Construction of efficient xylose utilizing Pichia pastoris for industrial enzyme production.

BACKGROUND: Cellulosic biomass especially agricultural/wood residues can be utilized as feedstock to cost-effectively produce fuels, chemicals and bulk industrial enzymes, which demands xylose utilization from microbial cell factories. While previous works have made significant progress in improving microbial conversion of xylose into fuels and chemicals, no study has reported the engineering of efficient xylose utilizing protein expression systems for the purpose of producing industrial enzymes. RESULTS: In this work, using Pichia pastoris as an example, we demonstrated the successful engineering of xylose metabolizing ability into of protein expression systems. A heterologous XI (xylose isomerase) pathway was introduced into P. pastoris GS115 by overexpressing the Orpinomyces spp. XI or/and the endogenous XK (xylulokinase) gene, and evolutionary engineering strategies were also applied. Results showed that the XI pathway could be functionally expressed in P. pastoris. After 50 generation of sequential batch cultivation, a set of domesticated recombinant P. pastoris strains with different performance metrics on xylose were obtained. One evolved strain showed the highest xylose assimilation ability, whose cell yield on xylose can even be comparable to that on glucose or glycerol. This strain also showed significantly increased ß-mannanase production when cultured on xylose medium. Furthermore, transcription analysis of xylose pathway genes suggested that overexpression of XI and XK might be the key factors affecting effective xylose assimilation. CONCLUSIONS: To our best knowledge, this study is the first work demonstrating the construction of efficient xylose utilizing P. pastoris strains, thus providing a basis for using cellulosic biomass for bulk industrial enzyme production.

[Study on infrared spectrum change of Ganoderma lucidum and its extracts].

From the determination of the infrared spectra of four substances (original ganoderma lucidum and ganoderma lucidum water extract, 95% ethanol extract and petroleum ether extract), it was found that the infrared spectrum can carry systematic chemical information and basically reflects the distribution of each component of the analyte. Ganoderma lucidum and its extracts can be distinguished according to the absorption peak area ratio of 3 416-3 279, 1 541 and 723 cm(-1) to 2 935-2 852 cm(-1). A method of calculating the information entropy of the sample set with Euclidean distance was proposed, the relationship between the information entropy and the amount of chemical information carried by the sample set was discussed, and the authors come to a conclusion that sample set of original ganoderma lucidum carry the most abundant chemical information. The infrared spectrum set of original ganoderma lucidum has better clustering effect on ganoderma atrum, Cyan ganoderma, ganoderma multiplicatum and ganoderma lucidum when making hierarchical cluster analysis of 4 sample set. The results show that infrared spectrum carries the chemical information of the material structure and closely relates to the chemical composition of the system. The higher the value of information entropy, the much richer the chemical information and the more the benefit for pattern recognition. This study has a guidance function to the construction of the sample set in pattern recognition.

[Similar spectrum of infrared spectrum and its application in identification of Chinese herbs].

In the present paper, the similar spectra of 18 samples, which include Astragalus, red-blue Astragalus and Codonopsis, were obtained in the range of 1600-700 cm(-1). The result showed that all kinds of herbs have their own characteristic similar spectra, and 18 samples can be identified according to the characteristic similar spectra. Furthermore, three correlation coefficients of 93 ganoderma samples were calculated which is in the range of 1560-1502, 1460-1421 and 1319-1260 cm(-1) according to the information of similar spectrum of infrared spectrum of ganoderma. Without priori knowledge of the classification of these samples, the K-means cluster analysis can successfully divide them into four classes, i.e., Ganoderma lucidum and Ganoderma sinensis, Ganoderma atrum, Ganoderma aoshiba, Ganoderma multiplicatum. This result is consistent with the result of morphological classification.

Super-Long SERS Active Single Silver Nanowires for Molecular Imaging in 2D and 3D Cell Culture Models.

Establishing a systematic molecular information analysis strategy for cell culture models is of great significance for drug development and tissue engineering technologies. Here, we fabricated single silver nanowires with high surface-enhanced Raman scattering activity to extract SERS spectra in situ from two-dimensional (2D) and three-dimensional (3D) cell culture models. The silver nanowires were super long, flexible and thin enough to penetrate through multiple cells. A single silver nanowire was used in combination with a four-dimensional microcontroller as a cell endoscope for spectrally analyzing the components in cell culture models. Then, we adopted a machine learning algorithm to analyze the obtained spectra. Our results show that the abundance of proteins differs significantly between the 2D and 3D models, and that nucleic acid-rich and protein-rich regions can be distinguished with satisfactory accuracy.

Injectable Magnesium-Zinc Alloy Containing Hydrogel Complex for Bone Regeneration.

