Selection and Validation of Reference Genes for Normalization of RT-qPCR Analysis in Developing or Abiotic-Stressed Tissues of Loquat (Eriobotrya japonica)

Loquat(Eriobotrya japonica Lindl.)is a subtropical evergreen fruit tree that produces fruits with abundant nutrients and medicinal components.Confirming suitable reference genes for a set of loquat samples before qRT-PCR experiments is essential for the accurate quantification of gene expression.In this study,eight candidate reference genes were selected from our previously published RNA-seq data,and primers for each candidate reference gene were designed and evaluated.The Cq values of the candidate reference genes were calculated by RT-qPCR in 31 different loquat samples,including 12 subgroups of developing or abiotic-stressed tissues.Different combinations of stable reference genes were screened according to a comprehensive rank,which was synthesized from the results of four algorithms,including the geNorm,NormFinder,BestKeeper andΔCt methods.The screened reference genes were verified by normalizing EjLGA1 in each subgroup.The obtained suitable combinations of reference genes for accurate normalization were GAPDH,EF1αand ACT for floral development;GAPDH,UBCE and ACT for fruit setting;EF1α,GAPDH and eIF2B for fruit ripening;ACT,EF1αand UBCE for leaves under heat stress;eIF2B,UBCE and EF1αfor leaves under freezing stress;EF1α,TUA and UBCE for leaves under salt stress;ACT,EF1αand eIF2B for immature pulp under freezing stress;ACT,UBCE and eIF2B for immature seeds under freezing stress;EF1α,eIF2B and UBCE for both immature pulp and seeds under freezing stress;UBCE,TUB and TUA for red-fleshed fruits under cold-storage stress;eIF2B,RPS3 and TUB for white-fleshed fruits under coldstorage stress;and eIF2B,UBCE and RPS3 for both red-and white-fleshed fruits under cold-storage stress.This study obtained different combinations of stable reference genes for accurate normalization in twelve subgroups of developing or abiotic-stressed tissues in loquat.To our knowledge,this is the first report to obtain stable reference genes for normalizing gene expression of abiotic-stressed tissues in E.japonica.The use of the three most stable reference genes could increase the reliability of future quantification experiments.

The influence of lipid-extraction on theδ^(13)C of mesopelagic and demersal fish in the South China Sea:modification and application of lipid normalization models

Mesopelagic fish,the most important daily vertically migrating community in the oceans,are characterized by high lipid content which may obscure the interpretation of stable isotopes analysis.Demersal fish,which are important consumers in the food web dominated by mesopelagic fish,also have a high lipid content.Here we collected 127 fish samples from the South China Sea and evaluated the effect of lipid contents on△δ^(13)C of mesopelagic and demersal fish.In lipid-extracted mesopelagic fish,the C/N content ratio(<5.5)shows a clear correlation withΔδ^(13)C(the offset of bulk and lipid-extractedδ^(13)C values),especially in non-migratory and semi-migratory species;these values were less correlation in demersal fish.Based on our results,we suggest that mesopelagic and demersal fish in different regions of the South China Sea should be studied separately using appropriate correction models and less fit for the traditional model.Moreover,the C/N content ratio should be used cautiously for establishing the lipid normalization model,especially for the fish in migratory mesopelagic fish and demersal fish.Our results also reveal that mesopelagic fish across nearby regions could be analyzed together.The new models described here can be applied in future studies of mesopelagic and demersal fish in the South China Sea.

Supervised Feature Learning for Offline Writer Identification Using VLAD and Double Power Normalization

As an indispensable part of identity authentication,offline writer identification plays a notable role in biology,forensics,and historical document analysis.However,identifying handwriting efficiently,stably,and quickly is still challenging due to the method of extracting and processing handwriting features.In this paper,we propose an efficient system to identify writers through handwritten images,which integrates local and global features from similar handwritten images.The local features are modeled by effective aggregate processing,and global features are extracted through transfer learning.Specifically,the proposed system employs a pre-trained Residual Network to mine the relationship between large image sets and specific handwritten images,while the vector of locally aggregated descriptors with double power normalization is employed in aggregating local and global features.Moreover,handwritten image segmentation,preprocessing,enhancement,optimization of neural network architecture,and normalization for local and global features are exploited,significantly improving system performance.The proposed system is evaluated on Computer Vision Lab(CVL)datasets and the International Conference on Document Analysis and Recognition(ICDAR)2013 datasets.The results show that it represents good generalizability and achieves state-of-the-art performance.Furthermore,the system performs better when training complete handwriting patches with the normalization method.The experimental result indicates that it’s significant to segment handwriting reasonably while dealing with handwriting overlap,which reduces visual burstiness.

