Endoscopic Nasobiliary Drainage Comparable with Endoscopic Biliary Stenting as a Preoperative Drainage Method for Malignant Hilar Biliary Obstruction: A Multicenter Retrospective Study.

INTRODUCTION: Preoperative endoscopic biliary drainage (PEBD) for malignant hilar biliary obstruction (MHBO) is widely accepted. Recent PEBD consists of endoscopic nasobiliary drainage (ENBD), conventional endoscopic biliary stenting (CEBS) with plastic stents across the papilla, and endoscopic biliary inside stenting (EBIS) with plastic stents above the papilla, while ENBD is the primary procedure in Asian countries. Thus, we aimed to compare the efficacy of ENBD with those of CEBS and EBIS as a means of PEBD for MHBO. METHODS: We retrospectively identified patients with MHBO who underwent upfront surgery between January 2011 and December 2018 in a multicenter setting. The outcome measures were cumulative dysfunction of PEBD, risk factors for PEBD dysfunction, and adverse events. RESULTS: We analyzed a total of 219 patients, comprising 163 males (74.4%); mean age, 69.7 (±7.6) years; Bismuth-Corlette (BC) classification I, II, IIIa, IIIb, and IV in 68, 49, 43, 30, and 29 patients, respectively; and diagnosis of hilar cholangiocarcinoma and gallbladder cancer in 188 and 31 patients, respectively. PEBD procedures were performed in 160 patients with ENBD, 31 patients with CEBS, and 28 patients with EBIS. PEBD dysfunction occurred in 58 patients (26.5%), and the cumulative dysfunction rates were not significantly different among PEBD methods (p = 0.60). Multivariate analysis showed that BC-IV was significantly associated with the occurrence of PEBD dysfunction (hazard ratio = 2.10, p = 0.02). The adverse event rates were not significantly different among PEBD groups (p = 0.70). CONCLUSION: ENBD as a means of PEBD for MHBO is comparable with CEBS and EBIS in rates of dysfunction and adverse events.

Biosynthesis of D-lactic acid from lignocellulosic biomass.

D-lactic acid is a versatile and important industrial chemical that can be applied in the synthesis of thermal-resistant poly-lactic acid. Biosynthesis of D-lactic acid can be achieved by a variety of microorganisms, including lactic acid bacteria, yeast, and fungi; however, the final product yield, optical purity, and the utilization of both glucose and xylose are restricted. Consequently, engineered microbial systems are essential to attain high titer, productivity, and complete utilization of sugars. Herein, we critically evaluate the promising wild-type microorganisms, as well as genetically modified microorganisms to produce enantiomerically pure D-lactic acid, particularly from renewable lignocellulosic biomass. In addition, innovative bioreactor operation, metabolic flux analysis, and recent genetic engineering methods for targeted microbial D-lactic acid synthesis will be discussed.

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes.

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the ß-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE: Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.

Effective treatment of inhaled corticosteroid and bronchodilator for "lymphocytic interstitial pneumonia" in primary Sjögren's syndrome.

We present a case of an 86-year-old woman who visited our hospital with a one-year history of exertional dyspnea (modified medical research council dyspnea scale; mMRC grade 2). Despite the absence of any smoking or dust exposure history, multiple cystic lesions were apparent in both lungs on her CT scan. We suspected Sjögren's syndrome-associated lymphocytic interstitial pneumonia (LIP) due to her additional symptoms of dry mouth and eyes. Her respiratory function test showed a restrictive disorder with a forced vital capacity (FVC) of 1.23 L (70.3 % predicted), forced expiratory volume in 1 s (FEV1) of 0.88 L, and FEV1/FVC of 71.5 %. The flow-volume curve showed a downward convex, suggesting peripheral airway obstruction. We initiated a daily inhalation treatment regimen comprising vilanterol 25 µg and fluticasone furoate 200 µg. One month later, at the follow-up visit, the clinical diagnosis of Sjögren's syndrome with LIP was made by positive SS-A and SS-B antibodies in the initial blood work, a Saxon test that confirmed decreased salivary secretion, and a confirmed diagnosis of dry eyes by her ophthalmologist. We noted improvement in FVC of 1.45 L (+17.8 %) and FEV1 to 0.99 L (+12.5 %) in the subsequent respiratory function test, along with alleviation of her symptoms. The present case represents the first report of LIP treated exclusively with inhaled corticosteroids and bronchodilators, highlighting a potential therapeutic approach, particularly for elderly patients vulnerable to immunosuppressive therapies.

