Platelet volume indices and von Willebrand factor levels in blood exposed to polymer- or heparin-coated membrane oxygenators.

INTRODUCTION: To understand the behavior of platelet volume indices and the von Willebrand factor (VWF), in vitro experiments using whole human blood were performed with extracorporeal circulation (ECC) circuits, including membrane oxygenators coated with acrylate copolymer (ACP) or immobilized heparin (IHP). METHODS: Heparinized blood was circulated through two distinct experimental circuits: an ACP-coated reservoir and tubes, as well as membranes coated with either ACP or IHP (comprising five pieces of each type). The platelet distribution width, mean platelet volume (MPV), platelet large cell ratio (P-LCR), VWF quantity (VWFQ), and VWF activity (VWFA) were measured at 0, 8, 16, 24, and 32 h in each experiment. A two-way analysis of variance (ANOVA) was performed to determine whether the coating type or circulation duration affected the transition of each measurement. RESULTS: Two-way ANOVA indicated that the transitions of MPV, P-LCR, and VWFA were significantly affected by the circulation duration (p = 0.030, 0.001, and <0.001, respectively) and that the transitions of VWFQ and VWFA were significantly affected by the coating type (p = 0.022 and 0.006, respectively). Factor interactions between the coating type and circulation duration were not observed for each transition (p > 0.05). CONCLUSIONS: Our findings suggest that P-LCR is a good index for platelet activation in blood-circulating ECC and that VWFA and VWFQ are significantly attenuated in blood-circulating ECC with ACP-coated membranes, indicating the advantage of IHP coating regarding platelet activation.

Paraburkholderia sabiae administration alters zebrafish anxiety-like behavior via gut microbial taurine metabolism.

Interventions to the gut microbiome manipulate the gut-brain axis and could be useful in the treatment of anxiety and depression. In this study, we demonstrated that administration of the bacterium Paraburkholderia sabiae reduces anxiety-like behavior in adult zebrafish. P. sabiae administration increased the diversity of the zebrafish gut microbiome. Linear discriminant analysis Effect Size (LEfSe) analysis revealed that the populations of Actinomycetales including Nocardiaceae, Nocardia, Gordoniaceae, Gordonia, Nakamurellaceae, and Aeromonadaceae were reduced, whereas those of Rhizobiales including Xanthobacteraceae, Bradyrhizobiaceae, Rhodospirillaceae, and Pirellulaceae were increased in the gut microbiome. Functional analysis using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) predicted that P. sabiae administration altered taurine metabolism in the zebrafish gut, and we demonstrated that P. sabiae administration increased the taurine concentration in the brain. Since taurine functions as an antidepressant neurotransmitter in vertebrates, our results suggest that P. sabiae could improve anxiety-like behavior in zebrafish via the gut-brain axis.

Rare UV-resistant cells in clonal populations of Escherichia coli.

Water disinfection is one of the most important applications of ultraviolet light-emitting diodes (UV-LEDs), though bacterial regrowth remains a serious problem. In this study, we showed that UV-resistant cells, though rare, exist in an Escherichia coli clonal population. The UV-resistance of stationary phase cells was higher than that of exponential phase cells. Regrowth cell populations showed identical UV sensitivity before and after UV treatment, indicating that UV resistance is not acquired genetically, but is generated stochastically. The characteristics of these UV-resistant cells are similar to those of non-heritable antibiotic-resistant cells, termed persisters. The induction of persister formation increased the number of viable cells after UV treatment. The toxin-antitoxin system gene hipA (high persistence A) is a key factor in persister cell formation. We observed that hipA was strongly expressed in the stationary phase cells, while regrowth cells after UV treatment lost hipA expression, suggesting that the regrowth cells lost their persistence. Compared to UV batch radiation, we demonstrated that intermittent UV irradiation, which included the induction of regrowth between UV treatments, significantly reduced the number of viable E. coli cells.

The expression of alternative sigma-I7 factor induces the transcription of cellulosomal genes in the cellulolytic bacterium Clostridium thermocellum.

