Insulin receptor substrate 2 (IRS-2) modulates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which controls the suppression of gluconeogenic genes; IRS-2 is also a critical node of insulin signaling. Because of the high homology between pig and human IRS-2, we investigated the expression pattern and function of porcine IRS-2. QPCR and immunoblotting were used to detect the IRS-2 expression level in different tissues. There were high IRS-2 levels in the cerebral cortex, hypothalamus, and cerebellum in the central nervous system. In peripheral tissues, IRS-2 was expressed at relatively high levels in the liver. Immunohistochemistry analysis revealed that IRS-2 was mainly distributed in the hypothalamus and cerebral cortex. Furthermore, IRS-2 knockdown porcine hepatocytes and porcine aortic endothelial cells (PAECs) were generated. The IRS-2 knockdown induced abnormal expression of genes involved in glycolipid metabolism in hepatocytes and reduced the antiatherosclerosis ability in PAECs. In addition, we disrupted IRS-2 in porcine embryonic fibroblasts (PEFs) using the CRISPR/Cas9 genome editing system, before finally generating IRS-2 knockout embryos by somatic cell nuclear transfer (SCNT). Taken together, our results indicate that IRS-2 might be a valuable target to establish diabetes and vascular disease models in the pig.
Sperm-mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind exoge- nous DNA and transfer it into oocytes by fertilization. However, SMGT is still undergoing opti- mization to improve its efficiency to produce transgenic animals. The acrosome reaction is neces- sary for spermatozoa to carry the exogenous DNA into oocytes. In this study, the effect of the acrosome reaction on the efficiency of spermatozoa carrying exogenous DNA was evalua- ted. The results showed that the efficiency of the acrosome reaction was significantly higher (p<0.05) after incubation with 50 μmol/L progesterone compared to incubation without proges- terone. It was significantly higher (p<0.05) in the 20, 40, and 60 min of progesterone treatment groups than in the 0 min treatment group. The spermatozoa were further incubated with cyanine dye Cy5 labeled DNA (Cy5-DNA) for 30 min at 37°C, and positive fluorescence signals were detected after the acrosome reaction was induced by progesterone at concentrations of 0 and 50 μmol/L for 40 min. The percentage of positive Cy5-DNA signals in spermatozoa was 96.61±2.06% and 97.51±2.03% following exposure to 0 and 50 μmol/L progesterone, respective- ly. The percentage of partial spermatozoa heads observed following combination with Cy5-DNA was 39.73±3.03% and 56.88±3.12% following exposure to 0 and 50 μmol/L progesterone, respec- tively. The ratio of positively stained spermatozoa combined with exogenous DNA showed no reduction after the acrosome reaction. These results suggest that the acrosome reaction might not be the key factor affecting the efficiency of SMGT.
Osteocalcin is a major non-collagenous component of the bone extracellular matrix and is considered to be an indicative factor of osteoblast differentiation. In the present study, we detected osteocalcin expression in different antler areas and growth phases by immunohisto- chemistry. Osteocalcin was highly expressed in all areas during the mineralization period and in mesenchymal cell and chondrocyte areas during the rapid growth period. The nucleotide sequence of the osteocalcin gene in sika deer antler was determined. The open reading frame was 303 bp encoding a protein of 100 amino acids. The estimated molecular mass of osteocalcin was 10.38 kDa and the theoretical isoelectric point was 5.37. The osteocalcin gene with a 6× His-tag at the C-terminus was cloned into the pGEX-4T1 vector and expressed in Escherichia coli under optimal conditions. The recombinant soluble protein fused with GST was purified with Ni-NTA resin. The purified osteocalcin protein exhibited a significant increase in HA adhesion and promoted antler chondrocyte proliferation. Osteocalcin is an important factor in regulating the rapid growth and differentiation of deer antlers.
In this study, medium-carbon steel was subjected to warm deformation experiments on a Gleeble 3500 thermosimulator machine at temperatures of 550°C and 650°C and strain rates of 0.001 s–1 to 1 s–1. The warm deformation behavior of martensite and the effects of strain rate on the microstructure of ultrafine grained medium-carbon steel were investigated. The precipitation behavior of Fe3C during deformation was analyzed and the results showed that recrystallization occurred at a low strain rate. The average ultrafine ferrite grains of 500 ± 58 nm were fabricated at 550°C and a strain rate of 0.001 s–1. In addition, the size of Fe3C particles in the ferrite grains did not show any apparent change, while that of the Fe3C particles at the grain boundaries was mainly affected by the deformation temperature. The size of Fe3C particles increased with the increasing deformation temperature, while the strain rate had no significant effect on Fe3C particles. Moreover, the grain size of recrystallized ferrite decreased with an increase in the strain rate. The effects of the strain rate on the grain size of recrystallized ferrite depended on the deformation temperature and the strain rate had a prominent effect on the grain size at 550°C deformation temperature. Finally, the deformation resistance apparently decreased at 550°C and strain rate of 1 s–1 due to the maximum adiabatic heating in the material.
Ludwigite is the main available boron-bearing resource in China. In order to enrich the theory system and optimize its utilization processes, this paper study the mechanism and kinetics on non-isothermal decomposition of ludwigite in inert atmosphere by means of thermal analysis. Results show that, the decomposition of serpentine and szajbelyite is the main cause of mass loss in the process. At the end of decomposition, hortonolite and ludwigite are the two main phases in the sample. The average E value of structural water decomposition is 277.97 kJ/mol based on FWO method (277.17 kJ/mol based on KAS method). The results is proved to be accurate and reliable. The mechanism model function of structural water decomposition is confirmed by Satava method and Popescu method. The form of the most probable model function is G(α) = (1 – α)–1 – 1 (integral form) and f (α) = (1 – α)2 (differential form), and its mechanism is chemical reaction. This is verified by the criterion based on activation energy of model-free kinetics analysis.
The friction and wear properties of 201HT aluminum alloys and the corresponding competitive coupons were tested on an electrohydraulic servo face friction and wear testing machine (MM-U10G). The microstructures of the competitive coupons were investigated by scanning electron microscopy (SEM) and consequently the corresponding friction and wear mechanisms were studied. The results demonstrated that: (1) the best competitive material of friction and wear performance of the 201HT was the 201HTC. (2) the 201HTC modified by carbon following the initial mill for oil storage of the micro-groove to be produced, increased the corresponding lubrication performance reduced the friction coefficient and wear rate effectively. (3) the 201HT-201HTC could obtain both better friction and wear mainly due to the initial process of grinding following the 201HT plastic deformation occurred in the surface and the formation of a series of re-melting welding points, whereas the 201HT material hardness would be similar to the 201HTC material hardness, which led into the competitive material friction and wear performance improvement.