Effect of lime pretreatment of brown midrib sorghums.

The effect of lime pretreatment of brown midrib sorghums on enzymatic saccharification was investigated. Under most of the pretreatment conditions, the saccharification yields of bmrs were higher than those of the normal counterparts. This result suggests that bmr is useful to reduce pretreatment costs, because the amount of lime necessary for the pretreatment of biomass can reduced by using bmr mutants.

Development of a gene transfer system for the mycelia of Flammulina velutipes Fv-1 strain.

To develop a gene transformation method for Flammulina velutipes, we constructed a vector with hph gene under control of the trp1 gene promoter. The vector was integrated into protoplast derived from mycelia by the calcium-polyethylene glycol method, as it has not been reported for F. velutipes. Transformation efficiency was much improved when transformation was performed by the restriction enzyme mediated integration method.

Transcriptome analysis of the brown rot fungus Gloeophyllum trabeum during lignocellulose degradation.

Brown rot fungi have great potential in biorefinery wood conversion systems because they are the primary wood decomposers in coniferous forests and have an efficient lignocellulose degrading system. Their initial wood degradation mechanism is thought to consist of an oxidative radical-based system that acts sequentially with an enzymatic saccharification system, but the complete molecular mechanism of this system has not yet been elucidated. Some studies have shown that wood degradation mechanisms of brown rot fungi have diversity in their substrate selectivity. Gloeophyllum trabeum, one of the most studied brown rot species, has broad substrate selectivity and even can degrade some grasses. However, the basis for this broad substrate specificity is poorly understood. In this study, we performed RNA-seq analyses on G. trabeum grown on media containing glucose, cellulose, or Japanese cedar (Cryptomeria japonica) as the sole carbon source. Comparison to the gene expression on glucose, 1,129 genes were upregulated on cellulose and 1,516 genes were upregulated on cedar. Carbohydrate Active enZyme (CAZyme) genes upregulated on cellulose and cedar media by G. trabeum included glycoside hyrolase family 12 (GH12), GH131, carbohydrate esterase family 1 (CE1), auxiliary activities family 3 subfamily 1 (AA3_1), AA3_2, AA3_4 and AA9, which is a newly reported expression pattern for brown rot fungi. The upregulation of both terpene synthase and cytochrome P450 genes on cedar media suggests the potential importance of these gene products in the production of secondary metabolites associated with the chelator-mediated Fenton reaction. These results provide new insights into the inherent wood degradation mechanism of G. trabeum and the diversity of brown rot mechanisms.

Cloning and transcript analysis of multiple genes encoding the glycoside hydrolase family 6 enzyme from Coprinopsis cinerea.

We searched the genome database of the basidiomycete Coprinopsis cinerea (Coprinus cinereus) and found five genes encoding the glycoside hydrolase family 6 (GH6) enzyme, CcCel6A, CcCel6B, CcCel6C, CcCel6D, and CcCel6E, designated in order of increasing locus number (CC1G_01107.1, CC1G_04166.1, CC1G_08276.1, CC1G_08277.1, and CC1G_10605.1). The amino acid sequence of CcCel6A suggests a two-domain structure consisting of an N-terminal family 1 carbohydrate-binding module (CBM1) and a GH6 catalytic domain, while the other genes lack CBM1. The transcripts of CcCel6A were observed at the active growth stage in cellulose culture, whereas they were absent from glucose culture. Cellobiose strongly induced transcription of CcCel6A. On the other hand, transcripts of CcCel6B, -D, and -E were detected in both glucose and cellulose cultures, and transcription of them was induced weakly by cellobiose. The transcript level of CcCel6C was not influenced by glucose or cellobiose.

Multiple iron reduction by methoxylated phenolic lignin structures and the generation of reactive oxygen species by lignocellulose surfaces.

