A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao

Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhuangdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction（q PCR） based on 18 S r DNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression（R2 = 0.91） was generated based on cycle thresholds value（Ct） versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using q PCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abundance, category 3 brown tide blooms（〉200 000 cells/m L） occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms（35 000–200 000 cells/m L） in August and all stations had category 1 blooms（〉0 to ≤35 000 cells/m L） in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.

Confusing finding of quantitative fluorescent polymerase chain reaction analysis in invasive prenatal genetic diagnosis:A case report

BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.

Correlation Between the Clinical Severity, Bacterial Load, and Inflammatory Reaction in Children with Mycoplasma Pneumoniae Pneumonia

Given the lack of defining features in the clinical manifestations and radiographic findings for children with mycoplasma pneumoniae pneumonia(MPP),quantitative polymerase chain reaction(qPCR)has become a useful diagnostic method.This study was performed to explore the relationship between the qPCR findings,clinical symptoms,and inflammatory markers in children with MPP.Four hundred children with MPP have been enrolled in this retrospective analysis.All clinical and analytical information,including mycoplasma pneumoniae(MP)PCR results,has been collected.Based on the PCR results,the patients were divided into groups with load values(copy number)<105(54 cases),2105 and<106(71 cases),2106 and<107(112 cases),>107 and<108(114 cases),and>108(49 cases).The clinical features(including symptoms and signs)and inflammatory indicators were compared among the groups.The incidence of high fever(above 39℃),thermal peak during the entire hospitalization period,fever duration,days of hospitalization,and plasma lactate dehydrogenase(LDH)levels were statistically correlated with the MP PCR load value in children with MPP.The analysis of relevance degree showed the correlative order as a thermal peak of hospitalization>duration of fever>period of hospitalization>LDH value>C-reactive protein value.The host immune response was significantly greater in the complication group than in the non-complication group.

Establishment of a new quantitative detection approach to adefovir-resistant HBV and its clinical application

AIM:To establish the more feasible and sensitive assessment approach to the detection of adefovir (ADV) resistance-associated hepatitis B virus (HBV) quasispecies.METHODS: Based on the characteristics of rtA181V/T and rtN236T mutations, a new approach based on real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was established for the detection of ADV-resistant HBV quasispecies, total HBV DNA, rtA181 and rtN236 mutations in blood samples from 32 chronic hepatitis B (CHB) patients with unsatisfactory curative effect on ADV and compared with routine HBV DNA sequencing.RESULTS: Both the sensitivity and specificity of this new detection approach to ADV-resistant HBV quasispecies were 100%, which were much higher than those of direct HBV DNA sequencing. The approach was able to detect 0.1% of mutated strains in a total plasmid population. Among the 32 clinical patients, single rtA181 and rtN236T mutation and double rtA181T and rtN236T mutations were detected in 20 and 8, respectively, while ADV-resistant mutations in 6 (including, rtA181V/T mutation alone in 5 patients) and no associated mutations in 26.CONCLUSION: This new approach is more feasible and efficient to detect ADV-resistant mutants of HBV and ADV-resistant mutations before and during ADV treatment with a specificity of 100% and a sensitivity of 100%.

Rapid prenatal diagnosis of trisomy 21 by fluorescent quantitative multiplex polymerase chain reaction

Trisomy 21, also named Down syndrome was the most frequent autosomal aneuploidy and the most common cause of mental retardation. Fifty percent patients had congenital heart malformation. Every 20 minutes one case of trisomy 21 was born, and the incidence rate was 1 in 600 to 800 newborns in China.1 In two thirds of cases with trisomy 21, there was a spontaneous abortion, so the actual incidence was higher than that obtained postnatally.

