Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/<Emphasis Type="Italic">FRT</Emphasis> site-specific recombination system

Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.     

CTSB promotes porcine preadipocytes differentiation by degrading fibronectin and attenuating the Wnt/β-catenin signaling pathway

The process of preadipocytes differentiation plays a vital role in adipose tissue expansion and many factors are involved in this event. Cathepsin B (CTSB), secreted from lysosome, has been reported in regulating a variety of physiological processes. In this study, we demonstrated CTSB promotes lipid accumulation and adipogenic genes expression in porcine primary preadipocytes by degrading fibronectin (Fn), a key component of extracellular matrix. Lithium chloride (LiCl) is an activator of Wnt/β-catenin signaling through stabilizing β-catenin. We found that CTSB can relieve the anti-adipogenic effects of LiCl, indicating that CTSB could impact Wnt/β-catenin signaling pathway. Interestingly, Fn is an important target gene of Wnt/β-catenin. So we considered that CTSB promote preadipocytes differentiation by suppressing these two pathways.     

通过优化表达元件及培养基组分提高地衣芽胞杆菌中碱性丝氨酸蛋白酶产量

【背景】碱性丝氨酸蛋白酶(Subtilisin)是一种具有广泛用途的工业酶制剂。【目的】旨在通过优化启动子、信号肽及培养基组分来提高地衣芽胞杆菌中碱性丝氨酸蛋白酶产量。【方法】以地衣芽胞杆菌BL10为出发菌株,构建了含有4种不同类型启动子(PbacA、P43、PaprE和PsrfA)及4种不同类型信号肽(SPVpr、SPSacB、SPSacC和SPAprE)的碱性丝氨酸蛋白酶表达菌株,并在获得高产菌株的基础上进行培养基优化。【结果】4种启动子的表达水平为PbacAPaprEP43PsrfA,4种信号肽的分泌效率为SPAprESPSacCSPSacBSPVpr。其中,菌株BL10/pPbacA-aprE产生最高的碱性丝氨酸蛋白酶酶活(275.21 U/mL),相比于出发菌株BL10/pHY-aprE (167.98 U/mL)提高了64%。随后,通过对发酵培养基成分进行优化并结合正交优化,获得了一种高产碱性丝氨酸蛋白酶的培养基(g/L):玉米淀粉40.0,豆粕50.0,(NH4)2SO4 4.0,K2HPO4 3.0,CaCO3 1.0。最后,碱性丝氨酸蛋白酶酶活提高到747.37 U/mL,是初始酶活的4.45倍。【结论】为工业化高产碱性丝氨酸蛋白酶提供了一种有效策略。     

Body mass and behavior in Swiss mice subjected to continuous or discontinuous food restriction and refeeding

Food restriction (FR) is hypothesized to decrease body fat content of an animal and thus prevent obesity. However, the response of energy budget to a continuous (CFR) or discontinuous FR (DFR) remains inconsistent. In the present study, effects of CFR or DFR and refeeding on energy budget and behavior were examined in male Swiss mice. CFR significantly decreased the energy expenditure associated with basal metabolic rate (BMR) and activity behavior, but not sufficiently to compensate for energy deficit and thus resulted in lower body mass and fat content. DFR mice had a significantly higher food intake on ad libitum days and showed increases in BMR and activity after 4 weeks’ DFR, which might resulted in lower body mass and less body fat than controls. After being refed ad libitum, both CFR and DFR mice had similar body mass, BMR, and behavioral patterns to controls but had 95% and 75% higher fat content. This suggested that not only CFR but also DFR would be a significant factor in the process of obesity for animals that were refed ad libitum. It also indicated that food restriction interrupted many times by periods of ad libitum feeding had the same long-term effects like continuous underfeeding.     