Chelator-mediated Fenton chemistry is capable of reducing non-stochiometric amounts of iron via hydroquinone oxidation. These types of reactions have previously been demonstrated to be promoted by some lignocellulose degrading fungi in generating hydroxyl radicals to permit lignified plant cell wall deconstruction. Here we demonstrate that lignocellulose surfaces, when exposed by chemical treatment or fragmentation, can promote a similar multi-oxidative mechanism in the presence of iron. Iron reduction by lignin surfaces permits the generation of hydroxyl radicals in the cell wall to help explain fungal non-enzymatic cell wall deconstruction, and it also provides an explanation for certain phenomenon such as the anthropogenic generation of formaldehyde by wood. The mechanism also provides a basis for the generation of electrons by lignin that are required by certain fungal redox enzymes active in plant cell wall degrading systems. Overall, the data demonstrate that iron found naturally in lignocellulose materials will promote the oxidation of phenolic lignin compounds in the naturally low pH environments occurring within lignified plant cell walls, and that this activity is promoted by cell wall fragmentation.

Effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides.

The effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides were investigated. A air-dried biomass was ground by ball-milling, and the powder was separated into four fractions by passage through a series of sieves with mesh sizes 250-355 microm, 150-250 microm, 63-150 microm, and <63 microm. Each fraction was hydrolyzed with commercially available cellulase and beta-glucosidase. The yield of monosaccharides increased as the crystallinity of the substrate decreased. The addition of xylanase increased the yield of both pentoses and glucose. Delignification by the sodium chlorite method improved the initial rate of hydrolysis by cellulolytic enzymes significantly, resulting in a higher yield of monosaccharides as compared with that for untreated samples. When delignified M. sinensis was hydrolyzed with cellulase, beta-glucosidase, and xylanase, hemicellulose was hydrolyzed completely into monosaccharides, and the conversion rate of glucan to glucose was 90.6%.

Domino Reconstruction of the Portal Vein Using the External Iliac Vein and an ePTFE Graft in Pancreatic Surgery.

BACKGROUND: In the portal vein resection of long distance, an interposition by autologous vein is mandatory. External iliac vein (EIV) has been used, but harvesting the EIV is associated with severe venous congestion of the affected lower extremity. We have reconstructed the EIV using a ringed expanded polytetrafluoroethylene (ePTFE) graft. METHODS: Thirteen patients underwent this surgery. The right EIV was used for reconstructing the portal vein, and the retrieved portion of EIV was interposed by the ePTFE graft. We evaluated size and length of the graft, graft patency, girth of thigh, time for reconstruction of EIV, and graft infection. RESULTS: ePTFE grafts of 8 or 10 mm in diameter were used. The length of ePTFE graft used was 4.4 ± 0.5 cm. Graft patency was kept in 76.9% patients. Graft obstruction was encountered in three patients, and the girth of right thigh increased by about 10 cm. Time for reconstruction of EIV was 29.5 ± 6.8 min. Graft infection did not occur in any patients. CONCLUSIONS: Reconstruction of the EIV using a ringed ePTFE graft seems to be a feasible option for preventing the swelling of the affected lower extremity after procurement of EIV for repairing the portal vein.

Relationship Between Increased Fucosylation and Metastatic Potential in Colorectal Cancer.

BACKGROUND: Fucose is utilized for the modification of different molecules involved in blood group determination, immunological reactions, and signal transduction pathways. We have recently reported that enhanced activity of the fucosyltransferase 3 and/or 6 promoted TGF-ß-mediated epithelial mesenchymal transition and was associated with increased metastatic potential of colorectal cancer (CRC), suggesting that fucose is required by CRC cells. With this in mind, we examined requirement of L-fucose in CRC cells and developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specific to CRC. METHODS: In this study, we first examined the expression of fucosylated proteins in 50 cases of CRC by immunochistochemical staining with biotinylated Aleuria aurantia lectin (AAL). Then we carried out an L-fucose uptake assay using three CRC cell lines. Finally, we developed fucose-bound nanoparticles as vehicles for the delivery of an anticancer drug, SN38, and examined tumor growth inhibition in mouse xenograft model (n = 6 mice per group). All statistical tests were two-sided. RESULTS: We found a statistically significant relationship between vascular invasion, clinical stage, and intensity score of AAL staining (P≤ .02). L-fucose uptake assay revealed that L-fucose incorporation, as well as fucosylated protein release, was high in cells rich in fucosylated proteins. L-fucose-bound liposomes effectively delivered Cy5.5 into CRC cells. The excess of L-fucose decreased the efficiency of Cy5.5 uptake through L-fucose-bound liposomes, suggesting an L-fucose receptor dependency. Intravenously injected, L-fucose-bound liposomes carrying SN38 were successfully delivered to CRC cells, mediating efficient tumor growth inhibition (relative tumor growth ratio: no treatment group [NT], 8.29 ± 3.09; SN38-treated group [SN38], 3.53 ± 1.47; liposome-carrying, SN38-treated group [F0], 3.1 ± 1.39; L-fucose-bound, liposome-carrying, SN38-treated group [F50], 0.94 ± 0.89; F50 vs NT,P= .003; F50 vs SN38,P= .02, F50 vs F0,P= .04), as well as prolonging survival of mouse xenograft models (log-rank test,P< .001). CONCLUSIONS: Thus, fucose-bound liposomes carrying anticancer drugs provide a new strategy for the treatment of CRC patients.

