Positive regulation of the PhcB neighbouring regulator PrhX on expression of the type III secretion system and pathogenesis in Ralstonia solanacearum.

Ralstonia solanacearum PhcB and PhcA control a quorum-sensing (QS) system that globally regulates expression of about one third of all genes, including pathogenesis genes. The PhcB-PhcA QS system positively regulates the production of exopolysaccharide (EPS) and negatively regulates hrp gene expression, which is crucial for the type III secretion system (T3SS). Both EPS and the T3SS are essential for pathogenicity. The gene rsc2734 is located upstream of a phcBSR operon and annotated as a response regulator of a two-component system. Here, we demonstrated that RSc2734, hereafter named PrhX, positively regulated hrp gene expression via a PrhA-PrhIR-PrhJ-HrpG signalling cascade. Moreover, PrhX was crucial for R. solanacearum to invade host roots and grow in planta naturally. prhX expression was independent of the PhcB-PhcA QS system. PrhX did not affect the expression of phcB and phcA and the QS-dependent phenotypes, such as EPS production and biofilm formation. Our results provide novel insights into the complex regulatory network of the T3SS and pathogenesis in R. solanacearum.

Insights into the metabolic specificities of pathogenic strains from the Ralstonia solanacearum species complex.

All the strains grouped under the species Ralstonia solanacearum represent a species complex responsible for many diseases on agricultural crops throughout the world. The strains have different lifestyles and host range. Here, we investigated whether specific metabolic pathways contribute to strain diversification. To this end, we carried out systematic comparisons on 11 strains representing the diversity of the species complex. We reconstructed the metabolic network of each strain from its genome sequence and looked for the metabolic pathways differentiating the different reconstructed networks and, by extension, the different strains. Finally, we conducted an experimental validation by determining the metabolic profile of each strain with the Biolog technology. Results revealed that the metabolism is conserved between strains, with a core metabolism composed of 82% of the pan-reactome. The three species composing the species complex could be distinguished according to the presence/absence of some metabolic pathways, in particular, one involving salicylic acid degradation. Phenotypic assays revealed that the trophic preferences on organic acids and several amino acids such as glutamine, glutamate, aspartate, and asparagine are conserved between strains. Finally, we generated mutants lacking the quorum-sensing-dependent regulator PhcA in four diverse strains, and we showed that the phcA-dependent trade-off between growth and production of virulence factors is conserved across the R. solanacearum species complex. IMPORTANCE Ralstonia solanacearum is one of the most important threats to plant health worldwide, causing disease on a very large range of agricultural crops such as tomato or potato. Behind the R. solanacearum name are hundreds of strains with different host range and lifestyle, classified into three species. Studying the differences between strains allows to better apprehend the biology of the pathogens and the specificity of some strains. None of the published genomic comparative studies have focused on the metabolism of the strains so far. We developed a new bioinformatic pipeline to build high-quality metabolic networks and used a combination of metabolic modeling and high-throughput phenotypic Biolog microplates to look for the metabolic differences between 11 strains across the three species. Our study revealed that genes encoding enzymes are overall conserved, with few variations between strains. However, more variations were observed when considering substrate usage. These variations probably result from regulation rather than the presence or absence of enzymes in the genome.

Insight into the uptake, accumulation, and metabolism of the fungicide phenamacril in lettuce (Lactuca sativa L.) and radish (Raphanus sativus L.).

