Enrichment and MALDI-TOF MS Analysis of Phosphoinositides in Brain Tissue.

Triazolium cyclodextrin click cluster (+CCC) is an ideal scaffold to specifically bind phosphoinositides (PIPs) via multivalent electrostatic interaction. A new enrichment material, triazolium cyclodextrin click cluster-magnetic agarose bead conjugate (+CCC-MAB), was synthesized and applied to the PIP enrichment of brain tissue. The enriched sample was analyzed using MALDI-TOF MS in negative ion mode without any derivatization. The PIP extract of brain tissue is known to contain abundant lipid interferences. By employing magnetic pull-down separation using +CCC-MAB, we effectively removed the weak-binding interferences in the PIP extract, thereby improving the signal-to-noise ratio (S/N) of the PIPs. Our +CCC-MAB-based PIP enrichment enabled us to analyze 16 PIP species in brain tissue. Six species with high S/N were assigned by MS/MS, while the remaining 10 species with low S/N were characterized by an empirical selection guide based on the biological relevance of PIPs. We conclude that +CCC-MAB-based PIP enrichment is a promising MALDI sample preparation method for specific PIP analysis in brain tissue.

A ligand-independent Tie2-activating antibody reduces vascular leakage in models of Clarkson disease.

Vascular dysfunction resulting from endothelial hyperpermeability is a common and important feature of critical illness due to sepsis, trauma, and other conditions associated with acute systemic inflammation. Clarkson disease [monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome (ISCLS)] is a rare, orphan disorder marked by spontaneous and recurrent episodes of hypotensive shock and peripheral edema due to widespread vascular leakage in peripheral tissues. Mortality from acute flares approaches 30% due to lack of effective therapies. We evaluated a monoclonal antibody (4E2) specific for the endothelial receptor tyrosine kinase Tie2 in ISCLS models. 4E2 activated Tie2 in ISCLS patient-derived endothelial cells and reduced baseline and proinflammatory mediator-induced barrier dysfunction. 4E2 also reduced mortality and/or vascular leakage associated with systemic histamine challenge or influenza infection in the SJL/J mouse model of ISCLS. These findings support a critical role for Tie2 dysregulation in ISCLS and highlight a viable therapeutic approach to this catastrophic disorder.

An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone.

Tumor progression is intimately associated with the vasculature, as tumor proliferation induces angiogenesis and tumor cells metastasize to distant organs via blood vessels. However, whether tumor invasion is associated with blood vessels remains unknown. As glioblastoma (GBM) is featured by aggressive invasion and vascular abnormalities, we characterized the onset of vascular remodeling in the diffuse tumor infiltrating zone by establishing new spontaneous GBM models with robust invasion capacity. Normal brain vessels underwent a gradual transition to severely impaired tumor vessels at the GBM periphery over several days. Increasing vasodilation from the tumor periphery to the tumor core was also found in human GBM. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) showed a spatial correlation with the extent of vascular abnormalities spanning the tumor-invading zone. Blockade of VEGFR2 suppressed vascular remodeling at the tumor periphery, confirming the role of VEGF-VEGFR2 signaling in the invasion-associated vascular transition. As angiopoietin-2 (ANGPT2) was expressed in only a portion of the central tumor vessels, we developed a ligand-independent tunica interna endothelial cell kinase 2 (Tie2)-activating antibody that can result in Tie2 phosphorylation in vivo. This agonistic anti-Tie2 antibody effectively normalized the vasculature in both the tumor periphery and tumor center, similar to the effects of VEGFR2 blockade. Mechanistically, this antibody-based Tie2 activation induced VE-PTP-mediated VEGFR2 dephosphorylation in vivo. Thus, our study reveals that the normal-to-tumor vascular transition is spatiotemporally associated with GBM invasion and may be controlled by Tie2 activation via a novel mechanism of action.

Preparation and Characterization of Polybutylene Succinate Reinforced with Pure Cellulose Nanofibril and Lignocellulose Nanofibril Using Two-Step Process.

