Quantitative RT-PCR Method Research Articles

Abstract 1035: PLEK2 promotes epithelial mesenchymal transition through increasing Snail1 in lung cancer cells

Abstract Pleckstrin 2 (PLEK2), one of the pleckstrin homology domain proteins, have been suggested to modulate actin rearrangement and cell spreading in T lymphocytes. Recently studies also implicated that PLEK2 paly an importance role in human myeloproliferative neoplasms, melanoma, and lung cancers. PLEK2 mRNA genes were related to poor overall survival in lung adenocarcinoma patients. However, the biological functions of PLEK2 in lung cancer is not clear. We detected the expression of PLEK2 in various lung adenocarcinoma cell lines using quantitative RT-PCR methods. Molecular manipulations were performed to investigate the roles of PLEK2 in lung cancer cells. Over-expression of PLEK2 in lung cancer cells exhibited markedly promoting in the proliferation and anchorage independent growth, whereas reduced of PLEK2 was found to suppress lung cancer cell progression. This studies also indicated that PLEK2 may play an important role in the epithelial-mesenchymal transition (EMT) features, including a morphological change, increased invasive and migratory ability, reduction the epithelial marker expression and up-regulated EMT regulators. Our results suggested that PLEK2 expression induce lung cancer cell EMT phenomenon through up-regulation Snail1 expression. These results will provide information and help us to understand the roles of PLEK2 in lung cancer cells. Citation Format: En-Chu Liu, Tsai-Tu Lee, Shey-Lin Wu, Tai-Lin Lee, Meng-Feng Tsai. PLEK2 promotes epithelial mesenchymal transition through increasing Snail1 in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1035.

Wounding induces tomato Ve1 R-gene expression.

In tomato, Ve1 gene expression is induced specifically by physical damage or plant wounding, resulting in a defense/stress cascade that mimics responses during Verticillium colonization and wilt. In tomato, Verticillium resistance is determined by the Ve gene locus, which encodes two leucine-rich repeat-receptor-like proteins (Ve1, Ve2); the Ve1 gene is induced differentially while Ve2 is constitutively expressed throughout disease development. These profiles have been observed even during compatible Verticillium interactions, colonization by some bacterial pathogens, and growth of transgenic tomato plants expressing the fungal Ave1 effector, suggesting broader roles in disease and/or stress. Here, we have examined further Ve gene expression in resistant and susceptible plants under abiotic stress, including a water deficit, salinity and physical damage. Using both quantitative RT-PCR and label-free LC-MS methods, changes have been evaluated at both the mRNA and protein levels. The results indicate that Ve1 gene expression responds specifically to physical damage or plant wounding, resulting in a defense/stress cascade that resembles observations during Verticillium colonization. In addition, the elimination or reduction of Ve1 or Ve2 gene function also result in proteomic responses that occur with wilt pathogen and continue to be consistent with an antagonistic relationship between the two genes. Mutational analyses also indicate the plant wounding hormone, systemin, is not required, while jasmonic acid again appears to play a direct role in induction of the Ve1 gene.

Nestin is highly expressed in foetal spinal cord isolated from placenta previa patients and promotes inflammation by enhancing NF-κB activity

Purpose: Nestin is expressed in various tissues of the embryo in patients with placenta previa, while the regulatory mechanism still unknown.Materials and methods: All participants terminated pregnancy. Among them, 75 patients with placenta previa were assigned to the case group and 80 healthy pregnant women with normal placenta were assigned to the control group. Expression of nestin and CDK5 in foetal spinal cord tissues was detected by Western blot and quantitative real-time RT-PCR methods. The enzyme-linked immunosorbent assay (ELISA) was used to determine the serum expression of some pro-inflammatory cytokines in placenta previa patients. The interaction between nestin and CDK5 was evaluated by immunoprecipitation and siRNA inhibition of nestin was performed to estimate its effect on NF-κB activity in foetal spinal cord tissues.Results: Along with increased expression of nestin and CDK5 in foetal spinal cord tissues in the case group, IL-1β, IL-6, TNF-α and IFN-γ were increased in the serum of placenta previa patients. siRNA inhibition analysis indicated that nestin interacted with CDK5 and regulated NF-κB activity in foetal spinal cord tissues.Conclusions: Nestin is highly expressed and the interaction between nestin and CDK5 might lead to the progress of placenta previa through its regulation on NF-κB.

