Novel Split-Luciferase-Based Genetically Encoded Biosensors for Noninvasive Visualization of Rho GTPases

Rho family GTPases are critical regulators of many important cellular processes and the dysregulation of their activities is implicated in a variety of human diseases including oncogenesis and propagation of malignancy. The traditional methods, such as “pull-down” or two-hybrid procedures, are poorly suited to dynamically evaluate the activity of Rho GTPases, especially in living mammalian cells. To provide a novel alternative approach to analyzing Rho GTPase-associated signaling pathways in vivo, we developed a series of bioluminescent biosensors based on the genetically engineered firefly luciferase. These split-luciferase-based biosensors enable non-invasive visualization and quantification of the activity of Rho GTPases in living subjects. The strategy is to reasonably split the gene of firefly luciferase protein into two inactive fragments and then respectively fuse the two fragments to Rho GTPase and the GTPase-binding domain (GBD) of the specific effector. Upon Rho GTPase interacting with the binding domain in a GTP-dependent manner, these two luciferase fragments are brought into close proximity, leading to luciferase reconstitution and photon production in the presence of the substrate. Using these bimolecular luminescence complementation (BiLC) biosensors, we successfully visualized and quantified the activities of the three best characterized Rho GTPases by measuring the luminescence in living cells. We also experimentally investigated the sensitivity of these Rho GTPase biosensors to upstream regulatory proteins and extracellular ligands without lysing cells and doing labor-intensive works. By virtue of the unique functional characteristics of bioluminescence imaging, the BiLC-based biosensors provide an enormous potential for in vivo imaging of Rho GTPase signaling pathways and high-throughput screening of therapeutic drugs targeted to Rho GTPases and (or) upstream molecules in the near future.     

Removal and Recovery of Toxic Silver Ion Using Deep-Sea Bacterial Generated Biogenic Manganese Oxides

Products containing silver ion (Ag⁺) are widely used, leading to a large amount of Ag⁺-containing waste. The deep-sea manganese-oxidizing bacterium Marinobacter sp. MnI7-9 efficiently oxidizes Mn²⁺ to generate biogenic Mn oxide (BMO). The potential of BMO for recovering metal ions by adsorption has been investigated for some ions but not for Ag⁺. The main aim of this study was to develop effective methods for adsorbing and recovering Ag using BMO produced by Marinobacter sp. MnI7-9. In addition, the adsorption mechanism was determined using X-ray photoelectron spectroscopy analysis, specific surface area analysis, adsorption kinetics and thermodynamics. The results showed that BMO had a higher adsorption capacity for Ag⁺ compared to the chemical synthesized MnO₂ (CMO). The isothermal absorption curves of BMO and CMO both fit the Langmuir model well and the maximum adsorption capacities at 28°C were 8.097 mmol/g and 0.787 mmol/g, for BMO and CMO, respectively. The change in enthalpy (ΔH^θ) for BMO was 59.69 kJ/mol indicating that it acts primarily by chemical adsorption. The change in free energy (ΔG^θ) for BMO was negative, which suggests that the adsorption occurs spontaneously. Ag⁺ adsorption by BMO was driven by entropy based on the positive ΔS^θ values. The Ag⁺ adsorption kinetics by BMO fit the pseudo-second order model and the apparent activation energy of E_a is 21.72 kJ/mol. X-ray photoelectron spectroscopy analysis showed that 15.29% Ag⁺ adsorbed by BMO was transferred to Ag(0) and meant that redox reaction had happened during the adsorption. Desorption using nitric acid and Na₂S completely recovered the Ag. The results show that BMO produced by strain MnI7-9 has potential for bioremediation and reutilization of Ag⁺-containing waste.     

DNA-guided genome editing tool

正Technologies for targeted modification of eukaryotic genomes are of great value for elucidating and manipulating gene functions in fundamental and applied biological research.In the past two decades,genome editing tools have evolved quickly from random DNA mutagenesis-inducing chemicals or ionizing radiation to programmable sequencespecific nucleases(SSNs)with the CRISPR/Cas technology as the most significant breakthrough(Gaj et al.,2013).     