The fungal species Fusarium can cause devastating disease in agricultural crops. Phenamacril is an extremely specific cyanoacrylate fungicide and a strobilurine analog that has excellent efficacy against Fusarium. To date, information on the mechanisms involved in the uptake, accumulation, and metabolism of phenamacril in plants is scarce. In this study, lettuce and radish were chosen as model plants for a comparative analysis of the absorption, accumulation, and metabolic characteristics of phenamacril from a polluted environment. We determined the total amount of phenamacril in the plant-water system by measuring the concentrations in the solution and plant tissues at frequent intervals over the exposure period. Phenamacril was readily taken up by the plant roots with average root concentration factor ranges of 60.8-172.7 and 16.4-26.9 mL/g for lettuce and radish, respectively. However, it showed limited root-to-shoot translocation. The lettuce roots had a 2.8-12.4-fold higher phenamacril content than the shoots; whereas the radish plants demonstrated the opposite, with the shoots having 1.5 to 10.0 times more phenamacril than the roots. By the end of the exposure period, the mass losses from the plant-water systems reached 72.0% and 66.3% for phenamacril in lettuce and radish, respectively, suggesting evidence of phenamacril biotransformation. Further analysis confirmed that phenamacril was metabolized via hydroxylation, hydrolysis of esters, demethylation, and desaturation reactions, and formed multiple transformation products. This study furthers our understanding of the fate of phenamacril when it passes from the environment to plants and provides an important reference for its scientific use and risk assessment.

Barrier-disrupted skin: Quantitative analysis of tape and cyanoacrylate stripping efficiency by multiphoton tomography.

Numerous studies have employed tape stripping (TS) or cyanoacrylate stripping (CS) to induce skin barrier disruption of the stratum corneum (SC) in human and porcine skin. However, the thickness of the remaining SC and the respective changes of the skin permeability have been rarely quantified. By using high-resolution multiphoton tomography, about 5 µm thick SC was found remaining on human skin after the performance of 30 times TS or 2 times CS. 50 tape strips or 4 times CS removed the entire human SC, but on porcine skin 2-3 µm thick SC was still left. TS can only reach the transition zone between the SC and the stratum granulosum because of the limited adhesion, whereas CS was able to remove viable skin layers. Permeation investigations on porcine skin revealed that the apparent permeability coefficient of the hydrophilic nitroxide spin 2,5,5-Tetramethyl-1-pyrrolidinyloxy-3-carboxylic acid increased 15-, 18-, and 21-fold when the SC amount remaining in the skin was 30%, 16%, and 8%, respectively. It is recommended to use at most 30 times TS or 3 times CS to obtain ex vivo barrier-disrupted skin that mimics diseased skin. The study provides quantitative information for the utility of TS and CS in skin penetration research.

Mucoadhesive properties of low molecular weight chitosan- or glycol chitosan- and corresponding thiomer-coated poly(isobutylcyanoacrylate) core-shell nanoparticles.

The aim of the present work was to evaluate the mucoadhesive properties of poly(isobutyl cyanoacrylate) (PIBCA) nanoparticles (NPs) coated with Low Molecular Weight (LMW) chitosan (CS)- and glycol chitosan (GCS)-based thiomers as well as with the corresponding LMW unmodified polysaccharides. For this purpose, all the CS- and GCS-based thiomers were prepared under simple and mild conditions starting from the LMW unmodified polymers CS and GCS. The resulting NPs were of spherical shape with diameters ranging from 400 to 600nm and 187 to 309nm, for CS- and GCS-based NPs, respectively. The mucoadhesive characteristics of these core shell NPs were studied in Ussing chambers measuring the percentage of NPs stuck on the mucosal of fresh intestinal tissue after 2h of incubation. Moreover, incubation of nanoparticle formulations with the intestinal tissue induced changes in transmucosal electrical resistance which were measured to gain information into the opening of tight junctions and to control the integrity of the mucosa. Thus, it was found that PIBCA NPs coated with the GCS-Glutathione conjugate (GCGPIBCA NPs) possessed the most favorable mucoadhesive performances. Moreover, both GCGPIBCA- and GCS-N-acetyl-cysteine (GCNPIBCA)-core-shell NPs might induced an enlargement of the epithelial cell tight junctions. In conclusion, coating of PIBCA NPs with GCS-based thiomers may be useful for improving the mucoadhesive and permeation properties of these nanocarriers.

