Enhancing xylanase production in the thermophilic fungus Myceliophthora thermophila by homologous overexpression of Mtxyr1.

The xylanase regulator 1 protein in Myceliophthora thermophila ATCC42464 (MtXyr1) is 60 % homologous with that of Trichoderma reesei. However, MtXyr1's regulatory role on cellulolytic and xylanolytic genes in M. thermophila is unknown. Herein, MtXyr1 was overexpressed under the control of the MtPpdc (pyruvate decarboxylase) promoter. Compared with the wild type, the extracellular xylanase activities of the transformant cultured in non-inducing and inducing media for 120 h were 25.19- and 9.04-fold higher, respectively. The Mtxyr1 mRNA level was 300-fold higher than in the wild type in corncob-containing medium. However, the filter paper activity and endoglucanase activities were unchanged in corncob-containing medium and glucose-containing medium. The different zymograms between the transformant and the wild type were analyzed and identified by mass spectrometry as three xylanases of the glycoside hydrolase (GH) family 11. Thus, overexpression of xyr1 resulted in enhanced xylanase activity in M. thermophila. Xylanase production could be improved by overexpressing Mtxyr1 in M. thermophila.

Truncation of a mannanase from Trichoderma harzianum improves its enzymatic properties and expression efficiency in Trichoderma reesei.

To obtain high expression efficiency of a mannanase gene, ThMan5A, cloned from Trichoderma harzianum MGQ2, both the full-length gene and a truncated gene (ThMan5AΔCBM) that contains only the catalytic domain, were expressed in Trichoderma reesei QM9414 using the strong constitutive promoter of the gene encoding pyruvate decarboxylase (pdc), and purified to homogeneity, respectively. We found that truncation of the gene improved its expression efficiency as well as the enzymatic properties of the encoded protein. The recombinant strain expressing ThMan5AΔCBM produced 2,460 ± 45.1 U/ml of mannanase activity in the culture supernatant; 2.3-fold higher than when expressing the full-length ThMan5A gene. In addition, the truncated mannanase had superior thermostability compared with the full-length enzyme and retained 100 % of its activity after incubation at 60 °C for 48 h. Our results clearly show that the truncated ThMan5A enzyme exhibited improved characteristics both in expression efficiency and in its thermal stability. These characteristics suggest that ThMan5AΔCBM has potential applications in the food, feed, paper, and pulp industries.

Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters.

BACKGROUNDS: The fungus Trichoderma reesei is an important workhorse for expression of homologous or heterologous genes, and the inducible cbh1 promoter is generally used. However, constitutive expression is more preferable in some cases than inducible expression that leads to production of unwanted cellulase components. In this work, constitutive promoters of T. reesei were screened and successfully used for high level homologous expression of xylanase II. RESULTS: The transcriptional profiles of 13 key genes that participate in glucose metabolism in T. reesei were analyzed by quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). The results indicated that the mRNA levels of pdc (encoding pyruvate decarboxylase) and eno (encoding enolase) genes were much higher than other genes under high glucose conditions. Recombinant T. reesei strains that homologously expressed xylanase II were constructed by using the promoters of the pdc and eno genes, and they respectively produced 9266 IU/ml and 8866 IU/ml of xylanase activities in the cultivation supernatant in a medium with high glucose concentration. The productivities of xylanase II were 1.61 g/L (with the pdc promoter) and 1.52 g/L (with the eno promoter), approximately accounted for 83% and 82% of the total protein secreted by T. reesei, respectively. CONCLUSIONS: This work demonstrates the screening of constitutive promoters by using RT-qPCR in T. reesei, and has obtained the highest expression of recombinant xylanase II to date by using these promoters.

Using a consensus acupoints regimen to explore the relationship between acupuncture sensation and lumbar spinal postoperative analgesia: A retrospective analysis of prospective clinical cooperation.

OBJECTIVE: This study evaluated the effectiveness of acupuncture treatment on postoperative pain in patients with degenerative lumbar spine disease, and explored the relationship between the postoperative analgesic effect of acupuncture and the sensation of acupuncture experienced by the patients. METHODS: This retrospective study analyzed the medical records of 97 patients who had undergone an operation by the same surgeon due to degenerative lumbar disease. These patients were divided into acupuncture group (n = 32), patient-controlled analgesia (PCA) group (n = 27), and oral analgesia group (n = 38) according to the different postoperative analgesic methods. During their hospitalization, patients completed daily evaluations of their pain using a visual analogue scale (VAS), and injection times of supplemental meperidine were recorded. Also, the Chinese version of the Massachusetts General Hospital Acupuncture Sensation Scale (C-MASS) was used in the acupuncture group. RESULTS: Each of the three treatment groups showed significant reductions in postoperative pain, as shown by reduced VAS scores. The acupuncture group, however, had less rebound pain (P < 0.05) than the other two groups. Both the acupuncture and PCA groups experienced acute analgesic effects that were superior to those in the oral analgesia group. In addition, the higher the C-MASS index on the second day after surgery, the lower the VAS score on the fourth day after surgery. There was also a significant difference in the "dull pain" in the acupuncture sensation. CONCLUSION: The results demonstrated that acupuncture was beneficial for postoperative pain and discomfort after simple surgery for degenerative spinal disease. It is worth noting that there was a disproportionate relevance between the patient's acupuncture sensation and the improvement of pain VAS score.

