Generation and characterization of a breast carcinoma model by PyMT overexpression in mammary epithelial cells of tree shrew, an animal close to primates in evolution.

The tree shrew is becoming an attractive experimental animal model for human breast cancer owing to a closer relationship to primates/humans than rodents. Tree shrews are superior to classical primates because tree shrew are easier to manipulate, maintain and propagate. It is required to establish a high-efficiency tree shrew breast cancer model for etiological research and drug assessment. Our previous studies suggest that 7,12-dimethylbenz(a)anthracene (DMBA) and medroxyprogesterone acetate (MPA) induce breast tumors in tree shrews with a low frequency (<50%) and long latency (∼ 7-month), making these methods less than ideal. We induced mammary tumors in tree shrew (Tupaia belangeri chinensis) by injection of lentivirus expressing the PyMT oncogene into mammary ducts of 22 animals. Most tree shrews developed mammary tumors with a latency of about three weeks, and by 7 weeks all injected tree shrews had developed mammary tumors. Among these, papillary carcinoma is the predominant tumor type. One case showed lymph node and lung metastasis. Interestingly, the expression levels of phosphorylated AKT, ERK and STAT3 were elevated in 41-68% of PyMT-induced mammary tumors, but not all tumors. Finally, we observed that the growth of PyMT-induced tree shrew mammary tumors was significantly inhibited by Cisplatin and Epidoxorubicin. PyMT-induced tree shrew mammary tumor model may be suitable for further breast cancer research and drug development, due to its high efficiency and short latency.

Open achilles tendon defects: a 12-year study on the injury mechanisms, reconstructions, and late complications.

BACKGROUND: Open injuries of the Achilles tendon, which can be complicated by skin and bone injuries, continue to be a great challenge for surgeons. This study aims to report our experience with treatment of open Achilles tendon defects, focusing on the injury mechanisms, soft tissue coverage and late complications. METHODS: A retrospective review was performed on 31 patients with open Achilles tendon defects between 1999 and 2011. The analyzed factors were injury mechanisms, surgeries, and long follow-up complications. The defect lengths of the Achilles tendons in the study ranged from 1 to 11 cm and the soft tissue defects ranged from 3 × 3 to 12 × 10 cm. Nine types of flaps were used for the coverage of concomitant skin defects. RESULTS: Motorcycle spoke injuries were the most common cause of injury. There was no complete flap loss or rerupture of the reconstructed Achilles tendon. At the latest follow-up, all limbs were preserved and all the patients had regained full walking abilities. The algorithm of one-stage reconstruction was established, according to the defect length of the Achilles tendon and the defect size of skin. Late complications included maximum dorsiflexion loss and failure of heel raising ability on the single reconstructed foot. CONCLUSION: Open Achilles tendon defects are characteristic of concurrent skin and bone injuries and the reconstruction protocols of the different tissues should not be separated.

Efficacy of combined vancomycin and fosfomycin against methicillin-resistant Staphylococcus aureus in biofilms in vivo.

Infection by methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. The aim of this study was to demonstrate the in vivo bactericidal effects of a combination of vancomycin (VAN) and fosfomycin (FOS) against MRSA in a rat carboxymethyl cellulose-pouch biofilm model. The results of the time-kill assay showed that the combination therapy was capable of killing at low minimal inhibitory concentrations (MIC) (½ × MIC VAN +1 × MIC FOS and 1 × MIC VAN + 1 × MIC FOS). In the in vivo study, a synergistically bactericidal effect was observed when using the combination therapy on MRSA embedded in the mature biofilm model. In comparison with the untreated control group and the groups receiving either VAN or FOS alone, the rats treated with combination therapy had lower MRSA colony counts in exudates from the pouch, lower white blood cell and neutrophil counts, and C-reactive protein (CRP) in peripheral blood. Furthermore, histological analysis of the pouch wall indicated combination therapy resulted in disappearance of biofilm-like structures, marked decrease in necrosis, and formation of granular tissue. In conclusion, the combination of VAN with FOS had a synergistic bactericidal effect on chronic MRSA infection embedded in biofilm, providing an alternative approach to treating this condition.

[The change in Toll-like receptor 4 gene expression in skeleton of endotoxemia mice].

OBJECTIVE: To explore the changes in gene expression of bone in endotoxemia in mice. METHODS: Lipopolysaccharide (LPS) was injected intraperitoneally to reproduce an endotoxemia model in mice. Forty-eight mice were randomly divided into eight groups: normal group, 4, 6, 8, 12, 24, 48 and 72 hours after LPS injection groups, with 6 mice in each group. To evaluate endotoxemia whole blood was collected for leukocytic count, eye sockets blood was obtained for liver and renal functions tests. mRNA expression level of Toll-like receptor 4 (TLR4) in bone was determined by reverse transcription-polymerase chain reaction (RT-PCR) assay. The pathological changes of bone and tissue slides were prepared with hematoxylin and eosin (HE) staining for observing under microscope. RESULTS: Compared with normal group, leukocyte count of 4-hour LPS-treated group was significantly decreased (P<0.01). However, after 4 hours, leukocyte count became higher gradually and remained at high level at 72 hours compared with that of normal group (P<0.05). Compared with normal group, alanine aminotransferase (ALT) level reached to a high level in LPS-treated groups at 4 hours and 6 hours (both P<0.05), and then decreased gradually showing a tendency to return to normal level after 8 hours (P>0.05). Blood urea nitrogen (BUN) was increased at 6 hours (P<0.05), reaching to the highest level at 8 hours (P<0.05) and tended to become normal level after 12 hours (P>0.05). Six hours after LPS treatment, the expression of TLR4 mRNA was enhanced (P<0.01) and reached the peak at 24 hours (P<0.01), and it remained at a high level at 72 hours (P<0.05). However, there were no significant pathological changes in bone structure after LPS treatment. CONCLUSION: Expression level of TLR4 mRNA in bone is significantly increased in endotoxemia mice.

[Generation of bone morphogenetic protein 4 conditional RNA interference mice].

To explore bone morphogenetic protein 4 (BMP4) function in the developing bone, a BMP4 conditional RNA interference (CRNAi) vector was constructed based on the pBSK/U6 vector with a LoxPneo cassette. The transgene fragment targeting bmp4 was obtained by Kpn and Afl double digestion and was purified before being microinjected into fertilized eggs from FVB/NJ mice. BMP4CRNAi transgenic mice were genotyped by PCR. And the PCR positive mice were crossed with Col2a1-Cre transgenic mice, whose Cre recombinase was specifically expressed in osteo-chondro-progenitor cells. Bmp4 mRNA expression in primary chondrocytes were examined by semi-quantitive RT-PCR to determine RNA interference efficiency. Results showed that BMP4(CRNAi) mice and BMP4 (Col2a1-CRNAi) mice were produced successfully, and bmp4 knockdown efficiency in primary chondrocytes of BMP4 Col2a1-CRNAi mice was 81%. This transgenic mouse line provides excellent model for studying the role of BMP4 in chondrocyte development, and BMP4CRNAi mouse may be a good model for studying the role of BMP4 in different cells, tissues and organs through crossing with different Cre transgenic mice.