This study reports the preparation of a polybutylene succinate (PBS) film reinforced with pure cellulose nanofibril (PCNF) and lignocellulose nanofibril (LCNF) by a two-step process that consists of solvent dispersion and twin-screw extrusion. Compared to the conventional one-step process, this method offered improved mechanical properties. The addition of 5% CNF increased the tensile properties up to 18.8%. Further, the effect of the lignin content was also studied by using LCNF as a reinforcement. The LCNF was prepared with and without a deep eutectic solvent (DES) pretreatment to gain LCNF with a lignin content that varied between 5, 19, and 30%. The mechanical properties results show that a 5% addition of LCNF to the PBS matrix increased its tensile strength and elastic modulus. Further, the morphological and thermal properties of the composites were also studied in detail.

Wet-Spun Composite Filaments from Lignocellulose Nanofibrils/Alginate and Their Physico-Mechanical Properties.

Lignocellulose nanofibrils (LCNFs) with different lignin contents were prepared using choline chloride (ChCl)/lactic acid (LA), deep eutectic solvent (DES) pretreatment, and subsequent mechanical defibrillation. The LCNFs had a diameter of 15.3-18.2 nm, which was similar to the diameter of commercial pure cellulose nanofibrils (PCNFs). The LCNFs and PCNFs were wet-spun in CaCl2 solution for filament fabrication. The addition of sodium alginate (AL) significantly improved the wet-spinnability of the LCNFs. As the AL content increased, the average diameter of the composite filaments increased, and the orientation index decreased. The increase in AL content improved the wet-spinnability of CNFs but deteriorated the tensile properties. The increase in the spinning rate resulted in an increase in the orientation index, which improved the tensile strength and elastic modulus.

Preparation and Characterization of Cellulose Acetate Film Reinforced with Cellulose Nanofibril.

In this study, cellulose acetate (CA)/cellulose nanofibril (CNF) film was prepared via solvent casting. CNF was used as reinforcement to increase tensile properties of CA film. CNF ratio was varied into 3, 5, and 10 phr (parts per hundred rubbers). Triacetin (TA) and triethyl citrate (TC) were used as two different eco-friendly plasticizers. Two different types of solvent, which are acetone and N-methyl-2-pyrrolidone (NMP), were also used. CA/CNF film was prepared by mixing CA and CNF in acetone or NMP with 10% concentration and stirred for 24 h. Then, the solution was cast in a polytetrafluoroethylene (PTFE) dish followed by solvent evaporation for 12 h at room temperature for acetone and 24 h at 80 °C in an oven dryer for NMP. The effect of solvent type, plasticizers type, and CNF amount on film properties was studied. Good dispersion in NMP was evident from the morphological study of fractured surface and visible light transmittance. The results showed that CNF has a better dispersion in NMP which leads to a significant increase in tensile strength and elastic modulus up to 38% and 65%, respectively, compared with those of neat CA. CNF addition up to 5 phr loading increased the mechanical properties of the film composites.

Esterification of Lignin Isolated by Deep Eutectic Solvent Using Fatty Acid Chloride, and Its Composite Film with Poly(lactic acid).

In this study, the effect of lignin esterification with fatty acid chloride on the properties of lignin and lignin/poly(lactic acid) (PLA) composites was investigated. Lignocellulose (Pinus densiflora S. et Z.) was treated using a deep eutectic solvent (DES) with choline chloride (ChCl)/lactic acid (LA). From the DES-soluble fraction, DES-lignin (DL) was isolated by a regeneration process. Lignin esterification was conducted with palmitoyl chloride (PC). As the PC loading increased for DL esterification, the Mw of esterified DL (EDL) was increased, and the glass transition temperature (Tg) was decreased. In DL or EDL/PLA composite films, it was observed that EDL/PLA had cleaner and smoother morphological characteristics than DL/PLA. The addition of DL or EDL in a PLA matrix resulted in a deterioration of tensile properties as compared with neat PLA. The EDL/PLA composite film had a higher tensile strength and elastic modulus than the DL/PLA composite film. DL esterification decreased water absorption with lower water diffusion coefficients. The effect of lignin esterification on improving the compatibility of lignin and PLA was demonstrated. These results are expected to contribute to the development of high-strength lignin composites.