Triptolide prevents bone loss via suppressing osteoclastogenesis through inhibiting PI3K-AKT-NFATc1 pathway

Bone loss (osteopenia) is a common complication in human solid tumour. In addition, after surgical treatment of gynaecological tumour, osteoporosis often occurs due to the withdrawal of oestrogen. The major characteristic of osteoporosis is the low bone mass with micro-architectural deteriorated bone tissue. And the main cause is the overactivation of osteoclastogenesis, which is one of the most important therapeutic targets. Inflammation could induce the interaction of RANKL/RANK, which is the promoter of osteoclastogenesis. Triptolide is derived from the traditional Chinese herb lei gong teng, presented multiple biological effects, including anti-cancer, anti-inflammation and immunosuppression. We hypothesized that triptolide could inhibits osteoclastogenesis by suppressing inflammation activation. In this study, we confirmed that triptolide could suppress RANKL-induced osteoclastogenesis in bone marrow mononuclear cells (BMMCs) and RAW264.7 cells and inhibited the osteoclast bone resorption functions. PI3K-AKT-NFATc1 pathway is one of the most important downstream pathways of RANKL-induced osteogenesis. The experiments in vitro indicated that triptolide suppresses the activation of PI3K-AKT-NFATc1 pathway and the target point located at the upstream of AKT because both NFATc1 overexpression and AKT phosphorylation could ameliorate the triptolide suppression effects. The expression of MDM2 was elevated, which demonstrated the MDM-p53-induced cell death might contribute to the osteoclastogenesis suppression. Ovariectomy-induced bone loss and inflammation activation were also found to be ameliorated in the experiments in vivo. In summary, the new effect of anti-cancer drug triptolide was demonstrated to be anti-osteoclastogenesis, and we demonstrated triptolide might be a promising therapy for bone loss caused by tumour.     

Chemical Composition and Antioxidant Activity of Essential Oil of Chinese Propolis

The chemical composition and in vitro antioxidant activity of the essential oil of propolis (EOP) collected from 25 locations in China was investigated. Steam‐distillation extraction was used to extract the EOP, and chemical composition was identified by GC/MS. The antioxidant activities of EOP were also measured. The result showed that a total of 406 compounds were detected in EOP. The major compounds of Chinese EOP were cedrol, γ‐eudesmol, benzyl alcohol, phenethyl alcohol, 2‐methoxy‐4‐vinylphenol, 3,4‐dimethoxystyrene and guaiol. Principal component analysis revealed the significant correlation between EOP compositions and their origins, and certain correlation was detected between EOP and their color. Linear discriminant analysis showed that 88 % and 84 % of the propolis samples were predicted correctly as the groupings identified by climatic zone and the color, respectively. Furthermore, the differences of antioxidant activities of EOP were significant. EOP of Shandong had the strongest antioxidant activities, whereas EOP of Guangdong, Yunnan and Hunan showed the poorest.     

Isoliquiritigenin ameliorates caerulein‐induced chronic pancreatitis by inhibiting the activation of PSCs and pancreatic infiltration of macrophages

Chronic pancreatitis (CP) is characterized by persistent inflammation of the pancreas that results in progressive loss of the endocrine and exocrine compartment owing to atrophy and/or replacement with fibrotic tissue. Currently, the clinical therapeutic scheme of CP is mainly symptomatic treatment including pancreatic enzyme replacement, glycaemic control and nutritional support therapy, lacking of specific therapeutic drugs for prevention and suppression of inflammation and fibrosis aggravating in CP. Here, we investigated the effect of isoliquiritigenin (ILG), a chalcone‐type dietary compound derived from licorice, on pancreatic fibrosis and inflammation in a model of caerulein‐induced murine CP, and the results indicated that ILG notably alleviated pancreatic fibrosis and infiltration of macrophages. Further in vitro studies in human pancreatic stellate cells (hPSCs) showed that ILG exerted significant inhibition on the proliferation and activation of hPSCs, which may be due to negative regulation of the ERK1/2 and JNK1/2 activities. Moreover, ILG significantly restrained the M1 polarization of macrophages (RAW 264.7) via attenuation of the NF‐κB signalling pathway, whereas the M2 polarization was hardly affected. These findings indicated that ILG might be a potential anti‐inflammatory and anti‐fibrotic therapeutic agent for CP.