Matrix metalloproteinase-13, NF-κB p65 and interleukin-1ß are associated with the severity of knee osteoarthritis.

Knee osteoarthritis (KOA) is a prevalent disease, especially in the elderly. The present study examined the expression of matrix metalloproteinase-13 (MMP-13), NF-κBp65 and interleukin (IL)-lß in the synovial tissues of KOA patients and the role of MMP-13 and the NF-κBp65 signalling pathway in KOA pathogenesis. A total of 100 KOA patients were enrolled in our hospital from December 2015 to December 2017 and were classified into either a mild KOA group (Outerbridge grade 1 and 2) or a severe KOA group (Outerbridge grade 3 and 4). Non-OA patients were included as controls. Synovial tissues from patients in both groups were collected for detection of the mRNA and protein expression of MMP-13, NF-κBp65 and IL-lß. Synovial tissue slices were subjected to haematoxylin and eosin staining and immunohistochemistry (SP method). Cartilage tissues were observed under a light microscope after Safranin O-fast green staining. Reverse transcription-quantitative PCR and western blot analyses demonstrated that the expression of MMP-13, NF-κBp65 and IL-lß in the mild and severe groups were substantially upregulated compared with the control group (all P<0.05). A positive correlation between MMP-13 and NF-κBp65 expression in the KOA synovial tissues was identified (P<0.05). Immunohistochemistry revealed that the expression of MMP-13 and NF-κBp65 was related to the severity of KOA (MMP-13: severe, 92.54%; moderate, 76.52%; control: 32.14%; and NF-κBp65: severe, 85.56%; moderate, 48.12%; control: 28.32%). This evidence indicated that the severity of KOA was related to MMP-13 and NF-κBp65 expression. The NF-κB signalling pathway may be activated during OA progression, which could upregulate the expression of MMP-13 and IL-1ß and accelerate the deterioration of articular cartilage.

Improving Performance and Operational Stability of Porcine Interferon-α Production by Pichia pastoris with Combinational Induction Strategy of Low Temperature and Methanol/Sorbitol Co-feeding.

Various induction strategies were investigated for effective porcine interferon-α (pIFN-α) production by Pichia pastoris in a 10 L fermenter. We found that pIFN-α concentration could be significantly improved with the strategies of low-temperature induction or methanol/sorbitol co-feeding. On this basis, a combinational strategy of induction at lower temperature (20 °C) with methanol/sorbitol co-feeding has been proposed for improvement of pIFN-α production. The results reveal that maximal pIFN-α concentration and antiviral activity reach the highest level of 2.7 g/L and 1.8 × 10(7) IU/mg with the proposed induction strategy, about 1.3-2.1 folds higher than those obtained with other sub-optimal induction strategies. Metabolic analysis and online multi-variable measurement results indicate that energy metabolic enrichment is responsible for the performance enhancement of pIFN-α production, as a large amount of ATP could be simultaneously produced from both formaldehyde oxidation pathway in methanol metabolism and tricarboxylic acid (TCA) cycle in sorbitol metabolism. In addition, the proposed combinational induction strategy enables P. pastoris to be resistant to high methanol concentration (42 g/L), which conceivably occur associating with the error-prone methanol over-feeding. As a result, the proposed combinational induction strategy simultaneously increased the targeted protein concentration and operational stability leading to significant improvement of pIFN-α production.

Enhancing pIFN-α production and process stability in fed-batch culture of Pichia pastoris by controlling the methanol concentration and monitoring the responses of OUR/DO levels.

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor using an in situ methanol electrode-based feeding process with the control level of methanol concentration linearly increased to 10 g l⁻¹ for the first 20 h and maintained at 10 g l⁻¹ for the rest of expression phase. With this two-stage control process, the highest pIFN-α concentration reached a level of 1.81 g l⁻¹, which was 1.5-fold of that in the previous constant 10 g l⁻¹ induction experiments. There is an improvement of the pIFN-α productivity from more distribution of carbon flux to protein expression. The pIFN-α expression stability could be further enhanced by a simple on-line fault diagnosis method for methanol overfeeding based on oxygen uptake rate changing patterns. By implementing corrective action of feeding glycerol after fault detection, the production yield increased to twice the amount it would have been without the diagnosis.

Methanol/sorbitol co-feeding induction enhanced porcine interferon-α production by P. pastoris associated with energy metabolism shift.

The production of porcine interferon-α (pIFN-α) by Pichia pastoris was largely enhanced when adopting sorbitol/methanol co-feeding induction strategy at 30 °C in a 10-L fermentor. Analysis of energy regeneration pattern and carbon metabolism revealed that major energy metabolism energizing pIFN-α synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle under the methanol/sorbitol co-feeding induction strategy. The sorbitol/methanol co-feeding induction strategy weakened formaldehyde dissimilatory pathway and repressed the accumulation of toxic metabolite-formaldehyde, reduced theoretical oxygen consumption rate and oxygen supply requirement, and increased energy/methanol utilization efficiency so that more methanol could be effectively used for pIFN-α synthesis. As a result, pIFN-α antiviral activity reached a highest level of 1.8 × 10(7) IU/mL which was about 10- to 200-folds of those obtained under pure methanol induction at 20 and 30 °C, respectively.

Generalised additive modelling approach to the fermentation process of glutamate.

In this work, generalised additive models (GAMs) were used for the first time to model the fermentation of glutamate (Glu). It was found that three fermentation parameters fermentation time (T), dissolved oxygen (DO) and oxygen uptake rate (OUR) could capture 97% variance of the production of Glu during the fermentation process through a GAM model calibrated using online data from 15 fermentation experiments. This model was applied to investigate the individual and combined effects of T, DO and OUR on the production of Glu. The conditions to optimize the fermentation process were proposed based on the simulation study from this model. Results suggested that the production of Glu can reach a high level by controlling concentration levels of DO and OUR to the proposed optimization conditions during the fermentation process. The GAM approach therefore provides an alternative way to model and optimize the fermentation process of Glu.