An Evaluation Method of Human Gut Microbial Homeostasis by Testing Specific Fecal Microbiota

Research on microecology has been carried out with broad perspectives in recent decades,which has enabled a better understanding of the gut microbiota and its roles in human health and disease.It is of great significance to routinely acquire the status of the human gut microbiota;however,there is no method to evaluate the gut microbiome through small amounts of fecal microbes.In this study,we found ten predominant groups of gut bacteria that characterized the whole microbiome in the human gut from a large-sample Chinese cohort,constructed a real-time quantitative polymerase chain reaction(qPCR)method and developed a set of analytical approaches to detect these ten groups of predominant gut bacterial species with great maneuverability,efficiency,and quantitative features.Reference ranges for the ten predominant gut bacterial groups were established,and we found that the concentration and pairwise ratios of the ten predominant gut bacterial groups varied with age,indicating gut microbial dysbiosis.By comparing the detection results of liver cirrhosis(LC)patients with those of healthy control subjects,differences were then analyzed,and a classification model for the two groups was built by machine learning.Among the six established classification models,the model established by using the random forest algorithm achieved the highest area under the curve(AUC)value and sensitivity for predicting LC.This research enables easy,rapid,stable,and reliable testing and evaluation of the balance of the gut microbiota in the human body,which may contribute to clinical work.

Exploring the regulatory mechanism of tRNA-derived fragments 36 in acute pancreatitis based on small RNA sequencing and experiments

BACKGROUND Acute pancreatitis(AP)is a disease featuring acute inflammation of the pancreas and histological destruction of acinar cells.Approximately 20%of AP patients progress to moderately severe or severe pancreatitis,with a case fatality rate of up to 30%.However,a single indicator that can serve as the gold standard for prognostic prediction has not been discovered.Therefore,gaining deeper insights into the underlying mechanism of AP progression and the evolution of the disease and exploring effective biomarkers are important for early diagnosis,progression evaluation,and precise treatment of AP.AIM To determine the regulatory mechanisms of tRNA-derived fragments(tRFs)in AP based on small RNA sequencing and experiments.METHODS Small RNA sequencing and functional enrichment analyses were performed to identify key tRFs and the potential mechanisms in AP.Reverse transcription quantitative polymerase chain reaction(RT-qPCR)was conducted to determine tRF expression.AP cell and mouse models were created to investigate the role of tRF36 in AP progression.Lipase,amylase,and cytokine levels were assayed to examine AP progression.Ferritin expression,reactive oxygen species,malondialdehyde,and ferric ion levels were assayed to evaluate cellular ferroptosis.RNA pull down assays and methylated RNA immunoprecipitation were performed to explore the molecular mechanisms.RESULTS RT-qPCR results showed that tRF36 was significantly upregulated in the serum of AP patients,compared to healthy controls.Functional enrichment analysis indicated that target genes of tRF36 were involved in ferroptosisrelated pathways,including the Hippo signaling pathway and ion transport.Moreover,the occurrence of pancreatic cell ferroptosis was detected in AP cells and mouse models.The results of interference experiments and AP cell models suggested that tRF-36 could promote AP progression through the regulation of ferroptosis.Furthermore,ferroptosis gene microarray,database prediction,and immunoprecipitation suggested that tRF-36 accelerated the progression of AP by recruiting insulin-like growth factor 2 mRNA binding protein 3(IGF2BP3)to the p53 mRNA m6A modification site by binding to IGF2BP3,which enhanced p53 mRNA stability and promoted the ferroptosis of pancreatic follicle cells.CONCLUSION In conclusion,regulation of nuclear pre-mRNA domain-containing protein 1B promoted AP development by regulating the ferroptosis of pancreatic cells,thereby acting as a prospective therapeutic target for AP.In addition,this study provided a basis for understanding the regulatory mechanisms of tRFs in AP.

Correlations among core species corresponding to the clinical staging of periodontitis

The correlation between microbiota plays a vital role in the progression of periodontal disease.This study investigated the in situ interaction networks between periodontal pathogens in periodontal and peri-implant disease.We used quantitative real-time polymerase chain reaction and Pearson’s correlation coefficients to quantify the copy numbers and correlations of four oral core species—Fusobacterium nucleatum,Porphyromonas gingivalis,Prevotella intermedia,and Streptococcus gordonii—from 80 subgingival sites(healthy and with periodontitis or gingivitis)in patients with periodontitis,and 68 subgingival sites(healthy and with periodontitis,gingivitis,peri-implantitis,or peri-implant mucositis)in patients with implants.The highest bacterial counts were observed for Porphyromonas gingivalis and Prevotella intermedia at all the sites.Within the same cohorts,the bacterial loads were greater at diseased sites than at healthy sites.Bacterial counts did not differ among clinical sites in the same group(P>0.05)but differed between periodontitis and peri-implant mucositis sites in the two groups.Porphyromonas gingivalis,F.nucleatum,and Prevotella intermedia had strong correlations at gingivitis and healthy sites and moderate correlations at periodontitis sites in patients with periodontitis.In patients with implants,Prevotella intermedia,F.nucleatum,and S.gordonii had strong correlations only at peri-implantitis sites.Also,based on metagenomic analysis,F.nucleatum and Prevotella intermedia were significantly correlated at the subgingival plaque in peri-implantitis and periodontitis samples.Our results suggest that variations in microbe-microbe interactions in subgingival plaque reflect changes in the progression of periodontal disease,providing a new perspective for understanding the mechanisms of periodontitis and peri-implantitis.

