Improving the fermentable sugar yields of wheat straw by high-temperature pre-hydrolysis with thermophilic enzymes of Malbranchea cinnamomea.

BACKGROUND: Enzymatic hydrolysis is a key step in the conversion of lignocellulosic polysaccharides to fermentable sugars for the production of biofuels and high-value chemicals. However, current enzyme preparations from mesophilic fungi are deficient in their thermostability and biomass-hydrolyzing efficiency at high temperatures. Thermophilic fungi represent promising sources of thermostable and highly active enzymes for improving the biomass-to-sugar conversion process. Here we present a comprehensive study on the lignocellulosic biomass-degrading ability and enzyme system of thermophilic fungus Malbranchea cinnamomea N12 and the application of its enzymes in the synergistic hydrolysis of lignocellulosic biomass. RESULTS: Malbranchea cinnamomea N12 was capable of utilizing untreated wheat straw to produce high levels of xylanases and efficiently degrading lignocellulose under thermophilic conditions. Temporal analysis of the wheat straw-induced secretome revealed that M. cinnamomea N12 successively degraded the lignocellulosic polysaccharides through sequential secretion of enzymes targeting xylan and cellulose. Xylanase-enriched cocktail from M. cinnamomea N12 was more active on native and alkali­pretreated wheat straw than the commercial xylanases from Trichoderma reesei over temperatures ranging from 40 to 75 °C. Integration of M. cinnamomea N12 enzymes with the commercial cellulase preparation increased the glucose and xylose yields of alkali­pretreated wheat straw by 32 and 166%, respectively, with pronounced effects at elevated temperature. CONCLUSIONS: This study demonstrated the remarkable xylanase-producing ability and strategy of sequential lignocellulose breakdown of M. cinnamomea N12. A new process for the hydrolysis of lignocellulosic biomass was proposed, comprising thermophilic enzymolysis by enzymes of M. cinnamomea N12 followed with mesophilic enzymolysis by commercial cellulases. Developing M. cinnamomea N12 as platforms for thermophilic enzyme mixture production will provide new perspectives for improved conversion yields for current biomass saccharification schemes.

An unusual crystal structure of ferric-enterobactin bound FepB suggests novel functions of FepB in microbial iron uptake.

Iron acquisition by siderophores is critical for the survival of most bacteria. Enterobactin is a kind of catechol siderophore that exhibits the highest affinity to iron atoms secreted by E. coli and several other species of Enterobacteriaceae. The periplasmic binding protein (PBP) FepB can transport ferric-enterobactin (Fe-Ent) from the outer membrane to the membrane-associated ATP-binding cassette transport system in E. coli. To elucidate this process, we solved the crystal structure of FepB in complex with Fe-Ent at a resolution of 1.8 Å. Consistent with previously reported NMR results, our crystal structure shows that, similar to the other type III PBPs, the FepB structure was folded with separated globular N- and C-termini linked by a long α-helix. Additionally, the structure showed that the Fe-Ent bound to the cleft between the N- and C-terminal domains. Exceptionally, FepB differs from the other known siderophore binding PBPs in that it forms a trimer by capturing four Fe-Ents that can each contribute to FepB trimerization. Dynamic light-scattering experiments are consistent with the structural observations and indicate that FepB forms a trimer in a Fe-Ent-dependent manner.

Genome analysis of enterovirus 71 strains differing in mouse pathogenicity.

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD) and is occasionally associated with severe neurological diseases. The investigation of virulence determinants of EV71 is rudimentary. Therefore, it is important to understand the relationship between EV71 virulence and genomic information. In this study, a series of analyses about full-length genomic sequence were performed on six EV71 strains isolated from HFMD patients with either severe or mild clinical symptoms. A one-day-old BALB/c mouse model was used to study the infection characteristics. Results showed all six strains were of the subgenogroup C4a. Viral full-length genomic sequence analysis showed that a total of 40 nucleotide differences between strains of highly and low virulence were revealed. Among all mutations, three nucleotide mutations were found in the untranslated region. A mutation, nt115, at internal ribozyme entry site (IRES) caused RNA secondary structural change. The other 37 mutations were all located in the open reading frame resulting in 8 amino acid mutations. Importantly, we discovered that a mutation of amino acid (Asn1617 → Asp1617) in the 3C proteinase (3C(pro)) of highly and low pathogenic strains could lead to conformational change at the active center, suggesting that this site may be a virulence determinant of EV71.

MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation.

