 over-expression was correlated with poor prognosis. Honokiol significantly abolished HDAC3 activity (Y298) via inhibition of NFκBp65/CEBPb signaling, which was reversed by the over-expression of plasmids of NFκBp65/CEBPb. Honokiol-inhibited NFκBp65/CEBPb activation could be reversed by 4-phenylbutyric acid (a chemical chaperone) and calpain-2 gene silencing. Honokiol increased ER stress markers and inhibited EMT-associated epithelial markers, but decreased Wnt/β-catenin activity. Suppression of HDAC3 by both Honokiol and HDAC3 gene silencing decreased cell migration and invasion in vitro and metastasis in vivo. Conclusions and Implications: Honokiol acts by suppressing HDAC3-mediated EMT and metastatic signaling. By prohibiting HDAC3, metastatic dissemination of gastric cancer may be blocked.

Background and purpose Histone deacetylase (HDAC) inhibitors (HDIs) can modulate the epithelial-mesenchymal transition (EMT) progression and inhibit the migration and invasion of cancer cells. Emerging as a novel class of anti-cancer drugs, HDIs are attracted much attention in the field of drug discovery. This study aimed to discern the underlying mechanisms of Honokiol in preventing the metastatic dissemination of gastric cancer cells by inhibiting HDAC3 activity/expression. Experimental approach Clinical pathological analysis was performed to determine the relationship between HDAC3 and tumor progression. The effects of Honokiol on pharmacological characterization, functional, transcriptional activities, organelle structure changes, and molecular signaling were analyzed using binding assays, differential scanning calorimetry, luciferase reporter assay, HDAC3 activity, ER stress response element activity, transmission electron microscopy, immune-blotting, and Wnt/β-catenin activity assays. The in vivo effects of Honokiol on peritoneal dissemination were determined by a mouse model and detected by PET/CT tomography. Key results HDAC3 over-expression was correlated with poor prognosis. Honokiol significantly abolished HDAC3 activity (Y298) via inhibition of NFκBp65/CEBPβ signaling, which could be reversed by the over-expression of plasmids of NFκBp65/CEBPβ. Treatments with 4-phenylbutyric acid (a chemical chaperone) and calpain-2 gene silencing inhibited Honokiol-inhibited NFκBp65/CEBPβ activation. Honokiol increased ER stress markers and inhibited EMT-associated epithelial markers, but decreased Wnt/β-catenin activity. Suppression of HDAC3 by both Honokiol and HDAC3 gene silencing decreased cell migration and invasion in vitro and metastasis in vivo. Conclusions and implications Honokiol acts by suppressing HDAC3-mediated EMT and metastatic signaling. By prohibiting HDAC3, metastatic dissemination of gastric cancer may be blocked. Graphical abstract Conceptual model showing the working hypothesis on the interaction among Honokiol, HDAC3, and ER stress in the peritoneal dissemination of gastric cancer. Honokiol targeting HDAC3 by ER stress cascade and mitigating the peritoneal spread of gastric cancer. Honokiol-induced ER stress–activated calpain activity targeted HDAC3 and blocked Tyr298 phosphorylation, subsequently blocked cooperating with EMT transcription factors and cancer progression. The present study provides evidence to demonstrate that HDAC3 is a positive regulator of EMT and metastatic growth of gastric cancer cells. The findings here imply that overexpressed HDAC3 is a potential therapeutic target for honokiol to reverse EMT and prevent gastric cancer migration, invasion, and metastatic dissemination. • Honokiol significantly abolished HDAC3 activity on catalytic tyrosine 298 residue site. In addition, Honokiol-induced ER stress markedly inhibited HDAC3 expression via inhibition of NFκBp65/CEBPβ signaling. • HDAC3, which is a positive regulator of metastatic gastric cancer cell growth, can be significantly inhibited by Honokiol. • Opportunities for HDAC3 inhibition may be a potential therapeutic target for preventing gastric cancer metastatic dissemination.