Urine exosome mRNA-based test for monitoring kidney allograft rejection:Effects of sample transportation and storage,and interference substances

BACKGROUND Exosomes are 30-150 nm nanovesicles with sophisticated nucleic acids cargo,actively secreted by all cells within human body,and found in abundance in all body fluids,including urine.These extracellular vesicles have tremendous potential for next generation diagnostics,theoretically enabling noninvasive assessment of organ and tissue function via liquid biopsy analysis.AIM Recently,feasibility of an exosomal molecular test was demonstrated for postorgan transplant monitoring:Analysis of urine-derived exosomal mRNA cargo allowed early detection of kidney allograft rejection.Here,we further studied urine-derived exosomes and their mRNA content as a highly promising diagnostic modality.This included stability studies of urine samples and exosomal mRNA upon transportation from the point of collection to a centralized testing facility,short-term storage of urine at different conditions upon receipt till the point molecular assay is performed,and effects of various potentially interfering substances on the downstream quantitative polymerase chain reaction(qPCR)assay.METHODS The urine specimens were stored at various conditions and pre-processed in different ways.Next,samples were passed through the columns to capture all extracellular vesicles,the vesicles were lysed to release their content and the exosomal RNA was purified on the mini-columns,reverse transcription was performed,next pre-amplification,followed by a qPCR analysis for a panel of RESULTS To ensure exosomal RNA integrity,the harvested urine specimens should be shipped refrigerated,by overnight delivery.Urine can next be stored at the test site for up to 1 wk at 4°C,and long term should be frozen at-80°C.Urine specimens must be centrifuge at low G-force to deplete cells and debris,to ensure consistent top results in downstream molecular assays.All commonly used medications(tacrolimus,cyclosporin A,mycophenolic acid,everolimus,sirolimus,ascomycin,teriflunomide)were tested and confirmed that they do not cause assay interference.CONCLUSION mRNA from urine-derived exosomes was shown to be stable across a broad range of conditions and produced accurate results when analyzed via qPCR assay for detection of kidney allograft rejection.We identified the most optimal conditions for every step of the process,ensuring pre-analytical sample integrity and robust qPCR results.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.

Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction

Backgroud Amniotic fluid （AF） supernatant contains cell-free fetal DNA （cffDNA） fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction （QF-PCR） to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat （STR） fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93％ （26/28; confidence interval,CI：77％-98％） and the specificity was 100％ （26/26; CI：88％-100％）.The determination rate of the origin of the extra chromosome was 69％.The sensitivity and the specificity of the assay in the euploid were 100％ （27/27）.Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.

Magnesium effects on behavior and substance P mRNA expression in the midbrain of a rat migraine model