Log‐gamma linear‐mixed effects models for multiple outcomes with application to a longitudinal glaucoma study

Glaucoma is a progressive disease due to damage in the optic nerve with associated functional losses. Although the relationship between structural and functional progression in glaucoma is well established, there is disagreement on how this association evolves over time. In addressing this issue, we propose a new class of non‐Gaussian linear‐mixed models to estimate the correlations among subject‐specific effects in multivariate longitudinal studies with a skewed distribution of random effects, to be used in a study of glaucoma. This class provides an efficient estimation of subject‐specific effects by modeling the skewed random effects through the log‐gamma distribution. It also provides more reliable estimates of the correlations between the random effects. To validate the log‐gamma assumption against the usual normality assumption of the random effects, we propose a lack‐of‐fit test using the profile likelihood function of the shape parameter. We apply this method to data from a prospective observation study, the Diagnostic Innovations in Glaucoma Study, to present a statistically significant association between structural and functional change rates that leads to a better understanding of the progression of glaucoma over time.     

Experimental study of the synergistic effect and network regulation mechanisms of an applied combination of BMP-2, VEGF,and TGF-β1 on osteogenic differentiation

The study aimed to explore the osteogenic effect induced by the combined use of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1), attain the best combination for osteogenic quality and efficiency, and explore the network regulation mechanisms of induced osteogenesis. MC3T3-E1 cells were cultured in vitro, and BMP-2, VEGF, and TGF β1 were added to osteogenic induction mediums in different combinations to conduct experiments. At 7 and 14 days, the alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining of the applied BMP-2 and VEGF combination were deeper and the quantitative analysis were higher than those of the other groups. After optimizing the time–effect relationship of the combined application, with BMP-2, VEGF, and TGF-β1 adding in the early stage and BMP-2 and VEGF adding in the late, the ALP and ARS staining of these groups were deeper and the quantitative analyses were meaningfully higher than the BMP-2 and VEGF combination group at 7 and 14 days. The expression of the RUNX2 gene and the Smad1 signaling pathway in the optimized combination group was also significantly higher. The results demonstrate that the combination of BMP-2, VEGF, and TGF-β1 applied according to the time–effect relationship can significantly promote osteogenic differentiation mainly through the classical BMP-receptor-Smad signal pathway.     

Aspermia,associated with a presumably balanced X/autosomal translocation

Summary A new case of X/autosome translocation in a male patient is described. Azoospermia and Klinefelter like stigmata can be explained as a consequence of the balanced translocation, or by disturbed X-chromosomal inactivation during spermiogenesis.

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Zusammenfassung Es wird über einen neuen Fall einer X/Autosom-Translokation beim Mann berichtet. Azoospermie und Klinefelter-ähnliche Stigmata können unmittelbar auf die balancierte Translokation zurückgeführt werden oder Folge einer durch die Translokation gestörten X-chromosomalen Inaktivierung während der Spermiogenese sein.

     

高纤维食物对黄毛鼠摄食和消化的影响

动物的消化生理特征和消化对策可决定其营养生态位。为揭示黄毛鼠适应高纤维食物的消化对策,在高纤维食物饲喂10 d和20 d时,以食物平衡法测定摄食量、粪便量及相应的能量学参数。在取食高纤维食物10 d时,摄食量无显著变化,但粪便量显著增加,消化率显著下降;至20 d时,摄食量显著增加,消化率仍低于对照组,但消化能与对照组无差异。高纤维组动物的体重在第10 d时显著下降,但在第20 d时未进一步下降。该结果表明,黄毛鼠能够通过增加摄食量和降低消化率的消化对策适应高纤维食物,此外,降低体重使总能量需求减少,也是其适应低质量食物的对策之一。