The twin tail of ornamental goldfish is known to be caused by a nonsense mutation in one chordin paralogue gene. Our previous molecular studies in goldfish revealed that the ancestral chordin gene was duplicated, creating the chdA and chdB genes, and the subsequent introduction of a stop codon allele in the chdA gene ( chdA E127X) caused the twin‐tail morphology. The chdA E127X allele was positively selected by breeders, and the allele was genetically fixed in the ornamental twin‐tail goldfish population. However, little is known about the evolutionary history of the chdB paralogue, begging the question: are there the functionally distinct alleles at the chdB locus, and if so, how did they evolve? To address these questions, we conducted molecular sequencing of the chdB gene from five different goldfish strains and discovered two alleles at the chdB gene locus; the two alleles are designated chdB 1 and chdB 2. The chdB 1 allele is the major allele and was found in all investigated goldfish strains, whereas the chdB 2 allele is minor, having only been found in one twin‐tail strain. Genetic analyses further suggested that these two alleles are functionally different with regard to survivability ( chdB 1 > chdB 2). These results led us to presume that in contrast to the chdA locus, the chdB locus has tended to be eliminated from the population. We also discuss how the chdB 2 allele was retained in the goldfish population, despite its disadvantageous function. This study provides empirical evidence of the long‐term retention of a disadvantageous allele under domesticated conditions.

The twin‐tail of ornamental goldfish provides unique evolutionary evidence that the highly conserved midline localization of axial skeleton components can be changed by artificial selection. This morphological change is known to be caused by a nonsense mutation in one of the recently duplicated chordin genes, which are key players in dorsal–ventral (DV) patterning. Since all of the multiple twin‐tail ornamental goldfish strains share the same mutation, it is reasonable to presume that this mutation occurred only once in domesticated goldfish. However, zebrafish with mutated szl gene (another DV patterning‐related gene) also exhibit twin‐tail morphology and higher viability than dino/chordin‐mutant zebrafish. This observation raises the question of whether the szl gene mutation could also reproduce the twin‐tail morphology in goldfish. Here we show that goldfish have at least two subfunctionalized szl genes, designated szlA and szlB, and depletion of these genes in single‐fin goldfish was able to reproduce the bifurcated caudal fin found in twin‐tail ornamental goldfish. Interestingly, several phenotypes were observed in szlA‐depleted fish, while low expressivity of the twin‐tail phenotype was observed in szlB‐depleted goldfish. Thus, even though szl gene mutations may produce twin‐tail goldfish, these szl gene mutations might not be favorable for selection in domestic breeding. These results highlight the uniqueness and rarity of mutations that are able to cause large‐scale morphological changes, such as a bifurcated axial skeleton, with high viability and expressivity in natural and domesticated populations.

Characterizations for the comprehensive viscoelastic behaviors of materials were conducted in this research. For considering the multiaxial viscoelastic behaviors of the polymeric constituents in finite element models for package stress and deformation simulations, it is required to construct the mathematical models of the viscoelastic bulk and shear relaxation moduli. In order to obtain the consistent viscoelastic functions, the interconversion relations were developed and applied as a new fitting procedure. By using the consistently fitted values of viscoelastic bulk and shear relaxation moduli, the constitutive behaviors of organic packaging materials by using time-dependent viscoelastic models for warpage predictions could be more accurate.