BACKGROUND： Substance P participates in pain transmission and modulation, suggesting a close association with migraine headaches. The clinical application of magnesium has been effective in treating migraines, and the action mechanisms underlying migraines correlate with substance P expression. OBJECTIVE： To analyze different magnesium doses on behavior and substance P mRNA expression in the midbrain of a rat migraine model, and to determine the action pathway of migraine treatment using magnesium. DESIGN, TIME AND SETTING： A completely randomized, controlled, animal experiment was performed at the Experimental Animal Center and Central Laboratory in the Second Hospital of Jilin University between 2007 and 2008. MATERIALS： Magnesium sulfate （25%） was supplied by Tianjin Pharmaceutical Jiaozuo, China. Nitroglycerin was provided by Shanxi Kangbao Biological, China. Substance P primer sequence was synthesized by TaKaRa Biotechnology （Dalian）, China. METHODS： A total of 36 healthy, adult, Wistar rats were randomly assigned to 6 groups： control, migraine, low- and high-dose magnesium sulfate treated, and low- and high-dose magnesium sulfate control, with 6 rats in each group. Migraines were induced by subcutaneous injection of 10 mg/kg nitroglycerin in the migraine and low- and high-dose magnesium sulfate treated groups, and 2 mL/kg physiological saline was administered to rats in the control and low- and high-dose of magnesium sulfate control groups. Five minutes following administration, rats in low-dose groups were intraperitoneally injected with 100 mg/kg magnesium sulfate, while those in high-dose groups were injected with 300 mg/kg magnesium sulfate. No interventions were administered to the control and migraine groups. MAIN OUTCOME MEASURES： At 2 hours after nitroglycerin injection, substance P mRNA expression in the rat midbrain was measured by real-time quantitative polymerase chain reaction. At 60-90 minutes after nitroglycerin injection, behavioral changes of pain were analyzed in the experimental rats. RESULTS： The migraine group exhibited significantly lower levels of substance P mRNA expression compared with the control group （P 〈 0.05）. Following magnesium sulfate injection, substance P mRNA expression increased, compared with the migraine and control groups （P 〈 0.05）. In the low- and high-dose magnesium sulfate treated groups, pain behavior was remarkably ameliorated, compared with the migraine group （P 〈 0.05）, particularly with the high-dose injection （P 〈 0.05）. CONCLUSION： Magnesium relieved pain behaviors in a rat migraine model in a dose-dependent manner, and the therapeutic effect was achieved in conjunction with increased substance P expression in the midbrain.

Exosomal mi R-182 regulates the effect of RECK on gallbladder cancer

BACKGROUND As the most common biliary malignancy,gallbladder cancer(GC)is an elderlybiased disease.Although extensive studies have elucidated the molecular mechanism of microRNA 182(miR-182)and reversion-inducing-cysteine-rich protein with kazal motifs(RECK)in various cancers,the specific role of exosomal miR-182 and RECK in GC remains poorly understood.AIM To explore the relationship between exosomal miR-182/RECK and metastasis of GC.METHODS Paired GC and adjacent normal tissues were collected from 78 patients.Quantitative polymerase chain reaction was employed to detect miR-182 and exosomal miR-182 expression,and Western blotting was conducted to determine RECK expression.In addition,the effects of exosomal miR-182/RECK on the biological function of human GC cells were observed.Moreover,the double luciferase reporter gene assay was applied to validate the targeting relationship between miR-182 and RECK.RESULTS Compared with normal gallbladder epithelial cells,miR-182 was highly expressed in GC cells,while RECK had low expression.Exosomal miR-182 could be absorbed and transferred by cells.Exosomal miR-182 inhibited RECK expression and promoted the migration and invasion of GC cells.CONCLUSION Exosomal miR-182 can significantly promote the migration and invasion of GC cells by inhibiting RECK;thus miR-182 can be used as a therapeutic target for GC.

Spontaneous Proliferation in Organotypic Cultures of Mouse Cochleae

Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regeneration. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse cochlea explants obtained from up to 12 postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine (BrdU) staining and expression of proliferation cell nuclear antigen (PCNA) evaluated with real-time quantitative poly-merase chain reaction(RT-PCR). Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow-down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed.

Gene expression changes of urokinase plasminogen activator and urokinase receptor in rat testes at postnatal stages

Aim： To investigate the gene expression changes of urokinase plasminogen activator （uPA）/urokinase receptor （uPAR） in rat testes at postnatal stages and explore the effects of uPA/uPAR system on the rat spermatogenesis. Methods： The mRNAs of uPA and uPAR in rat testes were measured by using real-time quantitative polymerase chain reaction （PCR） at postnatal days 0, 5, 10, 15, 21, 28, 35, 42, 49 and 56, respectively. Results： The tendencies of uPA and uPAR mRNA expression were similar at most postnatal stages except for Do. The expression of uPAR mRNA in rats testes was relatively higher than that of uPA at postnatal Do, and both were decreased until D21, increased obviously at postnatal D28, reached a peak at postnatal D35, then declined sharply at postnatal D42 and retained at a low level afterwards. Conclusion： The uPA/uPAR system may be strongly linked to spermiation and spermatogenesis via regulating germ cell migration and proliferation, as well as promoting the spermiation and detached residual bodies from the mature spermatids.

