藜科植物藜和灰绿藜实时荧光定量PCR内参基因的选择

选择合适的内参基因是实时荧光定量PCR(qRT-PCR)研究的关键,目前对藜科耐盐(盐生)植物胁迫相关基因的表达分析中所用内参基因的报道较为有限.该研究利用GeNorm、NormFinder和BestKeeper 3个内参基因分析软件,对已选择过的β-TUBULIN、β-ACTIN、GAPDH 3个常用候选内参基因进行了比较分析,筛选出在NaCl和PEG胁迫下藜和灰绿藜中表达相对稳定的内参基因.结果表明:GeNorm、NormFinder内参软件分析在NaCl和PEG胁迫下,GAPDH是藜和灰绿藜中均共同稳定表达的内参基因,同时在藜和灰绿藜中也有各自表达较稳定的内参基因,β-ACTIN在藜中稳定表达,β-TUBULIN则在灰绿藜中稳定表达.对相同科不同种的植物内参基因表达差异进行比较,内参基因在相同科中具有相同稳定表达的内参;对相同胁迫下两种不同植物内参基因表达稳定性进行分析,内参基因的选择需根据实际的实验材料和实验条件而定.基于3个分析软件对以上3个常用内参基因的分析结果,初步确定了在藜科植物藜和灰绿藜中相对稳定的内参基因,为藜和灰绿藜胁迫相关基因的定量表达分析提供了参考依据.     

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

An experimental set-up which enabled non-invasive, real-time reactive oxygen species (ROS) visualization on a whole plant level was constructed. In the test organism, Lemna minor L. (common duckweed), apoplastic and symplastic oxidative stress was evaluated by exposure to menadione (50 μM), menadione (50 μM) + ascorbate (100 μM) or neither for control. Menadione (50 μM) caused a statistically significant increase in H₂DCFDA fluorescence in the apoplast after 60 minutes of exposure. The addition of ascorbate (100 μM) in the test medium significantly decreased apoplastic oxidative stress. 50 μM menadione caused an increase in symplastic H₂DCFDA fluorescence in 57% of fronds. The exposure of L. minor plants to both menadione and ascorbate decreased the rate of fluorescence intensity accumulation in the symplast to control levels. The method has proven to be quick and straightforward and could be applied to a range of chemicals in various physiological and toxicological plant studies. The advantages of the set-up and different possible artefacts are discussed.     

Validation of Zebrafish （Danio redo） Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR （qRT-PCR） is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The fl-actin, EFlot and Rpll3ot genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1 or, Rpll3α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

不同氮素供应下水稻酚类物质代谢关键酶基因差异表达

运用实时荧光定量PCR技术探讨了不同供氮条件下强化感与弱化感水稻苯丙烷代谢途径中9个关键酶基因的表达差异。结果表明,与正常氮素供应相比,低氮胁迫引起强化感水稻‘P1312777’中与酚类代谢途径相关的9个关键酶基因表达量均上调,表达量增幅在1.9～5.4倍之间,且以PAL基因上调倍数最大。而弱化感水稻‘Lemont’则相反,只有2个基因(苯丙氨酸裂解酶基因和肉桂酰CoA基因)表达上调,但上调倍数分别是强化感水稻对应的基因的22%和74%,其余的7个基因表达均下调,降幅在29%～72%之间,表明低氮胁迫诱发的水稻化感抑草能力增强与其体内酚类物质合成代谢增强有关。     

A novel in vitro co-culture model to examine contact formation between astrocytic processes and cerebral vessels

Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels.     

The shear modulus of lower-leg muscles correlates to intramuscular pressure

Shear wave elastography (SWE) is emerging as an innovative tool to evaluate muscle properties and function. It has been shown to correlate with both passive and active muscle forces, and is sensitive to physiological processes and pathological conditions. Similarly, intramuscular pressure (IMP) is an important parameter that changes with passive and active muscle contraction, body position, exercise, blood pressure, and several pathologies. Therefore, the objective of this study was to quantify the dependency of shear modulus within the lower-leg muscles on IMP in healthy individuals. Nineteen healthy individuals (age: Mean age ± SD, 23.84 ± 6.64 years) were recruited. Shear modulus was measured using ultrasound SWE on the tibialis anterior (TA) and peroneus longus (PL) muscles using pressure cuff inflation around the thigh at 40 mmHg, 80 mmHg, and 120 mmHg. Changes in IMP were verified using a catheter connected to a blood pressure monitor. It was found that IMP was correlated to TA and PL shear modulus (spearman's rank correlation = 0.99 and 0.99, respectively). Applying a gradual increase of cuff pressure from 0 to 120 mmHg increased the shear modulus of the TA and PL muscles from 15.83 (2.46) kPa to 21.88 (4.33) kPa and from 9.64 (1.97) kPa to 12.88 (5.99) kPa, respectively. These results demonstrate that changes of muscle mechanical properties are dependent on IMP. This observation is important to improve interpretation of ultrasound elastograms and to potentially use it as a biomarker for more accurate diagnosis of pathologies related to increased IMP.     

Abdominal girth as an index of muscle tension during abdominal hollowing: Selecting the optimal training intensity for the transversus abdominis muscle

The abdominal hollowing technique is used for training the transversus abdominis (TrA). However, the optimal intensity of hollowing is still unclear. The objective of the present study is to verify the validity of estimating the tension of the TrA by measuring the girth of the abdomen with a tape and to determine the optimum intensity of hollowing to effectively train the TrA. Sixteen healthy males performed hollowing with an intensity of 0%, 25%, 50%, 75%, and 100%, estimated from the girth of the abdomen. The shear elastic modulus was measured for the rectus abdominis (RA), external oblique (EO), internal oblique (IO), and TrA at all intensities via ultrasonic shear wave elastography. The shear elastic modulus was considered as the index of the tension of the abdominal muscles at each intensity, and the ratio of the TrA to RA, EO, and IO respectively was calculated as the index of TrA selectivity. As the intensity of hollowing increased, the girth of abdomen decreased and tension of all the four muscles increased. The ratio of TrA to the RA, EO, and IO did not exhibit a significant variation among hollowing intensities of 25% to 100%. It is rational to estimate the tension of the TrA by measuring the girth of the abdomen. Moreover, considering both TrA contraction intensity and selectivity, abdominal hollowing performed at maximum intensity was effective for the maximum contraction training of the TrA.