Roles of four targets in the pathogenesis of graves' orbitopathy.

Graves' orbitopathy (GO) is an autoimmune disease that involves complex immune systems. The mainstays of clinical management for this disease are surgery, targeted drugs therapy, and no-targeted drugs drug therapy. targeted drugs can improve therapeutic efficacy and enhance the quality of life for GO patients. However, as a second-line treatment for GO, targeted drugs such as tocilizumab and rituximab have very limited therapeutic effects and may be accompanied by side effects. The introduction of Teprotumumab, which targets IGF-IR, has made significant progress in the clinical management of GO. The pathophysiology of GO still remains uncertain as it involves a variety of immune cells and fibroblast interactions as well as immune responses to relevant disease targets of action. Therfore, learning more about immune response feedback pathways and potential targets of action will assist in the treatment of GO. In this discussion, we explore the pathogenesis of GO and relevant work, and highlight four potential targets for GO: Interleukin-23 receptor (IL-23 R), Leptin receptor (LepR), Orbital fibroblast activating factors, and Plasminogen activator inhibitor-1 (PAI-1). A deeper understanding of the pathogenesis of GO and the role of potential target signaling pathways is crucial for effective treatment of this disease.

[Three ways for protein aggregation and the control strategies].

Protein aggregation is a critical issue in the production of biopharmaceuticals. During protein production, transport and storage, various factors can lead to protein aggregation. With the in-depth study, different ways of protein aggregation and various influencing factors were identified. This includes physical and chemical factors, translation modifications and protein structure. Since protein aggregation exerts major impact on the activity and homogeneity of proteins, it is of great importance to study the ways of protein aggregation and how to control it to obtain high-quality proteins. The review focuses on three ways of protein aggregation, namely 3D domain swapping, salt bridge formation, and oxidative stress, as well as methods to control protein aggregation during protein production, transport and storage. This may facilitate reducing the loss caused by the formation of protein aggregation and improving the purity and homogeneity of protein in research and commercial production.

Anti-Inflammatory Effects of a Mytilus coruscus α-d-Glucan (MP-A) in Activated Macrophage Cells via TLR4/NF-κB/MAPK Pathway Inhibition.

The hard-shelled mussel (Mytilus coruscus) has been used as Chinese traditional medicine for thousands of years; however, to date the ingredients responsible for the various beneficial health outcomes attributed to Mytilus coruscus are still unclear. An α-d-Glucan, called MP-A, was isolated from Mytilus coruscus, and observed to exert anti-inflammatory activity in THP-1 human macrophage cells. Specifically, we showed that MP-A treatment inhibited the production of inflammatory markers, including TNF-α, NO, and PGE2, inducible NOS (iNOS), and cyclooxygenase-2 (COX-2), in LPS-activated THP-1 cells. It was also shown to enhance phagocytosis in the analyzed cells, but to severely inhibit the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear translocation of NF-κB P65. Finally, MP-A was found to exhibit a high binding affinity for the cell surface receptor TLR4, but a low affinity for TLR2 and dectin-1, via surface plasmon resonance (SPR) analysis. The study indicates that MP-A suppresses LPS-induced TNF-α, NO and PEG2 production via TLR4/NF-κB/MAPK pathway inhibition, and suggests that MP-A may be a promising therapeutic candidate for diseases associated with TNF-α, NO, and/or PEG2 overproduction.

Effect of Co-overexpression of Nisin Key Genes on Nisin Production Improvement in Lactococcus lactis LS01.

Nisin is a small antimicrobial peptide produced by several subset strains of Lactococcus lactis. To improve nisin yield in the producer L. lactis LS01, we proposed a successive fusion of nisA with nisRK and nisFEG into a single shuttle expression vector pMG36e under the control of the native strong constitutive promoter p32. Subsequently, the recombinant vectors were transplanted into the producer cell through electroporation. Nisin productivity was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assays. Expression of nisin peptide was detected by agar diffusion bioassay, and the transcriptional levels of the target genes involved in nisin biosynthesis were investigated via semi-quantitative reverse transcription PCR expression analysis using 16S ribosomal RNA (rRNA) as an internal control. Results suggested that the introduction of empty plasmid did not affect nisin production of L. lactis LS01, whereas by our rational construction and screening, the engineered strain co-overexpressing nisA, nisRK, and nisFEG achieved a maximum increment in bioactive nisin production with a yield of 2470 IU/ml in shake flasks and 4857 IU/ml in 1.0-l fermenters, which increased by approximately 66.3 and 52.6% (P < 0.05), respectively, compared with that of the original strain under the given fermentation conditions. Meanwhile, the transcriptional analysis revealed that the expression of most of these multicopy genes except nisE at transcriptional level were upregulated in the two recombinant strains (LS01/pAR and LS01/pARF), possibly contributing to the improved nisin production. Therefore, this study would provide a potential strategy to improve the economic benefits of nisin manufacture for large-scale industrial production.