Viperin is an anti-viral protein, induced by viral infection. In this study, we examined whether over-expression of viperin in fish muscle could inhibit bacterial growth. We first obtained the cDNA sequence of tilapia viperin, through RT-PCR-mediated cloning and sequencing. The cDNA sequence was similar to those of several fish viperins in GenBank, and it was predicted to encode the conserved domain of radical S-adenosylmethionine superfamily proteins. Phylogenetic analysis revealed that tilapia viperin was most closely related to viperin of Sciaenops ocellatus, Coreoperca kawamebari, and C. whiteheadi. Expression of tilapia viperin was significantly up-regulated in the kidney, liver, spleen, and gills upon challenge with lipopolysaccharide (LPS) and poly(I:C) in a time- and dose-dependent manner. Injection of Vibrio vulnificus (204) and Streptococcus agalactiae (SA47) bacteria into tilapia resulted in significant induction of viperin expression in the whole body, kidney, liver, and spleen. Electrotransfer of a viperin-expressing plasmid into zebrafish muscles decreased bacterial numbers and altered expression of immune-related genes. These data indicate that such altered expression may account for the improvement in bacterial clearance following electroporation of viperin, suggesting that fish viperin has antiviral and antibacterial activities.

Twin-tail goldfish strains are examples of drastic morphological alterations that emerged through domestication. Although this mutation is known to be caused by deficiency of one of two duplicated chordin genes, it is unknown why equivalent mutations have not been observed in other domesticated fish species. Here, we compared the chordin gene morphant phenotypes of single-tail goldfish and common carp (close relatives, both of which underwent chordin gene duplication and domestication). Morpholino-induced knockdown depleted chordin gene expression in both species; however, while knockdown reproduced twin-tail morphology in single-tail goldfish, it had no effect on common carp morphology. This difference can be explained by the observation that expression patterns of the duplicated chordin genes overlap completely in common carp, but are sub-functionalized in goldfish. Our finding implies that goldfish drastic morphological changes might be enhanced by the subsequent occurrence of three different types of evolutionary event (duplication, sub-functionalization and selection) in a certain order.

Background An outbreak of Acinetobacter baumannii in neurological intensive care unit was detected, causing nosocomial infection on 6 cases and colonization on 5 cases. Methods Five major clones of A baumannii circulating in the intensive care unit were detected by pulse-field gel electrophoresis (PFGE). The outbreak was controlled by aggressive intervention with disinfection of equipments and environment, and audit of hand-washing practice by the staff in this intensive care unit. Results The A baumannii could be isolated from equipments and the environment, including the chart folders 5/8 (62.5%), work trolley 1/2 (50.0%), ambulance bag of patient 1/3 (33.3%), hands of healthcare-workers 7/22 (31.8%), surfaces of monitors 1/4 (25.0%), bottle of suction fluid 1/4 (25.0%); but not isolated from the hands of healthcare-workers after disinfection with wash practice. Conclusions Different strains of A baumannii may cause infection, exist in medical environment, and colonize the patients at the same time. It may develop into true infection anytime. We conclude that hand hygiene as well as disinfection of equipments and the environment are the two most important factors to control and prevent the outbreak of A baumannii .

PurposeThe purpose of this paper is to investigate the policy consequences of expensing stock‐based compensation in Taiwan.Design/methodology/approachData were collected on listed firms from 2006 to 2008 and a goodness‐of‐fit of accounting earnings valuation model was used to investigate the incremental information content of expensing stock‐based compensation. In addition, two sensitivity indexes were used to investigate the sensitivity between compensation and firm performance before and after income statement recognition of stock‐based compensation.FindingsIt was found that the association between earnings and abnormal returns is stronger after expensing compensation. In addition, the relationship between compensation variables, especially stock compensation, and firm performance is stronger after 2008, indicating that expensing compensation reinforces the relationship between compensation and performance.Practical implicationsThe findings suggest that disclosure and recognition are not substitutes. The findings also have implications for standard setters and for investors attempting to mitigate managers' self‐interested behavior.Originality/valueThe accounting treatment of employee stock‐based compensation is a controversial issue among academics, regulators, managers, auditors, and investors. This paper investigates the incremental information content of the new accounting standard and explores whether the relationship between compensation and firm performance has become more transparent than before.