The composition of cellulosomal carbohydrate-active enzymes (CAZymes) secreted from a cellulolytic bacterium Clostridium thermocellum varies depending on the cellulosic substrate used during cultivation. C. thermocellum detects the polysaccharides in cellulosic material via anti-sigma factors and expresses the appropriate CAZyme gene via alternative sigma factors, SigIs. Previous studies on the regulation of CAZyme gene expression via SigIs in C. thermocellum have been conducted in vitro or in a heterologous host, because of the limited genetic tools available for C. thermocellum. To characterize the in vivo function of SigIs, in the present study, we established a sigI7 gene expression strain of C. thermocellum. Transcriptome analysis of this strain revealed that SigI7 induced the expression of cellulosomal CAZyme genes and cellulosomal scaffold genes. However, there was a decrease in the degradation ability of the exoproteome from the sigI7 expression strain; the product of the downregulated gene, Clo1313_1002, rescued the activity of the C. thermocellum exoproteome from the sigI7 expression strain. In this study, we demonstrate the in vivo function of SigI7 and discuss the CAZymes that are important for cellulosic biomass degradation by C. thermocellum.

Metabolome Analysis of Constituents in Membrane Vesicles for Clostridium thermocellum Growth Stimulation.

The cultivation of the cellulolytic bacterium, Clostridium thermocellum, can have cost-effective cellulosic biomass utilizations, such as consolidated bioprocessing, simultaneous biological enzyme production and saccharification. However, these processes require a longer cultivation term of approximately 1 week. We demonstrate that constituents of the C. thermocellum membrane vesicle fraction significantly promoted the growth rate of C. thermocellum. Similarly, cell-free Bacillus subtilis broth was able to increase C. thermocellum growth rate, while several B. subtilis single-gene deletion mutants, e.g., yxeJ, yxeH, ahpC, yxdK, iolF, decreased the growth stimulation ability. Metabolome analysis revealed signal compounds for cell-cell communication in the C. thermocellum membrane vesicle fraction (ethyl 2-decenoate, ethyl 4-decenoate, and 2-dodecenoic acid) and B. subtilis broth (nicotinamide, indole-3-carboxaldehyde, urocanic acid, nopaline, and 6-paradol). These findings suggest that the constituents in membrane vesicles from C. thermocellum and B. subtilis could promote C. thermocellum growth, leading to improved efficiency of cellulosic biomass utilization.

Using SPME-GC/REMPI-TOFMS to Measure the Volatile Odor-Active Compounds in Freshly Cooked Rice.

The volatile odor-active compounds of cooked rice were evaluated using a method that combined solid-phase microextraction (SPME) with gas chromatography-resonance-enhanced multiphoton ionization time-of-flight mass spectrometry (GC/REMPI-TOFMS). An SPME fiber was held at the upper levels of the cooked rice and given an extraction time of 5 min. By using a nanosecond ultraviolet (266 nm) pulsed laser for ionization, two compounds, 4-vinylphenol and indole, which are considered to be important for the characteristic flavor of cooked rice, could be detected from all types of cultivars measured in the present study-nonglutinous rice, glutinous rice, and aromatic rice. In the case of fresh nonglutinous rice, the amounts of introduction for 4-vinylphenol and indole to GC were ca. 70 and 20 pg, respectively. While both peak areas decreased with increases in the time needed to maintain warmth, the decreasing behaviors differed slightly with a noteworthy rapid decrease for indole. For nonglutinous rice, the peak areas for 4-vinylphenol were almost the same, whether it was fresh (measured within 1 month from harvest) or aged (measured 6-12 months after harvest), but those of indole significantly decreased following storage. We also found differences among cultivars: the peak area for 4-vinylphenol in nonglutinous rice was somewhat strong; the peak area for indole was intensely strong in glutinous rice; however, the peak areas for both 4-vinylphenol and indole were weak in aromatic rice. Volatile odor-active compounds were detected in a sensitive and time-resolved manner; therefore, the proposed method could be useful for differentiating varieties of cooked rice from the viewpoints of cooking conditions, freshness, and cultivar types.

Biocompatibility of a polymer-coated membrane possessing a hydrophilic blood-contacting layer: Adsorption-related assessment.