Abrupt occlusion of right gastroepiploic artery as an angiographic evidence of gastrointestinal hemorrhage

Angiography plays an important role in both diagnosis and treatment of gastrointestinal (GI) bleeding; however, the sensitivity is low for diagnosis. We report a case of a 38-year-old woman who presented with recurrent upper GI bleeding following central pancreatectomy. Multiple selective arteriograms failed to reveal any active bleeding or other common signs of bleeding. There was an abrupt occlusion of the right gastroepiploic artery initially interpreted to be a surgical ligation. Upon direct superselective injection near the occlusion, an area of frank contrast extravasation was demonstrated immediately beyond the occlusion. The underlying vessel was embolized with n-butyl cyanoacrylate without recurrent bleeding up to 3-month follow-up (AU)

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Cellular uptake and intracellular degradation of poly(alkyl cyanoacrylate) nanoparticles.

BACKGROUND: Poly(alkyl cyanoacrylate) (PACA) nanoparticles have shown promise as drug carriers both to solid tumors and across the blood-brain barrier. Efficient drug delivery requires both high cellular uptake of the nanoparticles and release of the drug from the nanoparticles. Release of hydrophobic drugs from PACA nanoparticles is primarily governed by nanoparticle degradation, and this process has been poorly studied at the cellular level. Here we use the hydrophobic model drug Nile Red 668 (NR668) to investigate intracellular degradation of PACA nanoparticles by measuring changes in NR668 fluorescence emission and lifetime, as the spectral properties of NR668 depend on the hydrophobicity of the dye environment. We also assess the potential of poly(butyl cyanoacrylate) (PBCA) and poly(octyl cyanoacrylate) (POCA) nanoparticles for intracellular drug delivery in the prostate cancer cell line PC3 and rat brain endothelial cell line RBE4 and the role of endocytosis pathways in PACA nanoparticle uptake in those cell lines. RESULTS: Fluorescence lifetime imaging, emission spectra analysis and Förster resonance energy transfer indicated that the intracellular degradation was in line with the degradation found by direct methods such as gas chromatography and scanning electron microscopy, showing that PBCA has a faster degradation rate compared to POCA. The combined P(BCA/OCA) nanoparticles had an intermediate degradation rate. The uptake of POCA and PBCA nanoparticles was much higher in RBE4 than in PC3 cells. Endocytosis inhibition studies showed that both clathrin- and caveolin-mediated endocytosis were involved in PACA nanoparticle uptake, and that the former played a predominant role, particularly in PC3 cells. CONCLUSIONS: In the present study, we used three different optical techniques to show that within a 24-hour period PBCA nanoparticles degraded significantly inside cells, releasing their payload into the cytosol, while POCA nanoparticles remained intact. This indicates that it is possible to tune the intracellular drug release rate by choosing appropriate monomers from the PACA family or by using hybrid PACA nanoparticles containing different monomers. In addition, we showed that the uptake of PACA nanoparticles depends not only on the monomer material, but also on the cell type, and that different cell lines can use different internalization pathways.

Analogs of cinnamic acid benzyl amide as nonclassical inhibitors of activated JAK2 kinase.