Detecting HB-1 Expression Level in Bone Marrow of Acute Leukemia Patients by Real-Time Fluorescence Quantitative RT-PCR

To investigate the expression level of HB-1 gene in patients with acute lymphoblastic leukemia (ALL) and the significance of HB-1 gene in monitoring of minimal residual disease (MRD). The method of real-time fluorescence quantitative RT-PCR (Taqman probe) was established to detect the expression levels of HB-1 gene; then the sensitivity, specificity and repeatability of this assay were evaluated and verified. The HB-1 gene expression levels in bone marrow of 183 cases of ALL, 70 cases of acute myeloid leukemias (AML), 52 cases of non-malignant hematologic diseases and 24 healthy hematopoietic stem cell donors were detected. The correlation of HB-1 level with diagnosis and relapse was analyzed by detecting bone marrow samples of 33 B-ALL. The sensitivity of this assay reached the 10-4 level. The coefficient of variation for inter-batch and inter-tube of HB-1 were 6.79% and 4.80%, respectively. It was found that HB-1 gene specifically expressed in acute B lymphoblastic leukemia. The median expression levels of HB-1 gene in newly diagnosed and relapsed B-ALL patients were statistically significantly higher than those in ALL in complete remission(CR), newly diagnosed T-ALL, newly diagnosed AML, non-malignant hematologic diseases, and healthy hematopoietic stem cell donors(33.0% vs 0.68%, 0.07%, 0.02%, 0.58% and 0, respectively) (P<0.01). No statistical differences were found between newly diagnosed T-ALL, newly diagnosed AML, non-malignant hematologic diseases and healthy donors (P>0.05). The expression level of HB-1 gene declined sharply when B-ALL patients reached complete remission (0-7.99%, with median level 0.68%), but increased when relapsed (7.69%, 8.08% and 484.0% in 3 relapsed samples), which was in accordance with results of flow cytometry. HB-1 gene specifically expressed in acute B lymphoblastic leukemia cells. The established real-time fluorescence quantitative RT-PCR assay shows good sensitivity, specificity and repeatability, thus, can be used as a biological marker in the clinical detection, monitoring MRD and predicting of early relapse for B-ALL patients.

High dose of histone deacetylase inhibitors affects insulin secretory mechanism of pancreatic beta cell line.

Histone deacytylase inhibitors (HDACis) inhibit the deacetylation of the lysine residue of proteins, including histones, and regulate the transcription of a variety of genes. Recently, HDACis have been used clinically as anti-cancer drugs and possible anti-diabetic drugs. Even though HDACis have been proven to protect the cytokine-induced damage of pancreatic beta cells, evidence also shows that high doses of HDACis are cytotoxic. In the present study, we, therefore, investigated the eff ect of HDACis on insulin secretion in a pancreatic beta cell line. Pancreatic beta cells MIN6 were treated with selected HDACis (trichostatin A, TSA; valproic acid, VPA; and sodium butyrate, NaB) in medium supplemented with 25 mM glucose and 13% heat-inactivated fetal bovine serum (FBS) for indicated time intervals. Protein expression of Pdx1 and Mafa in MIN6 cells was demonstrated by immunohistochemistry and immunocytochemistry, expression of Pdx1 and Mafa genes was measured by quantitative RT-PCR method. Insulin release from MIN6 cells and insulin cell content were estimated by ELISA kit. Superoxide production in MIN6 cells was measured using a Total ROS/Superoxide Detection System. TSA, VPA, and NaB inhibited the expression of Pdx1 and Mafa genes and their products. TSA treatment led to beta cell malfunction, characterized by enhanced insulin secretion at 3 and 9 mM glucose, but impaired insulin secretion at 15 and 25 mM glucose. Th us, TSA induced dysregulation of the insulin secretion mechanism. TSA also enhanced reactive oxygen species production in pancreatic beta cells. Our results showed that HDACis caused failure to suppress insulin secretion at low glucose concentrations and enhance insulin secretion at high glucose concentrations. In other words, when these HDACis are used clinically, high doses of HDACis may cause hypoglycemia in the fasting state and hyperglycemia in the fed state. When using HDACis, physicians should, therefore, be aware of the capacity of these drugs to modulate the insulin secretory capacity of pancreatic beta cells.

