Relationship Between Rumen Microbial Composition and Fibrolytic Isozyme Activity During the Biodegradation of Rice Straw Powder Using Rumen Fluid.

Rumen fibrolytic microorganisms have been used to increase the rate of lignocellulosic biomass biodegradation; however, the microbial and isozymatic characteristics of biodegradation remain unclear. Therefore, the present study investigated the relationship between rumen microorganisms and fibrolytic isozymes associated with lignocellulosic biomass hydrolysis. Rice straw, a widely available agricultural byproduct, was ground and used as a substrate. The biodegradation of rice straw powder was performed anaerobically in rumen fluid for 48 h. The results obtained revealed that 31.6 and 23.3% of cellulose and hemicellulose, respectively, were degraded. The total concentration of volatile fatty acids showed a 1.8-fold increase (from 85.4 to 151.6| |mM) in 48 h, and 1,230.1| |mL L-1 of CO2 and 523.5| |mL L-1 of CH4 were produced. The major isozymes identified by zymograms during the first 12| |h were 51- and 140-kDa carboxymethyl cellulases (CMCases) and 23- and 57-kDa xylanases. The band densities of 37-, 53-, and 58-kDa CMCases and 38-, 44-, and 130-kDa xylanases increased from 24 to 36 h. A microbial ana-lysis indicated that the relative abundances of Prevotella, Fibrobacter, and Bacteroidales RF16 bacteria, Neocallimastix and Cyllamyces fungi, and Dasytricha and Polyplastron protozoa were related to fibrolytic isozyme activity. The present results provide novel insights into the relationships between fibrolytic isozymes and rumen microorganisms during lignocellulose biodegradation.

Effect of body posture on stability and balance of occlusal contacts.

OBJECTIVE: To evaluate the effect of body posture on occlusal contact. METHODS: A total of 30 healthy subjects were evaluated. T-Scan™ III was used to analyze the center of occlusal force (COF) and occlusal force distribution while subjects remained supine (SP), upright sitting with the head fixed (UP-HFI), upright sitting with the head free (UP-HFR), and natural standing (NS). RESULTS: The total trajectory length of COF was significantly longer in NS than in SP, UP-HFI, and UP-HFR. The COF area was significantly larger in UP-HFR than in SP and UP-HFI and also significantly larger in NS than in SP, UP-HFI, and UP-HFR. The anteroposterior occlusal force distribution (AOD) in NS shifted significantly forward, compared to SP, UP-HFI, and UP-HFR. AOD in UP-HFI and UP-HFR shifted significantly forward, compared to the SP position. CONCLUSION: Changes in body posture affect the stability and anteroposterior balance of occlusal contacts.

Change of Endoglucanase Activity and Rumen Microbial Community During Biodegradation of Cellulose Using Rumen Microbiota.

Treatment with rumen microorganisms improves the methane fermentation of undegradable lignocellulosic biomass; however, the role of endoglucanase in lignocellulose digestion remains unclear. This study was conducted to investigate endoglucanases contributing to cellulose degradation during treatment with rumen microorganisms, using carboxymethyl cellulose (CMC) as a substrate. The rate of CMC degradation increased for the first 24 h of treatment. Zymogram analysis revealed that endoglucanases of 52 and 53 kDa exhibited high enzyme activity for the first 12 h, whereas endoglucanases of 42, 50, and 101 kDa exhibited high enzyme activities from 12 to 24 h. This indicates that the activities of these five endoglucanases shifted and contributed to efficient CMC degradation. Metagenomic analysis revealed that the relative abundances of Selenomonas, Eudiplodinium, and Metadinium decreased after 12 h, which was positively correlated with the 52- and 53-kDa endoglucanases. Additionally, the relative abundances of Porphyromonas, Didinium, unclassified Bacteroidetes, Clostridiales family XI, Lachnospiraceae and Sphingobacteriaceae increased for the first 24 h, which was positively correlated with endoglucanases of 42, 50, and 101 kDa. This study suggests that uncharacterized and non-dominant microorganisms produce and/or contribute to activity of 40, 50, 52, 53, and 101 kDa endoglucanases, enhancing CMC degradation during treatment with rumen microorganisms.