This study investigates the anti-MCF-7 breast cancer cell effects and the underlying pharmacological activity and mechanism of taiwanin A, a major lignan isolated from Taiwania cryptomerioides. Our results show that taiwanin A time-dependently induced reactive oxygen species level and DNA damage in MCF-7 cells, which were likely activated kinases ataxia telangiectasia mutated (ATM) and checkpoint kinase (Chk). Taiwanin A could also up-regulate p53, phosphorylated p53, p21(Cip1), and p27(Kip1) and down-regulate the G(2)/M checkpoint cyclin-dependent kinase1 (Cdk1)-cyclin A/B, leading to induction of G(2)/M cell-cycle arrest in MCF-7 cells. Blockade of p53 gene expression by siRNA further demonstrated that the cell-cycle arrest induced by taiwanin A was p53-dependent. The FasL/Fas-mediated apoptotic signaling cascade was involved in taiwanin A-induced apoptosis via activation of caspases-10 and -7 (but not caspase-8), and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). In contrast, mitochondria-initiated apoptotic pathway was not involved. This is the first report to delineate novel mechanism of the action of taiwanin A against MCF-7 cells, suggesting this lignan may have value for development as an anti-breast cancer agent.

The present paper introduced a non-destructive TDR (Time Domain Reflectomerty Analysis) methodology, an essential E-FA (Electrical Failure Analysis) technique for separating the fault isolation and identifying failure mode of advanced 2.5D IC package. The package consists of TSV and u-bump staked packaging structure. This methodology has been shown to be applicable for allocating the defect within a package. The I/V curve tracing, TDR (Time Domain Reflectometry) and the LIT (Lock-in thermography) as the useful package level and die level non-destructive techniques on fault isolation have been performed to overcome some of the difficulties. In this paper, the methodology and application of TDR and LIT on open and short/leakage failure isolations in 2.5D IC packages have been presented. The analysis procedure and results on both of fault isolations as well physical analyses to further inspection the root cause have also been discussed in detail from some case studies and the conclusion are presented at the end.

Orchids have profound diversity of specialized pollination and ecological strategies and provide a rich setting for studying evolutionary relationships and molecular biology. The sophisticated orchid flower morphology includes two whorls of perianth segments, three sepals and three petals, one of which is modified as labellum (or lip). In addition, the male and female reproductive organs are fused to form a gynostemium. In order to study gene expression in orchid reproductive organs, a cDNA library of mature flower buds of Phalaenopsis equestris, a native diploid species of Phalaenopsis in Taiwan, was constructed. A total of 5593 expressed sequence tags (ESTs) from randomly selected clones were identified and characterized. Cluster analysis allowed the identification of a unigene set of 3688 sequences. This abundance of transcripts with predicted cellular roles was functionally characterized by the BLASTX matches to known proteins. Comparison of the relative EST frequencies based on functional categories among floral tissues of five species including P. equestris, Acorus Americanus, Asparagus officinalis, Oryza sativa and Arabidopsis thaliana was performed. The most highly transcribed genes in Phalaenopsis floral buds are those coding for RNA-dependent RNA polymerase of Cymbidium mosaic virus, followed by heat shock protein genes. A total 217 putative transcription factor related ESTs were identified. C3H and trihelix families occupied 25% of transcribed transcription factor genes, indicating that the profile of the transcription factors in orchid flower buds is polarized. The extensive analysis of the genes in floral organs adds to the growing repertoire of known plant genes and may also reveal unique features of the reproductive organs of orchids.

