Improving production of N-glycosylated recombinant proteins by leaky Escherichia coli.

Escherichia coli has been considered as a promising host for the production of N-glycosylated therapeutic proteins and glycoconjugate vaccines. In this study, we developed a simple and efficient strategy for improving the production of N-glycosylated recombinant proteins by combining auto-induction with the use of a leaky E. coli strain. A leaky E. coli strain, designated as CLM37-Δlpp, was engineered by deleting the Braun's lipoprotein (lpp) gene of E. coli strain CLM37. Three distinct acceptor model N-glycosylated proteins, glyco-tagged human tenth fibronectin type III domain (FN3-Gly), enhanced green fluorescent protein (eGFP-Gly), and scFv of vascular endothelial growth factor receptor 3 (scFv-VEGFR3-Gly) were then expressed in CLM37-Δlpp, which carried an N-glycosylation machinery from Campylobacter jejuni for the investigation of glycoprotein production. As much as 75%, 65%, and 60% of the glycosylated FN3-Gly, eGFP-Gly, and scFv-VEGFR3-Gly, respectively, were found in the culture medium. The yields of glycosylated FN3-Gly, eGFP-Gly, and scFv-VEGFR3-Gly were 106 ± 7.4 mg/L, 65 ± 2.5 mg/L, and 62 ± 4.3 mg/L, respectively, which were more than three folds the corresponding yields obtained when these proteins were expressed in CLM37, the unmodified strain. The results suggested that this simplified approach could improve the production of N-glycosylated proteins with E. coli to facilitate large-scale production.

Esthetic evaluation of facial cheek volume: A study using 3D stereophotogrammetry.

OBJECTIVES: To investigate the influence of cheek volume on facial esthetics judged by orthodontists and non-specialists. MATERIALS AND METHODS: A 25-year-old female's natural and smiling face was captured by 3D stereophotogrammetry. Cheek volume of the 3D image was altered to different degrees three-dimensionally. For the natural and smiling face, seven groups of facial images were created: decreased grade I/II/III, increased grade I/II/III, and the original one. Thirty orthodontists and 30 nonspecialists were invited to perform esthetic evaluation of the original and transformed images using a questionnaire. Data were calculated with one-way analysis of variance (least significant difference test) and independent samples t test. RESULTS: Compared to nonspecialists, orthodontists gave lower esthetic scores to the decreased grade III facial images (maximum deformation degree: 7.500 mm and 7.327 mm in natural and smiling face-oriented image groups, respectively). The decreased grade III facial images also received the highest age ranks. However, the increased grade III facial images received the lowest scores and highest age ranks from nonspecialists (maximum deformation degree: 6.994 mm and 5.300 mm in natural and smiling face-oriented image groups, respectively) ( P < .01). CONCLUSIONS: Orthodontists and nonspecialists showed different esthetic evaluation of varied cheek volume. The influence of cheek volume in orthodontic diagnostic analysis needs further consideration.

YAP regulates periodontal ligament cell differentiation into myofibroblast interacted with RhoA/ROCK pathway.

During orthodontic tooth movement (OTM), periodontal ligament cells (PDLCs) receive the mechanical stimuli and transform it into myofibroblasts (Mfbs). Indeed, previous studies have demonstrated that mechanical stimuli can promote the expression of Mfb marker α-smooth muscle actin (α-SMA) in PDLCs. Transforming growth factor ß1 (TGF-ß1), as the target gene of yes-associated protein (YAP), has been proven to be involved in this process. Here, we sought to assess the role of YAP in Mfbs differentiation from PDLCs. The time-course expression of YAP and α-SMA was manifested in OTM model in vivo as well as under tensional stimuli in vitro. Inhibition of RhoA/Rho-associated kinase (ROCK) pathway using Y27632 significantly reduced tension-induced Mfb differentiation and YAP expression. Moreover, overexpression of YAP with lentiviral transfection in PDLCs rescued the repression effect of Mfb differentiation induced by Y27632. These data together suggest a crucial role of YAP in regulating tension-induced Mfb differentiation from PDLC interacted with RhoA/ROCK pathway.

Angiopoietin-2 attenuates angiotensin II-induced aortic aneurysm and atherosclerosis in apolipoprotein E-deficient mice.

