Selection of reliable reference genes for gene expression studies in peach using real-time PCR

<p>Abstract</p> <p>Background</p> <p>RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach <it>(Prunus persica </it>L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach.</p> <p>Results</p> <p>In this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (<it>ACT</it>), cyclophilin (<it>CYP2</it>), RNA polymerase II (<it>RP II</it>), phospholipase A2 (<it>PLA2</it>), ribosomal protein L13 (<it>RPL13</it>), glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), 18S ribosomal RNA (<it>18S rRNA</it>), tubblin beta (<it>TUB</it>), tubblin alpha (<it>TUA</it>), translation elongation factor 2 (<it>TEF2</it>) and ubiquitin 10 (<it>UBQ10</it>). All eleven reference genes displayed a wide range of C_qvalues in all samples, indicating that they expressed variably. The stability of these genes except for <it>RPL13 </it>was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results.</p> <p>Conclusion</p> <p>Our study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, <it>TEF2</it>, <it>UBQ10 </it>and <it>RP II </it>were found to be the most suitable reference genes with a very high statistical reliability, and <it>TEF2 </it>and <it>RP II </it>for the other sample series, while <it>18S rRNA</it>, <it>RPL13 </it>and <it>PLA2 </it>were unsuitable as internal controls. <it>GAPDH </it>and <it>ACT </it>also performed poorly and were less stable in our analysis. To achieve accurate comparison of levels of gene expression, two or more reference genes must be used for data normalization. The combinations of <it>TEF2</it>/<it>UBQ10/RP II </it>and <it>TEF2/RP II </it>were suggested for use in all samples and subsets, respectively.</p

EXTRACTION AND ISOLATION OF ALKALOIDS OF SOPHORA ALOPECUROIDES AND THEIR ANTI-TUMOR EFFECTS IN H22 TUMOR-BEARING MICE

Background: Alkaloids of Sophora alopecuroides have good biological activity, and are widely used in clinical settings, which not only have pharmacological activities of anti-cancer, cancer suppression, as well as the inhibition, and killing of various microorganisms; but also possess extensive pharmacological effects on immune system, nervous system and cardiovascular system. The objective of this paper was to extract and isolate total alkaloids of Sophora alopecuroides (TASA), and to study their anti-tumor effects in H22 tumor-bearing mice. Materials and Methods: TASA were extracted and isolated using thin-layer chromatography, and column chromatography; and the isolated compounds were analyzed using nuclear magnetic resonance. The inhibitory effects of TASA on tumor in H22-bearing mice were determined by MTT assay. Results: Three compounds were isolated from Sophora alopecuroides L., which were matrine, oxymatrine and sophoridine, respectively. Meanwhile, mouse H22 sarcoma model was established and different doses of TASA apparently inhibited solid H22-tumor in mice; it inhibited the thymus, and spleen to some extent; the degree of inhibition was more obvious for the spleen. Conclusion: TASA has an anti-tumor effect in H22 tumor-bearing mice

Functional piezocrystal characterisation under varying conditions

Piezocrystals, especially the relaxor-based ferroelectric crystals, have been subject to intense investigation and development within the past three decades, motivated by the performance advantages offered by their ultrahigh piezoelectric coefficients and higher electromechanical coupling coefficients than piezoceramics. Structural anisotropy of piezocrystals also provides opportunities for devices to operate in novel vibration modes, such as the d36 face shear mode, with domain engineering and special crystal cuts. These piezocrystal characteristics contribute to their potential usage in a wide range of low- and high-power ultrasound applications. In such applications, conventional piezoelectric materials are presently subject to varying mechanical stress/pressure, temperature and electric field conditions. However, as observed previously, piezocrystal properties are significantly affected by a single such condition or a combination of conditions. Laboratory characterisation of the piezocrystal properties under these conditions is therefore essential to fully understand these materials and to allow electroacoustic transducer design in realistic scenarios. This will help to establish the extent to which these high performance piezocrystals can replace conventional piezoceramics in demanding applications. However, such characterisation requires specific experimental arrangements, examples of which are reported here, along with relevant results. The measurements include high frequency-resolution impedance spectroscopy with the piezocrystal material under mechanical stress 0–60 MPa, temperature 20–200 °C, high electric AC drive and DC bias. A laser Doppler vibrometer and infrared thermal camera are also integrated into the measurement system for vibration mode shape scanning and thermal conditioning with high AC drive. Three generations of piezocrystal have been tested: (I) binary, PMN-PT; (II) ternary, PIN-PMN-PT; and (III) doped ternary, Mn:PIN-PMN-PT. Utilising resonant mode analysis, variations in elastic, dielectric and piezoelectric constants and coupling coefficients have been analysed, and tests with thermal conditioning have been carried out to assess the stability of the piezocrystals under high power conditions

A multi-isotope approach towards constraining the origin of large-scale Paleoproterozoic B-(Fe) mineralization in NE China

