DNA Transfection of Bone Marrow Stromal Cells Using Microbubble-Mediated Ultrasound and Polyethylenimine: An In Vitro Study

Non-viral vector transfection efficiency is an issue affecting the clinical application of stem cell gene therapy. This study makes use of the synergistic effect of combining ultrasound (US) with microbubbles (MB) and polyethylenimine (PEI) to increase DNA transfection efficiency, which will enhance the efficiency of gene transfer to bone marrow stromal cells (BMSCs). The optimal parameters for primary-cultured rat-BMSC DNA transfection were examined. The study was arranged based on uniform design. Using a construct containing hepatocyte growth factor (HGF) tagged with enhanced green fluorescent protein (pEGFP-HGF) as example, the mixture of BMSCs, MB, and PEI:DNA complex were exposed to US with frequency of 1 MHz and 10 % duty cycle pulses. Other factors such as acoustic intensity (Q), MB dosage, and total treatment time (T) were also tested. The results were analyzed by regression analysis. Using the best match of parameters, Q = 0.6 W/cm², MB = 10⁶/ml, T = 30 s, different groups were compared. The cooperativity of MB-mediated US and PEI enhanced the gene transfection efficiency by nearly 38-times compared to the DNA without US group. Furthermore, the expression of HGF protein was confirmed by Western blot. The eGFP could be not only seen mainly at the cytoplasm, but also seen in the nucleus in a small proportion of the cells (<10 %) for up to 7 observed days. The transfected BMSCs maintained their capability of multi-directional differentiation and reproductive activity. Our results provide useful information in establishing a novel non-viral transfection method, which may be applied to clinical application in stem cell gene therapy.     

yeaI基因对奶牛源大肠杆菌NJ17生物学特性的影响

c-di-GMP是细菌中广泛存在的第二信使,可通过效应蛋白参与调控细菌的生物被膜形成、运动性和毒力等生物学特性。YeaI因含有能结合c-di-GMP分子的EGEVF基序,可能作为c-di-GMP效应蛋白发挥作用。[目的] 研究yeaI基因缺失对奶牛源大肠杆菌临床分离株NJ17生物学特性的影响。[方法] 构建NJ17的yeaI缺失株（NJ17ΔyeaI）及回复株cNJ17ΔyeaI,分析yeaI对NJ17生物学特性（如生长特性、生物被膜形成能力和对小鼠乳腺上皮细胞（EpH₄-Ev）的黏附）的影响。[结果] 成功构建NJ17的yeaI缺失株（NJ17ΔyeaI）及其回复株（cNJ17ΔyeaI）;与野生株NJ17相比,缺失株NJ17ΔyeaI生长特性及耐药性无显著变化,生物被膜形成能力显著下降,运动性显著升高（P<0.05）;透射电镜检测结果表明,yeaI缺失影响NJ17菌毛和鞭毛的形成;实时定量PCR（qPCR）结果显示,yeaI基因显著抑制NJ17鞭毛基因filG和motB的转录水平（P<0.05）;血清杀菌实验表明,yeaI缺失能显著增强其抵抗血清杀菌作用（P<0.05）;对EpH₄-Ev细胞黏附实验表明,yeaI缺失对NJ17黏附性无显著影响（P>0.05）。[结论] yeaI对奶牛源大肠杆菌NJ17的生物学特性具有重要的调控作用。     

A 1,2‐propylene oxide sensor utilizing cataluminescence on CeO2 nanoparticles

A simple and sensitive gas sensor was proposed for the determination of 1,2‐propylene oxide (PO) based on its cataluminescence (CTL) by oxidation in the air on the surface of CeO₂ nanoparticles. The luminescence characteristics and optimal conditions were investigated in detail. Under optimized conditions, the linear range of the CTL intensity versus the concentration of PO was 10–150 ppm, with a correlation coefficient (r) of 0.9974 and a limit of detection (S/N = 3) of 0.9 ppm. The relative standard deviation for 40 ppm PO was 1.2% (n = 7). There was no or only weak response to common foreign substances including acetone, formaldehyde, ethyl acetate, acetic acid, chloroform, propanol, carbon tetrachloride, ether and methanol. There was no significant change in the catalytic activity of the sensor for 100 h. The proposed method was simple and sensitive, with a potential of detecting PO in the environment and industry. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of the Microcirculation in a Rabbit Hemorrhagic Shock Model Using Laser Doppler Imaging

