Improving the biotransformation of phytosterols to 9α-hydroxy-4-androstene-3,17-dione by deleting embC associated with the assembly of cell envelope in Mycobacterium neoaurum.

The conversion of low value-added phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is an important step in the steroid pharmaceutical industry. However, the highly dense cell envelope with extremely low permeability largely affects the overall transformation efficiency. Here, we preliminarily located the key gene embC required for the synthesis of lipoarabinomannan from lipomannan in Mycobacterium neoaurum. The genetic manipulation of embC indicated that it might be the only functional enzyme catalyzing the above synthesis process. The deficiency of lipoarabinomannan led to a significantly increased cell permeability, which in turn caused the enhanced uptake capacity of cells. The sterol substrate conversion efficiency of mycobacterial cells was increased by about 52.4 % after 72-h conversion. Ultimately, the absence of embC increased the productivity from 0.0927 g/L/h to 0.1031 g/L/h, as confirmed by a resting cell system. This study verified the feasibility of improving the efficiency of the microbial conversion system through the cell envelope engineering strategy.

Differentiation of nestin­negative human hair follicle outer root sheath cells into neurons in vitro.

A specialized quiescent population of hair follicle stem cells, residing in the hair follicle outer root sheath cells (ORSCs), has previously demonstrated pluripotency for differentiation into neural stem cells (NSCs). A previous study indicated that nestin­positive hair follicle ORSCs are able to differentiate into neurons. However, little has been reported on the isolation of nestin­negative human ORSCs and whether they can successfully differentiate into neurons in vitro. In the present study, nestin­positive ORSCs were significantly reduced with a prolonged incubation time in vitro. Following 9 days of primary culture, nestin­expressing ORSCs disappeared entirely, and ORSCs remained nestin­negative following 5 days of subculture. Notably, nestin was identified in ORSCs following a three­step process of neuro­induction. In addition, neruodevelopmental markers were detected in the ORSC­derived nestin­positive spherical cell mass, including the induction of the neuronal specific markers growth associated protein­43, neurotensin receptor­3 and p75 neurotrophin receptor, and also the gliocyte markers, glial fibrillary acidic protein and S100. These sphere­forming cells did not express the mature neuron­associated markers neurofilament medium, neuronal nuclei and neuron­specific enolase, which suggested that sphere­forming cells may preferentially differentiate into neural stem cell­like cells as opposed to mature neurons or neurogliocyte. In conclusion, ORSC­driven neural differentiation may be a suitable treatment strategy for neurodegenerative diseases and may possess an important value in regenerative medicine.

EPO improves the proliferation and inhibits apoptosis of trophoblast and decidual stromal cells through activating STAT-5 and inactivating p38 signal in human early pregnancy.

The erythropoietin (EPO) belongs to the family of angiogenic factors, which is regulated by Hypoxia-inducible factor- 1α (HIF-1α). As known, EPO are expressed in human villi and decidua, but the function is not clear. In this study, we investigated the expression and roles of HIF-1α, EPO and its receptor (EPOR) in the biological functions of trophoblast and decidual stromal cell (DSC) in human early pregnancy. The expression of EPO, EPOR and HIF-1α was evaluated in the villi and deciduas by RT-PCR and immunohistochemistry. Thereafter, we silenced HIF-1α expression in HTR-8/SVneo cell line and decidual stromal cells (DSCs). The effects of EPO on the proliferation and apoptosis of trophoblasts and DSCs, and activation of signal molecules were investigated by BrdU proliferation assay, flow cytometry and western blot, respectively. We have observed that the HIF-1α silence results in the lower expression of EPO in trophoblasts and DSCs. The anti-EPO neutralizing antibody can inactivate the phosphorylation of STAT5 and activate p38 of these cells in a dosage-dependent manner. Furthermore, the expressions of EPO, EPOR and HIF-1α in the villi and decidua from the unexplained miscarriage were significantly lower than that of the normal early pregnancy. This study suggests that HIF-1α may regulate the expression of EPO, which plays a favorable regulatory role in the proliferation and survival of human first-trimester trophoblast cells and DSCs via inactivating p38 and activating STAT5 in an autocrine manner, while the inadequate EPO expression at maternal-fetal interface may lead to pregnancy wastage in humans.

[Design and experimental study of an epiphyseal slide-traction plate].

OBJECTIVE: To develop an epiphyseal slide-traction plate in child, which can supply the fracture a sufficient internal fixation, and will not restrain the growth of epiphyses. Animal experiments were carried out with the plates to compare the slide-traction with traditional plate. METHODS: Develop a slide-traction plate for the configuration of the femur condylus of children. Thirty adolescent goats in the experiment were divided into control group (12 goats) and plate group (18 goats). In plate group, right femurs of goats were fixed with common plates and the left femurs with slide-traction plates. All the goats were given X-ray examination at different time after surgery. And the goats were sacrificed at 3 and 6 month, histological method and electron microscopy were performed to evaluate the development of epiphyseal plate. RESULTS: The both femurs of the goats in control group have no difference in evidence in length at all time we examined. And the both femurs of the goats fixed with plates have no difference in evidence in length at 1 day after surgery. However, the both femurs of the goats fixed with plates have difference in evidence in length at 1 month, 2 month, 3 month, 6 month after surgery. The increased length of the femurs at I month, 2 month, 3 month, 6 month after surgery was also compared with the length at 1 day after surgery, there was difference in evidence between the right femurs of the control group and the femurs were fixed with common plates, but no difference in evidence between the left femurs of the normal control group and the femurs were fixed with slide-traction plate (P > 0.05). More thicker epiphyseal plate were found in the left femurs than the right femurs of the group fixed with plates at 3 and 6 month after surgery (P < 0.01). In the plate group, safranine O staining showed epiphyseal plates at the left femurs had more fuscous staining than the right femurs at 3 and 6 month after surgery and electron microscopy also found that the cells of the epiphyseal plates of left femurs were more eugenic than the right femurs at 3 and 6 month after surgery. CONCLUSION: The epiphyseal slide-traction plate can slide with the growth of epiphyses, which is suitable for fixation of the fracture in this part.