Angiogenesis and inflammation are implicated in aortic aneurysm and atherosclerosis and regulated by angiopoietin-2 (Angpt2). The effect of Angpt2 administration on experimental aortic aneurysm and atherosclerosis was examined. Six-month-old male apolipoprotein E deficient (ApoE-/-) mice were infused with angiotensin II (AngII) and administered subcutaneous human Fc-protein (control) or recombinant Angpt2 (rAngpt2) over 14 days. Administration of rAngpt2 significantly inhibited AngII-induced aortic dilatation and rupture of the suprarenal aorta (SRA), and development of atherosclerosis within the aortic arch. These effects were blood pressure and plasma lipoprotein independent and associated with Tie2 activation and down-regulation of monocyte chemotactic protein-1 (MCP-1) within the SRA. Plasma concentrations of MCP-1 and interleukin-6 were significantly lower in mice receiving rAngpt2. Immunostaining for the monocyte/macrophage marker MOMA-2 and the angiogenesis marker CD31 within the SRA were less in mice receiving rAngpt2 than controls. The percentage of inflammatory (Ly6Chi) monocytes within the bone marrow was increased while that in peripheral blood was decreased by rAngpt2 administration. In conclusion, administration of rAngpt2 attenuated angiotensin II-induced aortic aneurysm and atherosclerosis in ApoE-/- mice associated with reduced aortic inflammation and angiogenesis. Up-regulation of Angpt2 may have potential therapeutic value in patients with aortic aneurysm and atherosclerosis.

Influence of apolipoprotein E, age and aortic site on calcium phosphate induced abdominal aortic aneurysm in mice.

OBJECTIVE: To assess relevant features of abdominal aortic aneurysms (AAA) induced by calcium phosphate within a mouse model. Specifically we investigated: (1) whether apolipoprotein E deficiency and older age promoted AAA formation, and (2) whether the local application of calcium phosphate affected the size of distant aortic segments. METHODS: AAA was induced by application of calcium phosphate to the infra-renal aortas of 3 and 7 month old male mice. AAA induction was assessed by calculating expansion of the infra-renal aortic diameter over 1-4 weeks. Aortic samples were assessed to quantify calcification, macrophages infiltration, elastic lamellar degradation and apoptosis. Blood pressure was measured by the tail cuff method, and plasma concentrations of total cholesterol, low density lipoprotein and very low density lipoprotein cholesterol, and pro-inflammatory cytokines were measured using commercially available kits. The maximum diameters of the aortic arch, thoracic and supra-renal aorta at sacrifice were measured by morphometry and the mean maximal diameter of these three aortic segments was calculated. RESULTS: The median expansion of the infra-renal aorta 2 weeks after AAA induction was significantly greater in apolipoprotein E deficient (ApoE(-/-)) mice than in age- and gender-matched wild type controls [275.8% (IQR 193.8%-348.5%) versus 94.7% (IQR 47.8%-163.4%), P = 0.02]. The greater aortic expansion in ApoE(-/-) mice was associated with aortic calcification, macrophage infiltration, elastic lamellar degradation and apoptosis of cells in the media and adventitia. The plasma low density lipoprotein/very low density lipoprotein cholesterol concentrations 2 weeks after AAA induction were positively correlated with the expansion of the infra-renal aorta induced by calcium phosphate. The median expansion of the infra-renal aorta 2 weeks after AAA induction was similar in 3 and 7 month old wild type mice. The local administration of calcium phosphate was associated with an increase in the mean maximal diameter of distant aortic segments, but not associated with changes in the concentrations of pro-inflammatory markers in either the plasma or the spleen. CONCLUSION: This study suggests that apolipoprotein E deficiency, but not age, predisposes to AAA induced within the calcium phosphate model. Increased AAA expansion in ApoE(-/-) mice was associated with calcification, macrophage infiltration, elastic lamellar degradation, and cell apoptosis. Local application of calcium phosphate also promoted dilation of distant aortic segments.

Impaired acetylcholine-induced endothelium-dependent aortic relaxation by caveolin-1 in angiotensin II-infused apolipoprotein-E (ApoE-/-) knockout mice.

OBJECTIVE: The angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE(-/-)) mouse model is widely used to study atherosclerosis and abdominal aortic aneurysm. An increase in blood pressure has been reported in this model however the underlying mechanism has not been fully explored. In this study, we investigated whether vasomotor dysfunction develops in AngII-infused ApoE(-/-) mice and the underlying mechanism involved. METHODS: ApoE(-/-) mice were infused with vehicle (distilled water) or AngII subcutaneously for 14 days. Blood pressure and heart rate were measured using the non-invasive tail cuff method. Aortic vascular reactivity and expression of key proteins (endothelial nitric oxide synthase (eNOS), phospho-eNOS and caveolin-1) were assessed using tension myography and Western blotting respectively. Plasma nitric oxide (NO) level was estimated using a colorimetric assay. RESULTS: AngII infusion caused a time-dependent increase in blood pressure (P<0.001). Aortas from AngII-infused mice were significantly less responsive to acetylcholine-induced endothelium-dependent relaxation when compared to aortas from mice infused with vehicle control (P<0.05). Contractile responses to phenylephrine (P<0.01) and potassium chloride (P<0.001) were significantly enhanced in aortas from AngII-infused mice. eNOS phosphorylation was significantly decreased in the aorta of AngII-infused mice (P<0.05). Aortic caveolin-1 protein expression was significantly increased in AngII-infused mice (P<0.05). Plasma nitrate/nitrite level was significantly reduced in AngII-infused mice (P<0.05). Pharmacological disruption of caveolae using methyl-ß-cyclodextrin (MßCD) in isolated aortas from AngII-infused mice caused a significant leftward shift of the acetylcholine-induced relaxation concentration-response curve when compared to vehicle control (P<0.05). CONCLUSION: Upregulation of caveolin-1 protein expression and reduced NO bioavailability contributes to aortic endothelial dysfunction in AngII-infused ApoE(-/-) mice.