Preparation and Properties of Wet-Spun Microcomposite Filaments from Various CNFs and Alginate.

We aimed to improve the mechanical properties of alginate fibers by reinforcing with various cellulose nanofibrils (CNFs). Pure cellulose nanofibril (PCNF), lignocellulose nanofibril (LCNF) obtained via deep eutectic solvent (DES) pretreatment, and TEMPO-oxidized lignocellulose nanofibril (TOLCNF) were employed. Sodium alginate (AL) was mixed with PCNF, LCNF, and TOLCNF with a CNF content of 5-30%. To fabricate microcomposite filaments, the suspensions were wet-spun in calcium chloride (CaCl2) solution through a microfluidic channel. Average diameters of the microcomposite filaments were in the range of 40.2-73.7 µm, which increased with increasing CNF content and spinning rate. The tensile strength and elastic modulus improved as the CNF content increased to 10%, but the addition of 30% CNF deteriorated the tensile properties. The tensile strength and elastic modulus were in the order of LCNF/AL > PCNF/AL > TOLCNF/AL > AL. An increase in the spinning rate improved the tensile properties.

Preparation and Characteristics of Wet-Spun Filament Made of Cellulose Nanofibrils with Different Chemical Compositions.

In this study, wet-spun filaments were prepared using lignocellulose nanofibril (LCNF), with 6.0% and 13.0% of hemicellulose and lignin, respectively, holocellulose nanofibril (HCNF), with 37% hemicellulose, and nearly purified-cellulose nanofibril (NP-CNF) through wet-disk milling followed by high-pressure homogenization. The diameter was observed to increase in the order of NP-CNF ≤ HCNF < LCNF. The removal of lignin improved the defibrillation efficiency, thus increasing the specific surface area and filtration time. All samples showed the typical X-ray diffraction pattern of cellulose I. The orientation of CNFs in the wet-spun filaments was observed to increase at a low concentration of CNF suspensions and high spinning rate. The increase in the CNF orientation improved the tensile strength and elastic modulus of the wet-spun filaments. The tensile strength of the wet-spun filaments decreased in the order of HCNF > NP-CNF > LCNF.

Guiding Engineering Student Teams' Ethics Discussions with Peer Advising.

This study explores how peer advising affects student project teams' discussions of engineering ethics. Peer ethics advisors from non-engineering disciplines are expected to provide diverse perspectives and to help engineering student teams engage and sustain ethics discussions. To investigate how peer advising helps engineering student teams' ethics discussions, three student teams in different peer advising conditions were closely observed: without any advisor, with a single volunteer advisor, and with an advising team working on the ethics advising project. Micro-scale discourse analysis based on cognitive ethnography was conducted to find each team's cultural model of understanding of engineering ethics. Cultural-historical activity theory (CHAT) analysis was also conducted to see what influenced each team's cultural model. In cultural model, the engineering team with an ethics advising team showed broader understanding in social implications of engineering. The results of CHAT analysis indicated that differences in rules, community, and division of labor among three teams influenced the teams' cultural models. The CHAT analysis also indicated that the peer advisors working on the ethics advising project and the engineering team working on engineering design project created a collaborative environment. The findings indicated that collaborative environment supported peer ethics advising to facilitate team discussions of engineering ethics.

Effect of Lignin Plasticization on Physico-Mechanical Properties of Lignin/Poly(Lactic Acid) Composites.

Kraft lignin (KL) or plasticized KL (PKL)/poly(lactic acid) (PLA) composites, containing different lignin contents and with and without the coupling agent, were prepared in this study using twin-screw extrusion at 180 °C. Furthermore, ε-caprolactone and polymeric diphenylmethane diisocyanate (pMDI) were used as a plasticizer of KL and a coupling agent to improve interfacial adhesion, respectively. It was found that lignin plasticization improved lignin dispersibility in the PLA matrix and increased the melt flow index due to decrease in melt viscosity. The tensile strength of KL or PKL/PLA composites was found to decrease as the content of KL and PKL increased in the absence of pMDI, and increased due to pMDI addition. The existence of KL and PKL in the composites decreased the thermal degradation rate against the temperature and increased char residue. Furthermore, the diffusion coefficient of water in the composites was also found to decrease due to KL or PKL addition.

