mRNA Extraction Research Articles

Evaluation of the Effect of Probiotics Isolated from Traditional Dairy Products on the Expression of Stx1 and Stx2 Genes of Verotoxigenic Escherichia coli (VTEC) in Laboratory Conditions

Background: Lactobacilli are commonly used organisms in the production of probiotics. Objectives: The present study was conducted to examine the effect of probiotics isolated from dairy products on the expression of Stx1 and Stx2 genes in verotoxigenic E. coli (VTEC). Methods: This experimental study was conducted during April and January 2016 in East Azerbaijan, Iran. At first, Hundred samples of traditional dairy products were collected. Then, using standard phenotypic and genotypic methods, the Lactobacillus casei (L. casei) and lactobacillus (L. plantarum) were studied. Subsequently, these strains were examined along with the pathogenic bacteria strain of Escherichia coli (E. coli) (O157: H7) by neighboring cultivation. After extraction of mRNA, the expression of these genes was determined using real- time PCR. Results: The results revealed that L. casei reduces the expression of Shiga toxin (Stx1 and Stx2) genes more compared to L. plantarum (P = 0.001). Moreover, it was found that both Lactobacilli reduce the expression of Stx1 genes more than Stx2. Conclusions: Probiotics could be used to prevent and control intestinal diseases caused by E. coli.

Analysis of Endoplasmic Reticulum (ER) Stress Induced during Somatic Cell Nuclear Transfer (SCNT) Process in Porcine SCNT Embryos.

ABSTRACTThis study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.

Human beta defensin-1 is involved in the susceptibility to adeno-tonsillar hypertrophy

IntroductionInnate immunity molecules are known to play a pivotal role in the homeostasis of the oral mucosa, permitting the presence of commensal microflora and, at the same time, providing a first line of defense against pathogens attempting to invade the oral cavity.Tonsils represent the local immune tissue in oral cavity, being able to provide a non-specific response to pathogens; however, in the presence of microbes or foreign materials present in the mouth tonsils could became infected and develop chronic inflammation, thus leading to hypertrophy.The etiology of the disease is multifactorial depending upon environmental and host factors, the latter including molecules of mucosal innate immunity. MethodsNinety-five children with adeno-tonsillar hypertrophy subjected to adeno-tonsillectomy were recruited at the pediatric otorhinolaryngology service of the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste (Italy).The specimen discarded from the surgery were used for genomic DNA extraction and genotyping, for mRNA extraction and gene expression analysis, finally the samples were cut and used to prepare slides to perform immunohistochemistry. ResultsFunctional polymorphisms within DEFB1 gene, encoding the human beta defensin-1 (hBD-1), were analyzed finding association between DEFB1 rare haplotypes and susceptibility to adeno-tonsillar hypertrophy. DEFB1 mRNA expression was detected in the tonsils and the hBD-1 protein was localized at the epithelia of tonsils mainly in the proximity of the basal lamina. ConclusionOur findings lead us to hypothesize an involvement of hBD-1 mediated innate immunity in the modulation of the susceptibility towards adeno-tonsillar hypertrophy development.

Investigation of the changes in the expression levels of MOZ gene in colorectal cancer tissues.

MOZ is one of the most important histone acetyltransferases (HATs) that has an effective role in gene expression. It is an important partner in chromosomal rearrangement that usually occurs in hematological malignancies such as leukemia. Besides these malignancies, its role in solid tumors has been reported. In the present study, we aimed to quantify of MOZ messenger RNA (mRNA) expression in colorectal cancer (CRC) tissues from a northwest population of Iran and consequently to assess the effect of MOZ in CRC. Tumorous and adjacent non-tumorous tissues recruited from 26 patients with CRC. mRNA extraction and complementary DNA (cDNA) synthesis were performed from these tissues, at the next step quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) was carried out. Finally, expression levels were statistically analyzed using IBM SPSS Statistics 24.0 software and independent t-test. Statistical significance was considered as P≤0.05. The results showed significantly higher expression of MOZ in the majority of CRC tissues compared to normal colorectal tissues (P=0.048). There were no significant correlations between expression levels of MOZ and clinical parameters of patients (P>0.05). Our data showed that dysregulation of MOZ is potentially involved in the pathogenesis of CRC and we could suggest that there is a straight relationship between tumor formation and MOZ expression. These results showed possible role of MOZ as a prognostic factor in the said population.