Scaffold-based analogs of cinnamic acid benzyl amide (CABA) exhibit pleiotropic effects in cancer cells, and their exact molecular mechanism of action is under investigation. The present study is part of our systemic analysis of interactions of CABA analogs with their molecular targets. These compounds were shown to inhibit Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and JAK2/signal transducer and activator of transcription 5 (STAT5) signaling and thus are attractive scaffolds for anticancer drug design. To identify the potential mechanisms of action of this class of compounds, direct interactions of the selected CABA analogs with JAK2 kinase were examined. Inhibition of JAK2 enzymatic activity was assessed, and molecular modeling studies of selected compounds-(E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide (WP1065), (E)-2-cyano-N-[(S)-1-phenylbutyl]- 3-(3-bromopyridin-2-yl)acrylamide (WP1130), and (E)-2-cyano-N-[(S)-1,4-diphenylbutyl]-3-(3-bromopyridin-2-yl)acrylamide (WP1702)-in the JAK2 kinase domain were used to support interpretation of the experimental data. Our results indicated that the tested CABA analogs are nonclassical inhibitors of activated (phosphorylated) JAK2, although markedly weaker than clinically tested ATP-competitive JAK2 inhibitors. Relatively small structural changes in the studied compounds affected interactions with JAK2, and their mode of action ranged from allosteric-noncompetitive to bisubstratecompetitive. These results demonstrated that direct inhibition of JAK2 enzymatic activity by the WP1065 (half-maximal inhibitory concentration [IC50] = 14.8 µM), WP1130 (IC50 = 3.8 µM), and WP1702 (IC50 = 2.9 µM) potentially contributes, albeit minimally, to suppression of the JAK2/STAT signaling pathways in cancer cells and that additional specific structural modifications may amplify JAK2-inhibitory effects.

Poly(styrene-alt-maleic anhydride)-based diblock copolymer micelles exhibit versatile hydrophobic drug loading, drug-dependent release, and internalization by multidrug resistant ovarian cancer cells.

Amphiphilic diblock copolymers of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) and poly(styrene-alt-maleic anhydride)-b-poly(butyl acrylate) (PSMA-b-PBA) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerizations. Polymers were well-controlled with respect to molecular weight evolution and polydispersity indices (PDI < 1.2). Additionally, RAFT allowed for control of diblock compositions (i.e., ratio of hydrophilic PSMA blocks to hydrophobic PS/PBA blocks) and overall molecular weight, which resulted in reproducible self-assembly of diblocks into micelle nanoparticles with diameters of 20-100 nm. Parthenolide (PTL), a hydrophobic anticancer drug, was loaded and released from the micelles. The highest loading and prolonged release of PTL was observed from predominantly hydrophobic PSMA-b-PS micelles (e.g., PSMA100-b-PS258), which exhibited the most ordered hydrophobic environment for more favorable core-drug interactions. PSMA100-b-PS258 micelles were further loaded with doxorubicin (DOX), as well as two hydrophobic fluorescent probes, nile red and IR-780. While PTL released quantitatively within 24 h, DOX, IR-780, and nile red showed release over 1 week, suggesting stronger drug-core interactions and/or hindrance due to less favorable drug-solvent interactions. Finally, uptake and intracellular localization of PSMA100-b-PS258 micelles by multidrug resistant (MDR) ovarian cancer cells was observed by transmission electron microscopy (TEM). Additionally, in vitro analyses showed DOX-loaded PSMA-b-PS micelles exhibited greater cytotoxicity to NCI/ADR RES cells than equivalent free DOX doses (75% reduction in cell viability by DOX-loaded micelles compared to 40% reduction in viability by free DOX at 10 µM DOX), likely due to avoidance of MDR mechanisms that limit free hydrophobic drug accumulation. The ability of micelles to achieve intracellular delivery via avoidance of MDR mechanisms, along with the versatility of chemical constituents and drug loading and release rates, offer many advantages for a variety of drug delivery applications.

Right ventricular plasticity in a porcine model of chronic pressure overload.

