FUNDC1 Mediated Mitophagy in Epileptic Hippocampal Neuronal Injury Induced by Magnesium-Free Fluid.

Mitophagy plays a key role in epileptic neuronal injury, and recent studies have shown that FUNDC1 plays an important role in regulating mitophagy. However, the specific effect of FUNDC1 on neuronal damage in epilepsy is unknown. In this study, we investigated the role of FUNDC1 in mitophagy and neuronal apoptosis using a hippocampal neuronal culture model of acquired epilepsy (AE) in vitro. We found that mitophagy levels were significantly increased in this model, as indicated by elevated LC3A/B ratios. FUNDC1 overexpression using lentiviral vectors enhanced mitophagy, whereas FUNDC1 down-regulation using lentiviral vectors impaired this process. Overexpression of FUNDC1 significantly decreased AE-induced superoxide anion, enhanced cell viability, reduced oxidative stress, and reduced neuronal apoptosis in epileptic hippocampus, while FUNDC1 down-regulation caused the opposite effect. In conclusion, we demonstrated that FUNDC1 is an important modulator of AE-induced neuronal apoptosis by controlling mitophagy function.

SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF-1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. qRT-PCR and flow cytometry were used to explore eGFP expression levels. And gene copy number was also detected by qPCR, respectively. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in CHO cells.

Development and characterizations of a miniature capillary electrophoresis mass spectrometry system.

A miniature capillary electrophoresis mass spectrometry (CE/MS) system has been developed in this work. A 100% electrical driven miniaturized CE device was integrated with a miniature MS instrument, which has a discontinuous atmospheric pressure interface (DAPI) for coupling with atmospheric pressure ionization sources. A nanoelectrospray ionization (nano-ESI) source was developed with a sheath liquid interface for coupling the miniature CE and the MS system. A systematic characterization and optimization of the analytical performance have been done. The analysis of isobaric peptides and avoiding charge competition effects in nano-ESI sources have been demonstrated.

[In vitro effect of alendronate on chondrocytes and articular cartilage and subchondral bone in rabbit anterior cruciate ligament transection model].

OBJECTIVE: To examine the effects of alendronate (ALN) on IL-1beta-stimulated chondrocyte of rabbit in vitro and on cartilage and subchondral bone in rabbit osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT). METHODS: The chondrocytes from articular surface of healthy 3-month-old Japanese White rabbits were obtained by the method of enzyme digestion and cultured in vitro. The third generation chondrocytes were assigned into three groups: the chondrocytes were cultured in DMEM medium with 10 ng/mL IL-1beta for 2 days, subsequently with (ALN group, group A1) or without (IL-1beta group, group B1) 1 x 10(-6) mol/L ALN for 3 days; the chondrocytes in vacant group (group C1) were cultured in DMEM medium for 5 days. The expression of Col II and MMP-13 were analyzed by immunocytochemical staining observation and real time RT-PCR test. Another twenty-four 3-month-old male Japanese White rabbits were randomized into three groups (n = 8 per group). The OA model was made by ACLT in ACLT+ALN group (group A2) and ACLT group (group B2); the joint cave was sutured after exposure of ACL in sham group (group C2). After 4 days, the rabbits of group A2 received the subcutaneous injection of ALN at a dosage of 10 microg/(kg x d) for 8 weeks. Rabbits of group B2 and C2 received equal normal saline treatment. After 8 weeks, the rabbits were executed. The macro-pathologic changes of right knee joints were observed, so were the histological changes of femoral condyles. Expression levels of Col II and MMP-13 were detected by immunohistochemical staining. The bone histomorphometry analysis was applied to subchondral bone of proximal tibia. RESULTS: In vitro, the Col II immunocytochemical staining showed intensely positive staining in group C1, and the intensity of staining was slightly decreased in group A1, but the intensity of Col II immunocytochemical staining was extremely lower in the group B1. The integrated absorbance (IA) value for Col II in group A1 was significantly higher than that of group B1 (P < 0.05), but there was no significant difference between group A1 and group C1 (P > 0.05). Immunocytochemical detection of MMP-13 showed intense staining in group B1, and the intensity of staining was slightly decreased in group A1, but no MMP-13 expression was detected in the group C1. The IA value for MMP-13 in group A1 was significantly lower than that of group B1 (P < 0.05), but significantly higher than that of group C1 (P <0.05). The real time RT-PCR analysis showed significantly higher mRNA levels of Col II in group A1 than in group B1 (P < 0.05), but there was no significant difference between group A1 and group C1 (P > 0.05). The MMP-13 mRNA level of the chondrocytes in group A1 was significantly lower than that of group B1 (P < 0.05), but significantly higher than that of group C1 (P < 0.05). In vivo, the gross appearance of surface of knee joint showed that there was no ulcer in group C2, and there was some ulcers in group A2, but many and all layers ulcers in group B2. Mankin score of group A2 was significantly lower than that of group B2 (P < 0.05), but significantly higher than that of group C2 (P < 0.05). Immunohistochemical staining showed that Col II in articular cartilage was intensely staining in group C2, the intensity of staining was slightly decreased in group A2, and the intensity of Col II immunohistochemical staining was extremely low in group B2, but there was no significant difference between group A2 and group C2 (P > 0.05). The immunohistochemical staining for MMP-13 significantly increased in group B2, the intensity of staining was slightly decreased in group A2, and no MMP-13 expression was detectable in the group C2. The IA value for MMP-13 in group A2 was significantly lower than that of group B2 (P < 0.05), but significant higher than that of group C2 (P < 0.05). Bone histomorphometry showed that in group B2 percent trabecular area and trabecular thickness markedly decreased compared with those in group A2 and group C2 (P < 0.05), but there was no significant difference between group A2 and group C2 (P > 0.05). The trabecular separation, percent labeled perimeter and bone formation rate were significantly elevated in group B2 compared with those in group A1 and group C2 (P < 0.05), but there was no significant difference between group A2 and group C2 (P > 0.05). No significant difference was evident on trabecular number and mineral apposition rate values among groups A2, B2, and C2 (P > 0.05). CONCLUSION: For rabbits OA induced by ACLT, the subcutaneous injections of alendronate can inhibit cartilage degradation, prevent bone loss, and improve microarchitecture of subchondral bone. ALN can partially protect chondrocytes by inhibiting the expression of MMP-13 both in vitro and in vivo.

[Effect of calcitonin on articular cartilage of osteoarthritis].

OBJECTIVE: To review the effect of calcitonin on cartilage and subchondral bone of osteoarthritis. METHODS: Recent literatures about the effect of calcitonin on osteoarthritis was reviewed. RESULTS: Calcitonin could promote the synthesis of important cartilage matrix such as proteoglycans and collagen II, propelling the regeneration of cartilage and subchondral bone. CONCLUSION: Calcitonin can protect articular cartilage through promoting the synthesis of cartilage and inhibiting its degradation.