OBJECTIVE: Currently, the foreign surfaces of extracorporeal circulation devices are coated with an acrylate-based copolymer that creates a hydrophilic blood-contacting layer to enhance biocompatibility. Several reports of acrylate-based copolymer with respect to biocompatibility have been published; however, the adsorption of peptide compounds on acrylate-based copolymer-coated membranes still requires clarity. In this study, we aimed to understand the adsorption of several peptide compounds of various molecular weights, including albumin, lysozyme, and vancomycin, on acrylate-based copolymer-coated membranes using in vitro studies. METHODS: Six experimental circuits consisting of acrylate-based copolymer-coated tubes and membranes, and six comprising acrylate-based copolymer-coated tubes and non-coated membranes were prepared for comparison. An experimental solution, composed of albumin, lysozyme, vancomycin, and saline, was continuously stirred in a reservoir, recirculated in each experimental circuit, and then filtered. Concentrations of albumin, lysozyme, and vancomycin were measured after 0, 15, 30, 45, 60, 90, and 120 min of recirculation. Similar experiments were performed in all the prepared circuits. RESULTS: The ratio of measured values at each time point to those at 0 min was not significantly different between acrylate-based copolymer-coated and non-coated membranes for albumin and lysozyme, but differed significantly for vancomycin; the ratios were higher in acrylate-based copolymer-coated than in non-coated membranes. CONCLUSION: This study suggests that albumin is not adsorbed on either acrylate-based copolymer-coated or non-coated membranes, that lysozyme is not adsorbed on either membrane or is adsorbed at a similar rate on both membranes, and that vancomycin is less adsorbed on acrylate-based copolymer-coated membranes. Thus, acrylate-based copolymer coating could inhibit the adsorption of various peptide compounds.

Cellulosomes localise on the surface of membrane vesicles from the cellulolytic bacterium Clostridium thermocellum.

Membrane vesicles released from bacteria contribute to cell-cell communication by carrying various cargos such as proteins, nucleic acids and signaling molecules. Cellulolytic bacteria have been isolated from many environments, yet the function of membrane vesicles for cellulolytic ability has been rarely described. Here, we show that a Gram-positive cellulolytic bacterium Clostridium thermocellum released membrane vesicles, each approximately 50-300 nm in diameter, into the broth. The observations with immunoelectron microscopy also revealed that cellulosomes, which are carbohydrate-active enzyme complexes that give C. thermocellum high cellulolytic activity, localized on the surface of the membrane vesicles. The membrane vesicles collected by ultracentrifugation maintained the cellulolytic activity. Supplementation with the biosurfactant surfactin or sonication treatment disrupted the membrane vesicles in the exoproteome of C. thermocellum and significantly decreased the degradation activity of the exoproteome for microcrystalline cellulose. However, these did not affect the degradation activity for soluble carboxymethyl cellulose. These results suggest a novel function of membrane vesicles: C. thermocellum releases cellulolytic enzymes on the surface of membrane vesicles to enhance the cellulolytic activity of C. thermocellum for crystalline cellulose.

Physcomitrella STEMIN transcription factor induces stem cell formation with epigenetic reprogramming.

Epigenetic modifications, including histone modifications, stabilize cell-specific gene expression programmes to maintain cell identities in both metazoans and land plants1-3. Notwithstanding the existence of these stable cell states, in land plants, stem cells are formed from differentiated cells during post-embryonic development and regeneration4-6, indicating that land plants have an intrinsic ability to regulate epigenetic memory to initiate a new gene regulatory network. However, it is less well understood how epigenetic modifications are locally regulated to influence the specific genes necessary for cellular changes without affecting other genes in a genome. In this study, we found that ectopic induction of the AP2/ERF transcription factor STEMIN1 in leaf cells of the moss Physcomitrella patens decreases a repressive chromatin mark, histone H3 lysine 27 trimethylation (H3K27me3), on its direct target genes before cell division, resulting in the conversion of leaf cells to chloronema apical stem cells. STEMIN1 and its homologues positively regulate the formation of secondary chloronema apical stem cells from chloronema cells during development. Our results suggest that STEMIN1 functions within an intrinsic mechanism underlying local H3K27me3 reprogramming to initiate stem cell formation.