Relationship Between Increased Fucosylation and Metastatic Potential in Colorectal Cancer.

BACKGROUND: Fucose is utilized for the modification of different molecules involved in blood group determination, immunological reactions, and signal transduction pathways. We have recently reported that enhanced activity of the fucosyltransferase 3 and/or 6 promoted TGF-ß-mediated epithelial mesenchymal transition and was associated with increased metastatic potential of colorectal cancer (CRC), suggesting that fucose is required by CRC cells. With this in mind, we examined requirement of L-fucose in CRC cells and developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specific to CRC. METHODS: In this study, we first examined the expression of fucosylated proteins in 50 cases of CRC by immunochistochemical staining with biotinylated Aleuria aurantia lectin (AAL). Then we carried out an L-fucose uptake assay using three CRC cell lines. Finally, we developed fucose-bound nanoparticles as vehicles for the delivery of an anticancer drug, SN38, and examined tumor growth inhibition in mouse xenograft model (n = 6 mice per group). All statistical tests were two-sided. RESULTS: We found a statistically significant relationship between vascular invasion, clinical stage, and intensity score of AAL staining (P ≤ .02). L-fucose uptake assay revealed that L-fucose incorporation, as well as fucosylated protein release, was high in cells rich in fucosylated proteins. L-fucose-bound liposomes effectively delivered Cy5.5 into CRC cells. The excess of L-fucose decreased the efficiency of Cy5.5 uptake through L-fucose-bound liposomes, suggesting an L-fucose receptor dependency. Intravenously injected, L-fucose-bound liposomes carrying SN38 were successfully delivered to CRC cells, mediating efficient tumor growth inhibition (relative tumor growth ratio: no treatment group [NT], 8.29 ± 3.09; SN38-treated group [SN38], 3.53 ± 1.47; liposome-carrying, SN38-treated group [F0], 3.1 ± 1.39; L-fucose-bound, liposome-carrying, SN38-treated group [F50], 0.94 ± 0.89; F50 vs NT, P = .003; F50 vs SN38, P = .02, F50 vs F0, P = .04), as well as prolonging survival of mouse xenograft models (log-rank test, P < .001). CONCLUSIONS: Thus, fucose-bound liposomes carrying anticancer drugs provide a new strategy for the treatment of CRC patients.

Targeting Notch-1 positive acute leukemia cells by novel fucose-bound liposomes carrying daunorubicin.

Complete remission by induction therapy in acute myelogenous leukemia (AML) can be achieved due to improvements in supportive and optimized therapy. However, more than 20% of patients will still need to undergo salvage therapy, and most will have a poor prognosis. Determining the specificity of drugs to leukemia cells is important since this will maximize the dose of chemotherapeutic agents that can be administered to AML patients. In turn, this would be expected to lead to reduced drug toxicity and its increased efficacy. We targeted Notch-1 positive AML cells utilizing fucose-bound liposomes, since activation of Notch-1 is required for O-fucosylation. Herein, we report that intravenously injected, L-fucose-bound liposomes containing daunorubicin can be successfully delivered to AML cells that express fucosylated antigens. This resulted in efficient tumor growth inhibition in tumor-bearing mice and decreased proliferation of AML patient-derived leukemia cells. Thus, biological targeting by fucose-bound liposomes that takes advantage of the intrinsic characteristics of AML cells could be a promising new strategy for Notch-1 positive-AML treatment.