As an approach to improving Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase (Fsbeta-glucanase) for use in industry and to studying the structure-function relationship of the C-terminus in the enzyme, a C-terminally truncated ( approximately 10 kDa) Fsbeta-glucanase was generated using a PCR-based gene truncation method and then overexpressed in either Escherichia coli BL21(DE3) or Pichia pastoris strain X-33 host cells. The initial rate kinetics, protein folding, and thermostability of the wild-type and truncated glucanases were characterized. The truncated enzyme expressed in Pichia cells was found to be glycosylated and composed of two dominant polypeptide bands as judged by SDS-PAGE. An approximate 3-4-fold increase in the turnover rate (k(cat)), relative to that of the full-length enzyme, was detected for the purified truncated glucanases produced in E. coli (designated TF-glucanase) or Pichia host cells (designated glycosylated TF-glucanase). The glycosylated TF-glucanase is the most active known 1,3-1,4-beta-d-glucanase, with a specific activity of 10 800 +/- 200 units/mg. Similar binding affinities for lichenan (K(m) = 2.5-2.89 mg/mL) were detected for the full-length enzyme, TF-glucanase, and glycosylated TF-glucanase. Both forms of truncated glucanase retained more than 80% of their original enzymatic activity after a 10 min incubation at 90 degrees C, whereas the full-length enzyme possessed only 30% of its original enzymatic activity after the same treatment. This report demonstrates that deletion of the C-terminal region ( approximately 10 kDa) in Fsbeta-glucanase, consisting of serine-rich repeats and a basic terminal domain rich in positively charged amino acids, significantly increases the catalytic efficiency and thermotolerance of the enzyme.

The 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes (Fsbeta-glucanase) is classified as one of the family 16 glycosyl hydrolases. It hydrolyzes the glycosidic bond in the mixed-linked glucans containing beta-1,3- and beta-1,4-glycosidic linkages. We constructed a truncated form of recombinant Fsbeta-glucanase containing the catalytic domain from amino acid residues 1-258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme. The crystal structure of the truncated Fsbeta-glucanase was solved at a resolution of 1.7A by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein. The overall topology of the truncated Fsbeta-glucanase consists mainly of two eight-stranded anti-parallel beta-sheets arranged in a jellyroll beta-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules. Sequence comparison with other bacterial glucanases showed that Fsbeta-glucanase is the only naturally occurring circularly permuted beta-glucanase with reversed sequences. Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share approximately 70% sequence identity, than to the naturally occurring Fsbeta-glucanase of similar topology with 30% identity. This result suggests that protein structure relies more on sequence identity than topology. The high-resolution structure of Fsbeta-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability.

The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp 5 4 , Trp 1 0 5 , Trp 1 1 2 , Trp 1 4 1 , Trp 1 4 8 , Trp 1 6 5 , Trp 1 8 6 , Trp 1 9 8 , and Trp 2 0 3 in Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in K m value for lichenan was observed for W141F, W141H, and W203R mutant Fsβ-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k c a t relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively. In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k c a t , respectively. W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h. Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme. Structural modeling and kinetic studies suggest that Trp 5 4 , Trp 1 4 1 , Trp 1 4 8 , and Trp 2 0 3 play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.

The insulin-responsive glucose transporter, Glut4, exhibits a unique subcellular distribution such that in the absence of insulin >95% of the protein is stored within intracellular membranes. In response to insulin, Glut4 exhibits a large mobilisation to the plasma membrane. Studies of the amino acid motifs which regulate the unique trafficking of Glut4 have identified several key residues within the soluble cytoplasmic N- and C-terminal domains of Glut4. Of particular note is a Leu-498Leu-499 motif within the C-terminal domain that has been proposed to regulate both internalisation from the plasma membrane and sorting to an insulin-sensitive compartment. In this study, we have examined the role of the adjacent amino acids (Glu-491, Gln-492 and Glu-493) by their sequential replacement with Ala. Our results are consistent with the notion that Glu-491 and Glu-493 play an important role in the sub-endosomal trafficking of Glut4, as substitution of these residues with Ala results in increased levels of these proteins at the cell surface, reduced insulin-stimulated translocation and increased susceptibility to endosomal ablation. These residues, together with other identified sequences within the C-terminus of Glut4, are likely to be crucial targeting elements that regulate Glut4 subcellular distribution.