Enterovirus A71 (EV-A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV-A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV-A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV-A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV-A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV-A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID50 ) values calculated in viral proliferations (ranged from 10(7.6) to 10(7.8) ); the TCID50 being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV-A71Jinan1002 amplifies more efficiently in MA104, Hep-2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV-A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV-A71.

Application of ethanol improves the resin-dentin bond strength of a two-bottle, self-etching primer-adhesive.

PURPOSE: To investigate whether the application of ethanol to a two-step self-etching adhesive can improve its resin-dentin bond performance. METHODS: Four different ethanol concentrations were added to the adhesive of Clearfil SE Bond to create four ethanol primers (40, 60, 80 or 100% ethanol). 24 extracted third molars were divided into four groups. Each group corresponded to one of the four hydrophobic ethanol primer concentrations. The teeth in the control group were bonded with Clearfil SE Bond according to the manufacturer's instructions. The primed teeth in the experimental groups were treated with the ethanol primer prior to application of the solvent-free adhesive. Microtensile bond strengths (mTBS) were tested 24 hours after specimen preparation. Another 14 teeth were bonded using the same methods to evaluate mTBS after 1 year. Nanoleakage was evaluated under field-emission scanning electron microscopy before and after aging. RESULTS: In the 24-hour group, the mTBS in the 60% ethanol/40% adhesive primer group increased significantly (21.6%, P < 0.05) over the no ethanol control. After 12-month water storage, the bond strength of that experimental group was still higher than that of the control group (19.5%, P < 0.05). Before aging, the nanoleakage was clearly seen in the control group but hardly any was seen in the experimental group. After aging, the nanoleakage increase in the experimental group was much less than that in the control group.

Increased enzyme activities and fungal degraders by Gloeophyllum trabeum inoculation improve lignocellulose degradation efficiency during manure-straw composting.

The present study investigated the effect of brown-rot fungus Gloeophyllum trabeum inoculation on lignocellulose degradation, enzyme activities and fungal community during co-composting of swine manure and wheat straw. G. trabeum inoculation shortened the maturation period of composting from 39 to 30 days. Composting piles inoculated with G. trabeum showed a higher degree of maturity as indicated by 31.6% lower C/N ratio and 29.4% higher GI. The decomposition rate of cellulose, hemicellulose and lignin was increased by 181.1%, 49.4% and 109.4%, respectively, due to higher activities of filter paper enzyme, xylanase, manganese peroxidase and laccase. Redundancy analysis showed that inoculating G. trabeum influenced the succession of fungal communities by changing the main physicochemical parameters, resulting in the increased relative abundance of Aspergillus, Mycothermus and Melanocarpus. Pearson correlation analysis indicated that more dominant fungal genera were involved in the production of lignocellulose-degrading enzymes after G. trabeum inoculation.

Thermal pretreatment enhances the degradation and humification of lignocellulose by stimulating thermophilic bacteria during dairy manure composting.

This study investigated the effect of thermal pretreatment (TP) on the lignocellulose degradation and humification during dairy manure composting and the underlying microbial mechanism. The results showed that TP accelerated temperature rise and elevated composting temperature by increasing 26% initial content of simple organics. The degradation of cellulose, hemicellulose and lignin was 78, 10 and 109% higher in thermal pretreatment composting (TPC) than traditional composting (TC), respectively. Moreover, TP significantly improved the humification degree of composts, as indicated by 14 and 38% higher humus content and humification indexes in TPC, respectively. 16S rRNA sequencing showed that TP increased the relative abundance of thermophilic bacteria in TPC, of which Thermobifida, Planifilum, Truepera and Thermomonospora were potentially involved in lignocellulose biodegradation and humification. Canonical correspondence analysis revealed that TP changed the main factor determining the bacterial community evolution from dissolved organic carbon (DOC) in TC to temperature in TPC.

A Novel Enterovirus 71 (EV71) Virulence Determinant: The 69th Residue of 3C Protease Modulates Pathogenicity.