Acupuncture Effect and Mechanism for Treating Pain in Patients With Parkinson's Disease.

Non-motor symptoms of Parkinson's disease (PD) have been receiving increasing attention. Approximately half of patients with PD have experience PD-related pain. We investigated the effect and mechanism of acupuncture in patients with PD who have pain. PD patients with pain were divided into acupuncture group and control group. Nine patients completed acupuncture treatment; seven patients who received only an analgesic agent underwent resting-state functional magnetic resonance imaging (rs-fMRI) twice. fMRI was performed to evaluate the functional connectivity of the brain regions. After treatment, a decrease in total scores on the King's Parkinson's Disease Pain Scale (KPPS) and Unified Parkinson's Disease Rating Scale was observed in the acupuncture group (-46.2 and -21.6%, respectively). In the acupuncture group, increased connectivity was observed in four connections, one in the left hemisphere between the middle temporal gyrus (MTG) and precentral gyrus, and three in the right hemisphere between the postcentral gyrus and precentral gyrus, supramarginal gyrus and precentral gyrus, and MTG and insular cortex. A significant correlation was noted between the changes in functional connectivity and KPPS. The involved connection was between the left middle frontal gyrus and the right precentral gyrus (R = -0.698, P = 0.037). Acupuncture could relieve pain in PD patients by modulating brain regions related to both sensory-discriminative and emotional aspects. The present study might increase the confidence of users that acupuncture is an effective and safe analgesic tool that can relieve PD-related pain.

A longitudinal fixel-based analysis of white matter alterations in patients with Parkinson's disease.

INTRODUCTION: Disruption to white matter pathways is an important contributor to the pathogenesis of Parkinson's disease. Fixel-based analysis has recently emerged as a useful fiber-specific tool for examining white matter structure. In this longitudinal study, we used Fixel-based analysis to investigate white matter changes occurring over time in patients with Parkinson's disease. METHODS: Fifty patients with idiopathic Parkinson's disease (27 men and 23 women; mean age: 61.8 ± 6.1 years), were enrolled. Diffusion-weighted imaging and clinical examinations were performed at three different time points (baseline, first follow-up [after a mean of 24±2 months], and second follow-up [after a mean of 40 ± 3 months]). Additional 76 healthy control subjects (38 men and 38 women; mean age: 62.3 ± 5.5 years) were examined at baseline. The following fixel-based metrics were obtained: fiber density (FD), fiber bundle cross-section (FC), and a combined measure of both (FDC). Paired comparisons of metrics between three different time points were performed in patients. Linear regression was implemented between longitudinal changes of fixel-based metrics and the corresponding modifications in clinical parameters. A family-wise error corrected p < 0.05 was considered statistically significant. RESULTS AND DISCUSSIONS: Early degeneration in the splenium of corpus callosum was identified as a typical alteration of Parkinson's disease over time. At follow-up, we observed significant FDC reductions compared with baseline in white matter, noticeably in corpus callosum; tapetum; cingulum, posterior thalamic radiation, corona radiata, and sagittal stratum. We also identified significant FC decreases that reflected damage to white matter structures involved in Parkinson's disease -related pathways. Fixel-based metrics were found to relate with a deterioration of 39-item Parkinson's Disease Questionnaire, Unified Parkinson's Disease Rating Scale and activity of daily living. A Parkinson's disease -facilitated aging effect was observed in terms of white matter disruption. CONCLUSION: This study provides a thorough fixel-based profile of longitudinal white matter alterations occurring in patients with Parkinson's disease and new evidence of FC as an important role in white matter degeneration in this setting.

[Analysis of cellulase synthesis mechanism in Trichoderma reesei using red fluorescent protein].

Comprehensive analysis of the mechanism of metabolite synthesis in filamentous fungi is important for optimization of the filamentous fungus related industrial fermentation processes. In this work, the mechanism of cellulase synthesis in Trichoderma reesei was analyzed with red fluorescent protein (DsRed) as the reporting protein. The expression cassette for heterologous protein expression in T. reesei was constructed, through which the DsRed gene was inserted into the chromosomal DNA of T. reesei. The recombinant T. reesei strain, in which expression of DsRed was controlled by the promoter of cellobiohydrolase gene, was designated as T. reesei TR2. Expression of DsRed in T. reesei TR2 under different culture conditions was analyzed by using a fluorescent microscopy, and thereby the mechanism of cellulase gene expression in T. reesei could be interpreted. With induction of lactose, the pattern of change of red fluorescence in T. reesei TR2 was similar to that of the cellulase activity in the cultivation supernatant. As the culture aged, the red fluorescence in the mycelial increased. This was followed by a reduction in the end of the culture period because of death and autolysis of the mycelial. In the spatial aspect, the red fluorescence was distributed uniformly in the whole hypha after induction, indicating that all the three morphology including apical compartment, subapical compartment and hyphal compartment played a same role in cellulase synthesis. When T. reesei TR2 was cultivated without induction, faint red fluorescence appeared after a relative long period of cultivation, indicating that a small amount of cellulase was still synthesized without induction. This result was useful in explaining the mechanism of cellulase induction by insoluble cellulose.