Twin-tail goldfish possess a bifurcated caudal axial skeleton. The scarcity of this trait in nature suggests that a rare mutation, which drastically altered the mechanisms underlying axial skeleton formation, may have occurred during goldfish domestication. However, little is known about the molecular development of twin-tail goldfish. Here we show that the bifurcated caudal skeleton arises from a mutation in the chordin gene, which affects embryonic dorsal–ventral (DV) patterning. We demonstrate that formation of the bifurcated caudal axial skeleton requires a stop-codon mutation in one of two recently duplicated chordin genes; this mutation may have occurred within approximately 600 years of domestication. We also report that the ventral tissues of the twin-tail strain are enlarged, and form the embryonic bifurcated fin fold. However, unlike previously described chordin-deficient embryos, this is not accompanied by a reduction in anterior–dorsal neural tissues. These results provide insight into large-scale evolution arising from artificial selection., The ornamental twin-tail goldfish has a bifurcated caudal skeleton that arose during domestication, but the developmental mechanisms that generate this tail are unknown. Here, Abe et al. show that a mutation in the chordin gene affects embryonic dorsal–ventral patterning causing the bifurcated tail skeleton.

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Mullerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.

Piscidins are antimicrobial peptides (AMPs) that play important roles in helping fish resist pathogenic infections. Through comparisons of tilapia EST clones, the coding sequences of five piscidin-like AMPs (named TP1∼5) of Nile tilapia, Oreochromis niloticus, were determined. The complete piscidin coding sequences of TP1, -2, -3, -4, and -5 were respectively composed of 207, 234, 231, 270, and 195 bases, and each contained a translated region of 68, 77, 76, 89, and 64 amino acids. The tissue-specific, Vibrio vulnificus stimulation-specific, and Streptococcus agalactiae stimulation-specific expressions of TP2, -3, and -4 mRNA were determined by a comparative RT-PCR. Results of the tissue distribution analysis revealed high expression levels of TP2 mRNA in the skin, head kidneys, liver, and spleen. To study bacterial stimulation, S. agalactiae (SA47) was injected, and the TP4 transcript was upregulated by >13-fold (compared to the wild-type (WT) control, without injection) and was 60-fold upregulated (compared to the WT control, without injection) 24 h after the S. agalactiae (SA47) injection in the spleen and gills. Synthesized TP3 and TP4 peptides showed antimicrobial activities against several bacteria in this study, while the synthesized TP1, -2, and -5 peptides did not. The synthesized TP2, -3, and -4 peptides showed hemolytic activities and synthesized TP3 and TP4 peptides inhibited tilapia ovary cell proliferation with a dose-dependent effect. In summary, the amphiphilic α-helical cationic peptides of TP3 and TP4 may represent novel and potential antimicrobial agents for further peptide drug development.

Background Fibrobacter succinogenes 1,3-1,4-β- d -glucanase (Fsβ-glucanase) is the only naturally occurring circularly permuted β-glucanase among bacterial glucanases with reverse protein domains. We characterized the functional and structural significance of residues 200–209 located in the domain B of Fsβ-glucanase, corresponding to the major surface loop in the domain A region of Bacillus licheniformis glucanase. Methods Rational design approaches including site-directed mutagenesis, initial-rate kinetics, and structural modeling analysis were used in this study. Results Our kinetic data showed that D202N and D206N exhibited a 1.8- and 1.5-fold increase but G207N, G207−, F205L, N208G and T204F showed a 7.0- to 2.2-fold decrease, in catalytic efficiency (kcat/KM) compared to the wild-type enzyme. The comparative energy ΔΔGb value in individual mutant enzymes was well correlated to their catalytic efficiency. D206R mutant enzyme exhibited the highest relative activity at 50 °C over 10 min, whereas K200F was the most heat-sensitive enzyme. Conclusions This study demonstrates that Phe205, Gly207, and Asn208 in the Type II turn of the connecting loop may play a role in the catalytic function of Fsβ-glucanase. General significance Residues 200–209 in Fsβ-glucanase resided at the similar structural topology to that of Bacillus enzyme were found to play some similar catalytic function in glucanase.

