Optimizing a high-sensitivity NanoLuc-based bioluminescence system for in vivo evaluation of antimicrobial treatment.

Focal and systemic infections are serious threats to human health. Preclinical models enable the development of new drugs and therapeutic regimens. In vivo, animal bioluminescence (BL) imaging has been used with bacterial reporter strains to evaluate antimicrobial treatment effects. However, high-sensitivity bioluminescent systems are required because of the limited tissue penetration and low brightness of the BL signals of existing approaches. Here, we report that NanoLuc (Nluc) showed better performance than LuxCDABE in bacteria. However, the retention rate of plasmid constructs in bacteria was low. To construct stable Staphylococcus aureus reporter strains, a partner protein enolase (Eno) was identified by screening of S. aureus strain USA300 for fusion expression of Nluc-based luciferases, including Nluc, Teluc, and Antares2. Different substrates, such as hydrofurimazine (HFZ), furimazine (FUR), and diphenylterazine (DTZ), were used to optimize a stable reporter strain/substrate pair for BL imaging. S. aureus USA300/Eno-Antares2/HFZ produced the highest number of photons of orange-red light in vitro and enabled sensitive BL tracking of S. aureus in vivo, with sensitivities of approximately 10 CFU from mouse skin and 750 CFU from mouse kidneys. USA300/Eno-Antares2/HFZ was a powerful combination based on the longitudinal evaluation of the therapeutic efficacy of antibiotics. The optimized S. aureus Eno-Antares2/HFZ pair provides a technological advancement for the in vivo evaluation of antimicrobial treatment.

Visualization of the nanoscale assembly of type I collagen on PLA by AFM.

Collagen adsorption and the morphology of its assemblies at polymer surface play an important role in improving the biocompatibility of materials. In this study, the nanoscale organization of type I collagen on Polylactide (PLA) was observed directly by high-resolution atomic force microscopy. The results show that the supramolecular structure of adsorbed collagen was affected by the concentration of collagen solution, appropriate pH and electrolyte composition of the buffer. On PLA substrate, network structures formed in high humidity atmosphere. In addition, collagen formed well-oriented nano-patterns at nearly neutral pH and appropriate electrolyte composition. Particularly, the typical 65 nm D-periodicity of collagen fibers was observed in the presence of potassium ions. Our investigation provides useful insights into the regulation of collagen assembly by substrates and environmental conditions, which is important for understanding the mechanism of collagen adsorption and assembly on polymer surfaces. It also offers a potential way to create surfaces of bio-functioned and nano-patterned materials for biotechnological and biomedical applications.

[Predictive value of endometrial receptivity and pregnancy outcome by hysteroscopy examination at the phase of implantation window in unexplained infertile women].