Overexpression of osteoprotegerin promotes preosteoblast differentiation to mature osteoblasts.

OBJECTIVE: The hypothesis of the present study is that overexpression of osteoprotegerin (OPG) promotes preosteoblast maturation. MATERIALS AND METHODS: The preosteoblast cell line MC3T3-E1 was transfected with OPG overexpression. OPG expression was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blot. Changes in the transcription factors in OPG-expressing cells were assessed by real-time polymerase quantitative polymerase chain reaction (RT-qPCR). Alkaline phosphate (ALP) expression was measured by ELISA. RESULTS: The success of stable transfection of MC3T3-E1 cells with OPG overexpression was confirmed by MoFlow sorting followed by G418 selection. RT-qPCR showed that expression of RunX2, the most important osteoblast differentiation controlling factor, was suppressed. Smad1 and Akt1, as well as ALP, were upregulated in the OPG overexpressing cells. CONCLUSION: Results from the present study provide evidence that overexpression of OPG in preosteoblasts promotes its differentiation into mature osteoblasts.

Orthodontic mechanical tension effects on the myofibroblast expression of alpha-smooth muscle actin.

OBJECTIVE: To detect myofibroblast formation on the tension side during orthodontic tooth movement in vivo and myofibroblast expression of alpha-smooth muscle actin (alpha-SMA) induced by tension both in vivo and in vitro. MATERIALS AND METHODS: Fifty 6-week male rats were used in this in vivo study, and the right maxillary first molar was moved mesially, which served as the experimental group, and the left maxillary first molar served as the control. Rats were sacrificed at days 0, 3, 5, 7, and 14 after force loading. Myofibroblasts, identified with alpha-SMA, were examined through immunohistochemistry. For the in vitro study, human periodontal ligament (PDL) fibroblasts were obtained. Cyclic mechanical tension was applied to the fibroblasts for 0, 1, 3, 6, and 12 hours. Transmission electron microscopy was used to detect the ultrastructure of myofibroblasts. alpha-SMA mRNA gene expression was quantified by real-time quantitative PCR. The expression of alpha-SMA was detected by immunofluorescence and quantified by Western blotting. RESULTS: In vivo, the myofibroblasts expressing alpha-SMA were identified both in the experimental group and in the control group. The expressions of alpha-SMA were increased in the tension areas of the experimental group over time, and reached the maximum in day 14. In vitro, fibronexus junctions and actin microfilaments in the cells could be found with transmission electron microscopy. Cyclic mechanical tension could significantly induce alpha-SMA expression at 12 hours (P < .01) than the controls. CONCLUSIONS: Myofibroblasts existed in the PDL. The expressions of alpha-SMA in the myofibroblasts were significantly up regulated under tension both in vivo and in vitro.

Mechanical tensile stress effects on the expression of bone sialoprotein in bovine cementoblasts.

OBJECTIVE: To develop a new cementoblast culture method and to detect bone sialoprotein (BSP) expression in response to high and low mechanical tensile stress in cementoblast in vitro. MATERIALS AND METHODS: Cementoblasts were collected from the roots of newborn bovine teeth and were identified with cementum-derived attachment protein (CAP) antibody 3G9. Cell proliferation was evaluated by MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, and mineralization was confirmed by von Kossa staining. Mechanical tensile stress was applied in vitro to the cementoblast with the use of a uniaxial four-point bending system with 2000 or 4000 microstrains, at a frequency of 0.5 Hz for 3, 6, 12, 24, or 36 hours. BSP mRNA level was quantified by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: A large amount of cementoblast was observed to be expressing CAP. Cementoblasts had a proliferation tendency similar to that of osteoblasts but different from that of periodontal ligament (PDL) cells. Cementoblasts had the ability to become mineralized between osteoblasts and PDL cells. The mechanical tensile stress significantly up-regulated BSP mRNA expression, which reached a peak at 24 hours in both 2000 and 4000 microstrain groups (P < .01) and was tenfold and sixfold higher than that of controls, respectively. BSP expression dropped toward baseline levels at 36 hours in both groups. CONCLUSIONS: Mechanical tensile stress up-regulated the expression of BSP. Low mechanical tensile stress induced earlier and more intensive up-regulation of BSP mRNA; this might represent the optimal stimuli for cementoblast activity.