Robust Radiometric Normalization of the near Equatorial Satellite Images Using Feature Extraction and Remote Sensing Analysis

Relative radiometric normalization (RRN) minimizes radiometric differences among images caused by inconsistencies of acquisition conditions rather than changes in surface. Scale invariant feature transform (SIFT) has the ability to automatically extract control points (CPs) and is commonly used for remote sensing images. However, its results are mostly inaccurate and sometimes contain incorrect matching caused by generating a small number of false CP pairs. These CP pairs have high false alarm matching. This paper presents a modified method to improve the performance of SIFT CPs matching by applying sum of absolute difference (SAD) in a different manner for the new optical satellite generation called near-equatorial orbit satellite and multi-sensor images. The proposed method, which has a significantly high rate of correct matches, improves CP matching. The data in this study were obtained from the RazakSAT satellite a new near equatorial satellite system. The proposed method involves six steps: 1) data reduction, 2) applying the SIFT to automatically extract CPs, 3) refining CPs matching by using SAD algorithm with empirical threshold, and 4) calculation of true CPs intensity values over all image’ bands, 5) preforming a linear regression model between the intensity values of CPs locate in reverence and sensed image’ bands, 6) Relative radiometric normalization conducting using regression transformation functions. Different thresholds have experimentally tested and used in conducting this study (50 and 70), by followed the proposed method, and it removed the false extracted SIFT CPs to be from 775, 1125, 883, 804, 883 and 681 false pairs to 342, 424, 547, 706, 547, and 469 corrected and matched pairs, respectively.

Speaker Verification Based on Log-Likelihood Score Normalization

Due to differences in the distribution of scores for different trials, the performance of a speaker verification system will be seriously diminished if raw scores are directly used for detection with a unified threshold value. As such, the scores must be normalized. To tackle the shortcomings of score normalization methods, we propose a speaker verification system based on log-likelihood normalization (LLN). Without a priori knowledge, LLN increases the separation between scores of target and non-target speaker models, so as to improve score aliasing of “same-speaker” and “different-speaker” trials corresponding to the same test speech, enabling better discrimination and decision capability. The experiment shows that LLN is an effective method of scoring normalization.

基于TZ Normalization规整的话者确认阈值选取

针对说话人确认中,各目标话者模型输出评分分布不一致而导致系统确认阈值设置的困难,本文采取了通过评分规整确定系统最小检测代价函数(DCF)确认阈值的方法。在分析了已有的两种评分规整方法Z norm a l-ization和T norm a lization的基础上,提出了一种结合两者优点的组合规整方法——TZ norm a lization,并据此给出了一种阈值动态修正方法,有效地提高了系统的性能和阈值选取的鲁棒性。对历年的N IST(手机电话语音)评测语料库进行了实验,表明了该方法的有效性。

Application of well log normalization in coalfield seismic inversion

During the process of coal prospecting and exploration, different measurement time, different logging instruments and series can lead to systematic errors in well logs. Accordingly, all logging curves need to be normalized in the mining area. By studying well-logging normalization methods, and focusing on the characteristics of the coalfield, the frequency histogram method was used in accordance with the condition of the Guqiao Coal Mine. In this way, the density and sonic velocity at marker bed in the non-key well were made to close to those in the key well, and were eventually equal. Well log normalization was completed when this method was applied to the entire logging curves. The results show that the scales of logging data were unified by normalizing coal logging curves, and the logging data were consistent with wave impedance inversion data. A satisfactory inversion effect was obtained.

Evaluation of Two Absolute Radiometric Normalization Algorithms for Pre-processing of Landsat Imagery

In order to evaluate radiometric normalization techniques, two image normalization algorithms for absolute radiometric correction of Landsat imagery were quantitatively compared in this paper, which are the Illumination Correction Model proposed by Markham and Irish and the Illumination and Atmospheric Correction Model developed by the Remote Sensing and GIS Laboratory of the Utah State University. Relative noise, correlation coefficient and slope value were used as the criteria for the evaluation and comparison, which were derived from pseudo-invarlant features identified from multitemporal Landsat image pairs of Xiamen （厦门） and Fuzhou （福州） areas, both located in the eastern Fujian （福建） Province of China. Compared with the unnormalized image, the radiometric differences between the normalized multitemporal images were significantly reduced when the seasons of multitemporal images were different. However, there was no significant difference between the normalized and unnorrealized images with a similar seasonal condition. Furthermore, the correction results of two algorithms are similar when the images are relatively clear with a uniform atmospheric condition. Therefore, the radiometric normalization procedures should be carried out if the multitemporal images have a significant seasonal difference.