Glucose production from cellulose through biological simultaneous enzyme production and saccharification using recombinant bacteria expressing the ß-glucosidase gene.

Efficient cellulosic biomass saccharification technologies are required to meet biorefinery standards. Biological simultaneous enzyme production and saccharification (BSES), which is glucose production from cellulosic biomass by Clostridium thermocellum, can be a reliable cellulose saccharification technology for biorefineries. However, the current BSES processes require purified ß-glucosidase supplementation. In this study, recombinant bacteria expressing the ß-glucosidase gene were developed and directly applied to BSES. The engineered Escherichia coli expressing the thermostable ß-glucosidase gene from Thermoanaerobacter brockii exhibited 0.5 U/ml of ß-glucosidase activities. The signal peptide sequence of lytF gene from Bacillus subtilis was the most appropriate for the ß-glucosidase secretion from Brevibacillus choshinensis, and the broth exhibited 0.74 U/ml of ß-glucosidase activities. The engineered E. coli and B. choshinensis expressing the thermostable ß-glucosidase gene produced 47.4 g/L glucose and 49.4 g/L glucose, respectively. Glucose was produced by the hydrolysis of 100 g/L Avicel cellulose for 10 days through BSES, and the product yield was similar to that obtained through BSES with purified ß-glucosidase supplementation. Our findings indicate that the direct supplementation of ß-glucosidase using bacterial cells expressing ß-glucosidase gene or their broth was applicable to BSES, suggesting the potential of this process as a cost-effective approach to cellulose saccharification.

Characterization of lignocellulose particles during lignocellulose solubilization by Clostridium thermocellum.

Clostridium thermocellum is a candidate bacterium for lignocellulose utilization due to its efficient lignocellulose solubilization ability. It has been reported that C. thermocellum efficiently degrades purified cellulose substrates, but cannot completely degrade milled lignocellulose powders. Evaluation of cellulose and hemicellulose contents in a lignocellulose residue after the cultivation of C. thermocellum indicated that C. thermocellum degraded cellulose and hemicellulose equally. Microscopic observations demonstrated that C. thermocellum significantly degraded small-sized lignocellulose particles, but it only partially degraded the larger sized particles. The lignin content of the large-sized particles was higher than that of the small particles. The remained large-sized particles included vascular tissues. These results suggest that the lignified structures such as vascular tissues in milled lignocellulose were less susceptible to bacterial lignocellulose solubilization.

Single- and double-walled carbon nanotubes enhance atherosclerogenesis by promoting monocyte adhesion to endothelial cells and endothelial progenitor cell dysfunction.

BACKGROUND: The use of carbon nanotubes has increased lately. However, the cardiovascular effect of exposure to carbon nanotubes remains elusive. The present study investigated the effects of pulmonary exposure to single-walled carbon nanotubes (SWCNTs) and double-walled carbon nanotubes (DWCNTs) on atherosclerogenesis using normal human aortic endothelial cells (HAECs) and apolipoprotein E-deficient (ApoE-/-) mice, a model of human atherosclerosis. METHODS: HAECs were cultured and exposed to SWCNTs or DWCNTs for 16 h. ApoE-/- mice were exposed to SWCNTs or DWCNTs (10 or 40 µg/mouse) once every other week for 10 weeks by pharyngeal aspiration. RESULTS: Exposure to CNTs increased the expression level of adhesion molecule (ICAM-1) and enhanced THP-1 monocyte adhesion to HAECs. ApoE-/- mice exposed to CNTs showed increased plaque area in the aorta by oil red O staining and up-regulation of ICAM-1 expression in the aorta, compared with vehicle-treated ApoE-/- mice. Endothelial progenitor cells (EPCs) are mobilized from the bone marrow into the circulation and subsequently migrate to the site of endothelial damage and repair. Exposure of ApoE-/- mice to high-dose SWCNTs or DWCNTs reduced the colony-forming units of EPCs in the bone marrow and diminished their migration function. CONCLUSION: The results suggested that SWCNTs and DWCNTs enhanced atherosclerogenesis by promoting monocyte adhesion to endothelial cells and inducing EPC dysfunction.

