Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreER^T2, Col2a1-Cre; Col2a1-CreER^T2, Shh-Cre, Shh-CreER^T2, and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreER^T2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreER^T2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreER^T2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.     

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

The Combined Effects of Moss-Dominated Biocrusts and Vegetation on Erosion and Soil Moisture and Implications for Disturbance on the Loess Plateau,China

Biological soil crusts (BSCs, or biocrusts) have important positive ecological functions such as erosion control and soil fertility improvement, and they may also have negative effects on soil moisture in some cases. Simultaneous discussions of the two-sided impacts of BSCs are key to the rational use of this resource. This study focused on the contribution of BSCs while combining with specific types of vegetation to erosion reduction and their effects on soil moisture, and it addressed the feasibility of removal or raking disturbance. Twelve plots measuring 4 m × 2 m and six treatments (two plots for each) were established on a 15° slope in a small watershed in the Loess Plateau using BSCs, bare land (as a control, BL), Stipa bungeana Trin. (STBU), Caragana korshinskii Kom. (CAKO), STBU planted with BSCs (STBU+BSCs) and CAKO planted with BSCs (CAKO+BSCs). The runoff, soil loss and soil moisture to a depth of 3 m were measured throughout the rainy season (from June to September) of 2010. The results showed that BSCs significantly reduced runoff by 37.3% and soil loss by 81.0% and increased infiltration by 12.4% in comparison with BL. However, when combined with STBU or CAKO, BSCs only made negligible contributions to erosion control (a runoff reduction of 7.4% and 5.7% and a soil loss reduction of 0.7% and 0.3%). Generally, the soil moisture of the vegetation plots was lower in the upper layer than that of the BL plots, although when accompanied with a higher amount of infiltration, this soil moisture consumption phenomenon was much clearer when combining vegetation with BSCs. Because of the trivial contributions from BSCs to erosion control and the remaining exacerbated consumption of soil water, moderate disturbance by BSCs should be considered in plots with adequate vegetation cover to improve soil moisture levels without a significant erosion increase, which was implied to be necessary and feasible.     

Tongxinluo Protects against Hypertensive Kidney Injury in Spontaneously-Hypertensive Rats by Inhibiting Oxidative Stress and Activating Forkhead Box O1 Signaling

Hypertension is an independent risk factor for the progression of chronic renal failure, and oxidative stress plays a critical role in hypertensive renal damage. Forkbox O1(FoxO1) signaling protects cells against oxidative stress and may be a useful target for treating oxidative stress-induced hypertension. Tongxinluo is a traditional Chinese medicine with cardioprotective and renoprotective functions. Therefore, this study aimed to determine the effects of Tongxinluo in hypertensive renal damage in spontaneously hypertensive rats(SHRs)and elucidate the possible involvement of oxidative stress and FoxO1 signaling in its molecular mechanisms. SHRs treated with Tongxinluo for 12 weeks showed a reduction in systolic blood pressure. In addition to increasing creatinine clearance, Tongxinluo decreased urinary albumin excretion, oxidative stress injury markers including malondialdehyde and protein carbonyls, and expression of nicotinamide adenine dinucleotide phosphate oxidase subunits and its activity in SHR kidneys. While decreasing phosphorylation of FoxO1, Tongxinluo also inhibited the phosphorylation of extracellular signal-regulated kinase1/2 and p38 and enhanced manganese superoxide dismutase and catalase activities in SHR kidneys. Furthermore, histology revealed attenuation of glomerulosclerosis and renal podocyte injury, while Tongxinluo decreased the expression of α-smooth muscle actin, extracellular matrixprotein, transforming growth factor β1 and small mothers against decapentaplegic homolog 3,and improved tubulointerstitial fibrosis in SHR kidneys. Finally, Tongxinluo inhibited inflammatory cell infiltration as well as expression of tumor necrosis factor-α and interleukin-6. In conclusion, Tongxinluo protected SHRs against hypertension-induced renal injury by exerting antioxidant, antifibrotic, and anti-inflammatory activities. Moreover, the underlying mechanisms of these effects may involve inhibition of oxidative stress and functional activation of FoxO1 signaling.     

Hydroxysafflor yellow A (HYSA) inhibited the proliferation and differentiation of 3T3-L1 preadipocytes

Hydroxysafflor yellow A (HSYA), a main component of safflor yellow, has been demonstrated to prevent steroid-induced avascular necrosis of femoral head by inhibiting primary bone marrow-derived mesenchymal stromal cells adipogenic differentiation induced by steroid. In this study, we investigate the effect of HSYA on the proliferation and adipogenesis of mouse 3T3-L1 preadipocytes. The effects of HSYA on proliferation and differentiation of 3T3-L1 cells and its possible mechanism were studied by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide spectrophotometry, Oil Red O staining, intracellular triglyceride assays, real-time quantitative RT-PCR, transient transfection and dual luciferase reporter gene methods. HSYA inhibited the proliferation of 3T3-L1 preadipocytes and cell viability greatly decreased in a dose and time dependent manner. HSYA (1 mg/l) notably reduced the amount of intracellular lipid and triglyceride content in adipocytes by 21.3 % (2.13 ± 0.36 vs 2.71 ± 0.40, P < 0.01) and 22.6 % (1.33 ± 0.07 vs 1.72 ± 0.07, P < 0.01) on days 8 following the differentiation, respectively. HSYA (1 mg/l) significantly increased hormone-sensitive lipase (HSL) mRNA expression and promoter activities by 2.4- and 1.55-fold, respectively (P < 0.01), in differentiated 3T3-L1 adipocytes. HSYA inhibits the proliferation and adipogenesis of 3T3-L1 preadipocytes. The inhibitory action of HYSA on adipogenesis may be due to the promotion of lipolytic-specific enzyme HSL expression by increasing HSL promoter activity.     