Influence of Lignin and Polymeric Diphenylmethane Diisocyante Addition on the Properties of Poly(butylene succinate)/Wood Flour Composite.

Poly(butylene succinate) (PBS)/wood flour (WF) composites with different WF content were prepared by twin-screw extrusion at 160 °C. With increasing WF content, the tensile strength of the PBS/WF composite without polymeric diphenylmethane diisocyante (pMDI) decreased, while that of the composite with pMDI increased. The addition of kraft lignin (KL) deteriorated the tensile properties of the composites both with and without pMDI. The melt flow index (MFI) decreased with increasing WF content, but increased with increasing KL content. The addition of pMDI caused an increase in the melt viscosity of the PBS/WF and PBS/WF/KL composites, resulting in a decrease in the MFI. The composites had lower thermal stability than neat PBS. The exotherms of the PBS/WF (50/50) composite appeared at a higher temperature than that of the neat PBS, but the PBS/WF/KL (50/50/20) composites had similar exotherms as the neat PBS. The addition of KL caused a decrease in the crystallization rate of PBS.

Cyp7a1 is continuously increased with disrupted Fxr-mediated feedback inhibition in hypercholesterolemic TALLYHO/Jng mice.

TALLYHO/Jng (TH) mice reveal hypercholesterolemia at an early age before their plasma glucose levels have increased. The increased plasma cholesterol should be related to bile acids (BAs) metabolism, because cholesterol is the precursor of BAs and BAs change cholesterol metabolism in a feedback manner. We analyzed the BAs pool size, BAs composition, and expression levels of several proteins that have key roles in BAs synthesis, excretion, and reabsorption and compared them to those of C57BL/6 (B6) mice to study BAs metabolism in TH mice. TH mice exhibited an increased total BAs pool size, increased BAs content in the cecum feces, and an increased ratio of muricholic acid (MCA)/cholic acid (CA). The mRNA and protein levels of cholesterol 7 alpha-hydroxylase (Cyp7a1) and the ATP-binding cassette sub-family G member 5 (Abcg5) were elevated in the liver but not in the apical sodium bile acid transporter (Asbt) and organic solute transporters (Osts) in the ileum. These results indicate that synthesis and the excretion of BAs from the liver to the gallbladder might be elevated, but the reabsorption rate of BAs in the ileum might be reduced. The declined expression of fibroblast growth factor 15 (Fgf15) and fibroblast growth factor receptor 4 (Fgfr4) was respectively identified in the ileum and the liver, indicating the disrupted feedback inhibition of Cyp7a1. Consequently, hypercholesterolemia in TH mice might increase the BAs amounts via the interrupted Fxr/Fgf15/Fgfr4-mediated feedback regulation of Cyp7a1.

The Roles of Implicit Understanding of Engineering Ethics in Student Teams' Discussion.

Following previous work that shows engineering students possess different levels of understanding of ethics-implicit and explicit-this study focuses on how students' implicit understanding of engineering ethics influences their team discussion process, in cases where there is significant divergence between their explicit and implicit understanding. We observed student teams during group discussions of the ethical issues involved in their engineering design projects. Through the micro-scale discourse analysis based on cognitive ethnography, we found two possible ways in which implicit understanding influenced the discussion. In one case, implicit understanding played the role of intuitive ethics-an intuitive judgment followed by reasoning. In the other case, implicit understanding played the role of ethical insight, emotionally guiding the direction of the discussion. In either case, however, implicit understanding did not have a strong influence, and the conclusion of the discussion reflected students' explicit understanding. Because students' implicit understanding represented broader social implication of engineering design in both cases, we suggest to take account of students' relevant implicit understanding in engineering education, to help students become more socially responsible engineers.

Fatty Liver and Insulin Resistance in the Liver-Specific Knockout Mice of Mitogen Inducible Gene-6.