In Vitro Antiviral Activity of Commercial Products of Herbal Extracts Against Highly Pathogenic Avian Influenza (H5N1) Virus

Three commercial products of herbal extracts (Amphiflu-Ex®, Fyto-immune Plus® and Viu-Vet®) were tested for antiviral activity against two Egyptian highly pathogenic avian influenza subtype H5N1 isolates (isolated form backyard poultry) and upon sequencing and phylogenetic analysis, they belonged to clade 2.2.1.2., This antiviral assay for the products done by using chicken embryo fibroblast (CEF) cell culture through cytopathic effect (CPE) inhibition assay in two different layouts to elucidate post-exposure and pre-exposure antiviral activity of the products. The Amphiflu-Ex® at conc. 50 µl/ml showed significant virucidal activity (100% inhibition) in post-exposure assay for both viral isolates which was further confirmed by quantitative real time RT-PCR method showed 100% reduction in virus titers for both isolates and further investigation done on The Amphiflu-Ex® on conc. 20 µl/ml, the virus titers were decreased by 2.72-2.8 logs (Log 10 virus titers) and on conc 10 µl/ml, the virus titers were decreased by 1-1.27 logs for both isolates, The maximum non-toxic concentration (MNTC) for the three products estimated by cytotoxicity assay was 50 µl/ml and TCID50 used for both isolates were 106 copy/100 µl.

Association of serum miRNA6086 expression with postoperative anal fistula recurrence

To investigate the association between serum miRNA-6086 expression level and anal fistula recurrence. Clinical data and serum samples of 60 patients with anal fistula and mix hemorrhoid identified by pathology undergoing resection in our department from August 2015 to August 2016 were collected. In addition, serum samples of 20 patients matching with fistula group in age, gender and body weight and receiving only hemorroidectomy were collected as control during the same period. Serum miRNA6086 expression level was detected by real-time quantitative RT-PCR method, and the association of serum miRNA6086 expression level with clinicopathologic features was analyzed. Univariate ANOVA test and multivariate logistic regression analysis were performed to analyze the association between serum miRNA6086 expression level and anal fistula recurrence. The relative expression of serum miRNA6086 in fistula group was 65.85±15.57, which was significantly up-regulated for 4.87 folds of 13.52±7.32 in control group(P<0.05). In fistula group, 24 cases(40%) developed anal fistula recurrence, whose serum miRNA6086 expression was significantly higher compared to 36 cases without recurrence (74.06±12.92 vs. 60.38±14.90, P<0.05). No associations of serum miRNA6086 expression with age, gender, BMI, drug history, acute phase were observed (P>0.05), while association of serum miRNA6086 expression level with the type, number and position of anal fistula was significant (all P<0.05). Univariated analysis indicated that anal fistula type (χ2=6.890, P=0.009), anal fistula number (χ2=0.554, P<0.001) and serum miRNA6086 expression (χ2=11.390, P=0.001) were significantly associated with anal fistula recurrence. Multivariate logistic regression analysis showed that complex anal fistula (OR=4.75, 95%CI: 1.84 to 12.01, P=0.001) and high expression of serum miRNA6086 (OR=3.22, 95%CI:1.31 to 8.22, P=0.011) were independent risk factors of anal fistula recurrence. Up-regulated expression of serum miRNA6086 is associated to the anal fistula type and may be valuable in predicting the prognosis.

Polymorphisms and expression of genes encoding Argonautes 1 and 2 in autoimmune thyroid diseases