Mitochondrial DNA Copy Number in Peripheral Blood and Melanoma Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001). Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97). Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure).     

Using Geneswitch technolgy to teach sex differentiation for undergraduates

The development and evaluation of a two-session laboratory class, based on Geneswitch technology and sex determination in the fruit fly Drosophila melanogaster, is described. Geneswitch system allows conditional control of gene expression. A laboratory exercise has been devised for sophomores in order to illustrate how Geneswitch technology can be used to conditionally over-express the key sex determination gene transformer (tra) during development, and how this inhibits sexual differentiation in males, resulting in a lack of much of the external genitalia and a reduction in the size of the sex combs. The protocol is inexpensive and straightforward, at the meantime gives students a good understanding of how molecular biology technologies can change biological processes including development.     

基于多尺度协同的长沙市生态网络构建与层级优化

城市化背景下人类与自然环境的矛盾呈现出多尺度、层级化特征，而传统生态网络的构建方式较少考虑不同尺度下生态要素的关系，无法从区域落实到中心城区，难以形成系统性的解决方案。研究在综合梳理各尺度生态网络构建方法的基础上，以长沙市为例，基于形态学空间格局分析（Morphological Spatial Pattern Analysis，MSPA）、景观连通性原理和生态斑块重要性评价识别生态源地，并通过多层级生态阻力面的确定，综合运用最小费用路径（Least-cost path method，LCP）、电路理论、层级传导理论、尺度嵌套等方法对市域、都市区、中心城区的生态网络进行了协同构建和层级优化，最后基于不同尺度生态网络的特点应用并落实到多层级的国土空间规划体系中。研究结果表明：（1）长沙市域生态网络和都市区生态网络具有较好的层级嵌套特征；共识别两尺度生态叠合源地14个、生态叠合廊道15条，主要通过中心城区内的湘江、浏阳河和捞刀河部分河段与外围生态绿圈相衔接，形成"外环内楔"的空间格局。（2）确定市域重要廊道、市域潜在廊道、生态叠合廊道、都市区重要廊道、都市区潜在廊道的核心保护面积共501.14 km²，并提取位于生态廊道核心保护区范围内的生态夹点和生态障碍点，以进一步落实生态保护修复策略。（3）得到具有重要生态连通功能的中心城区生态绿道长度441.2 km，生态修复单元56个，并结合生态阻力值划分为5级进行针对性修复。（4）基于不同尺度生态网络的衔接、嵌套，最终构建"市域总体生态安全格局-都市区城市生态空间发展格局-以城市绿道为基础的中心城区生态修复单元"，并与不同层级的国土空间规划体系相对应。研究结果将为以大城市为中心的跨尺度生态系统修复和生态安全格局构建提供科学参考。     

Development and evaluation of Trichoderma asperellum preparation for control of sheath blight of rice (Oryza sativa L.)

Sheath blight, which is caused by Rhizoctonia solani, is a disease that majorly impacts rice production. A biocontrol agent used for control rice sheath blight must be sprayed on the stem at specific times during rice growth, a process that is labour-intensive and renders the antagonist vulnerable to environmental factors. In this study, Trichoderma asperellum T12 was used to produce preparation by solid-state fermentation using a surface-response method. Rice hull was selected as a carrier based on its ability to sustain the T12 floating in the water and protect T12 from ultraviolet irradiation. The production of a T12-based preparation required 32% wheat bran, 7% inoculum, 2.3 g kg^?1 (NH₄)₂SO₄ and 65% water content, with fermentation at 27.5°C for 30 days and agitation every six days. The preparation demonstrated 90% biocontrol efficacy and significantly (P > 0.05) increased the seed-set rate and 1000-grain weight as compared with the pathogen treatment. The population of Trichoderma on the surface of rice leaf sheath in the treatment applied with T12 preparation increased from 232 cfu (colony forming units) g^?1 fw (fresh weight) to 436 cfu g^?1 fw during rice growth stage, which was significantly (P > 0.05) higher than pathogen treatment. The population of R. solani on the leaf sheath increased from 41 cfu g^?1 fw to 271 cfu g^?1 fw in the pathogen treatment, while remained stable (P > 0.05) at level of 10–23 cfu g^?1 fw in T12 preparation applied treatment. Biocontrol of sheath blight by the addition of the preparation to the soil is effective and decreases the costs of agro-industrial waste disposal.