Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR

The accurate measurement of gene expression via quantitative real-time reverse transcription PCR（q RT-PCR）heavily relies on the choice of valid reference gene（s） for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of q RT-PCR to the HAB-forming group. In this study, six candidate reference genes,18 S ribosomal RNA（18S）, glyceraldehyde-3-phosphate dehydrogenase（GAPDH）, α-tubulin（TUA）, β-tubulin（TUB）, actin（ACT） and cytochrome oxidase subunit 1（COX1）, were evaluated for their expression stability with q RT-PCR and three statistical algorithms（Ge Norm, Norm Finder, and Best Keeper） for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages（resting cysts）, different growth stages, in darkness, exposed to abscisic acid（ABA） and exposed to temperature stress. The results indicated that TUA, 18 S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18 S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle,particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates regarding quantification of gene expression at different experimental scenarios, which will facilitate more accurate and widespread use of q RT-PCR in gene analysis of dinoflagellates and help to design primers targeting orthologous genes in other algal species.

Review of the Research Progress on Static Earth Gravity Field and Vertical Datum in China during 2019—2023

The contribution presents the representative research progress on global static gravity field modeling,regional geoid/quasigeoid determination,vertical datum study,as well as the theory,algorithm and software for gravity field study in China from 2019 to 2023,which are the highlights of the chapter 6“Progress in Earth Gravity Model and Vertical Datum”in the“2019—2023 China National Report on Geodesy”that submitted to the International Association of Geodesy(IAG).In addition,suggestions are proposed to promote the research in the fields of earth gravity field,geoid/quasigeoid and vertical datumin China according to trends of international geodesy and related disciplines.

附加IRI模型约束的全球电离层建模及定位精度分析

采用载波相位平滑伪距的全球电离层建模方法,确定了添加国际参考电离层(IRI)模型约束和不同数据选取策略的有效性和必要性。实验分析表明,采用P_(1)、P_(2)原始双频观测数据并添加IRI模型约束后,部分历元电离层建模的整体效果得到了显著提高,在观测数据不足的区域内定位误差在北、东、高方向上分别减小了0.260 m、0.146 m和0.103 m,验证了该方法的有效性;将C_(1)观测值作为低精度数据取代缺失P1观测值后,电离层建模的整体效果进一步提升,但损失了模型精度,在观测数据不足的区域内定位误差在北、东、高方向上分别增加0.524 m、0.111 m和1.549 m,说明了利用P_(1)、P_(2)双频观测数据的必要性。

Performance evaluation of neural network TEC forecasting models over equatorial low-latitude Indian GNSS station

Global Positioning System(GPS)services could be improved through prediction of ionospheric delays for satellite-based radio signals.With respect to latitude,longitude,local time,season,solar cycle and geomagnetic activity the Total Electron Content(TEC)have significant variations in both time and space.These temporal and spatial TEC variations driven by interplanetary space weather conditions such as solar and geomagnetic activities can degrade the communication and navigation links of GPS.Hence,in this paper,performance of TEC forecasting models based on Neural Networks(NN)have been evaluated to forecast(1-h ahead)ionospheric TEC over equatorial low latitude Bengaluru e12:97+N;77:59+ET,Global Navigation Satellite System(GNSS)station,India.The VTEC data is collected for 2009 e2016(8 years)during current 24 th solar cycle.The input space for the NN models comprise the solar Extreme UV flux,F10.7 proxy,a geomagnetic planetary A index(AP)index,sunspot number(SSN),disturbance storm time(DST)index,solar wind speed(Vsw),solar wind proton density(Np),Interplanetary Magnetic Field(IMF Bz).The performance of NN based TEC forecast models and International Reference Ionosphere,IRI-2016 global TEC model has evaluated during testing period,2016.The NN based model driven by all the inputs,which is a NN unified model(NNunq)has shown better accuracy with Mean Absolute Error(MAE)of 3.15 TECU,Mean Square Deviation(MSD)of 16.8 and Mean Absolute Percentage Error(MAPE)of 19.8%and is 1 e25%more accurate than the other NN based TEC forecast models(NN1,NN2 and NN3)and IRI-2016 model.NNunq model has less Root Mean Square Error(RMSE)value 3.8 TECU and highest goodness-of-fit(R2)with 0.85.The experimental results imply that NNunq/NN1 model forecasts ionospheric TEC accurately across equatorial low-latitude GNSS station and IRI-2016 model performance is necessarily improved as its forecast accuracy is limited to 69 e70%.