The microRNA (miRNA) biogenesis pathway is regulated by specific proteins and enzymes, including Dicer, Drosha, DGCR8, Exportin 5 and the Argonaute (AGO) family. In this study, we investigated the AGO family, which is the primary component of RISC (RNA-induced silencing complex) and directly binds to microRNA. We examined the association of polymorphisms in AGO family genes with AGO expression and with the development and prognosis of autoimmune thyroid diseases. We genotyped AGO1 rs636832A/G, AGO2 rs7005286C/T, AGO2 rs11166985A/G and AGO2 rs2292779C/G polymorphisms in 184 Graves’ disease (GD) patients, 195 Hashimoto’s disease (HD) patients and 122 healthy volunteers using the polymerase chain reaction-restriction fragment length polymorphism method. We also examined the expression of AGO1 and AGO2 mRNAs in peripheral blood mononuclear cells (PBMC) obtained from 52 GD patients, 41 HD patients, and 25 healthy volunteers using quantitative RT-PCR methods. The G allele of AGO1 rs636832 and the A allele of AGO2 rs11166985 polymorphisms were significantly more frequent in GD patients than in healthy controls. The A allele of AGO2 rs11166985 was also significantly more frequent in intractable GD patients than in controls. The C carrier (CC + CG genotypes) and C allele of AGO2 rs2292779 polymorphism were significantly more frequent in intractable GD patients than in patients with GD in remission. Expression of AGO1 mRNA in PBMC was significantly higher in AITD patient than in controls, and that of AGO2 mRNA in PBMC was significantly higher in intractable GD patients than in patients with GD in remission. Furthermore, the expression levels of both the AGO1 and AGO2 genes were significantly correlated with the proportions of Th17 cells in PBMC. In conclusion, the polymorphisms of the AGO1 and AGO2 genes, the expression levels of which correlated with the proportion of Th17 cells, were associated with the development and prognosis of GD. The AGO2 rs2292779 C carrier and C allele were associated with the intractability of GD.

Characterization of the dynamic change of microRNA expression in mice hypothalamus during the time of female puberty

Puberty onset is a milestone in sexual development. A tumor suppress gene (TSG) network had been reported to be involved in the regulation of female puberty onset. The observations in rodents and primates showed a potential link between microRNAs and puberty onset. To figure out what miRNAs play roles in this important biological process, profilings of microRNAs in the hypothalamus of female mice from three different pubertal stages, juvenile [postnatal day (P10)], early pubertal (P25) and pubertal (P30) were performed on the Affymetrix GeneChip miRNA 3.0 Arrays, the cerebral cortex (CTX) was used as a control tissue. 20 miRNAs were shown to be differentially expressed in hypothalamus (fold change > 1.5, P < 0.05), but not in CTX during the transition from juvenile to pubertal. Four of them were validated by real-time quantitative RT-PCR (qRT-PCR) method. 1018 genes were predicted as the targets of these miRNAs. Further bioinformatics analysis suggested that these target genes were involved in many important signaling pathways, especially in the cancer related pathways. We also found that about 90% of these target genes were expressed in the hypothalamus, as well as in the immortalized GnRH-producing GT1-7 cells, which provided additional evidence that these miRNAs could be female puberty onset related. Here we present a novel comprehensive data set of miRNA gene expression during the puberty onset; and it provides an important recourse for the future functional characterization of individual miRNAs and their targets in mouse hypothalamus and in GT1-7 cells.

Vitellogenin expression in the oilseed rape pest Meligethes aeneus.

When investigating insecticide resistance of pest insects, for example, the pollen beetle Meligethes aeneus, it is relevant to differentiate toxicological and molecular genetic data between male and female specimens. A molecular sex determination method would allow resistance testing to be run without prior sorting of the samples. A one-step quantitative RT-PCR method for quantification of the yolk protein vitellogenin expression in the pollen beetle was established. The expression level of vitellogenin relative to tubulin was determined. Pollen beetles were tested at different time points during their development to determine if vitellogenin is a reliable molecular marker for detection of sexually mature females. The differentiation between females and males by relative expression of vitellogenin to tubulin is conditional regarding the life cycle. Sexually mature females and males could easily be distinguished, whereas immature specimens could not be seperated. Vitellogenin expression is a successful marker for identification of sexually mature pollen beetles. Females from the spring populations showed vitellogenin expression when the temperature was above 10.2°C. Further, detailed observations of vitellogenin throughout the spring indicated a strong relationship between daily temperatures and vitellogenin expression, which is an indicator of oviposition ability.