Gelatin methacryloyl (GelMA) has been widely used in bone engineering. It can also be filled into the calvarial defects with irregular shape. However, lack of osteoinductive capacity limits its potential as a candidate repair material for calvarial defects. In this study, we developed an injectable magnesium-zinc alloy containing hydrogel complex (Mg-IHC), in which the alloy was fabricated in an atomization process and had small sphere, regular shape, and good fluidity. Mg-IHC can be injected and plastically shaped. After cross-linking, it contents the elastic modulus similar to GelMA, and has inner holes suitable for nutrient transportation. Furthermore, Mg-IHC showed promising biocompatibility according to our evaluations of its cell adhesion, growth status, and proliferating activity. The results of alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining, and real-time polymerase chain reaction (PCR) further indicated that Mg-IHC could significantly promote the osteogenic differentiation of MC3T3-E1 cells and upregulate the genetic expression of collagen I (COL-I), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2). Finally, after applied to a mouse model of critical-sized calvarial defect, Mg-IHC remarkably enhanced bone formation at the defect site. All of these results suggest that Mg-IHC can promote bone regeneration and can be potentially considered as a candidate for calvarial defect repairing.

High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules.

AIM: To construct and produce a recombinant bispecific humanized single-chain Fv (sFv) /Interleukin-2 (IL-2) fusion protein by using mammalian cells. METHODS: The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. RESULTS: The fusion protein was constructed and high efficiently expressed with yields up to 102 +/- 4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy (V(H)) and light (V(L)) domains of monoclonal antibody (mAb) 520C9 directed against the human HER-2/neu (c-erbB2) proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL-2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. CONCLUSION: The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.

Functional study of the upregulation of miRNA-27a and miRNA-27b in 3T3-L1 cells in response to berberine.

Berberine is the major active component of Rhizoma Coptidis derived from a traditional Chinese herbal medicine and is known to regulate micro (mi)RNA levels, although the mechanism for this action remains unknown. The present study confirmed that treatment of 3T3­L1 cells with berberine inhibited cell viability and differentiation in a dose­ and time­dependent manner, and significantly increased the mRNA expression levels of miRNA­27a and miRNA­27b. In addition, in 3T3­L1 cells treated with berberine, overexpression of miRNA­27a and miRNA­27b improved the berberine-mediated inhibition of cell differentiation and reduction of triglyceride contents. By contrast, miRNA­27a and miRNA­27b inhibitors attenuated the berberine­mediated inhibition of cell differentiation and reduction of triglyceride contents. Additionally, peroxisome proliferator­activated receptors (PPAR)­Î³ was confirmed to be a target of miRNA­27a in the 3T3­L1 cells. A dual­luciferase reporter assay indicated that the expression of PPAR­Î³ was negatively regulated by miRNA-27a. These findings may provide novel mechanistic insight into the antiobesity effects of certain compounds in traditional Chinese herbal medicine.

Effect of berberine on the ratio of high-molecular weight adiponectin to total adiponectin and adiponectin receptors expressions in high-fat diet fed rats.

OBJECTIVE: To assess the effects of berberine (BBR) on high-molecular weight (HMW) adiponectin and adiponectin receptors (adipoR1/adipoR2) expressions in high-fat (HF) diet fed rats. METHODS: Forty Wistar male rats were randomly assigned into a normal diet fed group and three HF diet (fat for 45% calories) fed groups (n=10 for each group). All rats underwent 12 weeks of feeding. After 4 weeks feeding, rats in the two of three HF diet fed groups were treated with 150 mg·kg-1·day-1 BBR (HF+LBBR group) and 380 mg·kg-1·day-1 BBR (HF+HBBR group) by gavage once a day respectively for the next 8 weeks while the rats in other groups treated with vehicle (NF+Veh and HF+Veh). Body weight and food intake were observed and recorded on daily basis. At the end of 12 weeks, the blood, liver, epididymal fat tissues and quadriceps femoris muscles were collected. Fasting insulin, plasma fasting glucose, serum free fatty acid (FFA), total adiponectin and HMW adiponectin levels were measured by enzyme linked immunosorbent assay method. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed to determine the insulinsensitizing. Meanwhile the homeostasis model assessment (HOMA) method was used to determine insulin resistance (HOMA-IR). The expressions of adipoR1, adipoR2 and adenosine monophophate activated protein kinase (AMPK) phosphorylation level in skeletal muscle and liver tissue were detected by Western blot. Liver and kidney toxicity were evaluated during treatment. RESULTS: The body weight of rats in high- or low-dose BBR group reduced as well as HOMA-IR, FFA concentrations and fasting insulin levels decreased compared with HF+Veh group (P<0.05). BBR also increased the ratio of HMW to total adiponectin in high fat-fed rats compared with rats in the HF+Veh group. High- and low-dose BBR increased adipoR1 expression in skeletal muscle by over 6- and 2-fold (P<0.05), respectively, and high-dose BBR also increased adipoR2 expression in liver tissue by over 2-fold (P<0.05). BBR significantly increased AMPK phosphorylation in HF diet rats compared with normal diet rats (P<0.05). The ratio of HMW to total adiponectin was inversely correlated with HOMA-IR (r=-0.52, P=0.001). Meantime, no liver and kidney toxicity was found in high fat-fed rats that treated by BBR. CONCLUSION: Berberine may improve insulin resistance by increasing the expression of adiponectin receptors and the ratio of HMW to total adiponectin.