Biological oxidation of Mn(II) coupled with nitrification for removal and recovery of minor metals by downflow hanging sponge reactor.

Biogenic manganese oxides (bio-MnO2) have been shown to absorb minor metals. Bioreactor cultivation of heterotrophic manganese oxidizing bacteria (MnOB), which produce bio-MnO2 via oxidation of Mn (II), can be expected to be involved in a promising system for removal and recovery of minor metals from wastewater. However, MnOB enrichment in wastewater treatment is difficult. This study investigated whether MnOB can be cultivated when coupled with nitrification in a system in which soluble microbial products (SMP) from nitrifiers are provided to MnOB as a substrate. A downflow hanging sponge (DHS) reactor was applied for MnOB cultivation with ammonium (NH4⁺) and Mn (II) continuously supplied. During long-term operation, Mn (II) oxidation was successfully established at a rate of 48 g Mn m⁻³ d⁻¹ and bio-MnO2 that formed on the sponges were recovered from the bottom of the reactor. The results also revealed that Ni and Co added to the influent were simultaneously removed. Microbial 16S rRNA gene clone analysis identified nitrifiers supporting MnOB growth and showed that only one clone of Bacillus subtilis, which was affiliated with a known MnOB cluster, was present, suggesting the existence of other novel bacteria with the ability to oxidize Mn (II).

Community composition of known and uncultured archaeal lineages in anaerobic or anoxic wastewater treatment sludge.

Microbial systems are widely used to treat different types of wastewater from domestic, agricultural, and industrial sources. Community composition is an important factor in determining the successful performance of microbial treatment systems; however, a variety of uncultured and unknown lineages exist in sludge that requires identification and characterization. The present study examined the archaeal community composition in methanogenic, denitrifying, and nitrogen-/phosphate-removing wastewater treatment sludge by Archaea-specific 16S rRNA gene sequencing analysis using Illumina sequencing technology. Phylotypes belonging to Euryarchaeota, including methanogens, were most abundant in all samples except for nitrogen-/phosphate-removing wastewater treatment sludge. High levels of Deep Sea Hydrothermal Vent Group 6 (DHVEG-6), WSA2, Terrestrial Miscellaneous Euryarchaeotal Group, and Miscellaneous Crenarchaeotic Group were also detected. Interestingly, DHVEG-6 was dominant in nitrogen-/phosphate-removing wastewater treatment sludge, indicating that unclear lineages of Archaea still exist in the anaerobic wastewater treatment sludges. These results reveal a previously unknown diversity of Archaea in sludge that can potentially be exploited for the development of more efficient wastewater treatment strategies.

Phosphate recovery as concentrated solution from treated wastewater by a PAO-enriched biofilm reactor.

Phosphorus recovery from wastewaters and its recycling are of importance for sustaining agricultural production. During the conventional enhanced biological phosphorus removal process, phosphorus is removed by withdrawing excess sludge from wastewater. However, excess sludge disposal is costly and energy intensive. A proposed novel process for phosphorus recovery from sewage treatment will result in no excess sludge if a polyphosphate accumulating organisms (PAOs) enrichment biofilm can be applied to effluents containing phosphate. This process allows the recovery of phosphate as phosphate-concentrated solutions by controlling PAOs to absorb and release phosphate. A reactor consisting of a modified trickling filter with a synthetic substrate (5 mg P L⁻¹) was operated to form a PAO-enriched biofilm. As a result of the enrichment, the concentration of phosphate of >100 mg P L⁻¹ was successfully achieved. During this experiment, no sludge withdrawal was carried out over the duration of the operation of 255 days. To highlight the new process, the principle of enriching PAOs on biofilm and concentrating phosphate from treated sewage is explained, and a discussion on phosphate recovery performance is given.