Deoxynivalenol impair skin barrier function through the down regulation of filaggrin and claudin 1/8 in HaCaT keratinocyte

Deoxynivalenol (DON), a typical mycotoxin, is a substance that is biosynthesized mainly by the Fusarium species. It is usually found in wheat and other grains grown in the field. When it enters the human body, it causes severe diarrhea, abdominal pain, vomiting, and even death. In addition, DON is known to induce inflammation of the small and large intestine, and is also associated with the occurrence of cancer. However, until recently, the effects of DON on the human skin were unknown. To investigate how DON affects HaCaT, human immortalized keratinocytes, we used CCK-8 assay and a quantitative real-time RT-PCR method to detect changes in the expression of tight junctions and skin cell regulatory proteins. The CCK-8 assay was performed to determine the growth inhibitory concentration of keratinocytes by DON. DON affected the cell survival rate from 1 μM in a concentration dependent manner, with the minimum set as 1 μM and the maximum as 4 μM for all experiments. DON inhibited the mRNA expression of filaggrin by up to 71% and SERPINA1 up to 75%. The expression of AQP3 was reduced by up to 93% compared to the untreated control group. This may cause problems in the pH control function of the skin and weaken the function of moisturizing. In addition, in the presence of DON, the gene expression of claudin 1 and claudin 8, which are important proteins in the regulation of intercellular skin barrier, decreased by up to 47 and 80%, respectively. Snail/ Slug, suppressors of the claudin gene expression, each increased up to 625 and 974%, respectively. Also, the MMP9 gene increased by up to 515% in a concentrationdependent manner, perhaps causing a weakness of the barrier function of the skin. These results suggest that DON may causing the development of atopic skin by impairing the skin barrier and pH control of skin, as well as intestinal inflammation diseases. Therefore, particular attention should be paid to DON contamination during the development of cosmetic ingredients using grains.

P1.17-003 Identification of Differentially Expressed Genes between Thymoma and Paraneoplastic Thymic Tissue

Our study tries to demonstrate the underlying genetic mechanisms of tumorigenesis of thymoma and understand the related features: association with myasthenia gravis, and histologic variability. This study uses CapitalBio mRNA microarray analysis of 31 cases of thymoma (including 5 cases of type AB, 6 B1-type cases, 12 B2-type cases, 5 B2B3-type cases, 3 type- B3 cases of; only 6 cases of thymoma were not associated with myasthenia gravis, 25 cases with myasthenia gravis). Some differentially expressed genes after comparisons between thymoma and the thymus tissue around tumor were identified preliminarily. Among them, 292 genes increased more than 2-fold, 2 genes more than 5-fold; on the other hand, 596 genes were decreased more than 2-fold, 115 genes more than 5-fold, 21 genes more than 10-fold, 6 genes more than 20-fold. Among these genes upregulated or downregulated, 6 driver genes, such as FANCI, NCAPD3, NCAPG, OXCT1, EPHA1 and MCM2, were identified. We selected 2-fold upregulated and 2-fold downregulated genes to generate a supervised clustering heat map. 6 distinct clusters were identified. In cluster 1, two were type B2 tumors; in cluster 6, three were type B2/B3 tumors. KEGG database analysis, utilized for research and education, found that the pathogenesis of thymoma might be associated with several signaling pathways, which provides important information for revealing genetic mechanisms of thymoma; By comparing with genetic differences of thymoma with myasthenia gravis and without, 4 genes （PNISR, NBPF14, PIK3IP1和RTCA）were upregulated more than 2-fold, more than 30 genes were downregulated more than 2-fold, and 2 signaling pathways with more than 2-fold upregulated genes (TGF- beta signaling pathway and HTLV-I signaling pathway)were found. The study would be shed light on the molecular bases for selecting appropriate oncological management, predict prognosis and provide important information on the genetic background of thymoma. But Confirmation of the data will be performed using immunohistochemical and multiplex quantitative RT-PCR methods.

Immunohistochemical and quantitative RT-PCR methods to assess RANK expression in normal and neoplastic canine mammary gland.