Improvement of Natamycin Production by Cholesterol Oxidase Overexpression in Streptomyces gilvosporeus.

Natamycin is a widely used antifungal antibiotic. For natamycin biosynthesis, the gene pimE encodes cholesterol oxidase, which acts as a signalling protein. To confirm the positive effect of the gene pimE on natamycin biosynthesis, an additional copy of the gene pimE was inserted into the genome of Streptomyces gilvosporeus 712 under the control of the ermE* promoter (permE*) using intergeneric conjugation. Overexpression of the target protein engendered 72% and 81% increases in the natamycin production and cell productivity, respectively, compared with the control strain. Further improvement in the antibiotic production was achieved in a 1 L fermenter to 7.0 g/l, which was a 153% improvement after 120 h cultivation. Exconjugants highly expressing pimE and pimM were constructed to investigate the effects of both genes on the increase of natamycin production. However, the co-effect of pimE and pimM did not enhance the antibiotic production obviously, compared with the exconjugants highly expressing pimE only. These results suggest not only a new application of cholesterol oxidase but also a useful strategy to genetically engineer natamycin production.

Development, validation and influence factor analysis of a near-infrared method for the molecular weight determination of xanthan gum.

A practical molecular weight determination model of xanthan gum (XG), based on near-infrared (NIR) spectroscopy, was built in this study. Two sample measurement modules, integrating sphere module and fiber-optic probe module, were compared, and the best partial least square (PLS) regression model was based on fiber-optic probe module. The values of coefficient of determination in calibration (R(2)c), coefficient of determination in prediction (R(2)p), residual predictive deviation (RPD) and root mean square error of prediction (RMSEP) were 0.967, 0.975, 6.028 and 0.250×10(6)Da, respectively. The molecular weight range, linearity, accuracy and precision of the established method were also validated. Furthermore, influence factors on this method were discussed in order to establish an appropriate measurement protocol. Results showed that the proposed NIR method may be suitable for practical applications in manufacturing plants and probably be accepted as a good alternative approach for fast determination of molecular weight of XG in production process.

High level production of ß-galactosidase exhibiting excellent milk-lactose degradation ability from Aspergillus oryzae by codon and fermentation optimization.

A ß-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL(-1) with o-nitrophenyl-ß-D-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L(-1) in which the target protein (6.24 g L(-1)) occupied approximately 86 %. The recombinant ß-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the ß-galactosidase may have a good application potential in whey and milk industries.

Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb).

Oxygen deficiency is a critical factor during the fermentation production of natamycin. In order to alleviate oxygen limitation and enhance the yield of natamycin, the vgb gene, encoding Vitreoscilla hemoglobin (VHb) was inserted into pSET152 with its native promoter and integrated into the chromosome of Streptomyces gilvosporeus (S. gilvosporeus). The expression of VHb was determined by Western blotting. The activity of expressed VHb was confirmed by the observation of VHb-specific CO-difference spectrum with a maximal absorption at 419 nm for the recombinant. Integration of the empty plasmid pSET152 did not affect natamycin production of S. gilvosporeus. While the vgb-harboring strain exhibited high natamycin productivity, reaching 3.31 g/L in shake flasks and 8.24 g/L in 1-L fermenters. Compared to the wild strain, expression of VHb, increased the natamycin yield of the strain bearing vgb by 131.3 % (jar fermenter scale) and 175 % (shake flask scale), respectively, under certain oxygen-limiting condition. Addition of an extra copy of the vgb gene in S. gilvosporeus-vgb2 did not enhance the natamycin production obviously. These results provided a superior natamycin-producing strain which can be directly used in industry and a useful strategy for increasing yields of other metabolites in industrial strains.

Poly-γ-glutamate from Bacillus subtilis inhibits tyrosinase activity and melanogenesis.