Human enterovirus type 71 (EV71), the major causative agent of hand-foot-and-mouth disease, has been known to cause fatal neurological complications. Unfortunately, the reason for neurological complications that have been seen in fatal cases of the disease and the relationship between EV71 virulence and viral genetic sequences remains largely undefined. The 3C protease (3Cpro) of EV71 plays an irreplaceable role in segmenting the precursor polyprotein during viral replication, and intervening with host life activity during viral infection. In this study, for the first time, the 69th residue of 3C protease has been identified as a novel virulence determinant of EV71. The recombinant virus with single point variation, in the 69th of 3Cpro, exhibited obvious decline in replication, and virulence. We further determined the crystal structure of 3C N69D at 1.39 Ǻ resolution and found that conformation of 3C N69D demonstrated significant changes compared with a normal 3C protein, in the substrate-binding site and catalytic active site. Strikingly, one of the switch loops, essential in fixing substrates, adopts an open conformation in the 3C N69D-rupintrivir complex. Consistent with this apparent structural disruption, the catalytic activity of 3C N69D decreased sharply for host derived and viral derived substrates, detected for both in vitro and in vivo. Interestingly, in addition to EV71, Asp69 was also found in 3C proteases of other virus strains, such as CAV16, and was conserved in nearly all C type human rhinovirus. Overall, we identified a natural virulence determinant of 3C protease and revealed the mechanism of attenuated virulence is mediated by N69D substitution. Our data provides new insight into the enzymatic mechanism of a subdued 3C protease and suggests a theoretical basis for virulence determinantion of picornaviridae.

Selective demineralisation of dentine extrafibrillar minerals-A potential method to eliminate water-wet bonding in the etch-and-rinse technique.

OBJECTIVE: The present study tested the central hypothesis that selective demineralisation of dentine extrafibrillar minerals by lowering the phosphoric acid concentration improves the quality of the resin-dentine interface. METHODS: Dentine surfaces were etched with different concentrations of phosphoric acid (1, 5, 10, 20, 30 or 40wt%). Scanning electron microscopy was used to observe the micromorphology of the etched dentine surfaces. Energy dispersive X-ray analysis was performed to determine the residual Ca-content of the demineralised dentine matrix. Atomic force microscopy-based nanoindentation was used to analyse the nanomechanical properties of the treated dentine surfaces. The influence of H3PO4 concentration on resin-dentine bond strength was evaluated by microtensile bond strength testing. One-way ANOVA was used to compare the residual Ca-content ratio, reduced elastic modulus (Er) of the treated dentine surfaces and microtensile bond strength among groups. RESULTS: Collagen fibrils appeared to be wider in diameter after etching with 5% and 10% H3PO4. The partially-demineralized collagen scaffold retained part of its rigidity to maintain an uncollapsed three-dimensional structure. Etching with 1% H3PO4 resulted in the highest residual Ca-content ratio and Er of demineralised dentine matrix, followed by 5% H3PO4. Those values were all significantly higher than values derived from the other groups. Etching with 30% H3PO4 resulted in the lowest Ca-content ratio and Er. Using 5% H3PO4 as etchant resulted in the highest resin-dentine bond strength. CONCLUSIONS: Selective demineralisation of the dentine matrix may be achieved by lowering the H3PO4 concentration to 5wt%, to achieve better bonding performance. CLINICAL RELEVANCE: By retaining intrafibrillar minerals, more through air-drying of the partially demineralised collagen matrix may be accomplished without the need to worry about collapsing a mineral-free collagen matrix during air-drying. This may result in the elimination of water-wet bonding during the application of etch-and-rinse adhesives.

Genomic and immunologic factors associated with viral pathogenesis in a lethal EV71 infected neonatal mouse model.

Hand, foot and mouth disease (HFMD) caused by enterovirus 71 (EV71) has emerged as a major health problem in China and worldwide. The present study aimed to understand the virological features of EV71 and host responses resulting from EV71 infection. Six different EV71 strains were isolated from HFMD patients with severe or mild clinical symptoms, and were analyzed for pathogenicity in vitro and in vivo. The results demonstrated that the six virus strains exhibited similar cytopathogenic effects on susceptible MA104 cells. However, marked differences in histological and immunopathological changes were observed when mice were inoculated with the different virus strains. Thus, the viruses studied were divided into two groups, highly or weakly pathogenic. Two representative virus strains, JN200804 and JN200803 (highly and weakly pathogenic, respectively) were studied further to investigate pathogenicity-associated factors, including genetic mutations and immunopathogenesis. The present study has demonstrated that highly pathogenic strains have stable genome and amino acid sequences. Notably, the present study demonstrated that a highly pathogenic strain induced a significant increase of the bulk CD4 T cell levels at 3 days post­inoculation. In conclusion, the current study demonstrates that genomic and immunologic factors may be responsible for the multiple tissue damage caused by highly pathogenic EV71 infection.