Breath Sound Intensity during Tidal Breathing in COPD Patients.

OBJECTIVE: There is a discrepancy in the intensity of breath sounds in chronic obstructive pulmonary disease (COPD) patients between subjective studies, which have reported a diminished intensity, and objective studies using airflow-standardized measurements, which have not demonstrated a diminished intensity. We herein evaluated the breath sound intensity in COPD patients during tidal breathing in order to obtain clinically relevant results. METHODS: The subjects included 20 stable COPD patients and 20 normal controls. Microphones were attached to six sites on the chest wall, and breath sounds at the chest wall and airflow in the mouth were measured during resting tidal and deep tidal breathing. The octave-band power values of the breath sounds were subsequently calculated. RESULTS: 1. During resting breathing, the intensity of breath sounds during both inspiration and expiration was significantly greater in the COPD group than in the control group; the difference was prominent at higher frequency bands (>400 Hz). In addition, the power of the high frequency bands tended to be positively correlated with the CT visual emphysema scores but not the forced expiratory volume in one second, The airflow during resting breathing did not differ between the two groups. 2. During deep breathing, the intensity of inspiratory breath sounds at the dominant frequency band (200-400 Hz) was diminished over the upper and middle lung fields in the COPD group compared to that observed in the control group, while the intensity during expiration was not. The airflow during deep breathing was lower in the COPD group than in the control group. CONCLUSION: In the present study, the breath sound intensity in the COPD patients was diminished during deep inspiration due to a reduced airflow and increased during both resting inspiration and expiration.

Differential transcription of beta-glucosidase and cellobiose dehydrogenase genes in cellulose degradation by the basidiomycete Phanerochaete chrysosporium.

Transcriptional analysis of beta-glucosidase gene (bgl) and cellobiose dehydrogenase gene (cdh) in relation to cellobiose metabolism in the basidiomycete Phanerochaete chrysosporium was performed using real-time quantitative RT-PCR. Addition of glucose to cellulose-degrading culture significantly decreased the number of both transcripts. In contrast, addition of cellobiose repressed only transcription of bgl but no effect for that of cdh. Moreover, to investigate induction of the two genes, the mycelia grown on glucose medium were transferred to medium containing glucose, cellobiose or no carbon source. In cellobiose medium, the number of bgl transcripts was slightly lower, whereas that of cdh transcripts was 2.3-fold higher than those in glucose medium. Consequently, cellobiose represses transcription of bgl, whereas it induces that of cdh.

Molecular cloning and characterization of a cDNA encoding cellobiose dehydrogenase from the wood-rotting fungus Grifola frondosa.

Cloning of a cDNA encoding cellobiose dehydrogenase (CDH) from the wood-rotting fungus Grifola frondosa, which produces the edible maitake mushroom, was performed using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The CDH cDNA consisted of 2469 bp, including an open reading frame encoding the 18-amino acid signal peptide at the N-terminal region and the 750-amino acid mature protein with a predicted molecular mass of 79.6 kDa and a pI value of 4.32. Analysis of the amino acid sequence revealed that it contains a flavin-binding motif, two glucose-methanol-choline oxidoreductase motifs, and two possible residues for heme ligand binding (Met61 and His58). The amino acid sequence of G. frondosa CDH (GfrCDH) has a high degree of identity with three known CDHs from basidiomycetes, but not with two CDHs from ascomycetes. In addition, transcription of the CDH gene in G. frondosa grown on several carbon sources was analyzed by RT-PCR. mRNA of GfrCDH was detected from mycelia grown on cellobiose and cellulose, but not on glucose. Consequently, transcription of the GfrCDH gene seems to be promoted under conditions favoring cellulose degradation, and to be regulated by carbon catabolite repression.

Pulpal response to different pulp capping methods after pulp exposure by air abrasion.