基于Moment Normalization和M序列的二值水印算法

针对因特网上数字图像的版权保护、认证和完整性等问题,基于DCT变换、image moment normalization和m序列,提出了一种二值水印嵌入算法,实现了二值图像的嵌入和提取。根据m序列的伪随机性和抗干扰性能,使水印具有良好的隐蔽性和稳健性;使用了moment normalization能抵制各种几何攻击。实验表明该算法具有很好的鲁棒性、实用性和可操作性。

Selection of suitable internal controls for gene expression normalization in rats with spinal cord injury

There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research.In this study,we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes:Actb,Ankrd27,CypA,Gapdh,Hprt1,Mrpl10,Pgk1,Rictor,Rn18s,Tbp,Ubc,Ubxn11,and Ywhaz.Our results show that the expression of Ankrd27,Ubc,and Tbp were stable after spinal cord injury,while Actb was the most unstable internal control gene.Ankrd27,Ubc,Tbp,and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression.Target gene expression levels and changes over time were similar when Ankrd27,Ubc,and Tbp were used as internal controls but different when Actb was used as an internal control.We recommend that Ankrd27,Ubc,and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research.This study was approved by the Administration Committee of Experimental Animals,Jiangsu Province,China(approval No.20180304-008)on March 4,2018.

Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

Background： Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Results： Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were： beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A （cyclophilin A）, ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein--zeta polypeptide The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were （most to least stable） succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, sucdnate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Conclusions： BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.

Normalization of Hydrodynamic Coefficients in Morison Equation

The hydrodynamic coefficients C-d and C-m are not only dependent on the size of slender cylinder, its location in water, KC number and Re number, but also vary with environmental conditions, i.e., in regular waves or in irregular waves, in pure waves or in wave-current coexisting field. In this paper, the normalization of hydrodynamic coefficients for various environmental conditions is discussed. When a proper definition of KC number and proper characteristic values of irregular waves are used, a unified relationship between C-d, C-m and KC number for regular waves, irregular waves, pure waves and wave-current coexisting field can be obtained.

Identification of normalization factors for quantitative realtime RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

3D numerical study of tumor blood perfusion and oxygen transport during vascular normalization

The changes of blood perfusion and oxygen transport in tumors during tumor vascular normalization are studied with 3-dimensional mathematical modeling and numerical simulation. The models of tumor angiogenesis and vascular-disrupting are used to simulate ＂un-normalized＂ and ＂normalized＂ vasculatures. A new model combining tumor hemodynamics and oxygen transport is developed. In this model, the intravasculartransvascular-interstitial flow with red blood cell（RBC） delivery is tightly coupled, and the oxygen resource is produced by heterogeneous distribution of hematocrit from the flow simulation. The results show that both tumor blood perfusion and hematocrit in the vessels increase, and the hypoxia microenvironment in the tumor center is greatly improved during vascular normalization. The total oxygen content inside the tumor tissue increases by about 67%, 51%, and 95% for the three approaches of vascular normalization,respectively. The elevation of oxygen concentration in tumors can improve its metabolic environment, and consequently reduce malignancy of tumor cells. It can also enhance radiation and chemotherapeutics to tumors.

Face Orientation Normalization Using Eye Positions

Despite the fact that progress in face recognition algorithms over the last decades has been made, changing lighting conditions and different face orientation still remain as a challenging problem. A standard face recognition system identifies the person by comparing the input picture against pictures of all faces in a database and finding the best match. Usually face matching is carried out in two steps： during the first step detection of a face is done by finding exact position of it in a complex background （various lightning condition）, and in the second step face identification is performed using gathered databases. In reality detected faces can appear in different position and they can be rotated, so these disturbances reduce quality of the recognition algorithms dramatically. In this paper to increase the identification accuracy we propose original geometric normalization of the face, based on extracted facial feature position such as eyes. For the eyes localization lbllowing methods has been used： color based method, mean eye template and SVM （Support Vector Machine） technique. Experimental investigation has shown that the best results for eye center detection can be achieved using SVM technique. The recognition rate increases statistically by 28% using face orientation normalization based on the eyes position.

