通过形成类胚体诱导人羊水多能干细胞向心肌细胞分化

由人羊水中分离羊水多能性干细胞,通过形成类胚体诱导其向心肌细胞分化.取人羊水标本进行体外培养,分离得到人羊水干细胞,已连续传代培养至42代,采用免疫细胞化学、RT-PCR和流式细胞仪技术对羊水干细胞的生物学特性进行检测.取10～15代羊水干细胞,悬浮培养使其形成类胚体,进而向心肌细胞诱导分化.培养的羊水干细胞呈成纤维样,表达部分胚胎干细胞特异标志基因,悬浮培养可形成类胚体.类胚体碱性磷酸酶(AP)检测呈阳性,表达三胚层特异标志基因fgf5、ζ-globin和α-fetoprotein.羊水干细胞形成类胚体后进行诱导,得到α-actin阳性细胞,表达心肌细胞特异标志基因Tbx5、Nkx2.5、GATA4和α-MHC.试验结果表明,从人羊水标本中可分离得到具有胚胎干细胞特性的细胞,经初步检测确定为羊水干细胞,并能通过形成类胚体诱导其向心肌细胞分化.     

A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52 % amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55 % of the maximum activity when assayed at 40–75 °C, 23 % at 20 °C, 16 % at 85 °C, and even 8 % at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62 % xylanase activity and stability at the concentration of 3–30 % (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5–19.0, 15.3–19.0, 21.9–27.7, and 28.2–31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.     

Optimisation of exopolysaccharide production by Gomphidius rutilus and its antioxidant activities in vitro

The production conditions of the Gomphidius rutilus exopolysaccharides (GREP) in submerged culture were optimised, and the antioxidant activities of GREP in vitro were evaluated. The optimal culture medium constituents were determined as follows: 30 g/L sucrose, 3.0 g/L soybean meal, 0.25 g/L MgSO₄, 1.5 g/L K₂HPO₄, 0.5 g/L KH₂PO₄, 0.03 g/L ZnSO₄, and 0.01 g/L FeSO₄. The optimum parameters for the liquid fermentation were as follows: temperature, 25 °C; cultivation time, 6 d; initial pH, 8.0; volume of medium, 150 mL; and rotary speed, 180 rpm. GREP content and dry cell weight in optimised conditions were 540.1 ± 15.9 mg/L and 8.2 ± 0.3 g/L, respectively. GREP content under the optimised conditions was 2.5 times than that under the basic culture medium and initial conditions. GREP demonstrated positive antioxidant potential on superoxide anion radical, 1,1-diphenyl-2-picrylhydrazyl, and hydroxyl radical scavenging, and reducing power.     

Axial resolution enhancement of light‐sheet microscopy by double scanning of Bessel beam and its complementary beam

The side lobes of Bessel beam will create significant out‐of‐focus background when scanned in light‐sheet fluorescence microscopy (LSFM), limiting the axial resolution of the imaging system. Here, we propose to overcome this issue by scanning the sample twice with zeroth‐order Bessel beam and another type of propagation‐invariant beam, complementary to the zeroth‐order Bessel beam, which greatly reduces the out‐of‐focus background created in the first scan. The axial resolution can be improved from 1.68 μm of the Bessel light‐sheet to 1.07 μm by subtraction of the two scanned images across a whole field‐of‐view of up to 300 μm × 200 μm × 200 μm. The optimization procedure to create the complementary beam is described in detail and it is experimentally generated with a spatial light modulator. The imaging performance is validated experimentally with fluorescent beads as well as eGFP‐labeled mouse brain neurons.      

Development of enhancer trap lines for functional analysis of the rice genome

Enhancer trapping has provided a powerful strategy for identifying novel genes and regulatory elements. In this study, we adopted an enhancer trap system, consisting of the GAL4/VP16-UAS elements with GUS as the reporter, to generate a trapping population of rice. Currently, 31 443 independent transformants were obtained from two cultivars using Agrobacterium-mediated T-DNA insertion. PCR tests and DNA blot hybridization showed that about 94% of the transformants contained T-DNA insertions. The transformants carried, on average, two copies of the T-DNA, and 42% of the transformants had single-copy insertions. Histochemical assays of approximately 1000 T0 plants revealed various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. The expression pattern of the reporter gene in T1 families corresponded well with the T0 plants and segregated in a 3 : 1 Mendelian ratio in majority of the T1 families tested. The frequency of reporter gene expression in the enhancer trap lines was much higher than that in gene trap lines reported previously. Analysis of flanking sequences of T-DNA insertion sites from about 200 transformants showed that almost all the sequences had homology with the sequences in the rice genome databases. Morphologically conspicuous mutations were observed in about 7.5% of the 2679 T1 families that were field-tested, and segregation in more than one-third of the families fit the 3 : 1 ratio. It was concluded that GAL4/VP16-UAS elements provided a useful system for enhancer trap in rice.