Air abrasion is regaining popularity especially in the area of pediatric dentistry due to its ease of use and its advantages. Due to the lost of tactile information, while using this technique, there is an increased risk for pulpal exposure. On the other hand, Ca(OH)2 medicament has been proven to induce dentin bridge formation, but an adequate sealing seems to be even more important that the capping material used. The purpose of this study was two fold: to assess the pulpal response after pulpal exposure by air abrasion and to evaluate the healing potential after using Ca(OH)2 medicament or Liner Bond II as a capping agent. Two hundred sixteen teeth from mixed-bred dogs were used in this study. The teeth were divided into three groups, A) pulpal exposure by air-abrasion followed by sealing of the cavity with Liner Bond II, B) pulpal exposure by air-abrasion and Ca(OH)2 pulp capping and C) pulpal exposure by high-speed followed by air-abrasion and Ca(OH)2 pulp capping as a control group. The animals were sacrificed after 7, 14, 30 and 60 days and a histopathological evaluation was undertaken. After applying Analysis of Variance to compare the groups, it was observed that at earlier observation periods, the inflammatory criteria near the exposure site were different among the groups. As time elapsed, the inflammation was resolved in the pulp tissue, however, the odontoblastic layer and the dentin bridge formation had a highly statistically significant difference (p<0.001) among the various groups at all observation periods. In addition, a positive correlation was observed between the organization of the odontoblastic layer and the dentin bridge formation mainly after 30 days. It could be concluded that dentin bridge formation could be achieved with the use of Ca(OH) or Liner Bond II as capping agent with an adequate sealing. However, the formation is delayed especially when Liner Bond II is used as capping agent.

Targeting anticancer drug delivery to pancreatic cancer cells using a fucose-bound nanoparticle approach.

Owing to its aggressiveness and the lack of effective therapies, pancreatic ductal adenocarcinoma has a dismal prognosis. New strategies to improve treatment and survival are therefore urgently required. Numerous fucosylated antigens in sera serve as tumor markers for cancer detection and evaluation of treatment efficacy. Increased expression of fucosyltransferases has also been reported for pancreatic cancer. These enzymes accelerate malignant transformation through fucosylation of sialylated precursors, suggesting a crucial requirement for fucose by pancreatic cancer cells. With this in mind, we developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specifically to cancer cells. L-fucose-bound liposomes containing Cy5.5 or Cisplatin were effectively delivered into CA19-9 expressing pancreatic cancer cells. Excess L-fucose decreased the efficiency of Cy5.5 introduction by L-fucose-bound liposomes, suggesting L-fucose-receptor-mediated delivery. Intravenously injected L-fucose-bound liposomes carrying Cisplatin were successfully delivered to pancreatic cancer cells, mediating efficient tumor growth inhibition as well as prolonging survival in mouse xenograft models. This modality represents a new strategy for pancreatic cancer cell-targeting therapy.

Characterization of carbohydrate-binding cytochrome b562 from the white-rot fungus Phanerochaete chrysosporium.

cDNA encoding a hemoprotein similar to the cytochrome domain of extracellular flavocytochrome cellobiose dehydrogenase (CDH) was cloned from the white-rot fungus Phanerochaete chrysosporium. The deduced amino acid sequence implies that there is a two-domain structure consisting of an N-terminal cytochrome domain and a C-terminal family 1 carbohydrate-binding module (CBM1) but that the flavin-containing domain of CDH is not present. The gene transcripts were observed in cultures in cellulose medium but not in cultures in glucose medium, suggesting that there is regulation by carbon catabolite repression. The gene was successfully overexpressed in Pichia pastoris, and the recombinant protein was designated carbohydrate-binding cytochrome b562 (CBCyt. b562). The resonance Raman spectrum suggested that the heme of CBCyt. b562 is 6-coordinated in both the ferric and ferrous states. Moreover, the redox potential measured by cyclic voltammetry was similar to that of the cytochrome domain of CDH. These results suggest that the redox characteristics may be similar to those of the cytochrome domain of CDH, and so CBCyt. b562 may have an electron transfer function. In a binding study with various carbohydrates, CBCyt. b562 was adsorbed with high affinity on both cellulose and chitin. As far as we know, this is the first example of a CBM1 connected to a domain without apparent catalytic activity for carbohydrate; this CBM1 may play a role in localization of the redox protein on the surface of cellulose or on the fungal sheath in vivo.