We report in this work the effect of multiple reflow cycles on ball impact test (BIT) responses and fractographies obtained at an impact velocity of 500 mm/s on Sn-4Ag-0.5 Cu solder joints. Solder balls were mounted on Cu substrate pads with immersion tin surface finish, supplied by two vendors. For these particular test vehicles and test conditions, fracture near the interface between the interfacial Cu6Sn5 intermetallic compound (IMC) and Cu pad was identified as the only failure mode induced by BIT. Measurement results indicate that BIT characteristics in general degrade as the number of reflow cycles increases. Furthermore, scanning electron microscopy observations show that the thickness and grain size of interfacial Cu6Sn5 increase with increasing number of reflow cycles. This correlation confirms the familiar notion that a thicker Cu6Sn5 degrades the interfacial strength.

1,3-1,4-beta-D-Glucanases (EC 3.2.1.73) specifically hydrolyze beta-1,4-glycosidic bonds located prior to beta-1,3-glycosidic linkages in lichenan or beta-D-glucans. It has been suggested that truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (TFsbeta-glucanase) can accommodate five glucose rings in its active site upon enzyme-substrate interaction. In this study, 12 mutant enzymes were created by mutating the conserved residues Gln70, Asn72, Gln81 and Glu85 proposed to bind to substrate subsites +1 and +2 and the catalytic properties of these mutants were determined. The most significant change in catalytic activity was observed on mutation of Gln70, with a 299-fold and 498-fold lower k(cat)/K(m) for the mutants Q70A and Q70I, respectively, compared with the wild-type enzyme. Mutagenesis, kinetic and structural studies revealed that the conserved residues surrounding the active site of TFsbeta-glucanase at substrate subsites +1 and +2 play an important role in its catalytic function, with the following order of importance: Gln70Asn72Glu85Gln81. The crystal structure of mutant E85I was determined at 2.2 A resolution. Further analysis of the E85I mutant structure revealed that the loop located at the concave site moved approximately 2 A from its position in the native enzyme complex without changing the core structure.

Forecast of earnings is one of the major tasks for financial statement analysts, and sales forecast is the most important step in the process of predicting earnings. Taking one large cell phone company in Taiwan as example and by the use of monthly data to fit a proper VAR model, we found that, when forecasting monthly sales, number of clients is a significant forward-looking information for financial statement analysts of the mobile industry. Compared to a model with only past sales as predicting variable, the forecasts generated by the bi-variate (monthly sales and month-end number of clients) model in this paper reduce the forecasting error by almost 30%. The evidence from the two variable VARMA model also support our hypothesis that there is a bidirectional dynamic relationship between sales and number of clients, i.e., both sales and number of clients are leading indicators of one another. The purpose of requiring listed firms to publicly announce their monthly sales before the 10 th day of the following month in Taiwan is of course to provide investors and analysts with relevant information for them to make rational decision. Monthly sales numbers are relevant because users of this financial information can predict future sales based on their past history. In this paper we target on a large cell phone company in Taiwan to demonstrate that a similar timely disclosure of number of clients should also be required in additional to the monthly sales. Besides, the SEC should careful examine for all industries that whether more forward-looking information should be made available to the public.

Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.

Due to the influence of endocrine, menopausal women are easily affected by osteoporosis. Nowadays, with the popularity of preventive medicine, the proportion of the public willing to accept alternative therapies has increased. In this study alternative therapies, including Chinese medicine, diet, exercise, aromatherapy, acupressure and acupuncture were integrated, to prevent osteoporosis in menopausal women. It is expected that this study will enhance the quality of nursing and expand the prospects of nursing care in this field.