Posterior reversible encephalopathy syndrome with involvement of the cervical cord and medulla: a case report.

Posterior reversible encephalopathy syndrome (PRES) is a neurotoxic state, which is associated with symmetrical subcortical areas of vasogenic oedema that are preferentially parieto-occipital, and it typically resolves within a few weeks after appropriate treatment, We hereby report a case of a female with adrenal tumour presenting with PRES, who was featured by a very rare neuroimaging manifestation, the involvement of cervical cord and medulla.

Enhancement of the gene targeting efficiency of non-conventional yeasts by increasing genetic redundancy.

In contrast to model yeasts, gene targeting efficiencies of non-conventional yeasts are usually low, which greatly limits the research and applications of these organisms. In this study, we aimed to enhance the gene targeting efficiency of non-conventional yeasts by improving the fitness of mutant strains, particularly by increasing the genetic redundancy of host cells. To demonstrate this process, OCH1 gene deletion in Pichia pastoris was performed. Extra copies of the OCH1 gene on a helper plasmid were provided for the P. pastoris GS115 strain before the native OCH1 gene in the genomic DNA was knocked out. The redundancy in OCH1 gene significantly eliminated the growth defects of the och1 mutant and increased the deletion efficiency of the OCH1 gene by two orders of magnitude with the same length of homologous flanks. The same strategy was used to delete the KU70 and SGS1 genes. The targeting efficiencies of KU70 and SGS1 were increased by 1- and 23-fold, respectively. Therefore, this study provided an efficient strategy for the deletion of "stubborn" genes in non-conventional yeasts. This study further showed that cellular fitness is potentially an important factor that can limit the efficiency of gene targeting.

[Direct secretory expression of active microbial transglutaminase in Pichia pastoris].

Direct secretory expression of active microbial transglutaminase (MTG) using heterologous hosts is a promising strategy, although its production level still needs to be improved for industrial production. Pichia pastoris is one of the most efficient expression systems developed in recent years. In this study, secretory expression of active MTG was successfully achieved by co-expressing the pro sequence and mature MTG genes in P. pastoris. Furthermore, we optimized the copy number of pro/MTG expression cassettes and the fermentation conditions. MTG production level reached 7.3 U/mL in 1-liter fermentor through high density fermentation, providing the feasiblity for industrial scale preparation of MTG.

2,6-Bis[(2-hydroxy-3-methoxybenzylidene)hydrazinocarbonyl]pyridine monohydrate.

In the title compound, C(23)H(19)N(5)O(6).H(2)O, the two components are linked into complex chains by a combination of two independent O-H...O and two independent N-H...O hydrogen bonds. The complex chains are linked into a two-dimensional sheet network via pi-pi stacking interactions and C-H...O hydrogen bonds.

An improved gel-based DNA microarray method for detecting single nucleotide mismatch.

3-D polyacrylamide gel-based DNA microarray platforms provide a high capacity for nucleic acids immobilization and a solution-mimicking environment for hybridization. However, several technological bottlenecks still remain in these platforms, such as difficult microarray preparation and high fluorescent background, which limit their application. In this study, two new approaches have been developed to improve the convenience in microarray preparation and to reduce the background after hybridization. To control the polymerization process, solutions containing acrylamide-modified oligonucleotide, acrylamide, glycerol and ammonium persulfate are spotted onto a functionalized glass slide, and then the slide is transferred to a vacuum chamber with TEMED, so that TEMED is vaporized and diffused into the spots to induce polymerization. By applying an electric field across a hybridized microarray to remove the nonspecifically bound labeled targets, this approach can solve the problem of high fluorescent background of the gel-based microarray after hybridization. Experimental results show that our immobilization method can be used to construct high quality microarrays and exhibits good reproducibility. Moreover, the polymerization is not affected by PCR medium, so that PCR products can be used for microarray construction without being treated by commercial purification cartridges. Electrophoresis can improve the signal-to-noise significantly and has the ability to differentiate single nucleotide variation between two homozygotes and a heterozygote. Our results demonstrated that this is a reliable novel method for high-throughput mutation analysis and disease diagnosis.