Validation of internal control for gene expression study in soybean by quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p>Normalizing to housekeeping gene (HKG) can make results from quantitative real-time PCR (qRT-PCR) more reliable. Recent studies have shown that no single HKG is universal for all experiments. Thus, a suitable HKG should be selected before its use. Only a few studies on HKGs have been done in plants, and none in soybean, an economically important crop. Therefore, the present study was conducted to identify suitable HKG(s) for normalization of gene expression in soybean.</p> <p>Results</p> <p>All ten HKGs displayed a wide range of Ct values in 21 sample pools, confirming that they were variably expressed. GeNorm was used to determine the expression stability of the HGKs in seven series sets. For all the sample pools analyzed, the stability rank was <it>ELF1B</it>, <it>CYP2 </it>> <it>ACT11 </it>> <it>TUA </it>> <it>ELF1A </it>> <it>UBC2 </it>> <it>ACT2/7 </it>> <it>TUB </it>> <it>G6PD </it>> <it>UBQ10</it>. For different tissues under the same developmental stage, the rank was <it>ELF1B</it>, <it>CYP2 </it>> <it>ACT2/7 </it>> <it>UBC2 </it>> <it>TUA </it>> <it>ELF1A </it>> <it>ACT11 </it>> <it>TUB </it>> <it>G6PD </it>> <it>UBQ10</it>. For the developmental stage series, the stability rank was <it>ACT2/7</it>, <it>TUA </it>> <it>ELF1A </it>> <it>UBC2 </it>> <it>ELF1B </it>> <it>TUB </it>> <it>CYP2 </it>> <it>ACT11 </it>> <it>G6PD </it>> <it>UBQ10</it>. For photoperiodic treatments, the rank was <it>ACT11</it>, <it>ELF1B </it>> <it>CYP2 </it>> <it>TUA </it>> <it>ELF1A </it>> <it>UBC2 </it>> <it>ACT2/7 </it>> <it>TUB </it>> <it>G6PD </it>> <it>UBQ10</it>. For different times of the day, the rank was <it>ELF1A</it>, <it>TUA </it>> <it>ELF1B </it>> <it>G6PD </it>> <it>CYP2 </it>> <it>ACT11 </it>> <it>ACT2/7 </it>> <it>TUB </it>> <it>UBC2 </it>> <it>UBQ10</it>. For different cultivars and leaves on different nodes of the main stem, the ten HKGs' stability did not differ significantly. ΔCt approach and 'Stability index' were also used to analyze the expression stability in all 21 sample pools. Results from ΔCt approach and geNorm indicated that <it>ELF1B </it>and <it>CYP2 </it>were the most stable HKGs, and <it>UBQ10 </it>and <it>G6PD </it>the most variable ones. Results from 'Stability index' analysis were different, with <it>ACT11 </it>and <it>CYP2 </it>being the most stable HKGs, and <it>ELF1A </it>and <it>TUA </it>the most variable ones.</p> <p>Conclusion</p> <p>Our data suggests that HKGs are expressed variably in soybean. Based on the results from geNorm and ΔCt analysis, <it>ELF1B </it>and <it>CYP2 </it>could be used as internal controls to normalize gene expression in soybean, while <it>UBQ10 </it>and <it>G6PD </it>should be avoided. To achieve accurate results, some conditions may require more than one HKG to be used for normalization.</p

Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics

<p>Abstract</p> <p>Background</p> <p>Transgenic approaches provide a powerful tool for gene function investigations in plants. However, some legumes are still recalcitrant to current transformation technologies, limiting the extent to which functional genomic studies can be performed on. <it>Superroo</it>t of <it>Lotus corniculatus </it>is a continuous root cloning system allowing direct somatic embryogenesis and mass regeneration of plants. Recently, a technique to obtain transgenic <it>L. corniculatus </it>plants from <it>Superroot</it>-derived leaves through <it>A. tumefaciens-</it>mediated transformation was described. However, transformation efficiency was low and it took about six months from gene transfer to PCR identification.</p> <p>Results</p> <p>In the present study, we developed an <it>A. rhizogenes</it>-mediated transformation of <it>Superroot</it>-derived <it>L. corniculatus </it>for gene function investigation, combining the efficient <it>A. rhizogenes</it>-mediated transformation and the rapid regeneration system of <it>Superroot</it>. The transformation system using <it>A. rhizogenes </it>K599 harbouring pGFPGUS<it>Plus </it>was improved by validating some parameters which may influence the transformation frequency. Using stem sections with one node as explants, a 2-day pre-culture of explants, infection with K599 at OD₆₀₀= 0.6, and co-cultivation on medium (pH 5.4) at 22°C for 2 days enhanced the transformation frequency significantly. As proof of concept, <it>Superroot</it>-derived <it>L. corniculatus </it>was transformed with a gene from wheat encoding an Na⁺/H⁺antiporter (<it>TaNHX2</it>) using the described system. Transgenic <it>Superroot </it>plants were obtained and had increased salt tolerance, as expected from the expression of <it>TaNHX2</it>.</p> <p>Conclusion</p> <p>A rapid and efficient tool for gene function investigation in <it>L. corniculatus </it>was developed, combining the simplicity and high efficiency of the <it>Superroot </it>regeneration system and the availability of <it>A. rhizogenes</it>-mediated transformation. This system was improved by validating some parameters influencing the transformation frequency, which could reach 92% based on GUS detection. The combination of the highly efficient transformation and the regeneration system of <it>Superroot </it>provides a valuable tool for functional genomics studies in <it>L. corniculatus</it>.</p