BACKGROUND: Ventricular-arterial coupling is a measure of the relationship between ventricular contractility and afterload. We sought to determine the relationship between ventricular-arterial coupling and right ventricular (RV) remodeling in a novel porcine model of progressive pulmonary hypertension (PH). METHODS: Chronic PH was induced in pigs by ligation of the left pulmonary artery (PA) followed by 5 weekly injections of cyanoacrylate to progressively obstruct the right lower lobe arteries (PH group, n = 10). At 6 weeks, 5 PH animals underwent reperfusion of the left lung through conduit anastomosis to decrease RV afterload, whereas 5 other animals received no treatment. Five sham-operated piglets were used as controls. RV function was assessed using echocardiography and conductance catheterization. RV gene expression of beta-myosin heavy chain (ß-MHC) and B-type natriuretic peptide (BNP) were quantified by polymerase chain reaction. RESULTS: At 6 weeks, compared with controls, the PH group had higher mean PA pressure (32 ± 6 vs 14 ± 2 mm Hg, p < 0.01). The increase in RV elastance was insufficient to compensate for the increase in pulmonary arterial elastance in the PH group and altered ventricular-arterial coupling occurred (0.65 ± 0.16 vs 1.28 ± 0.14, p < 0.01). The degree of ventricular-arterial uncoupling was related to RV enlargement and systolic dysfunction. Ventricular-arterial uncoupling and increased RV mass index were associated with up-regulation of ß-MHC and BNP expression. CONCLUSIONS: Ventricular-arterial coupling is closely associated with ventricular remodeling and systolic function as well as contractile and BNP gene expression. Dynamic changes in myosin expression may determine RV work efficiency in PH.

Regulation of HGF expression by ΔEGFR-mediated c-Met activation in glioblastoma cells.

The hepatocyte growth factor receptor (c-Met) and a constitutively active mutant of the epidermal growth factor receptor (ΔEGFR/EGFRvIII) are frequently overexpressed in glioblastoma (GBM) and promote tumorigenesis. The mechanisms underlying elevated hepatocyte growth factor (HGF) production in GBM are not understood. We found higher, coordinated mRNA expression levels of HGF and c-Met in mesenchymal (Mes) GBMs, a subtype associated with poor treatment response and shorter overall survival. In an HGF/c-Met-dependent GBM cell line, HGF expression declined upon silencing of c-Met using RNAi or by inhibiting its activity with SU11274. Silencing c-Met decreased anchorage-independent colony formation and increased the survival of mice bearing intracranial GBM xenografts. Consistent with these findings, c-Met activation by ΔEGFR also elevated HGF expression, and the inhibition of ΔEGFR with AG1478 reduced HGF levels. Interestingly, c-Met expression was required for ΔEGFR-mediated HGF production, anchorage-independent growth, and in vivo tumorigenicity, suggesting that these pathways are coupled. Using an unbiased mass spectrometry-based screen, we show that signal transducer and activator of transcription 3 (STAT3) Y705 is a downstream target of c-Met signaling. Suppression of STAT3 phosphorylation with WP1193 reduced HGF expression in ΔEGFR-expressing GBM cells, whereas constitutively active STAT3 partially rescued HGF expression and colony formation in c-Met knockdown cells expressing ΔEGFR. These results suggest that the c-Met/HGF signaling axis is enhanced by ΔEGFR through increased STAT3-dependent HGF expression and that targeting c-Met in Mes GBMs may be an important strategy for therapy.

Graft uptake rates with isoamyl-2-cyanoacrylate in myringoplasty procedures: a 10-year retrospective study.

OBJECTIVE: To assess graft uptake rates with the use of cyanoacrylate adhesives in myringoplasty procedures. STUDY DESIGN: Case series with chart review. Setting. Tertiary care center. SUBJECTS AND METHODS: Five hundred forty-two patients selected with safe central perforations were divided into 4 groups based on perforation size and each divided into 2 subgroups depending on the presence or absence of preexisting pathology on the remnant tympanic membrane. Myringoplasty without ossicular reconstruction was done postaurally or by transcanal approach, with inlay graft placed and adhesive applied. Ear pack was removed on the seventh postoperative day. Graft uptake rates, graft uptake time (neotympanum intact and mobile), and postoperative sequelae were noted. RESULTS: Graft uptake was about 99% at 3 months postsurgery. Residual perforation was seen in 2 patients in group 2 and 1 in group 4 and thinned-out tympanum in 1 patient in group 4. Mean uptake time was 21 days. Neotympanum mobility was sluggish in 9 cases postoperatively in group 4 and in 2 cases in group 3. Patients with successful (neotympanum intact and mobile) graft uptake showed significant improvement. Postoperative inflammatory signs in the external auditory canal were seen in 3 cases. CONCLUSION: Cyanoacrylate use in myringoplasty obtains graft uptake rates of 99% compared with most studies, which report 80% to 90%. This also allows removal of the pack by the seventh day, allowing close follow-up of the graft and less patient discomfort. Importantly, it has not interfered with the formation of the neotympanum or compromised hearing improvement.