Carbohydrate-binding modules influence substrate specificity of an endoglucanase from Clostridium thermocellum.

Most cellulases contain carbohydrate-binding modules (CBMs) that largely contribute to their activity for insoluble substrates. Clostridium thermocellum Cel5E is an endoglucanase having xylanolytic activity. The Cel5E originally has a family 11 CBM preferentially binding to ß-1,4- and ß-1,3-1,4-mixed linkage glucans. In this study, we replaced the CBM with a different type of CBM, either a family 3 microcrystalline cellulose-directed CBM from Clostridium josui scaffoldin, or a family 6 xylan-directed CBM from Clostridium stercorarium xylanase 11A. Chimeric endoglucanases showed enhanced activity that was affected by CBM binding specificity. These chimeric enzymes could efficiently degrade milled lignocellulosic materials, such as corn hulls, because of heterologous components in the plant cell wall, indicating that diverse CBMs play roles in degradation of lignocellulosic materials.

Bacterial production and secretion of water-insoluble fuel compounds from cellulose without the supplementation of cellulases.

Achieving economic biofuel production from cellulosic biomass will require significant cost reductions. Enzymatic degradation of cellulosic biomass and distillation of water-soluble fuel compounds substantially increase the cost of biofuel production. Consolidated bioprocessing is a strategy to circumvent expensive biofuel production steps. Clostridium thermocellum is a promising bacterium for consolidated bioprocessing because it does not require the supplementation of lignocellulose-degrading enzymes. To produce water-insoluble fuel compounds, C. thermocellum was engineered to express a fatty acyl-acyl carrier protein reductase and an aldehyde-deformylating oxygenase from Synechococcus elongatus PCC 7942. Expression of the aldehyde-deformylating oxygenase gene was clearly detected, whereas only slight expression of the fatty acyl-acyl carrier protein reductase gene was detected. Cells expressing the fatty acyl-acyl carrier protein reductase and the aldehyde-deformylating oxygenase accumulated fatty aldehydes (higher alcohol precursors). After cultivation with cellulose, the higher alcohols, decanol and dodecanol, were detected in the organic solvent phase of the culture broth, indicating that the strain secreted the higher alcohols. These results suggest that the engineered C. thermocellum strain, expressing fatty acyl-acyl carrier protein reductase and aldehyde-deformylating oxygenase genes, directly produces and secretes higher alcohols from cellulose without the supplementation of cellulases. The higher alcohols can be collected by phase separation.

Cellulosomal carbohydrate-binding module from Clostridium josui binds to crystalline and non-crystalline cellulose, and soluble polysaccharides.

To understand the lignocellulose degradation activity of the Clostridium josui cellulosome, a carbohydrate-binding module of the scaffoldin CjCBM3 was characterized. CjCBM3 shows binding to crystalline cellulose, non-crystalline cellulose and soluble polysaccharides. The binding isotherm of CjCBM3 to acid-swollen cellulose is best fitted by the Langmuir two-site model, suggesting that there are two CjCBM3 binding sites on acid-swollen cellulose with different affinities. The second site shows lower affinity and larger binding capacity, suggesting that the cellulosome is directly targeted to the cellulose surface with high affinity, where larger amounts of the cellulosome bind to cellulose with low affinity.

EEG coherence analysis in never-medicated patients with panic disorder.

To investigate the functional abnormalities in the central nervous system (CNS) of patients with panic disorder (PD), we compared the electroencephalography (EEG) coherence values in 18 never-medicated PD patients with those in age-matched normal control subjects, and examined the relationships between EEG coherence values and both the duration of disease and the severity of panic attacks. EEG data were recorded in the resting state. The PD patients had lower coherence values with significant differences in F3-F4, C3-C4, P3-P4, F7-T5, and F8-T6. There were positive correlations for the higher alpha band between coherence values and both the duration of disease and the severity of panic attacks. These findings provide further evidence that PD patients have a lower degree of inter-hemispheric functional connectivity in the frontal region and intra-hemispheric functional connectivity in the bilateral temporal region, and that chronic condition or frequent panic attacks in PD patients may be related to the pathophysiological CNS changes.