OBJECTIVE: To explore predictive value of endometrial receptivity and pregnancy outcome by hysteroscopy examination at the phase of implantation window in unexplained infertile women. METHODS: From Oct. 2007 to Mar. 2009, 93 unexplained infertile women underwent hysteroscopy examination at 7 approximately 9 days after a spontaneous ovulation in Family Planning Research Institute of Guangdong Province. According to the endometrial glandular openings and vascular shape, 79 cases without pathological endometrial changes were divided into 60 cases in good endometrium group and 19 cases in poor endometrium group. The following clinical parameters were analyzed and compared between two groups, including endometrial configuration, thickness, secretion, the development and number of pinopodes, vascular distribution, and the level of sex hormone, leukemia inhibitory factor (LIF) and glycodelin in the uterine flushing, and pregnancy outcome. RESULTS: (1) There was no statistical difference in the level of serum estrogen and progesterone at the phase of implantation window, which were (518 +/- 176) pmol/L, (40 +/- 20) nmol/L in good group and (513 +/- 244) pmol/L, (37 +/- 19) nmol/L in poor group (P < 0.05). The endometrium thickness at periovulatroy and implantation window days (1.06 +/- 0.10) cm/(1.16 +/- 0.08) cm in good group did not show significant difference with (0.93 +/- 0.12) cm/(1.02 +/- 0.10) cm in poor group (P > 0.05). The proportion of type A, B and C endometrium at periovulatory days were 63% (12/19), 37% (7/19) and 0 (0/19) in good group and 23% (14/60), 77% (46/60) and 0 (0/60) in poor group. When compared with those of type A or B between two groups respectively, it all showed statistical difference (P < 0.05). However, at phase of implantation window, endometrium configurations were all type B at both groups. (2) 90% (17/19) of women in good group and 7% (4/60) of women in poor group showed normal endometrial secretion function, which showed significant differences (P < 0.01). (3) The percentage of fully developed pinopodes and abundant pinopodes [84% (16/19) and 90% (17/19)] in good group were significantly higher than 42% (25/60) and 57% (34/60) in poor group (P < 0.05). (4) The level of CD(34) expression and microvessel density [MVD; (40.1 +/- 1.2) positive unit (PU) and (21.7 +/- 4.0)/high power field (HP)] in good group were significantly higher than (18.1 +/- 1.3) PU and (8.5 +/- 1.3)/HP in poor group (P < 0.01). (5) The level of LIF and glycodelin in uterine flushing [(72 +/- 54) ng/L and (196 +/- 20) microg/L] in good group were significantly higher than (15 +/- 16) ng/L and (116 +/- 26) microg/L in poor group (P < 0.05). (6) The rate of clinical pregnancy, spontaneous abortion and term delivery were 74% (14/19), 0 (0/14) and 100% (14/14) in good group and 23% (14/60), 14% (2/14) and 86% (12/14) in poor group, the rate of clinical pregnancy and term delivery in good group were significantly increased when compared with those in poor group (P < 0.01). CONCLUSIONS: Hysteroscopy examination at the phase of implantation window could reflect the development of glandular openings and vasculature. It is a preferable method to evaluate the endometrial receptivity and predict pregnancy outcome.

[Ultrastructural dynamic observation on murine schistosomal hepatic fibrosis].

OBJECTIVE: To explore possible mechanisms of hepatic fibrosis by investigating the ultrastructural dynamic changes of liver tissue, especially several kinds of cells related to hepatic fibrosis. METHODS: Murine schistosomal hepatic fibrosis model was established by infecting mice with Schistosoma japonicum cercariae. Routine transmission electron microscopy was used to observe the liver tissue. H.E. staining was used for examining the pathological changes. RESULTS: H.E. staining showed that the model was established successfully. Ultrastructural observation showed that at the 6th week after infection, the necrosis of hepatocytes around the acute granulomas occurred; the number of sinusoidal endothelial fenestrae and vitamin A droplets in fat-storing cells decreased; large phagosomes and rough endoplasmic reticulum could be seen in the cytoplasm of Kupffer's cells. At the 8th week, steatosis was found in some hepatocytes, some microvilli emerged on a few inter-hepatocytic surfaces and the inter-hepatocytic spaces were enlarged. Large collagen fibrillar bundles filled in the perisinusoidal spaces, and capillarization of hepatic sinusoids was observed. Secretory vesicles filled with collagen fibrils appeared in the cytoplasm of fat-storing cells with large amount of collagenous fiber bundles surround the cells. Rough endoplasmic reticulum increased in Kupffer's cells. At the 10th week, fat-storing cells were activated and transformed into myofibroblasts. At the 12th week, the number of myofibroblasts decreased but that of fibroblasts and fiber cells increased. CONCLUSION: Activation of fat-storing cells and transformation from fat-storing cells into myofibroblasts are the critical link in the development of hepatic fibrogenesis following schistosome infection. Kupffer's cells, necrotic hepatocytes and sinusoidal endothelial cells may relate to the activation of fat-storing cells. Capillarization of hepatic sinusoids possibly accelerates the development of hepatic fibrosis.