Identification of covalent binding sites of ethyl 2-cyanoacrylate, methyl methacrylate and 2-hydroxyethyl methacrylate in human hemoglobin using LC/MS/MS techniques.

Acrylates are used in vast quantities, for instance in paints, adhesive glues, molding. They are potent contact allergens and known to cause respiratory hypersensitivity and asthma. Here we study ethyl 2-cyanoacrylate (ECA), methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA). There are only limited possibilities to measure the exposure to acrylates, especially for biological monitoring. The aim of the present study was to investigate the chemical structures of adducts formed after reaction of hemoglobin (Hb) with ECA, MMA, and HEMA. This information may be used to identify adducted Hb peptides for biological monitoring of exposure to acrylates. Hb-conjugates with ECA, MMA, and HEMA were synthesized in vitro. The conjugates were digested by trypsin and pronase E. Adducted peptides were characterized and analyzed by liquid chromatography and nano electro spray/hybrid quadrupole time-of-flight mass spectrometry (MS) as well as tandem quadrupole MS. The search for the adducted peptides was facilitated by visualizing the MS data by different computer programs. The results showed that ECA binds covalently to cysteines at the 104 position in the α and the position 112 in the ß-chains in Hb. MMA and HEMA bound to all the cysteines in both chains, Cys(104) in the α-chain and Cys(93) and 112 in the ß-chain. The full-length spectra of in un-digested Hb confirmed this binding pattern. There was no reaction with N-acetyl-L-lysine at physiological pH. The adducted peptides were possible to measure using LC/MS/MS in selected reaction monitoring mode. These peptides may be used for biological monitoring of exposure to ECA, MMA and HEMA.

Cyanoacrylate gluing increases the effectiveness of systemic antimicrobial treatment in sternal infection: experimental study in a rodent model.

BACKGROUND: Sternal infection is a serious complication of cardiac surgery requiring resternotomy and radical debridement. In this experimental study, we aimed to test our hypothesis that the use of cyanoacrylate gluing (application of an acrylic resin, a monomer of cyanoacrylate molecules, which rapidly polymerizes in the presence of water, forming long, strong chains and joining the bonded surfaces together) together with systemic antimicrobial therapy will provide synergy for the treatment of sternal infection caused by methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Forty Wistar albino rats were randomly divided into four groups: Group I, uncontaminated sham group; Group II, untreated contaminated control group; Group III, contaminated group receiving only systemic vancomycin therapy; Group IV, contaminated group treated with a combination of cyanoacrylate gluing and systemic vancomycin. Cyanoacrylate gluing was applied on the 3rd postoperative day and all rats alive at the end of 8th week were sacrificed. The degree of sternal infection was assessed histologically and also by quantitative culture analysis. RESULTS: Histological evaluation revealed that cyanoacrylate was degraded and replaced by connective tissue at the end of the 8th week. Culture analysis revealed that the average growth of microorganisms was significantly reduced in Groups III and IV. In Group IV, the reduction in the amount of growing microorganisms was found to be more pronounced and significantly lower than in Groups II and III. CONCLUSION: Our experimental model suggests that cyanoacrylate gluing provides significant synergy for systemic antimicrobial therapy. However, further clinical trials are required in order to use this treatment modality safely in patients, even though our study demonstrated successful results in the treatment of mediastinitis and sternal osteomyelitis in rats.

Low-density lipoprotein receptor-mediated endocytosis of PEGylated nanoparticles in rat brain endothelial cells.

Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to diffuse through the blood-brain barrier after intravenous administration. However, the mechanism of transport of these nanoparticles into brain has not yet been clearly elucidated. The development of a model of rat brain endothelial cells (RBEC) in culture has allowed investigations into this mechanism. A study of the intracellular trafficking of nanoparticles by cell fractionation and confocal microscopy showed that nanoparticles are internalized by the endocytic pathway. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of the nanoparticles. In contrast, chlorpromazine and NaN(3) pretreatment, which interferes with clathrin and energy-dependent endocytosis, caused a significant decrease of nanoparticle internalization. Furthermore, cellular uptake experiments with nanoparticles preincubated with apolipoprotein E and blocking of low-density lipoprotein receptors (LDLR) clearly suggested that the LDLR-mediated pathway was involved in the endocytosis of PEGPHDCA nanoparticles by RBEC.

Quantitative assessment of polymerization-binding mechanics of cyanoacrylates: model development and validation.

BACKGROUND AND PURPOSE: Although commonly acknowledged as paramount in significance, the mechanics of cyanoacrylate polymerization remain poorly characterized and quantified for clinical applications. This prompted development of a simplistic model for the systematic study of polymerization and binding behaviors of cyanoacrylates. METHODS: A sliding bed apparatus was constructed that linked a strain gage with a vessel that could be filled with liquid medium, cyanoacrylate, and a microcatheter. As the cyanoacrylate polymerized, the microcatheter was mechanically drawn away very slowly from the fixed vessel, resulting in the development of forces that were recorded to characterize the dynamics of polymerization and binding. Optimization of the model required manipulation of several variables that could influence polymerization. Three different formulations of cyanoacrylate were also tested to determine whether there are significant differences in polymerization dynamics. RESULTS: After experimenting with a few basic physical parameters of the test apparatus, consistent measurements of binding forces during cyanoacrylate polymerization could be recorded and measured. Polymerization produced a multiphasic pattern of binding forces, in which three distinct phases were observed. Furthermore, the rates of polymerization were significantly influenced by a variety of parameters, including the type of fluid within the model vessel, geometry of the model vessel, and rate of injection of acrylic into the well. Furthermore, there were significant differences in the pattern of dynamic binding forces among the various formulations of cyanoacrylate tested. CONCLUSIONS: A standardized bench top testing apparatus has been developed, which can consistently show dynamic binding related to polymerization of cyanoacrylates. This preliminary study shows a clear multiphasic pattern of polymerization binding, which may have important clinical implications. The apparatus may be useful for gaining better insight into a variety of clinically important phenomena related to cyanoacrylate polymerization.

Vitamin K administration to elderly patients with osteoporosis induces no hemostatic activation, even in those with suspected vitamin K deficiency.

The administration of menaquinone-4 (MK-4), one of subclasses of vitamin K2, significantly reduces bone loss in postmenopausal osteoporotic women. However, concerns have been raised about whether vitamin K administration alters the hemostatic balance by inducing a thrombotic tendency. We investigated were whether the administration of vitamin K in the form of MK-4 induced a thrombotic tendency in 29 elderly patients with osteoporosis (5 men, 24 women; age range 78.7+/-5.1 years). Patients were administered 45 mg/day (three times a day, 30 min after each meal) of MK-4 for 12 weeks. Blood samples were obtained from the patients at 0, 4 and 12 weeks after the start of MK-4 administration. A number of hemostatic parameters remained stable under the markedly increased plasma levels of MK-4. However, in patients with suspected vitamin K deficiency, whose plasma levels of vitamin K or factor VII were low, vitamin-K-dependent clotting factors such as factor VII and prothrombin were gradually increased after administration of MK-4. No changes in the sensitive molecular markers such as TAT and F1+2, which reflect the amount of thrombin generated in the blood stream, were observed, even in those patients with suspected vitamin K deficiency. These results indicate that MK-4 can be administered safely, with regard to maintaining the hemostatic balance, to osteoporotic patients receiving no anticoagulant therapy.