Mitogen inducible gene-6 (Mig-6) is a feedback inhibitor of epidermal growth factor receptor (EGFR) signaling pathway. The liver-specific knockout mice of the Mig-6 gene (Mig-6 d/d ) showed hepatomegaly and increased hypercholesterolemia. In this study, the biomarkers of insulin resistance and the effects of high-fat diets in the wild (Mig-6 f/f ) and Mig-6 d/d mice were analyzed. The fasting plasma concentrations of glucose, triglyceride, cholesterols, free fatty acids, and HOMA-IR were measured and the glucose tolerance and insulin resistance tests were performed in the 25-week-old Mig-6 f/f and the Mig-6 d/d mice. The protein levels of active insulin receptor, glucose 6-phosphatase, and phosphoenolpyruvate carboxykinase were analyzed in the liver and fat. The fasting plasma cholesterol and glucose concentration were higher in the Mig-6 d/d mice than the Mig-6 f/f mice with increased fat deposition in the liver. But the Mig-6 d/d mice had the improved glucose intolerance and insulin resistance without increased amount of phosphoinsulin receptor after insulin infusion in the liver. The hepatic concentration of phosphoenolpyruvate carboxykinase was increased in fasting Mig-6 d/d mice. The feeding of high-fat diet accelerated the plasma lipids profiles and HOMA-IR in the Mig-6 d/d mice but had no differential effects in oral glucose tolerance test and insulin tolerance test in both genotypes. These results suggest that the activated EGFR signaling might increase the fasting plasma glucose concentration through inducing the hepatic steatosis and the improved whole-body insulin resistance in the KO mice be caused by decreased adipogenesis in fat tissues.

Predicting postoperative total calcium requirements after parathyroidectomy in secondary hyperparathyroidism.

BACKGROUND/AIMS: To prevent hypocalcemia after parathyroidectomy (PTX), parenteral calcium is required in addition to oral calcitriol and calcium. After switching to oral calcium, patients can be discharged from the hospital. The aim of this study was to analyze the clinical characteristics and outcomes of PTX performed at a single Korean center and to investigate the associated laboratory factors used to analyze the total amount of postoperative calcium required. METHODS: We enrolled 91 hemodialysis patients undergoing PTX from November 2003 to December 2011. We collected clinical and laboratory data preoperatively, 12 and 48 hours postoperatively, at discharge, and 3 and 6 months postoperatively. RESULTS: In total, 59 patients underwent PTX with autotransplantation (AT), 6 underwent total PTX without AT, 11 underwent subtotal PTX, and 15 underwent limited PTX. Total PTX without AT showed the lowest recurrence rate. At all postoperative time points, the mean levels of serum calcium, phosphorus, and intact parathyroid hormone (iPTH) decreased significantly, compared with preoperative levels; however, alkaline phosphatase (ALP) increased significantly from 48 hours postoperatively to discharge (p < 0.001). On multiple linear regression analysis, the total amount of injected calcium during hospitalization showed a significant correlation with preoperative ALP (p < 0.001), preoperative iPTH (p = 0.037), and Δphosphorus at 48 hours (p < 0.001). We developed an equation for estimating the total calcium requirement after PTX. CONCLUSIONS: Preoperative ALP, preoperative iPTH, and Δphosphorus at 48 hours may be significant factors in estimating the postoperative calcium requirement. The formula for postoperative calcium requirement after PTX may help to predict the duration of postoperative hospitalization.

Effect of Light Absorption in InGaN/GaN Vertical Light-Emitting Diodes.

For evaluating the effect of light absorption in vertically structured thin film light-emitting diodes (VLEDs), we investigate the dependence of the efficiencies on the several specific parameters including thickness and doping concentration (N(D)) of the n-GaN layer, a design of hetero-structures of the n-GaN layer, and a number of pairs of multi-quantum wells (MQWs). Generally, there is a complementary relation between internal quantum efficiency (IQE) and light extraction efficiency (LEE). However, we confirmed that LEE determined by light absorption is more dominant than IQE in VLED structures with a textured surface, from numerical simulation and experimental results. Effect of light absorption is more prominent in the vertical chip with a textured surface than in that with a flat surface, because a travel length of light extracted from the textured surface is longer. Minimizing light absorption in VLEDs is a key technology for improving light output, and light absorption speaks for the index of enhancement by the general technologies for improving LEE.