Homologous constitutive expression of Xyn III in Trichoderma reesei QM9414 and its characterization.

Xylanase III (Xyn III), a specific endoxylanase that belongs to family 10 of the glycoside hydrolases, was overexpressed in Trichoderma reesei QM9414 using a constitutive strong promoter of the gene encoding pyruvate decarboxylase (pdc). The maximum recombinant xylanase activity achieved was 817.2 ± 65.2 U/mL in the transformant fermentation liquid. The productivities of Xyn III accounted for approximately 53% of the total protein secreted by the recombinant. The enzyme was optimally active at 60 °C and pH 6. The recombinant Xyn III was stable at pH 5-8. This is the first report on the homologous expression of xyn3 in T. reesei QM9414. The properties of Xyn III make it promising in a variety of industrial use.

Molecular cloning and heterologous expression of an acid-stable endoxylanase gene from Penicillium oxalicum in Trichoderma reesei.

An endoxylanase gene (PoxynA) that belongs to the glycoside hydrolase (GH) family 11 was cloned from a xylanolytic strain, Penicillium oxalicum B3-11(2). PoxynA was overexpressed in Trichoderma reesei QM9414 by using a constitutive strong promoter of the encoding pyruvate decarboxylase (pdc). The high extracellular xylanase activities in the fermentation liquid of the transformants were maintained 29~35-fold higher compared with the wild strain. The recombinant POXYNA was purified to homogeneity, and its characters were analyzed. Its optimal temperature and pH value were 50 degrees C and 5.0, respectively. The enzyme was stable at a pH range of 2.0 to 7.0. Using beechwood as the substrate, POXYNA had a high specific activity of 1,856 +/- 53.5 IU/mg. In the presence of metal ions, such as Cu2+, and Mg2+, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of Mn2+ and Fe2+. The recombinant POXYNA hydrolyzed birchwood xylan, beechwood xylan, and oat spelt xylan to produce short-chain xylooligosaccharides, xylopentaose, xylotriose, and xylobiose as the main products. This is the first report on the expression properties of a recombinant endoxylanase gene from Penicillium oxalicum. The properties of this endoxylanase make it promising for applications in the food and feed industries.

SLC1A2 variant is associated with essential tremor in Taiwanese population.

Essential tremor (ET), which is one of the most common movement disorders, may lead to severe interference in quality of life. The first genome-wide association study (GWAS) has identified an association of the LINGO1 variant (rs9652490) with ET in Americans and Europeans. Recently, a second GWAS that was performed in a European population has discovered a new variant (rs3794087) of the main glial glutamate transporter (SLC1A2) that increases the risk of ET with an odds ratio of about 1.4. SLC1A2 encodes for the major glial high-affinity glutamate reuptake transporter in the brain and is a potential ET susceptibility gene. Because replication in a different ethnic population is important for validating a finding, we conducted a case-control study to investigate the SLC1A2 variant in an Asian cohort with ET in Taiwan. A total of 542 subjects (273 ET patients and 269 controls) were included. The results showed that rs3794087 was associated with ET among the Taiwanese. The odds ratio was 1.37. Our results were similar to those of the second GWAS of ET in Europeans, and this confirms that SLC1A2 may be a good functional candidate gene for ET. A replication study in another independent population is of importance to validate this association.

Improving cellulase production in Trichoderma koningii through RNA interference on ace1 gene expression.

Ribonucleic acid interference (RNAi) inhibits the expression of target genes in a sequence-specific manner, and shows potential for gene knockdown in filamentous fungi, in which the locus-specific gene knockout occurs in low frequency. In this study, the function of the repressor of cellulase expression I (ACEI) was verified in Trichoderma koningii (T. koningii) YC01 through RNAi, and ace1- silenced strains with improved cellulase productivity were obtained. An expression cassette that transcribed the interfering double-stranded RNA (dsRNA) of ace1 was constructed and transformed into T. koningii, and the transformants, in which the expression of ace1 was successfully silenced, were selected. As a result of the ace1 gene silencing, the expression levels of the main cellulase and xylanase genes were elevated, and the enhanced production of total proteins, cellulase, and xylanase was observed in the cultivation. In addition, the downregulation of ace1 resulted in an increasing expression of xyr1, but no clear variation in the expression of cre1, which suggested that ACEI acted as a repressor of the xyr1 transcription, but was not involved in the regulation of the cre1 expression. The results of this work indicate that ace1 is a valid target gene for enhancing enzyme production in T. koningii, and RNAi is an appropriate tool for improving the properties of industrial fungi.