BDTS: Blockchain-based Data Trading System

Trading data through blockchain platforms is hard to achieve \textit{fair exchange}. Reasons come from two folds: Firstly, guaranteeing fairness between sellers and consumers is a challenging task as the deception of any participating parties is risk-free. This leads to the second issue where judging the behavior of data executors (such as cloud service providers) among distrustful parties is impractical in the context of traditional trading protocols. To fill the gaps, in this paper, we present a \underline{b}lockchain-based \underline{d}ata \underline{t}rading \underline{s}ystem, named BDTS. BDTS implements a fair-exchange protocol in which benign behaviors can get rewarded while dishonest behaviors will be punished. Our scheme requires the seller to provide consumers with the correct encryption keys for proper execution and encourage a rational data executor to behave faithfully for maximum benefits from rewards. We analyze the strategies of consumers, sellers, and dealers in the trading game and point out that everyone should be honest about their interests so that the game will reach Nash equilibrium. Evaluations prove efficiency and practicability.Comment: ICICS 2023 (Best Paper Award

Klinička primjena RT-PCR u otkrivanju DNK tuberkuloze u kombinaciji s TB-IGRA u dijagnostici plućne tuberkuloze s negativnim nalazom sputuma

The aim was to investigate detection of pulmonary alveolar lavage fluid tuberculosis DNA by real-time fluorescent polymerase chain reaction (RT-PCR) combined with clinical application of the sputum smear-negative pulmonary tuberculosis diagnosis with TB interferon-γ release assay (TB-IGRA). From October 2014 to October 2015, 632 outpatients and inpatients treated in our hospital were randomly selected, of which 459 patients as the research group managed with RT-PCR detection combined with TB-IGRA and 173 patients as the control group undergoing electronic bronchoscopy alveolar lavage fluid detection, with detection results statistically evaluated. The positive rate in the research group was 96.51%, i.e. significantly higher than that in the control group (66.47%), yielding a statistically significant difference (χ2=109.68, p=0.00). The true positive rate was 97.7% in the research group and 67.92% in the control group; the true positive rate was significantly higher in the research group patients as compared with the control group, yielding a statistically significant difference (χ2=112.04, p=0.00). The sensitivity and specificity, as well as Youden index were significantly higher in the research group as compared with the control group. In conclusion, TB DNA detection by RT-PCR combined with TB-IGRA is a very good method of diagnosing tuberculosis, and it can be implemented in clinical diagnosis of pulmonary tuberculosis.Cilj istraživanja bio je ispitati otkrivanje DNK tuberkuloze u plućnom alveolarnom ispirku pomoću lančane reakcije polimeraze u stvarnom vremenu s fluorescentnim bojama (RT-PCR) u kombinaciji s kliničkom primjenom dijagnostike plućne tuberkuloze s negativnim nalazom sputuma pomoću testa otpuštanja TB interferona-γ (TB-IGRA). Od listopada 2014. do listopada 2015. godine nasumce su odabrane 632 osobe bolnički i izvanbolnički liječene u našoj bolnici, od kojih je 459 uključeno u ispitnu skupinu podvrgnutu otkrivanju pomoću RT-PCR u kombinaciji s testom TB-IGRA, a 173 u kontrolnu skupinu podvrgnutu elektroničkom otkrivanju u bronhoskopskom alveolarnom ispirku; rezultati su statistički obrađeni. Stopa pozitivnih nalaza bila je 96,51% u ispitnoj skupini, odnosno značajno viša od one u kontrolnoj skupini (66,47%), uz statistički značajnu razliku (χ2=109,68; p=0,00). Stopa stvarno pozitivnih nalaza bila je 97,7% u ispitnoj skupini i 67,92% u kontrolnoj skupini, dakle značajno viša kod bolesnika ispitne skupine u usporedbi s kontrolnom skupinom, uz statistički značajnu razliku (χ2=112,04; p=0,00). Osjetljivost i specifičnost, kao i Youdenov indeks bili su značajno viši u ispitnoj skupini u usporedbi s kontrolnom skupinom. U zaključku, otkrivanje TB DNK pomoću RT-PCR u kombinaciji s TB-IGRA vrlo je dobra metoda za dijagnosticiranje tuberkuloze, koja se može primjenjivati u kliničkoj dijagnostici plućne tuberkuloze

Modeling and Analysis of Truck Mounted Concrete Pump Boom by Virtual Prototyping

By far there is lack of research on different working conditions between rigid and flexible dynamics of truck mounted concrete pump booms. First a 3D model has been established by using virtual prototyping technology of a 37 m long boom in Pro/Engineering software. Then the rigid body simulation model has been built. Next modal superimposition method is adopted to change the 4 rigid body booms into flexible ones. Kinematics law and dynamic characteristics of 4 common working conditions had been studied then. Next tip displacement and the first boom hydraulic cylinder force of the 4 working conditions between rigid and flexible models have been researched. Furthermore the first natural frequencies of the structure have been calculated. The results show that the frequency of the horizontal condition has the lowest of all and the roof condition has the largest of all. Besides the cylinder forces of the flexible model are larger than the corresponding rigid ones because of the flexible boom vibration. Finally an experiment has been done on a boom test rig which proved that the established simulation model is reasonable and the frequency results are correct. All of these provide design reference to mechanical manipulator as well as reducing product development cost of such mechanism

COVID-19 Epidemic Peer Support and Crisis Intervention Via Social Media

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.This article describes a peer support project developed and carried out by a group of experienced mental health professionals, organized to offer peer psychological support from overseas to healthcare professionals on the frontline of the COVID-19 outbreak in Wuhan, China. This pandemic extremely challenged the existing health care systems and caused severe mental distress to frontline healthcare workers. The authors describe the infrastructure of the team and a novel model of peer support and crisis intervention that utilized a popular social media application on smartphone. Such a model for intervention that can be used elsewhere in the face of current global pandemic, or future disaster response