The receptor activator of nuclear factor-kB ( RANK) gene is found in both human and murine mammary epithelial cells and in human cancer cell lines. We analyzed RANK expression in normal and proliferative canine mammary tissue samples ( n = 47) and cell lines ( n = 10), and identified its expression in epithelial cell populations. The correlation of RANK protein with clinicopathologic parameters was also studied. A double immunohistochemical method using RANK and p63 antibodies was applied to 33 tissue samples to analyze RANK protein expression and its possible co-expression with p63 protein, the latter used to identify myoepithelial (ME) cells (p63-positive) or luminal epithelial (LE) cells (p63-negative). RANK protein expression was found in ~75% of the tissue samples analyzed, at a similar level in all of the histologic types studied: dysplasias (4 of 4, 100%), malignant tumors (13 of 17, 76%), normal glands (12 of 17, 70%), and benign tumors (6 of 9, 67%). ME and LE cells expressed RANK protein at a similar level. A higher level of RANK protein expression was found in older animals (≥10 y, p = 0.027). Quantitative RT-PCR was applied to 6 ME (1 normal and 5 neoplastic) and 4 LE (1 normal and 3 neoplastic) primary cell lines. The RANK gene was found at similar expression levels in all canine mammary ME and LE cell lines studied. We found RANK expression in normal, dysplastic, and neoplastic canine mammary tissues and cell lines, in both ME and LE cell populations.

Extraction and RT-qPCR detection of enteroviruses from solid environmental matrixes: Method decision tree for different sample types and viral concentrations

Quantitative RT-PCR methods (RT-qPCR) are becoming increasingly desirable for the detection of enteric viruses in solid environmental matrixes such as sediments, soils and sewage sludge. However, effective methodologies that allow the extraction of high quality RNA ready for molecular quantification continue to be evaluated. In the present study, four different methods for enterovirus extraction from solid environmental matrixes were compared in terms of viral recovery and inhibitor removal. Three indirect methods based on glycine elution and concentration by ultracentrifugation were tested. The main differences between indirect methods were the sample to glycine buffer ratio, and the ultracentrifugation protocol applied. One commercial direct method was also tested. The indirect methods produced better results than the direct method. The ultracentrifugation led to viral losses in samples with high titers; however, as the virus concentration reduced, the ultracentrifugation became increasingly important for viral recovery. Two commercial RNA extraction kits were also evaluated and it was selected the most effective in removing RT-qPCR inhibitors. The results obtained allowed the development of a method decision tree with three versions that are suitable for different samples and viral concentrations.

Chinese Sacbrood virus infection in Asian honey bees (Apis cerana cerana) and host immune responses to the virus infection.

Chinese Sacbrood virus (CSBV) is a positive-stranded RNAvirus that infects both the European honey bee (Apis mellifera) and the Asian honey bee (A. cerana). However, CSBV has much more devastating effects on Asian honey bees than on European honey bees, posing a serious threat to the agricultural and natural ecosystems that rely on A. cerana for pollination service. Using quantitative RT-PCR method, we conducted studies to examine the CSBV infection in Asian honey bee colonies and immune responses of individual bees in response to CSBV infection. Our study showed that CSBV could cause infection in different developmental stages of workers including eggs, larvae, pupae, newly emerged workers, and foraging workers. In addition, evaluating the tissue tropism and transmission of CSBV in infected bees showed that CSBV was detected in the ovaries, spermatheca, and feces of queens as well as semen of drones of the same colonies, suggesting an existence of vertical transmission of CSBV in Asian honey bees. Further, the detection of CSBV in colony food suggests that healthy bees could pick the infection by the virus-contaminated food, and therefore, a possible existence of a food-borne transmission pathway of CSBV in Asian bee colonies. The expression analysis of transcripts (defensin, abaecin, apidaecin, and hymenoptaecin) involving innate antiviral immune pathways showed that CSBV infection could induce significant immune responses in infected bees. However, the immune responses to CSBV infection varied among different development stages with eggs exhibiting the lowest level of immune expression and forager workers exhibiting the highest level of immune gene expression. The results obtained in the study yield important insights into the mechanisms underlying disease pathogenesis of CSBV infections in Asian honey bees and provide valuable information for a rational design of disease control measures.

Recombinant human elafin promotes alveologenesis in newborn mice exposed to chronic hyperoxia.