The mould genus, Penicillium, is a significant source of environmental aero-allergens. A major allergen from Penicillium notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of Penicillium citrinum, followed by matrix-assisted laser-desorption ionization—time-of-flight MS analysis of the peptide digest. Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence. The cDNA encoded a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N- and C-terminal prosequences. The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi. The Pen n 18 coding sequence was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni2+-chelate affinity chromatography. On immunoblots, the purified recombinant protein specifically bound IgE from mould-allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P. citrinum, Pen c 18. When mapping of the allergenic epitopes was performed, at least nine different linear IgE-binding epitopes, located throughout the Pen n 18 protein, were identified. Of these, peptide C12, located in the N-terminal region of the molecule, was recognized by serum from 75% of the patients tested and therefore appears to be an immunodominant IgE-binding epitope.

AbGn is a glycotope-specific humanized monoclonal antibody recognizing Lewisa- like carbohydrate expressed in gastric and pancreatic cancer cells. While AbGn binding to normal tissues is restricted to only luminal surface of the epithelium in gastro-intestinal track and secretory glands, about 50% of gastric and pancreatic cancer samples tested express AbGn epitope and can be recognized by AbGn antibody. FACS analysis and confocal microscopic studies demonstrated extensive internalization of AbGn after binding to the cell surface. The internalized AbGn co-localized with lysosomal marker LAMP1, suggesting that AbGn-mediated internalization followed the lysosomal pathway. A novel antibody-drug conjugate (AbGn-ADC) was generated by linking AbGn to a dolastatin analogue, a potent inhibitor of microtubulin polymerization, through a dipeptide-based cleavable linker. This AbGn-ADC was evaluated for antitumor activity in vitro and in mouse xenograft models. AbGn-ADC eliminated cancer cell lines with nanomolar potency in vitro. No cell death could be detected in SW480 cells, a gastric cell line with no AbGn epitope expression, suggesting high selectivity of AbGn-ADC. When used to treat mice with established human gastric (SNU-16) or pancreatic (Panc.02.03B) cancer xenografts, AbGn-ADC significantly reduced the size of the established tumors (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2517. doi:1538-7445.AM2012-2517

CD326 is one of the first and prominent immunotherapeutic targets in cancer therapy due to its frequent and high-level expression on most carcinomas of various origins. The dominant role of cytotoxic T cells for tumor suppression and surveillance in vivo is well documented in literature. A novel bi-specific antibody AbGn-100 was constructed, in which the antigen-binding domain of an anti- human CD326 antibody was linked to the antigen-binding domain of an anti-human T cell receptor (TCR) ≤ chain antibody. Human serum albumin was incorporated into this construct to prolong the half life in vivo. This bi-specific antibody is able to direct T cells toward CD326-expressing cancer cells to execute T cell cytotoxic function after TCR cross-linking mediated by CD326 expressed on cancer cells while it remains harmless when either T cells or CD326-expressing cancer cells is absent. In vitro cytotoxicity assays with PBMCs from different donors have demonstrated potent (up to 60% above the background) cytotoxicity eliciting activity of AbGn-100 to lung (NCI-H358), ovarian (OMC3) and colorectal (COLO205) cancer cells lines. In the absence of cancer cells, the addition of AbGn-100 didn't trigger the up-regulation of T cell activation markers such as CD25 or CD69, nor did it increase the secretion of cytokines such as IL-2 and Interferon β, demonstrating the safety profile of AbGn-100. In vivo activity of AbGn-100 was tested with established lung (NCI-H358) and ovarian (OMC-3) cancer xenografts in NOD-SCID mice. AbGn-100, in the presence of human PBMC, significantly reduced the size of the established lung and ovarian tumors. Most notably, in some of the mice, tumor was not detectable after 50 days of treatment, suggesting the possibility of tumor eradication. PK studies in mice showed the half-life of AbGn-100 to be around 20 hours, which is substantially longer than conventional single-chain Fv bi-specific antibodies. These data support the potential of further development of AbGn-100 as therapeutics for the treatment of CD326-expressing solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3527. doi:1538-7445.AM2012-3527