The protocadherin alpha cluster is required for axon extension and myelination in the developing central nervous system

In adult mammals, axon regeneration after central nervous system injury is very poor, resulting in persistent functional loss. Enhancing the ability of axonal outgrowth may be a potential treatment strategy because mature neurons of the adult central nervous system may retain the intrinsic ability to regrow axons after injury. The protocadherin （Pcdh） clusters are thought to function in neuronal morphogenesis and in the assembly of neural circuitry in the brain. We cultured primary hippocampal neurons from E17.5 Pcdhα deletion （del-α） mouse embryos. After culture for 1 day, axon length was obviously shorter in del-α neurons compared with wild-type neurons. RNA sequencing of hippocampal E17.5 RNA showed that expression levels of BDNF, Fmod, Nrp2, OGN, and Sema3d, which are associated with axon extension, were significantly down-regulated in the absence of the Pcdhα gene cluster. Using transmission electron microscopy, the ratio of myelinated nerve fibers in the axons of del-α hippocampal neurons was significantly decreased; myelin sheaths of P21 Pcdhα-del mice showed lamellar disorder, discrete appearance, and vacuoles. These results indicate that the Pcdhα cluster can promote the growth and myelination of axons in the neurodevelopmental stage.

Expression of Stanniocalcin 2 in Breast Cancer and Its Clinical Sigmiicance

This study aims to explore the expression of stanniocalcin 2(STC2)gene in breast cancer and its clinical significance.Female patients with breast cancer from Zhongnan Hospital of Wuhan University admitted during March 2014 to October 2014 were enrolled in this study.All the tissues used in this experiment included 50 cases of breast cancer tissues and corresponding 50 cases of paracancer normal breast tissues with complete patients'information.The real-time quantitative polymerase chain reaction(qPCR)was applied to detect the expression of STC2 gene in 50 cases of breast cancer and paracancer normal breast tissues.The results showed that the expression level of STC2 gene in 50 cases of breast cancer tissues was significantly higher than that in paracancer normal breast tissues(P<0.001).The expression of STC2 gene was correlated with lymph node metastasis,distant metastasis,TNM stage and histological grade(P<0.001).The expression level of STC2 gene was significantly higher in breast cancer tissues with higher expression of Ki-67(P<0.001).The expression level of STC2 gene was significantly higher in estrogen receptor(ER)positive breast cancer tissues than in ER negative ones(P<0.001).However,different groups of age,pathological type,tumor size,PR expression and human epidermal growth factor receptor-2(HER2)expression did not show significant differences in STC2 expression(P>0.05).In conclusion,the abnormal overexpression of STC2 gene may play a role in the development and progression of breast cancer,and it can be used as an independent metastasis and prognostic factor of breast cancer.In addition,STC2 gene probably promotes the development and metastasis of breast cancer by interacting with estrogen and ER,and it may become a new direction for breast cancer endocrine therapy.

Insulin-like growth factor-1 mRNA isoforms and insulinlike growth factor-1 receptor mRNA expression in chronic hepatitis C

AIM: to evaluate the expression of different insulinlike growth factor(IGF)-1 mRNA isoforms and IGF-1 receptor(IGF-1R) mRNA in hepatitis C virus(HCV)-infected livers. METHODS: Thirty-four liver biopsy specimens from chronic hepatitis C(CH-C) patients were obtained before anti-viral therapy. Inflammatory activity(grading) and advancement of fibrosis(staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturer's instruction. Expression levels of IGF-1 mRNA isoforms(IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. RESULTS: The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms(P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2(r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA(r = 0.891; r = 0.821, respectively), with IGF-1B mRNA(r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA(r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA(r = 0.956; r = 0.869, respectively), and B with C isoforms(r = 0.868)(P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading(G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data(e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. CONCLUSION: Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile.