Ossicular chain reconstruction using a new tissue adhesive.

HYPOTHESIS: A new medical-grade cyanoacrylate tissue adhesive will improve the results of ossicular chain reconstruction in a rat model. BACKGROUND: An ideal tissue adhesive has long been awaited by otologists. Studies examining the older cyanoacrylates have demonstrated variable efficacy and toxicity. Octylcyanoacrylate is a new tissue adhesive that has many ideal properties for otologic surgery. METHODS: Thirteen female C-D rats were anesthetized, and preoperative auditory brainstem response (ABR) testing was performed. A left antrotomy was performed, and the incus was removed. In the adhesive group, the incus was dipped in octylcyanoacrylate and interposed between the tympanic membrane and the stapes; no adhesive was used in the control group. At 8 weeks, postoperative ABR was performed, the integrity of the ossicular chain inspected, and histopathologic analysis of the temporal bones performed. Statistical comparison of ABR results was performed with the Mann-Whitney test. RESULTS: Seven rats were randomized to the adhesive group and six to the control group, of which four survived. There were no histopathologic differences in the temporal bones of the animals other than the presence of mild foreign body reaction around the ossicular chain of the animals in the adhesive group. The ossicular chain was not intact in two of the four controls, whereas the rest were intact at 8 weeks. Postoperative air conduction ABR results (mean dB sound pressure level) (62.5 control versus 34.3 adhesive, p = 0.010) and air-bone gaps (47.5 control versus 18.9 adhesive, p = 0.008) were significantly better in the adhesive group. CONCLUSIONS: This new medical-grade tissue adhesive improves the hearing results of ossicular chain reconstruction, with no apparent histotoxicity in this animal model.

Development of intracoronary local adhesive delivery technique.

Acute coronary occlusion may occur in weak coronary atherosclerotic lesions, including dissection, ulceration or thrombus. In some cases of occlusion "bail-out" is performed by using recently developed New Devices. However, these have not yet completely solved the problem to this end, we designed a new method of coronary revascularization, the Intracoronary Local Adhesive Delivery Technique, utilizing antithrombotic and absorbable adhesive injected locally into the fragile and morbid arterial wall using a drug delivery PTCA catheter more flexible than the existing New Devices. This adhesive strengthened and hardened the lesions. In this study, we examined the efficacy of making an adhesive cylinder in arteries of similar size to the coronary, through acute animal experiments using the existing clinical adhesives and drug delivery PTCA catheters and 12 femoral arteries of adult goats. We were successful in forming firm tunnels along the inside of six arteries, infused with approximately 0.04 ml Cyanoacrylate. These tunnels were observed with intravascular ultrasound (IVUS) imaging and evaluated microscopically. These results suggest the feasibility of this method as a new approach for making synthetic resinous stents.

Cells involved in the capture of nanoparticles in hematopoietic organs.

The affinity of nanoparticles for hematopoietic organs could be valuable for the targeting of certain stimulating factors to those tissues, but this affinity should also be taken into account in the toxicological evaluation of those carriers, especially when they are loaded with antimitotic compounds such as doxorubicin. However, the cells responsible for the capture of the nanoparticles and their localization in these organs is an important point to know before trying to modulate the nanoparticle's tissue distribution. Thus, we have studied, in this paper, the capture, the localization, and the retention in the bone marrow and in the spleen of biodegradable poly(isohexyl cyanoacrylate) nanoparticles as well as of nonbiodegradable polystyrene nanoparticles. The histological localization of these nanoparticles has been completed by cytological localization with a method used in cytochemistry for the evaluation of intracellular accumulation of various substances, such as iron deposits in bone marrow sideroblasts. These data indicate that, in the bone marrow, after a quick passage through the endothelium, nanoparticles were dispersed throughout in the tissue and captured by all types of phagocytizing cells. In the spleen, nanoparticles were mainly localized in large angular capturing cells in the marginal zone of the lymphoid follicles.