Tomographic Study of Ionospheric Effects Associated with a Solar Eclipse

This paper studies the ionospheric effects associated with the solar eclipse of October 24th, 1995 by means of Computerized Ionospheric Tomography (CIT). Since the reconstructed profiles from experimental CIT are sporadically located in time, a time domain interpolation method based on Singular Value Decomposition (SVD) technique is proposed and applied to extract the ionospheric effects. The effects can be extracted by comparison analysis between the interpolated CIT profiles of the eclipse days and that of the reference day that are time-aligned. A series of figs have been obtained showing the attenuation of photonization effect at low altitudes and the weakening of plasma’s transportation process at high altitudes, etc. The photonization effect recovered to normal level soon after the last contact. The maximum electron density diminishing is observed about 2 h after the eclipse maximum and the effects seem vanished in the hours followed. Analysis on vertical TEC’s latitudinal-temporal variation gives similar conclusions.

Geomagnetic Orbit Determination Using Fuzzy Regulating Unscented Kalman Filter

Geomagnetic orbit determination fits for nanosatellites which pursue low cost and high-density ratio,but one of its disadvantages is the poor position accuracy introduced by magnetic bias.Here,a new method,named the fuzzy regulating unscented Kalman filter(FRUKF),is proposed.The magnetic bias is regarded as a random walk model,and a fuzzy regulator is designed to estimate the magnetic bias more accurately.The input of the regulator is the derivative of magnetic bias estimated from unscented Kalman filter(UKF).According to the fuzzy rule,the process noise covariance is adaptively determined.The FRUKF is evaluated using the real-flight data of the SWARMA.The experimental results show that the root-mean-square(RMS)position error is 3.1 km and the convergence time is shorter than the traditional way.

Recommendations for construction of a nonlinear international Terrestrial Reference Frame

The problems of ITRF2008,the latest International Terrestrial Reference Frame,are pointed out and analyzed as follows:(1) ITRF is not a mm-level Terrestrial Reference Frame;(2) the origin of ITRF is neither the Earth's center of mass (CM) nor the center of figure (CF);(3) the scale of ITRF is not a uniform system in the sense of the gravitational theory of relativity.These problems result from the linear hypothesis used in the establishment and maintenance of ITRF,which includes the linear hypothesis of the coordinates definition of the ITRF reference stations,and the seven coordinate transformation parameters (three translation parameters,three rotation parameters,and one scale parameter) when the ITRF combine solution is constructed.The linear hypothesis of the ITRF construction leads to the current terrestrial reference frame only at the cm-level,which cannot satisfy the requirements of monitoring mm-level crust movements as well as the global environment.This article points out that the construction of a mm-level Terrestrial Reference Frame is actually a leap from linear to nonlinear.Therefore,according to the main characteristics of nonlinear changes of the crust's deformation,the geocenter motion and the overall height fluctuation of the Earth,the new ITRF station coordinates definition and the new observation equations of combined solutions are constructed for the realization of a mm-level nonlinear ITRF,which can solve the problems of the current ITRF.

Theoretical calculation of tidal Love numbers of the Moon with a new spectral element method

The tidal Love numbers of the Moon are a set of nondimensional parameters that describe the deformation responses of the Moon to the tidal forces of external celestial bodies.They play an important role in the theoretical calculation of the Moon’s tidal deformation and the inversion of its internal structure.In this study,we introduce the basic theory for the theoretical calculation of the tidal Love numbers and propose a new method of solving the tidal Love numbers:the spectral element method.Moreover,we explain the mathematical theory and advantages of this method.On the basis of this new method,using 10 published lunar internal structure reference models,the lunar surface and lunar internal tidal Love numbers were calculated,and the influence of different lunar models on the calculated Love numbers was analyzed.Results of the calculation showed that the difference in the second-degree lunar surface Love numbers among different lunar models was within 8.5%,the influence on the maximum vertical displacement on the lunar surface could reach±8.5 mm,and the influence on the maximum gravity change could reach±6μGal.Regarding the influence on the Love numbers inside the Moon,different lunar models had a greater impact on the Love numbers h_(2) and l_(2) than on k_(2) in the lower lunar mantle and core.

Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV-2 RNAs in Clinical Specimens by Using an Internal Reference

Reverse transcription-polymerase chain reaction(RT-PCR)is an essential method for specific diagnosis of SARS-CoV-2 infection.Unfortunately,false negative test results are often reported.In this study,we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens.Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercialfluorescent RT-PCR kit targeting the ORF1 ab and N regions of SARS-CoV-2 genome.The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30(RPP30)in these specimens was also assessed by RT-PCR.Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed.By using the ROC-AUC analysis,we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity(95.03%–95.26%)and specificity(83.72%–98.55%)for respective combination of specimen type and ampli-fication reaction.Using these Ct cutoff values,false negative results could be reliably identified.Therefore,the presence of cellular materials,likely infected host cells,are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens.RPP30 could serve as an indicator for cellular content,or a surrogate indicator for specimen quality.In addition,our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.

Output Consensus for Heterogeneous Nonlinear Multi-Agent Systems Based on T-S Fuzzy Model

In this paper, the output consensus problem of general heterogeneous nonlinear multi-agent systems subject to different disturbances is considered. A kind of Takagi-Sukeno fuzzy modeling method is used to describe the nonlinear agents' dynamics. Based on the model, a distributed fuzzy observer and controller are designed based on parallel distributed compensation scheme and internal reference models such that the heterogeneous nonlinear multi-agent systems can achieve output consensus. Then a necessary and sufficient condition is presented for the output consensus problem. And it is shown that the consensus trajectory of the global fuzzy model is determined by the network topology and the initial states of the internal reference models. Finally, some simulations are given to illustrate and verify the effectiveness of the proposed scheme.

Multiplex ratiometric gold nanoprobes based on surface-enhanced Raman scattering enable accurate molecular detection and imaging of bladder cancer

Recently,surface-enhanced Raman scattering(SERS)has been successfully used in the non-invasive detection of bladder tumor(BCa).The internal standard method was considered as an effective ratiometric strategy for calibrating signal fluctuation originated from the interference of measurement conditions and samples.However,it is still difficult to detect the target mRNA quantitatively using the current ratiometric SERS nanosensors.In this study,we developed an internal reference based ratiometric SERS assay.Two kinds of molecular beacons(MB)carrying Raman reporter molecules were anchored on sea-urchinlike Au nanoclusters(AuNCs).Thymidine kinase1(TK1)MBs with hexachlorofluorescein(HEX)were used to capture tumor biomarker TK1 mRNA,and glyceraldehyde 3-phosphate dehydrogenase(GAPDH)MBs with 5(6)-carboxyfluorescein(FAM)were used to offer internal standard signals.The internal reference GAPDH MB can reflect the consistent content of the GAPDH mRNA in single cells.The ratiometric method(I745/I645)can more accurately reflect the content of target mRNA in single cells.The ratiometric nanoprobes had excellent stability(coefficient of variation:0.3%),high sensitivity(detection limit:3.4 pM),high specificity(capable of single-base mismatch recognition)and ribozyme-resistant stability.Notably,the nanoprobes can effectively distinguish BCa cells from normal cells,and it was easy to contour the single BCa cell using the ratiometric method.By combining asymmetric polymerase chain reaction(PCR)and ratiometric nanoprobes,it was easy to distinguish the SERS ratio(I_(745)/I_(645))as low concentration as 10-14 M.Further clinical detection in urine samples from patients with BCa confirmed its potential for early noninvasive diagnosis of BCa with the sensitivity of 80%and specificity of 100%,which is superior to the current urine cytological method.