The final publication is available at Elsevier via https://doi.org/10.1016/j.precamres.2017.01.030 © 2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Borate ore deposits occur predominantly in Phanerozoic evaporative sedimentary environments but are scarce in Precambrian strata. However, massive B- and Mg-rich borate deposits are abundant in the Paleoproterozoic strata of Northeast (NE) China. In addition, several of these borate deposits are dominated by Fe (e.g., >60% Fe2O3 content in the Wengquangou deposit). To constrain the origin of these unusual deposits, we obtained B, Fe, and Mg isotope data on the wall rocks and ores of the Mg-rich Houxianyu borate deposit and the Fe-rich Wengquangou borate deposit in NE China. The d11B values of the borate deposits (10.7 ± 4.4‰, n = 15) are higher than most types of igneous and non-evaporative sedimentary rocks, suggesting that B is of evaporative sedimentary origin. However, the borate deposits have a limited range of d56Fe values near 0‰ (0.05 ± 0.18‰, n = 24), which is similar to igneous rocks and thus points to a magmatic origin for the Fe. The d26Mg values of the ores and associated rocks ( 0.52 ± 0.34‰, n = 24) are intermediate between Mg-rich carbonate rocks and igneous rocks, suggesting a mixed sedimentary and magmatic origin for the Mg. Regional metamorphism and intense deformation modified the deposits significantly by promoting metasomatic alteration of the igneous rocks and evaporite minerals, thus resulting in mixing of magmatic and sedimentary-evaporative sources as well as recrystallization of the sedimentary borate precursors to metamorphic borate minerals. Hence, the B, Fe, and Mg isotope data together with geological and mineralogical observations indicate that formation of the Paleoproterozoic B-(Fe) deposits in NE China involved a three-stage process: enrichment of B and Mg by evaporative sedimentation, introduction of Fe and additional Mg by volcanism, and modification by regional metamorphism/metasomatism.NSFC || (No. 41203004) NSFC || (No. 4127306 CAGS Research Fund || (YYWF201603) NSERC Discovery Grant || (RGPIN-435930

Heat shock protein 90 promotes RNA helicase DDX5 accumulation and exacerbates hepatocellular carcinoma by inhibiting autophagy

Objective: Hepatocellular carcinoma (HCC), the main type of liver cancer, has a high morbidity and mortality, and a poor prognosis. RNA helicase DDX5, which acts as a transcriptional co-regulator, is overexpressed in most malignant tumors and promotes cancer cell growth. Heat shock protein 90 (HSP90) is an important molecular chaperone in the conformational maturation and stabilization of numerous proteins involved in cell growth or survival. Methods: DDX5 mRNA and protein expression in surgically resected HCC tissues from 24 Asian patients were detected by quantitative real-time PCR and Western blot, respectively. The interaction of DDX5-HSP90 was determined by molecular docking, immunoprecipitation, and laser scanning confocal microscopy. The autophagy signal was detected by Western blot. The cell functions and signaling pathways of DDX5 were determined in 2 HCC cell lines. Two different murine HCC xenograft models were used to determine the function of DDX5 and the therapeutic effect of an HSP90 inhibitor. Results: HSP90 interacted directly with DDX5 and inhibited DDX5 protein degradation in the AMPK/ULK1-regulated autophagy pathway. The subsequent accumulation of DDX5 protein induced the malignant phenotype of HCC by activating the β-catenin signaling pathway. The silencing of DDX5 or treatment with HSP90 inhibitor both blocked in vivo tumor growth in a murine HCC xenograft model. High levels of HSP90 and DDX5 protein were associated with poor prognoses. Conclusions: HSP90 interacted with DDX5 protein and subsequently protected DDX5 protein from AMPK/ULK1-regulated autophagic degradation. DDX5 and HSP90 are therefore potential therapeutic targets for HCC

GmFT2a, a Soybean Homolog of FLOWERING LOCUS T, Is Involved in Flowering Transition and Maintenance

BACKGROUND: Flowering reversion can be induced in soybean (Glycine max L. Merr.), a typical short-day (SD) dicot, by switching from SD to long-day (LD) photoperiods. This process may involve florigen, putatively encoded by FLOWERING LOCUS T (FT) in Arabidopsis thaliana. However, little is known about the potential function of soybean FT homologs in flowering reversion. METHODS: A photoperiod-responsive FT homologue GmFT (renamed as GmFT2a hereafter) was cloned from the photoperiod-sensitive cultivar Zigongdongdou. GmFT2a gene expression under different photoperiods was analyzed by real-time quantitative PCR. In situ hybridization showed direct evidence for its expression during flowering-related processes. GmFT2a was shown to promote flowering using transgenic studies in Arabidopsis and soybean. The effects of photoperiod and temperature on GmFT2a expression were also analyzed in two cultivars with different photoperiod-sensitivities. RESULTS: GmFT2a expression is regulated by photoperiod. Analyses of GmFT2a transcripts revealed a strong correlation between GmFT2a expression and flowering maintenance. GmFT2a transcripts were observed continuously within the vascular tissue up to the shoot apex during flowering. By contrast, transcripts decreased to undetectable levels during flowering reversion. In grafting experiments, the early-flowering, photoperiod-insensitive stock Heihe27 promotes the appearance of GmFT2a transcripts in the shoot apex of scion Zigongdongdou under noninductive LD conditions. The photothermal effects of GmFT2a expression diversity in cultivars with different photoperiod-sensitivities and a hypothesis is proposed. CONCLUSION: GmFT2a expression is associated with flowering induction and maintenance. Therefore, GmFT2a is a potential target gene for soybean breeding, with the aim of increasing geographic adaptation of this crop

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The Ninth Visual Object Tracking VOT2021 Challenge Results

acceptedVersionPeer reviewe