The aim of this study is to evaluate the feasibility of Laser Doppler imaging (LDI) for noninvasive and dynamic assessment of hemorrhagic shock in a rabbit model. A rabbit model of hemorrhagic shock was generated and LDI of the microcirculation in the rabbit ears was performed before and at 0, 30, 60, and 90 min after hemorrhage. The CCD (Charge Coupled Device) image of the ears, the mean arterial pressure (MAP) and the heart rate (HR) were monitored. The mean LDI flux was calculated. The HR of rabbits was significantly (p < 0.05) elevated and the MAP was decreased after hemorrhage, compared to the pre-hemorrhage level. Within the initial 30 min after hemorrhage, the perfusion flux lineally dropped down. In contrast, the MAP values did not differ significantly between the time points of 0 and 30 after hemorrhage (p > 0.05). Both the flux numbers and the red-to-blue color changes on LDI imaging showed the reduction of the microcirculation. LDI imaging is a noninvasive and non-contact approach to evaluate the microcirculation and may offer benefits in the diagnosis and treatment of hemorrhage shock. Further studies are needed to confirm its effectiveness in clinical practice.     

Activation of HIFa Pathway in Mature Osteoblasts Disrupts the Integrity of the Osteocyte/Canalicular Network

The hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, are the central mediators of the homeostatic response that enables cells to survive and differentiate in low-oxygen conditions. Previous studies indicated that disruption of the von Hippel-Lindau gene (Vhl) coincides with the activation of HIFα signaling. Here we show that inactivation of Vhl in mature osteoblasts/osteocytes induces their apoptosis and disrupts the cell/canalicular network. VHL-deficient (ΔVHL) mice exhibited a significantly increased cortical bone area resulting from enhanced proliferation and osteogenic differentiation of the bone marrow stromal cells (BMSCs) by inducing the expression of β-catenin in the BMSC. Our data suggest that the VHL/HIFα pathway in mature osteoblasts/osteocytes plays a critical role in the bone cell/canalicular network and that the changes of osteocyte morphology/function and cell/canalicular network may unleash the bone formation, The underlying mechanism of which was the accumulation of β-catenin in the osteoblasts/osteoprogenitors of the bone marrow.     

Geographic divergence of “Sulfolobus islandicus” strains assessed by genomic analyses including electronic DNA hybridization confirms they are geovars

Ten well-annotated genomes of “Sulfolobus islandicus” strains from different geographic locations have been released at the NCBI database. Whole genome based composition vector trees indicate that these strains show the same branching patterns as originally reported by multi-locus sequence analysis. To determine whether the ten strains meet the criteria for separate species, DNA–DNA hybridization (DDH) was performed in silico. DDH values of strains from the same geographic location, i.e., Iceland, Kamchatka and North America, ranged from 82.4 to 95.4 %, clearly qualifying them as members of the same species. The lowest DDH values found between locations ranged from 75.5 to 76.6 %, which exceed the 70 % DDH threshold for a species thereby indicating they are all members of the same species based on the currently accepted definition. The clear divergences of strains from the different geographic locations are sufficiently great to consider them as separate geovars. “S. islandicus” has not yet been validly named and a type strain has not been deposited in culture collections. We urgently recommend that those who study the organism fulfill the criteria of the International Code of Nomenclature of Bacteria in order to designate a type strain and to identify and deposit related strains of this species to make them available to the broader scientific community.