Oxygen isotopic compositions of asteroidal materials returned from Itokawa by the Hayabusa mission.

Meteorite studies suggest that each solar system object has a unique oxygen isotopic composition. Chondrites, the most primitive of meteorites, have been believed to be derived from asteroids, but oxygen isotopic compositions of asteroids themselves have not been established. We measured, using secondary ion mass spectrometry, oxygen isotopic compositions of rock particles from asteroid 25143 Itokawa returned by the Hayabusa spacecraft. Compositions of the particles are depleted in (16)O relative to terrestrial materials and indicate that Itokawa, an S-type asteroid, is one of the sources of the LL or L group of equilibrated ordinary chondrites. This is a direct oxygen-isotope link between chondrites and their parent asteroid.

Anti-ulcer agent teprenone inhibits hepatitis C virus replication: potential treatment for hepatitis C.

BACKGROUND: Previously we reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, inhibited hepatitis C virus (HCV) RNA replication. Furthermore, recent reports revealed that the statins are associated with a reduced risk of hepatocellular carcinoma and lower portal pressure in patients with cirrhosis. The statins exhibited anti-HCV activity by inhibiting geranylgeranylation of host proteins essential for HCV RNA replication. Geranylgeranyl pyrophosphate (GGPP) is a substrate for geranylgeranyltransferase. Therefore, we examined the potential of geranyl compounds with chemical structures similar to those of GGPP to inhibit HCV RNA replication. METHODS: We tested geranyl compounds [geranylgeraniol, geranylgeranoic acid, vitamin K(2) and teprenone (Selbex)] for their effects on HCV RNA replication using genome-length HCV RNA-replicating cells (the OR6 assay system) and a JFH-1 infection cell culture system. Teprenone is the major component of the anti-ulcer agent, Selbex. We also examined the anti-HCV activities of the geranyl compounds in combination with interferon (IFN)-α or statins. RESULTS: Among the geranyl compounds tested, only teprenone exhibited anti-HCV activity at a clinically achievable concentration. However, other anti-ulcer agents tested had no inhibitory effect on HCV RNA replication. The combination of teprenone and IFN-α exhibited a strong inhibitory effect on HCV RNA replication. Although teprenone alone did not inhibit geranylgeranylation, surprisingly, statins' inhibitory action against geranylgeranylation was enhanced by cotreatment with teprenone. CONCLUSIONS: The anti-ulcer agent teprenone inhibited HCV RNA replication and enhanced statins' inhibitory action against geranylgeranylation. This newly discovered function of teprenone may improve the treatment of HCV-associated liver diseases as an adjuvant to statins.

Removal of organic substances and oxidation of ammonium nitrogen by a down-flow hanging sponge (DHS) reactor under high salinity conditions.

A down-flow hanging sponge (DHS) reactor, constructed by connecting three identical treatment units in series, was fed with highly saline artificial coke-plant wastewater containing 1400 mg L(-1) of phenol in terms of chemical oxygen demand (COD) and 500 mg-NL(-1) of ammonium nitrogen. The COD was removed by the 1st unit, achieving 92% removal at an average COD loading rate of 3.0 kg-COD m(-3)d(-1) for all units, with oxidation of ammonium nitrogen occurring primarily in the two downstream units. Microbial assays of the different units of the reactor revealed greater numbers of nitrifying bacteria in the 2nd and 3rd units than in the 1st unit, corresponding with the observed ammonium oxidation pattern of the reactor. These findings suggest that a succession of microflora was successfully established along the DHS.

Efficient replication systems for hepatitis C virus using a new human hepatoma cell line.

