Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Diagnostic accuracy of real-time tissue elastography for breast cancer:a meta-analysis

Objective The present study aimed to determine the accuracy of real-time tissue elastography （RTE） for the diagnosis of breast cancer. Methods The search was conducted in the PubMed, Web of Science, Cochrane Library, and China Biology Medicine databases from inception through December 31, 2014, without language restrictions. The meta-analysis was conducted using STATA version 12.0 and Meta-Disc version 1.4. We calculated the summary statistics for sensitivity （Sen）, specificity （Spe）, positive and negative likelihood ratio （LR＋/LR–）, diagnostic odds ratio （DOR）, and summary receiver operating characteristic （SROC） curve. Results Ten studies that met al inclusion criteria were included in the meta-analysis. A total of 608 ma-lignant breast lesions and 1292 benign breast tumors were assessed. Al breast lesions were histological y confirmed after RTE. The pooled Sen was 0.83 （95% CI = 0.79–0.86）; the pooled Spe was 0.86 （95% CI = 0.84–0.88）. The pooled LR＋ was 9.87 （95% CI = 2.66–36.71）; the pooled LR– was 0.20 （95% CI = 0.17–0.23）. The pooled DOR of RTE for the diagnosis of breast cancer was 62.21 （95% CI = 33.88–114.24）. The area under the SROC curve was 0.9334 （standard error = 0.00125）. We found no evidence of publica-tion bias （t = –0.57, P = 0.582）. Conclusion RTE may have high diagnostic accuracy for the dif erential diagnosis of benign and malig-nant breast tumors. RTE may be a good tool for breast cancer diagnosis.

Soft Tissue Deformation Modeling in the Procedure of Needle Insertion:A Kriging‑Based Method

The simulation and planning system(SPS)requires accurate and real-time feedback regarding the deformation of soft tissues during the needle insertion procedure.Traditional mechanical-based models such as the finite element method(FEM)are widely used to compute the deformations of soft tissue.However,it is difficult for the FEM or other methods to find a balance between an acceptable image fidelity and real-time deformation feedback due to their complex material properties,geometries and interaction mechanisms.In this paper,a Kriging-based method is applied to model the soft tissue deformation to strike a balance between the accuracy and efficiency of deformation feedback.Four combinations of regression and correlation functions are compared regarding their ability to predict the maximum deformations of ten characteristic markers at a fixed insertion depth.The results suggest that a first order regression function with Gaussian correlation functions can best fit the results of the ground truth.The functional response of the Kriging-based method is utilized to model the dynamic deformations of markers at a series of needle insertion depths.The feasibility of the method is verified by investigating the adaptation to step variations.Compared with the ground truth of the finite element(FE)results,the maximum residual is less than 0.92 mm in the Y direction and 0.31 mm in the X direction.The results suggest that the Kriging metamodel provides real-time deformation feedback for a target and an obstacle to a SPS.

Alternative Approach for Studying Contractility of the Isolated Muscle Preparations in Real-Time Mode

The real-time control over the observed parameters is known to be necessary during the experiments with biological objects. The use of approaches, such as real-time operating systems, often requires significant material or human resources (operating time, the special administration and programming skills and etc.). In this paper we propose an alternative approach for the implementation of real-time mode in the experiments on muscle preparations (papillary muscles, trabeculae, walls of vessels) based on an external input-output cards with built-in analog-to-digital/digital-to-analog converters and signal processor. As a result, we developed a hardware-software complex for studying the mechanical properties of biological materials (muscles) in real-time mode. This solution has a convenient and easy-to-use user interface, and requires a minimum of computing resources and minimal cost of electricity.

Liver fibrosis index-based nomograms for identifying esophageal varices in patients with chronic hepatitis B related cirrhosis

BACKGROUND Esophageal varices(EV)are the most fatal complication of chronic hepatitis B(CHB)related cirrhosis.The prognosis is poor,especially after the first upper gastrointestinal hemorrhage.AIM To construct nomograms to predict the risk and severity of EV in patients with CHB related cirrhosis.METHODS Between 2016 and 2018,the patients with CHB related cirrhosis were recruited and divided into a training or validation cohort at The First Affiliated Hospital of Wenzhou Medical University.Clinical and ultrasonic parameters that were closely related to EV risk and severity were screened out by univariate and multivariate logistic regression analyses,and integrated into two nomograms,respectively.Both nomograms were internally and externally validated by calibration,concordance index(C-index),receiver operating characteristic curve,and decision curve analyses(DCA).RESULTS A total of 307 patients with CHB related cirrhosis were recruited.The independent risk factors for EV included Child-Pugh class[odds ratio(OR)=7.705,95%confidence interval(CI)=2.169-27.370,P=0.002],platelet count(OR=0.992,95%CI=0.984-1.000,P=0.044),splenic portal index(SPI)(OR=3.895,95%CI=1.630-9.308,P=0.002),and liver fibrosis index(LFI)(OR=3.603,95%CI=1.336-9.719,P=0.011);those of EV severity included Child-Pugh class(OR=5.436,95%CI=2.112-13.990,P<0.001),mean portal vein velocity(OR=1.479,95%CI=1.043-2.098,P=0.028),portal vein diameter(OR=1.397,95%CI=1.021-1.912,P=0.037),SPI(OR=1.463,95%CI=1.030-2.079,P=0.034),and LFI(OR=3.089,95%CI=1.442-6.617,P=0.004).Two nomograms(predicting EV risk and severity,respectively)were well-calibrated and had a favorable discriminative ability,with C-indexes of 0.916 and 0.846 in the training cohort,respectively,higher than those of other predictive indexes,like LFI(C-indexes=0.781 and 0.738),SPI(C-indexes=0.805 and 0.714),ratio of platelet count to spleen diameter(PSR)(C-indexes=0.822 and 0.726),King’s score(C-indexes=0.694 and 0.609),and Lok index(C-indexes=0.788 and 0.700).The areas under the curves(AUCs)of the two nomograms were 0.916 and 0.846 in the training cohort,respectively,higher than those of LFI(AUCs=0.781 and 0.738),SPI(AUCs=0.805 and 0.714),PSR(AUCs=0.822 and 0.726),King’s score(AUCs=0.694 and 0.609),and Lok index(AUCs=0.788 and 0.700).Better net benefits were shown in the DCA.The results were validated in the validation cohort.CONCLUSION Nomograms incorporating clinical and ultrasonic variables are efficient in noninvasively predicting the risk and severity of EV.