Elastase inhibitors reverse elastin degradation and abnormal alveologenesis and attenuate the lung structural abnormalities induced by mechanical ventilation with O2-rich gas. The potential of these molecules to improve endothelial function and to ameliorate severe bronchopulmonary dysplasia (BPD) during lung development is not yet understood. We sought to determine whether the intratracheal treatment of newborn mice with the elastase inhibitor elafin would prevent hyperoxia-induced lung elastin degradation and the cascade of events that cause abnormal alveologenesis. Newborn mice were exposed to 85% O2 for 3, 7, 14 or 21days. Recombinant human elafin was administered by intratracheal instillation from the first day every two days for 20days. We next used morphometric analyses, quantitative RT-PCR, immunostaining, Western blotting, and ELISA methods to assess the key variables involved in elastogenesis disruption and the potential signaling pathways noted below in recombinant human elafin-treated mouse pups that had been exposed to 85% O2. We found that impaired alveolar development and aberrant elastin production were associated with elevations in whole lung elastase levels in 85% O2-exposed lungs. Elafin attenuated the structural disintegration that developed in the hyperoxia-damaged lungs. Furthermore, elafin prevented the elastin degradation, neutrophil influx, activation of TGF-β1 and apoptosis caused by 85% O2 exposure. Pulmonary elastase plays an important role in disrupting elastogenesis during O2-induced damage, which is the result of a pulmonary inflammatory response. Elafin prevents these changes by inhibiting elastase and the TGF-β1 signalling cascade and may be a new therapeutic target for preventing O2-induced lung injury in neonates.

Blockade of the 5-HT transporter contributes to the behavioural, neuronal and molecular effects of cocaine.

The psychostimulant cocaine induces complex molecular, cellular and behavioural responses as a consequence of inhibiting presynaptic dopamine, noradrenaline and 5-HT transporters. To elucidate 5-HT transporter (SERT)-specific contributions to cocaine action, we evaluated cocaine effects in the SERT Met172 knock-in mouse, which expresses a SERT coding substitution that eliminates high-affinity cocaine recognition. We measured the effects of SERT Met172 on cocaine antagonism of 5-HT re-uptake using ex vivo synaptosome preparations and in vivo microdialysis. We assessed SERT dependence of cocaine actions behaviourally through acute and chronic locomotor activation, sensitization, conditioned place preference (CPP) and oral cocaine consumption. We used c-Fos, quantitative RT-PCR and RNA sequencing methods for insights into cellular and molecular networks supporting SERT-dependent cocaine actions. SERT Met172 mice demonstrated functional insensitivity for cocaine at SERT. Although they displayed wild-type levels of acute cocaine-induced hyperactivity or chronic sensitization, the pattern of acute motor activation was different, with a bias toward thigmotaxis. CPP was increased, and a time-dependent elevation in oral cocaine consumption was observed. SERT Met172 mice displayed relatively higher levels of neuronal activation in the hippocampus, piriform cortex and prelimbic cortex (PrL), accompanied by region-dependent changes in immediate early gene expression. Distinct SERT-dependent gene expression networks triggered by acute and chronic cocaine administration were identified, including PrL Akt and nucleus accumbens ERK1/2 signalling. Our studies reveal distinct SERT contributions to cocaine action, reinforcing the possibility of targeting specific aspects of cocaine addiction by modulation of 5-HT signalling.

Abstract 3780: Bio-competition-based smart NanoVelcro Chip for isolation and gene expression analysis of circulating tumor cells from prostate cancer patients