Poly-γ-glutamate (γ-PGA) has been considered as one of the most promising biomaterials with a wide range of applications, but there has been no report that directly shows the anti-tyrosinase and anti-melanogenesis properties of γ-PGA. In the present study, we investigated the inhibitory effects of γ- PGA with low molecular weight (Mw; lγ-PGA) and high Mw (hγ-PGA) on mushroom tyrosinase and murine tyrosinase activities and on melanogenesis in B16 melanoma cells. First, we showed that both lγ-PGA and hγ-PGA could effectively inhibit mushroom tyrosinase activities including monophenolase and diphenolase activities in a dose-dependent manner. Second, both lγ-PGA and hγ-PGA showed strong anti-tyrosinase activity and anti-melanogenesis in B16 melanoma cells. Third, both lγ-PGA and hγ-PGA inhibited forskolin-induced tyrosinase activity and melanogenesis by decreasing the levels of intracellular reactive oxygen species and nitric oxide while increasing the catalase activity in B16 cells. This is the first report on the anti-melanogenesis effect of γ-PGA, which suggests that γ-PGA could have a potential in the cosmetic skin whitening business, therapeutic applications and the food industry.

Intra-articular injection of xanthan gum reduces pain and cartilage damage in a rat osteoarthritis model.

The objective of this study was to evaluate the alleviative effect of intra-articular (IA) injection of xanthan gum (XG) on pain and cartilage degradation in a model of monosodium iodoacetate (MIA)-induced knee osteoarthritis (OA). The rheological study and hyaluronidase (HAse) degradation analysis of XG injection were presented. The effect of pain relief was determined by measurements of paw withdrawal threshold and weight bearing by hind limbs. The protective effect on the cartilage was evaluated by gross morphological observation and histological evaluation of knee joints. The effect was investigated in two protocols: a therapeutic treatment protocol, and a prophylactic treatment protocol. Our results showed that HAse had no effect on the rheological properties of XG injection. Local XG administration in both protocols could reduce OA pain and alleviate the joint cartilage degradation induced by MIA. IA injection of XG might be an effective method for OA treatment in human.

The protective effect of xanthan gum on interleukin-1ß induced rabbit chondrocytes.

We have previously shown that intra-articular injection of xanthan gum (XG) could protect the joint cartilage and reduce osteoarthritis progression. In this study, we investigated the preliminary cytotoxicity of XG on chondrocytes, evaluated the effects of XG on the proliferation and the protein expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinase-1 (TIMP-1) in interleukin-1ß (IL-1ß)-induced rabbit chondrocytes. Primary rabbit chondrocytes were cultured. After treatment with various concentrations of XG with or without 10 ng/mL IL-1ß, the proliferation of chondrocytes was evaluated using the MTT assay and the expression levels of MMPs and TIMP-1 were evaluated using ELISA. The results showed that XG alone displayed no adverse effects on cell viability and reversed significantly IL-1ß-reduced cell proliferation in a dose-dependent manner. Furthermore, XG showed a dose-dependent inhibition in the IL-1ß-induced release of MMPs while increasing TIMP-1 expression. These results strongly suggest that XG affords protection on IL-1ß induced rabbit chondrocytes.

Improvement of xylanase production by thermophilic fungus Thermomyces lanuginosus SDYKY-1 using response surface methodology.

Response surface methodology (RSM) was employed to optimize medium composition for the production of a thermostable xylanase from T. lanuginosus SDYKY-1 using economical carbon and nitrogen sources (soybean meal and corncobs, respectively). Plackett-Burman (P-B) design was applied to evaluate the effects of nine variables (powdered corncobs, soybean meal, Tween-80, CaCl(2), MgSO(4)·7H(2)O, FeSO(4), KH(2)PO(4), initial pH and inoculum culture volume). Corncobs, soybean meal and FeSO(4) were found to significantly influence on the xylanase production. The concentrations of these three factors were therefore optimized using central composite design and RSM. Adjusting the concentration of corncobs to 38.7g/L, soybean meal to 17.5g/L and FeSO(4) to 0.26g/L favored maximum xylanase production. Xylanase activity of 3078U/mL was obtained after optimization, which was a 144% increase that obtained before optimization (1264U/mL).

Improved poly-gamma-glutamic acid production by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) in Bacillus subtilis.

In order to alleviate oxygen limitation and improve the yield of poly-gamma-glutamic acid (gamma-PGA) during fermentation, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosome of Bacillus subtilis and expressed during gamma-PGA production. The activity of the expressed Vitreoscilla hemoglobin (VHb) was confirmed by CO-difference spectrum. Expression of VHb enhanced cell growth under high viscosity fermentation conditions 1.26-fold and increased the yield of gamma-PGA 2.07-fold. These results indicate that the expression of VHb could be advantageous in high viscosity fermentation media.

On-column refolding of an insoluble His6-tagged recombinant EC-SOD overexpressed in Escherichia coli.

The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.