Methionine inducing carbohydrate esterase secretion of Trichoderma harzianum enhances the accessibility of substrate glycosidic bonds.

BACKGROUND: The conversion of plant biomass into biochemicals is a promising way to alleviate energy shortage, which depends on efficient microbial saccharification and cellular metabolism. Trichoderma spp. have plentiful CAZymes systems that can utilize all-components of lignocellulose. Acetylation of polysaccharides causes nanostructure densification and hydrophobicity enhancement, which is an obstacle for glycoside hydrolases to hydrolyze glycosidic bonds. The improvement of deacetylation ability can effectively release the potential for polysaccharide degradation. RESULTS: Ammonium sulfate addition facilitated the deacetylation of xylan by inducing the up-regulation of multiple carbohydrate esterases (CE3/CE4/CE15/CE16) of Trichoderma harzianum. Mainly, the pathway of ammonium-sulfate's cellular assimilates inducing up-regulation of the deacetylase gene (Thce3) was revealed. The intracellular metabolite changes were revealed through metabonomic analysis. Whole genome bisulfite sequencing identified a novel differentially methylated region (DMR) that existed in the ThgsfR2 promoter, and the DMR was closely related to lignocellulolytic response. ThGsfR2 was identified as a negative regulatory factor of Thce3, and methylation in ThgsfR2 promoter released the expression of Thce3. The up-regulation of CEs facilitated the substrate deacetylation. CONCLUSION: Ammonium sulfate increased the polysaccharide deacetylation capacity by inducing the up-regulation of multiple carbohydrate esterases of T. harzianum, which removed the spatial barrier of the glycosidic bond and improved hydrophilicity, and ultimately increased the accessibility of glycosidic bond to glycoside hydrolases.

Two degradation strategies for overcoming the recalcitrance of natural lignocellulosic xylan by polysaccharides-binding GH10 and GH11 xylanases of filamentous fungi.

The recalcitrance of lignocellulose forms a strong barrier for the bioconversion of lignocellulosic biomass in chemical or biofuel industries. Filamentous fungi are major plant biomass decomposer, and capable of forming all the required enzymes. Here, they characterized the GH10 and GH11 endo-xylanases and a CE1 acetyl-xylan esterase (Axe1) from a superior biomass-degrading strain, Aspergillus fumigatus Z5, and examined how they interact in xylan degradation. Cellulose-binding (CBM1) domain inhibited GH10 xylanase activities for pure xylan, but afforded them an ability to hydrolyze washed corncob particles (WCCP). CBM1-containing GH10 xylanases also showed synergism with CBM1-containing Axe1 in WCCP hydrolysis, and this synergy was strictly dependent on the presence of their CBM1 domains. In contrast, GH11 xylanases had no CBM1, but still could bind xylan and hydrolyzed WCCP; however, no synergism displayed with Axe1. GH10 xylanases and GH11 xylanases showed a pronounced synergism in WCCP hydrolysis, which was dependent on the presence of the CBM1 in GH10 xylanases and absence from GH11 xylanases. They exhibit different mechanisms to bind to cellulose and xylan, and act in synergy when these two structures are intact. These findings will be helpful for the further development of highly efficient enzyme mixtures for lignocellulosic biomass conversion.

Genome-wide transcriptomic analysis of a superior biomass-degrading strain of A. fumigatus revealed active lignocellulose-degrading genes.

BACKGROUND: Various saprotrophic microorganisms, especially filamentous fungi, can efficiently degrade lignocellulose that is one of the most abundant natural materials on earth. It consists of complex carbohydrates and aromatic polymers found in the plant cell wall and thus in plant debris. Aspergillus fumigatus Z5 was isolated from compost heaps and showed highly efficient plant biomass-degradation capability. RESULTS: The 29-million base-pair genome of Z5 was sequenced and 9540 protein-coding genes were predicted and annotated. Genome analysis revealed an impressive array of genes encoding cellulases, hemicellulases and pectinases involved in lignocellulosic biomass degradation. Transcriptional responses of A. fumigatus Z5 induced by sucrose, oat spelt xylan, Avicel PH-101 and rice straw were compared. There were 444, 1711 and 1386 significantly differently expressed genes in xylan, cellulose and rice straw, respectively, when compared to sucrose as a control condition. CONCLUSIONS: Combined analysis of the genomic and transcriptomic data provides a comprehensive understanding of the responding mechanisms to the most abundant natural polysaccharides in A. fumigatus. This study provides a basis for further analysis of genes shown to be highly induced in the presence of polysaccharide substrates and also the information which could prove useful for biomass degradation and heterologous protein expression.

Integrating transcriptomics and biochemical analysis to understand the interactive mechanisms of the coexisting exposure of nanoplastics and erythromycin on Chlorella pyrenoidosa.