Diameter of parathyroid glands measured by computed tomography as a predictive indicator for response to cinacalcet in dialysis patients with secondary hyperparathyroidism.

BACKGROUND/AIMS: Cinacalcet is one of the important treatments of secondary hyperparathyroidism (SHPT). We evaluated the role of computed tomography (CT) for parathyroid glands (PTGs) to determine the response to cinacalcet therapy in dialysis patients. METHODS: In study 1, we compared the predictive cutoff values of the largest volume or diameter of PTGs on ultrasonography or CT for achievement of target intact parathyroid hormone (iPTH) level according to K/DOQI guideline after cinacalcet treatment in a single dialysis center. In study 2, the role of the cutoff diameter of PTGs on CT in predicting responsive to cinacalcet therapy was reevaluated in dialysis patients with SHPT in multiple centers. RESULTS: In study 1, among the total population of 26 patients, the number of patients with baseline iPTH over 600 pg/mL was 16 (61%). In study 2, it was 45 (54%), among 82 patients. In study 1, the number of PTGs equal to or larger than the cutoff value (≥ 11.2 mm) on CT, not ultrasonography, was significantly higher in non-responders than in responders (p=0.038). In study 2, the proportion of patients with PTGs ≥ 11.2 mm on CT was significantly higher in non-responders than responders (p=0.003). Multivariate analysis showed that pretreatment iPTH (odds ratio [OR] 1.498, p=0.003) and the existence of enlarged PTGs on CT (OR 8.940, p=0.015) were significant clinical factors affecting the response to cinacalcet. CONCLUSIONS: The diameter of PTGs on CT could predict the response to cinacalcet in dialysis patients with SHPT.

Transplantation of cyclic stretched fibroblasts accelerates the wound-healing process in streptozotocin-induced diabetic mice.

Mechanical stimulation is a known modulator of survival and proliferation for many cells, including endothelial cells, smooth muscle cells, and bone marrow-derived mesenchymal stem cells. In this study, we found that mechanical strain prevents apoptosis and increases the adhesive ability of dermal fibroblasts in vitro and thus confers the survival advantage in vivo after transplantation of fibroblasts into the full-thickness wound of diabetic mice. Cyclic stretch at a frequency of 0.5 Hz and maximum elongation of 20% stimulates cellular survival mediated by the activation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and the serine/threonine kinase Akt (AKT). Stretching of the fibroblasts increases the synthesis of extracellular matrix proteins and the formation of denser focal adhesion structures, both of which are required for fibroblast adhesion. The stretched fibroblasts also upregulate the expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α), which enhanced wound healing in vivo. Indeed, preconditioning with mechanical stretch allows better survival of the transplanted fibroblasts, when compared to unstretched control cells, in the wound environment of mice with streptozotocin-induced diabetes and thus accelerates the wound-healing process in these mice.

The association of Klotho polymorphism with disease progression and mortality in IgA nephropathy.

BACKGROUNDS: IgA nephropathy (IgAN) is the most common primary glomerulonephritis causing end stage renal disease (ESRD), and vasculopathy is known to involve disease progression. Klotho, a gene related to aging, has been reported to play a role in atherosclerosis and endothelial dysfunction. We investigated whether klotho gene polymorphism affect clinical course of IgAN. METHODS: The data registered for PREMIER study which enrolled the patients with biopsy proven IgAN were analyzed. Two single nucleotide polymorphisms for klotho gene, G395A of promoter region and C1818T of exon 4, were examined, and investigated the association klotho genotypes with the progression of IgAN and patient survival. RESULTS: Clinical data from 973 patients confirmed about survival were analyzed. The allele frequency was 0.830 and 0.170 for allele G and A, and 0.816 and 0.184 for allele C and T, which were complied with Hardy-Weinberg equilibrium (p=0.996 and 0.531 respectively). Death was observed more frequently in A-allele carriers of G395A polymorphism (0.7 vs. 2.6 %, GG vs. GA+AA, p=0.022). Renal survival in Kaplan-Meier survival curve was also worse in same group (p=0.04). CONCLUSION: Klotho gene polymorphism was associated with patient survival and disease progression of IgAN.