Optical-Elevation Data Co-Registration and Classification-Based Height Normalization for Building Detection in Stereo VHR Images

Building detection in very high resolution (VHR) images is crucial for mapping and analysing urban environments. Since buildings are elevated objects, elevation data need to be integrated with images for reliable detection. This process requires two critical steps: optical-elevation data co-registration and aboveground elevation calculation. These two steps are still challenging to some extent. Therefore, this paper introduces optical-elevation data co-registration and normalization techniques for generating a dataset that facilitates elevation-based building detection. For achieving accurate co-registration, a dense set of stereo-based elevations is generated and co-registered to their relevant image based on their corresponding image locations. To normalize these co-registered elevations, the bare-earth elevations are detected based on classification information of some terrain-level features after achieving the image co-registration. The developed method was executed and validated. After implementation, 80% overall-quality of detection result was achieved with 94% correct detection. Together, the developed techniques successfully facilitate the incorporation of stereo-based elevations for detecting buildings in VHR remote sensing images.

Identification and evaluation of reference genes for normalization in quantitative real-time PCR analysis in the premodel tree Betula luminifera

Betula luminifera is a commercial tree species that is emerging as a new model system for tree genomics research. A draft genomic sequence is expected to be publicly available in the near future, which means that an explosion of gene expression studies awaits. Thus, the work of selecting appropriate reference genes for q PCR normalization in different tissues or under various experimental conditions is extremely valuable. In this study, ten candidate genes were analyzed in B. luminifera subjected to different abiotic stresses and at various flowering stages.The expression stability of these genes was evaluated using three distinct algorithms implemented using ge Norm,Norm Finder and Best Keeper. The best-ranked reference genes varied across different sample sets, though RPL39,MDH and EF1 a were determined as the most stable by the three programs among all tested samples. RPL39 and EF1 a should be appropriate for normalization in N-starved roots,while the combination of RPL39 and MDH should be appropriate for N-starved stems and EF1 a and MDH should be appropriate in N-starved leaves. In PEG-treated（osmotic） roots, MDH was the most suitable, whereas EF1 a was suitable for PEG-treated stems and leaves. TUA was also stably expressed levels in PEG-treated plants. The combination of RPL39 and TUB should be appropriate for heat-stressed leaves and flowering stage. For reference gene validation, the expression levels of SOD and NFYA-3were investigated. This work will be beneficial to future studies on gene expression under different abiotic stress conditions and flowering status in B. luminifera.

Short-term normalization of ventricular repolarization by transcatheter ablation in a patient with suspected brugada syndrome

We report observations in a 30 year old male without structural heart disease who presented with recurrent nocturnal syncope and aborted sudden cardiac death. 12-lead ECG showed elevated ST in inferior leads and short coupled premature ventricular complexes (PVCs). Propafenone challenge suggested a diagnosis of an atypical Brugada syndrome. Two morphological types of PVGs and ventricular fibrillation (VF) were induced during propafenone challenge test. He underwent two ablation procedures in right ventricular inflow tract and left ventricular post-inferior septum region by pace-mapping, respectively. After ablation, VF could not be induced and the elevated ST segments normalized. Two subsequent propafenone challenge tests were also negative. Nonetheless, elevated ST segments and PVCs reappeared by one month follow-up. An implantable defibrillator was recommended, but the patient declined for financial reasons. Unfortunately, he suffered a sudden cardiac death at home 10 weeks post-ablation. These findings suggest that short-term normalization of ventricular repolarizatiion after ablation is possible in Brugada syndrome. However, the transient nature of this finding suggests that it is not a reliable indicator of protection against sudden cardiac death.

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

An Evolutionary Normalization Algorithm for Signed Floating-Point Multiply-Accumulate Operation

In the era of digital signal processing,like graphics and computation systems,multiplication-accumulation is one of the prime operations.A MAC unit is a vital component of a digital system,like different Fast Fourier Transform(FFT)algorithms,convolution,image processing algorithms,etcetera.In the domain of digital signal processing,the use of normalization architecture is very vast.The main objective of using normalization is to performcomparison and shift operations.In this research paper,an evolutionary approach for designing an optimized normalization algorithm is proposed using basic logical blocks such as Multiplexer,Adder etc.The proposed normalization algorithm is further used in designing an 8×8 bit Signed Floating-Point Multiply-Accumulate(SFMAC)architecture.Since the SFMAC can accept an 8-bit significand and a 3-bit exponent,the input to the said architecture can be somewhere between−(7.96872)_(10) to+(7.96872)_(10).The proposed architecture is designed and implemented using the Cadence Virtuoso using 90 and 130 nm technologies(in Generic Process Design Kit(GPDK)and Taiwan Semiconductor Manufacturing Company(TSMC),respectively).To reduce the power consumption of the proposed normalization architecture,techniques such as“block enabling”and“clock gating”are used rigorously.According to the analysis done on Cadence,the proposed architecture uses the least amount of power compared to its current predecessors.