Nanoplastics and antibiotics frequently co-exist in water polluted by algal blooms, but little information is available about interaction between substances. Erythromycin, as a representative of antibiotics, has been frequently detected in aquatic environments. This investigation attempted to reveal the interaction mechanism of nanoplastics and erythromycin on Chlorella pyrenoidosa. Results demonstrated that the joint toxicity of erythromycin and nanoplastics was dynamic and depended on nanoplastics concentration. Antagonistic effects of 1/2 or 1 EC50 erythromycin and nanoplastic concentration (10 mg/L) on the growth of C. pyrenoidosa was observed. The joint toxicity of 1/2 or 1 EC50 erythromycin and nanoplastic concentration (50 mg/L) was initially synergistic during 24-48 h and then turned to antagonistic during 72-96 h. Consequently, antagonistic effect was the endpoint for joint toxicity. Integration of transcriptomics and physiological biochemical analysis indicated that the co-existence of nanoplastics and erythromycin affected the signal transduction and molecular transport of algal cell membrane, induced intracellular oxidative stress, and hindered photosynthetic efficiency. Overall, this study provided a theoretical basis for evaluating the interactive mechanisms of nanoplastics and antibiotics.

Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost.

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by ß-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.

The functioning of a novel protein, Swollenin, in promoting the lignocellulose degradation capacity of Trichoderma guizhouense NJAU4742 from a proteomic perspective.

The functioning of a novel auxiliary enzyme, TgSWO from Trichoderma guizhouense NJAU4742, was investigated based on the proteomic analysis of wild-type (WT), knockout (KO) and overexpression (OE) treatments. The results showed that the cellulase and hemicellulase activities of OE and WT were significantly higher than those of KO. Simultaneously, tandem mass tag (TMT) analysis results indicated that cellulases and hemicellulases were significantly upregulated in OE, especially hydrophobin (HFB, A1A105805.1) and endo-ß-1,4-glucanases (A1A101831.1), with ratios of 43.73 and 9.88, respectively, compared with WT. The synergistic effect of TgSWO on cellulases increased the reducing sugar content by 1.45 times in KO + TgSWO (1.8 mg) compared with KO, and there was no significant difference between KO + TgSWO (1.2 mg) and WT. This study elucidated the function of TgSWO in promoting the lignocellulose degradation capacity of NAJU4742, which provides new insights into the efficient conversion of lignocellulose.

The Effectiveness and Safety of Thermocoagulation Radiofrequency Treatment of the Ophthalmic Division (V1) and/or Maxillary (V2) and Mandibular (V3) Division in Idiopathic Trigeminal Neuralgia: An Observational Study.

BACKGROUND: Trigeminal neuralgia (TN) is a pain appearing in the ophthalmic (V1), maxillary (V2), and mandibular (V3) trigeminal branches. Pharmacologic treatment is the first line for TN; however, many patients prefer to receive minimally invasive treatment rather than medicine because of intolerable side effects. Thermocoagulation radiofrequency (TRF) is a minimally invasive treatment that has been shown to effectively treat the maxillary (V2) and mandibular (V3) divisions, but the safety of TRF treatment of the ophthalmic (V1) division has been controversial. OBJECTIVE: This study was to observe the effectiveness and safety of TRF treatment of the ophthalmic (V1) division of trigeminal branches in idiopathic TN patients. STUDY DESIGN: An observational study. SETTING: All of patients received temperature controlled TRF, the effectiveness and safety of TRF was assessed by VAS and complications. METHODS: Eighty patients with ophthalmic division (V1) or ophthalmic division (V1) combined with maxillary (V2) or mandibular (V3) divisions of idiopathic TN were treated with step-increased temperature TRF for 6 minutes. At a pulse width of 20 ms, the temperature was titrated up 2 degrees from 60 degrees to 66 degrees every 60 seconds, and then another 66 degrees or 68 degrees for 2 minutes. Meanwhile, the tip of the cannula was turned 180 degrees with each temperature titration. Patients were assessed for pain relief and corneal reflex, numbness, and masticatory muscle weakness at one week, one month, and 3 months after the procedure. RESULTS: Eighty patients were successfully treated with temperature controlled TRF for ophthalmic (V1) division. Excellent pain relief was achieved in 79 of 80 patients (98.75%) after one week, one month, and 3 months, and 78 of 80 patients (97.5%) patients experienced tolerable numbness. Only one patient lost the corneal reflex, 14 experienced a corneal reflex that was mildly decreased, and 2 patients felt a foreign body sensation in the ipsilateral eye after TRF, but there were no corneal ulcers, incidences of blindness, or other complications. LIMITATIONS: This study is limited by being an observation study and a non-prospective trial with a short-term follow-up period. CONCLUSION: Temperature controlled TRF to the ophthalmic division (V1) of the semilular ganglion is effectiveness and safe in TN. KEY WORDS: Thermocoagulation radiofrequency, pulsed radiofrequency, trigeminal neuralgia, ophthalmic division, trigeminal ganglion, pain, numbness, corneal reflex.

[Evaluation of midazolam intravenous sedated patient's comfort degree in mandibular third molar surgery].

OBJECTIVE: To evaluate the clinical efficacy and safety of continuous low-flow intravenous infusion of midazolam sedation in mandibular third molar surgery. METHODS: Fifty healthy patients with symmetrically placed impacted bilateral mandibular third molars were included in this self controlled, randomized clinical study. Degree of comfort (their actual current anxiety level) was assessed using a visual analogue scale (VAS) of pain and anxiety. Patients' satisfaction and degree of amnesia were also evaluated. Vital signs and oxygen saturation were recorded. RESULTS: Low dose midazolam sedation obviously increased the degree of patients' comfort and satisfaction. Vital signs and oxygen saturation levels did not differ significantly between the two groups. CONCLUSIONS: Midazolam as an intravenous sedation agent in mandibular third molar surgery showed satisfactory effect on patients with mild dental fear.