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a serious global health problem. Cell culture-based persistent HCV RNA replication systems and infectious HCV production systems are widely used in HCV research. However, persistent HCV production systems have been developed only for HuH-7 hepatoma cells. Here we found a new human hepatoma cell line, Li23, that enables persistent HCV production and anti-HCV reagent assay. Li23's cDNA expression profile differed from HuH-7's, although the two cells had similar liver-specific expression profiles. We used HCV RNA with a specific combination of adaptive mutations to develop an HCV replicon system and genome-length HCV RNA replicating systems including a reporter assay system. Finally, Li23-derived cells persistently produced infectious virus of an HCV strain. Li23-derived cells are potentially useful for understanding the HCV life cycle and for finding antiviral targets.

HCV genotype 1b chimeric replicon with NS5B of JFH-1 exhibited resistance to cyclosporine A.

Cyclosporine A (CsA) is a well-characterized anti-HCV reagent. Recently it was reported that the genotype 2a JFH-1 strain was more resistant than genotype 1 HCV strains to CsA in a cell culture system. However, the JFH-1 responsible region for the resistance to CsA remains unclear. It was also demonstrated that in genotype 1b HCVs, NS5B interacts with cyclophilin (CyP). To clarify whether or not NS5B of JFH-1 is significant for CsA resistance, we developed a chimeric replicon with NS5B of JFH-1 in the genotype 1b backbone. The chimeric replicon was more resistant to CsA than the parental genotype 1b replicon. Furthermore, reduction of CyPA had a greater effect on HCV RNA replication and sensitivity to CsA than reduction of CyPB. Here, we demonstrated that NS5B of JFH-1 contributed to this strain's CsA-resistant phenotype. NS5B and CyPA are significant for determining HCV's sensitivity to CsA.

Oxidative stress induces anti-hepatitis C virus status via the activation of extracellular signal-regulated kinase.

UNLABELLED: Recently, we reported that beta-carotene, vitamin D(2), and linoleic acid inhibited hepatitis C virus (HCV) RNA replication in hepatoma cells. Interestingly, in the course of the study, we found that the antioxidant vitamin E negated the anti-HCV activities of these nutrients. These results suggest that the oxidative stress caused by the three nutrients is involved in their anti-HCV activities. However, the molecular mechanism by which oxidative stress induces anti-HCV status remains unknown. Oxidative stress is also known to activate extracellular signal-regulated kinase (ERK). Therefore, we hypothesized that oxidative stress induces anti-HCV status via the mitogen activated protein kinase (MAPK)/ERK kinase (MEK)-ERK1/2 signaling pathway. In this study, we found that the MEK1/2-specific inhibitor U0126 abolished the anti-HCV activities of the three nutrients in a dose-dependent manner. Moreover, U0126 significantly attenuated the anti-HCV activities of polyunsaturated fatty acids, interferon-gamma, and cyclosporine A, but not statins. We further demonstrated that, with the exception of the statins, all of these anti-HCV nutrients and reagents actually induced activation of the MEK-ERK1/2 signaling pathway, which was inhibited or reduced by treatment not only with U0126 but also with vitamin E. We also demonstrated that phosphorylation of ERK1/2 by cyclosporine A was attenuated with N-acetylcysteine treatment and led to the negation of inhibition of HCV RNA replication. We propose that a cellular process that follows ERK1/2 phosphorylation and is specific to oxidative stimulation might lead to down-regulation of HCV RNA replication. CONCLUSION: Our results demonstrate the involvement of the MEK-ERK1/2 signaling pathway in the anti-HCV status induced by oxidative stress in a broad range of anti-HCV reagents. This intracellular modulation is expected to be a therapeutic target for the suppression of HCV RNA replication.

Development of a hepatitis C virus relapse model using genome-length hepatitis C virus ribonucleic acid-harboring cells possessing the interferon-alpha-resistance phenotype.