Abstract Introduction and Objective: Circulating tumor cells (CTCs) are being used in efforts to identify important transcriptomic features such as androgen receptor (AR) splicing variants in prostate cancer (PCa) and other malignancies. The low abundance of CTCs and the fragility of the genetic materials create a need for tools that obtain high-quality signals with great efficiency. Poly(3,4-ethylenedioxythiophene) (PEDOT) is a stimulation-responsive nanomaterial which allows for rapid and gentle cell purification through the use of bio-competition between pheylboronic acid and carbohydrates. We hypothesized that a combination of PEDOT with the NanoVelcro cell affinity assay (NanoVelcro-PBA Chip) would yield a tool that could minimize contamination from white blood cells and maximize cell viably and molecular intactness. In this study, we benchmarked the efficiency of this platform for purification of CTCs from blood specimens and the feasibility of using this approach for detection of PCa-related RNA signatures from purified CTCs. Methods: The capture and release efficiency of NanoVelcro-PBA Chip was tested using PCa cell lines (LNCaP, PC3, 22Rv1) and artificial blood samples with PCa cells mixed with healthy donor blood. Variations on operating conditions were tested including the capturing antibodies on the NanoVelcro substrate, incubation time, sorbitol concentration, and bio-competition time. Impact was measured on process efficacy and cell viability. Blood specimens from 17 PCa patients were processed using NanoVelcro-PBA chip. Analysis focused detection of full length AR (AR-FL), AR splicing variant 7 (AR-V7), KLK3 (prostate-specific antigen, PSA), FOLH1 (prostate-specific membrane antigen, PSMA), and long-noncoding RNA SChLAP1 (second chromosome locus associated with prostate-1) using quantitative RT-PCR method. Results: The combination of NanoVelcro substrate, PEDOT nanomaterial, and capturing antibody exhibits the highest cell capture efficiency (up to 80%). The highest cell release efficiency and viability was achieved by bio-competition with 100 μmol/200μL sorbitol solution for 30 minutes. PCa-related RNA signals were detected in 16/17 CTC(+) PCa patients, and was detected more frequently in patients with metastatic disease and with higher expression level. Conclusions: We have developed a novel CTC purification platform, NanoVelcro-PBA Chip. This platform is capable of purifying CTCs with high efficiency and while retaining cell viability and molecular integrity. This in turn allows for detection of disease-relevant RNA signals. Further this new tool is being moved into clinical studies that will validate its performance in CTC purification and subsequent RNA detection. Citation Format: Jie-Fu Chen, Mo-Yuan Shen, Chun-Hao Luo, Shirley Cheng, Sangjun Lee, Shuang Hou, Edwin M. Posadas, Hsian-Rong Tseng, Hsiao-Hua Yu. Bio-competition-based smart NanoVelcro Chip for isolation and gene expression analysis of circulating tumor cells from prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3780. doi:10.1158/1538-7445.AM2017-3780

Abstract 1890: PLEK2 contributes to lung cancer progression through upregulation of NDRG1 expression

Abstract PLEK2 is one of the pleckstrin homology domain (PH domain) proteins. Recently studies suggested that PLEK2 may play a critical role in T lymphocyte migration through by PI3K/AKT dependent and independent pathways. However, the biological functions of PLEK2 in lung cancer is not clear. We detected the expression of PLEK2 in various lung adenocarcinoma cell lines using quantitative RT-PCR methods. The results indicated that PLEK2 expression significantly positive correction with cancer cell migration and invasion ability. Forced expression of PLEK2 in lung cancer cells exhibited markedly promoting in the proliferation rate, anchorage independent growth, cell motility and invasion, whereas knockdown of PLEK2 was found to suppress lung cancer cell progression. In addition, we also reported that PLEK2 expression induce lung cancer cell epithelial-mesenchymal transition (EMT) phenomenon, and involve in chemotherapeutic drug resistance through upregulation NDRG1 gene expression. Overexpression of NDRG1 in PLEK2-knockdown cell line restored cancer cell proliferation, migration, and invasion. These results will provide information and help us to understand the roles of NDRG1 in lung cancer cells. Citation Format: Yi-Pin Hsieh, Hui-Chia Liu, Hao-Yu Huang, Wan-Jiun Tang, Tai-Lin Lee, Meng-Feng Tsai. PLEK2 contributes to lung cancer progression through upregulation of NDRG1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1890. doi:10.1158/1538-7445.AM2017-1890

Diagnostics of the Presence of Viruses in the Potato and Soil

Abstract The paper presents a combined biological assay on indicator plants, a serological assay using ELISA and a molecular assay for detection of viruses that infect the potato. It also elaborates on the bioassay for PVY strain differentiation and for detection of TRV in tubers and soil samples. At IHAR-PIB Młochów Research Center, ELISA tests can be conducted for detection of PVY, PVA, PVM, PVS, PLRV, PVX, TRV and PMTV. Multiplex RT-PCR, real-time quantitative RT-PCR and sequencing methods for detection of PVY, TRV and PMTV are optimized based on a published protocol or developed in our laboratory. The primers for detection of PVY, TRV, PVM, PVS, PLRV, PVX, PMTV, PVA, AMV, CMV, PAMV, TBRV, and BMYV according to the published methods are listed. The maintenance and usage of 19 species of indicator plants are discussed.