Measurement of the expression levels of BLyS and its receptors mRNA in patients with systemic lupus erythematosus

Objective： To measure the expression levels of BLyS and its receptors mRNA in peripheral blood ronnonuclear cells （PBMC） using real-time fluorescence quantitative polymerase chain reaction（RFQ-PCR） method and to investigate the relationship between BLyS and its receptors mRNA expression and systemic lupus erythematosus （SLE）. Methods： Specific primers and TaqMan probe were designed, and RFQ-PCR was perfomed. According to the standard curve of plasmid DNA, the level of BLyS and its receptors mRNA expression in 23 patients with SLE and 23 healthy subjects were detemined. The ratio of the copy number of BLyS mRNA to that of β2-microgluobulin （β2M） mRNA and the ratio of the copy number of BLyS receptors mRNA to that of β2M mRNA were regarded as indicator for the levels of BLyS and BLyS mRNA expression. Results： The concentration of RFQ-PCR was in the range of 10 - 109 pg/ml, and the coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged from 2.40% to 10.12% and from 4,.26% to 12.29%, respectively. In 23 SLE patients, the level of BLyS and its receptors（BCMA, TACI, BAFF-R） mRNA were in the ranges of 1.27 ± 1.4,9, 0.64 ± 0.77, 0.83 ± 1.05 and 0.9±3 -1.37, respectively. The mean values were 1.38 ± 0.07, 0.70 ± 0.04, 0.91 ±0.06 and 1.15±0.12, respectively. In 23 healthy donors, the levels of BLyS and its receptors（BCMA, TACI, BAFF-R） mRNA were： 0.60±1.0, 0.55±0.80, 0.54±0.74 and 0.54± 0.77, respectively. The mean values were 0.83 ±0.13, 0.68±0.08, 0.65± 0.07 and 0.68 ± 0.06, respectively. Conclusion： This results suggest that BLyS, TACI and BAFF-R might be involved in the pathogenesis of SLE and the mRNA expression levels might be used as new markers for the diagnosis of SLE.

Value of miR-1271 and glypican-3 in evaluating the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization

BACKGROUND Hepatocellular carcinoma(HCC)is the third leading cause of cancer death,causing about 750000 deaths worldwide every year.Patients with advanced hepatocellular carcinoma will often only receive transcatheter arterial chemoembolization(TACE).Glypican-3(GPC3)is one of the most promising serum markers for HCC.Abnormal expression of miRNAs may be involved in the occurrence and development of tumor.AIM To explore the value of miR-1271 and GPC3 in evaluating the prognosis of patients with HCC after TACE.METHODS From January 2016 to December 2018,162 patients with advanced HCC who received TACE in our hospital were selected into the cancer group,and 162 patients who underwent physical examination during the same period were selected into the health group.The patients in the HCC group were treated with TACE.The changes of serum GPC3 and circulating miR-1271 in the HCC before and after TACE were analyzed.The expression of serum GPC3 was detected by enzyme-linked immunosorbent assay,and the expression of circulating miR-1271 was detected by real-time quantitative polymerase chain reaction.The methodological results of sensitivity,specificity,and accuracy of miR-1271 and GPC3 alone and joint detection of HCC were also evaluated.RESULTSThe level of serum GPC3 in patients with HCC was significantly higher than that in healthy controls.GPC3 levels were increased in both HCC patients and those treated with TACE compared with healthy controls.After TACE,the level of serum GPC3 was significantly lower than that before treatment(P<0.05),and the level of circulating miR-1271 was significantly higher than that before treatment(P<0.05).There were 112 cases(69.14%)with remission(complete remission+complete remission+stable disease)and 50 cases(30.86%)with relapse disease progression in HCC patients.After TACE,the miR-1271 level in patients with remission and relapse was lower than that in the healthy group,and the GPC3 level was higher than that in the healthy group,the differences were statistically significant(P<0.05).The miR-1271 of relapsed patients was lower than that of remission patients,and the level of GPC3 was higher than that of remission patients,and the difference was statistically significant(P<0.05).The sensitivity of combined detection of miR-1271 and GPC3 was significantly higher than that of single detection,and the difference was statistically significant(P<0.05);while the specificity of the two combined detections was lower than that of the single detection;and the accuracy was slightly higher than that of single detection,but the difference was not statistically significant.CONCLUSION The level of miR-1271 in patients with HCC was significantly increased and the level of GPC3 was decreased after TACE.Monitoring the levels of serum GPC3 and circulating miR-1271 has important clinical reference value for evaluating the prognosis of patients with HCC.The levels of serum GPC3 and circulating miR-1271 may help to determine tumor recurrence,evaluate survival status,and guide the next step of treatment.