AIM: The cure rate of current interferon (IFN) therapy is limited to approximately 50% and most of the relapses after therapy are caused by genotype-1. To develop a relapse model in cell culture, we attempted to obtain genome-length hepatitis C virus ribonucleic acid (HCV RNA) harboring cells possessing the IFN-alpha-resistance phenotype from previously established OR6 cells, which enabled the luciferase reporter assay for monitoring of HCV RNA replication. METHODS: The IFN-alpha-resistant HCV RNA-harboring cells and control cells were obtained by the treatment of OR6 cells with and without IFN-alpha, respectively. Then, we examined the relapse of HCV in IFN-alpha-resistant HCV RNA-harboring cells. RESULTS: Only type I IFN (alpha and beta) showed significantly different anti-HCV activity between IFN-alpha-resistant HCV RNA-harboring cells and control cells. There was no significant difference in the anti-HCV activity of IFN-gamma, fluvastatin, or cyclosporine A between the two types of cells. Furthermore, we showed that fluvastatin or cyclosporine A in combination with IFN-alpha could prevent the relapse after therapy in the IFN-alpha-resistant HCV RNA-harboring cells. CONCLUSION: We developed a HCV relapse model in cell culture using IFN-alpha-resistant HCV RNA-harboring cells. Thus anti-HCV reagents, which have a mechanism different from IFN-alpha, were shown to be useful for preventing a relapse of IFN-alpha-resistant HCV.

Quasi-solid-state optical logic devices based on redox polymer nanosheet assembly.

We report fabrication of a quasi-solid-state optical logic gate based on a polymer nanosheet photodiode. Two amphiphilic copolymers, p(DDA/Ru) and p(DDA/Fc), which respectively have a ruthenium dipyridyl complex and a ferrocene derivative as a redox molecule, are synthesized to produce redox polymer nanosheets. To construct polymer nanosheet photodiodes, two redox polymer nanosheets were assembled onto an indium-tinoxide (ITO) electrode in a tailor-made manner. The ITO electrode was connected with a counter electrode using an agarose gel electrolyte. The simple two-electrode system performs as a quasi-solid-state polymer nanosheet photodiode (QS-PNP). The photocurrent flow direction of QS-PNP can be controlled to be anodic or be cathodic by changing the deposition order of the redox polymer nanosheets. The anodic and cathodic QS-PNPs were applied to construct optical OR and XOR logic gates. The OR logic gate was fabricated by connecting the anodic and cathodic QS-PNP in a series; XOR logic was fabricated by connecting two anodic QS-PNPs in series. In optical logic gates, excitations of p(DDA/Ru) were used as input signals, and photocurrent was used as an output signal. These optical logic gates operate in a quasi-solid-state in a simple two-electrode configuration, which facilitates integration of the logic elements.

Flexible structure multiple modeling using irregular self-organizing maps neural network.

The MMSOM identification method, which had been presented by the authors, is improved to the multiple modeling by the irregular self-organizing map (MMISOM) using the irregular SOM (ISOM). Inputs to the neural networks are parameters of the instantaneous model computed adaptively at every instant. The neural network learns these models. The reference vectors of its output nodes are estimation of the parameters of the local models. At every instant, the model with closest output to the plant output is selected as the model of the plant. ISOM used in this paper is a graph of all the nodes and some of the weighted links between them to make a minimum spanning tree graph. It is shown in this paper that it is possible to add new models if the number of models is initially less than the appropriate one. The MMISOM shows more flexibility to cover the linear model space of the plant when the space is concave.

The DNA damage sensors ataxia-telangiectasia mutated kinase and checkpoint kinase 2 are required for hepatitis C virus RNA replication.