Copy number changes of target genes in chromosome 3q25.3-qter of esophageal squamous cell carcinoma: TP63is amplified in early carcinogenesis but down-regulated as disease progressed

AIM: By using comparative genomic hybridization, gain of 3q was found in 45-86% cases of esophageal squamous cell carcinoma (EC-SCC). Chromosome 3q25.3-qter is the minimal common region with several oncogenes found within this region. However, amplification patterns of these genes in EC-SCC have never been reported. The possible association of copy number changes of these genes with pathologic characteristics is still not clear. METHODS: Real-time quantitative PCR (Q-PCR) was performed to analyze the copy number changes of 13 candidate genes within this region in 60 primary tumors of EC-SCC, and possible association of copy number changes with pathologic characteristics was analyzed by statistics. Immunohistochemistry (IHC) study was also performed on another set of 111 primary tumors of EC-SCC to verify the association between TP63 expression change and lymph node metastasis status. RESULTS: The average copy numbers (±SE) per haploid genome of individual genes in 60 samples were (from centromere to telomere): SSR3: 4.19 (±0.69); CCNL1: 5.24 (±0.67); SMC4L1: 2.01 (±0.16); EVI1: 2.02 (±0.12); hTERC. 5.28 (±0.54); SKIL 2.71 (±0.14); EIF5A2. 1.95 (±0.12); ECT2: 9.18 (±1.68); PIK3CA: 8.13 (±1.17); EIF4G1: 1.07 (±0.05); 557: 3.07 (±0.25); TP63: 2.51 (±0.22); TFRC. 2.42 (±0.19). Four clusters of amplification were found: SSR3 and CCLN1 at 3q25.31; hTERC and SKIL at 3q26.2; ECT2 and PIK3CA at 3q26.31-q26.32; and 55T, TP63 and TFRC at 3q27.3-q29. Patients with lymph node metastasis had significantly lower copy number of TP63 in the primary tumor than those without lymph node metastasis. IHC study on tissue arrays also showed that patients with lymph node metastasis have significantly lower TP63 staining score in the primary tumor than those without lymph node metastasis. CONCLUSION: This study showed that different amplification patterns were seen among different genes within 3q25.3-qter in EC-SCC, and several novel candidate oncogenes (SSR3, SMC4L1, ECT2, and SST) were identified. TP63 is amplified in early stage of EC-SCC carcinogenesis but down-regulated in advanced stage of disease.

Atypical chemokine receptor CCRL2 is overexpressed in prostate cancer cells

Atypical chemokine receptors have recently emerged as important molecular players in health and diseases; they affect chemokine availability and function and impact a multitude of pathophysiological events, including the tumorigenesis process. This family of atypical receptors comprises five members: ACKR1/DARC, ACKR2/D6,ACKR3/CXCR7, ACKR4/CCRL1, and ACKR5/CCRL2. This work evaluated the differential expression of these receptors in prostate cancer using quantitative PCR. Further evaluation of CCRL2 at the protein level confirmed its overexpression in a metastatic cell line and in malignant prostatic tissues from patients. CCRL2, a presumed member of the atypical chemokine receptor family, plays a key role in lung dendritic cell trafficking to peripheral lymph nodes.Recent studies have reported the expression of CCRL2 in different human cancer cell lines and tissues. However, its function and expression in prostate cancer has not been previously addressed.

Current use of intraoperative ultrasound in modern liver surgery

Ultrasound plays an important role not only in preoperative diagnosis but also in intraoperative guidance for liver surgery.Intraoperative ultrasound(IOUS)has become an indispensable tool for modern liver surgeons,especially for minimally invasive surgeries,partially substituting for the surgeon’s hands.In fundamental mode,Doppler mode,contrast enhancement,elastography,and real-time virtual sonography,IOUS can provide additional real-time information regarding the intrahepatic anatomy,tumor site and characteristics,macrovascular invasion,resection margin,transection plane,perfusion and outflow of the remnant liver,and local ablation efficacy for both open and minimally invasive liver resections.Identification and localization of intrahepatic lesions and surrounding structures are crucial for performing liver resection,preserving the adjacent vital vascular and bile ducts,and sparing the functional liver parenchyma.Intraoperative ultrasound can provide critical information for intraoperative decision-making and navigation.Therefore,all liver surgeons must master IOUS techniques,and IOUS should be included in the training of modern liver surgeons.Further investigation of the potential benefits and advances in these techniques will increase the use of IOUS in modern liver surgeries worldwide.This study comprehensively reviews the current use of IOUS in modern liver surgeries.