Cellular responses to DNA damage are crucial for maintaining genome integrity, virus infection, and preventing the development of cancer. Hepatitis C virus (HCV) infection and the expression of the HCV nonstructural protein NS3 and core protein have been proposed as factors involved in the induction of double-stranded DNA breaks and enhancement of the mutation frequency of cellular genes. Since DNA damage sensors, such as the ataxia-telangiectasia mutated kinase (ATM), ATM- and Rad3-related kinase (ATR), poly(ADP-ribose) polymerase 1 (PARP-1), and checkpoint kinase 2 (Chk2), play central roles in the response to genotoxic stress, we hypothesized that these sensors might affect HCV replication. To test this hypothesis, we examined the level of HCV RNA in HuH-7-derived cells stably expressing short hairpin RNA targeted to ATM, ATR, PARP-1, or Chk2. Consequently, we found that replication of both genome-length HCV RNA (HCV-O, genotype 1b) and the subgenomic replicon RNA were notably suppressed in ATM- or Chk2-knockdown cells. In addition, the RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were suppressed in these knockdown cells after inoculation of the cell culture-generated HCV. Consistent with these observations, ATM kinase inhibitor could suppress the HCV RNA replication. Furthermore, we observed that HCV NS3-NS4A interacted with ATM and that HCV NS5B interacted with both ATM and Chk2. Taken together, these results suggest that the ATM signaling pathway is critical for HCV RNA replication and may represent a novel target for the clinical treatment of patients with chronic hepatitis C.

Flexible structure multiple modeling using irregular self-organizing maps neural network.

The MMSOM identification method, which had been presented by the authors, is improved to the multiple modeling by the irregular self-organizing map (MMISOM) using the irregular SOM (ISOM). Inputs to the neural networks are parameters of the instantaneous model computed adaptively at every instant. The neural network learns these models. The reference vectors of its output nodes are estimation of the parameters of the local models. At every instant, the model with closest output to the plant output is selected as the model of the plant. ISOM used in this paper is a graph of all the nodes and some of the weighted links between them to make a minimum spanning tree graph. It is shown in this paper that it is possible to add new models if the number of models is initially less than the appropriate one. The MMISOM shows more flexibility to cover the linear model space of the plant when the space is concave.

A new living cell-based assay system for monitoring genome-length hepatitis C virus RNA replication.

We previously developed a cell-based luciferase reporter assay system for monitoring genome-length hepatitis C virus (HCV) RNA replication (OR6 assay system). Here, we aimed to develop a new living cell-based reporter assay system using enhanced green fluorescent protein (EGFP). Genome-length HCV RNAs encoding EGFP were introduced into a subline of HuH-7 cells and G418 selection was performed. One cloned cell line, OGF7, was successfully selected from among the several G418-resistant cell lines obtained, and the robust expression of HCV RNA and proteins in OGF7 cells was confirmed. The fluorescent intensity of OGF7 cells was decreased by interferon-alpha treatment in a dose-dependent manner, and it correlated well with the HCV RNA concentration. We demonstrated that the interferon-alpha sensitivity in the OGF7 assay system measuring the fluorescent intensity was equivalent to that of the OR6 assay system, and that the OGF7 assay system was useful for quantitative evaluation of anti-HCV reagents. The OGF7 assay system is expected to be the most time-saving and inexpensive assay system for high-throughput screening of anti-HCV reagents.

A novel approach to sustained pseudoephedrine release: differentially coated mini-tablets in HPMC capsules.

We developed and optimized a novel pseudoephedrine hydrochloride (PSE) sustained-release dosage form. The system comprises immediate-release mini-tablets (IRMT) and sustained-release mini-tablets (SRMT) contained in a hydroxypropyl methylcellulose (HPMC) capsule. The IRMT contained PSE, excipients and low-substituted hydroxypropyl cellulose (a disintegrant), and the tablets were coated with HPMC, a water-soluble polymer. IRMT prepared with varying amounts of low-substituted hydroxypropyl cellulose all dissolved completely within the first 60min, so low-substituted hydroxypropyl cellulose content does not greatly influence PSE release. The SRMT contained only PSE and excipients, and were coated with a mixture of HPMC and the water-insoluble polymer ethylcellulose. The PSE release profile for the SRMT could be controlled by varying the thickness of the coat, and the lag time could be controlled by varying the amount of ethylcellulose present in the polymer coat. PSE was released immediately from our encapsulated mini-tablet system and release was sustained over an extended period of time: the PSE in the IRMT dissolved within 60min, whereas the PSE in the SRMT was released over 8-10h. This system can be modified to yield various extended drug-release profiles, thereby harnessing the benefits of both SRMT and IRMT.