Diminished androgen and estrogen receptors and aromatase levels in hypogonadal diabetic men: reversal with testosterone.

One-third of males with type 2 diabetes (T2DM) have hypogonadism, characterized by low total and free testosterone concentrations. We hypothesized that this condition is associated with a compensatory increase in the expression of androgen receptors (AR) and that testosterone replacement reverses these changes. We also measured estrogen receptor and aromatase expression. This is a randomized double-blind placebo-controlled trial. Thirty-two hypogonadal and 32 eugonadal men with T2DM were recruited. Hypogonadal men were randomized to receive intramuscular testosterone or saline every 2 weeks for 22 weeks. We measured AR, ERα and aromatase expression in peripheral blood mononuclear cells (MNC), adipose tissue and skeletal muscle in hypogonadal and eugonadal males with T2DM at baseline and after 22 weeks of treatment in those with hypogonadism. The mRNA expression of AR, ERα (ESR1) and aromatase in adipose tissue from hypogonadal men was significantly lower as compared to eugonadal men, and it increased significantly to levels comparable to those in eugonadal patients with T2DM following testosterone treatment. AR mRNA expression was also significantly lower in MNC from hypogonadal patients compared to eugonadal T2DM patients. Testosterone administration in hypogonadal patients also restored AR mRNA and nuclear extract protein levels from MNC to that in eugonadal patients. In the skeletal muscle, AR mRNA and protein expression are lower in men with hypogonadism. Testosterone treatment restored AR expression levels to that comparable to levels in eugonadal men. We conclude that, contrary to our hypothesis, the expression of AR, ERα and aromatase is significantly diminished in hypogonadal men as compared to eugonadal men with type 2 diabetes. Following testosterone replacement, there is a reversal of these deficits.

Development of COL3A1-Specific Real-Time PCR Assay for Clinical and Sport Detection Applications

PIIINP, whose gene is COL3A1, is a protein used as a biomarker, which is altered in various pathologies such as fatty liver of non-alcoholic origin and is also used to detect abuse of human recombinant growth hormone (rhGH) by elite athletes to improve their performance. Therefore, the study of the levels of this protein or its gene could be useful for the diagnosis of these pathologies, in addition to other uses. The objective of this work was the development of a new detection method of the COL3A1 gene based on the quantification of mRNA in real time. 10 healthy men volunteers were injected intravenously for 3 consecutive days with 0.026 mg-1kg-1day-1person-1 of rhGH, and serum and blood samples were collected for 7 days at different times. mRNA extraction was performed after lymphocyte purification following standard protocols, and samples were quantified by quantitative real-time PCR using TaqMan probes designed specifically for this gene. The results showed an increase in COL3A1 expression that was maintained during 7 days of sampling after the last administration, observing more sensitive and robust results when compared to quantification of the PIIINP protein in serum from the same individuals. After the trial set-up, a partial validation was carried out in which parameters such as linearity, inter and intra-assay variation, influence of sex and age, stability of blood samples storage at 4°C and amount of blood sample, were studied.

114 Insulin-Like Growth Factor (IGF)2 and IGF2 Receptor (IGF2R) Murine Blastocyst Transcriptional Response after High Gaseous Pressure Exposure at 8-Cell Stage

Insulin-like growth factor 2 (IGF2) is a pleiotropic hormone encoded by an imprinted gene expressed in the paternal allele of mammals, and acts in physiological responses including cell proliferation, differentiation, and development. It is mediated through the IGF1R signalling pathway, whereas the IGF2R, a maternally imprinted gene, acts on lysosomal IGF2 degradation. As imprinted genes, both Igf2 and Igf2r expressions are more susceptible to dysregulation by environmental factors. This study aimed to evaluate the effect of exposure of 8-cell-stage murine embryos to 16 MPa of high gaseous pressure (HGP) on the relative Igf2 and Igf2r mRNA abundance in resulting blastocysts following in vitro culture (IVC). Day-3 embryos were recovered from superovulated Mus musculus domesticus females. Eight-cell embryos were exposed to 16 MPa HGP for either 2 h (P1 group) or 4 h (P2 group), with a Control group not exposed to HGP. Immediately after recovery or HGP exposure, embryos were in vitro-cultured for 48 h in mKSOM medium supplemented with 0.4% BSA at 37.5°C, 5% CO2, 5% O2, 90% N2, and saturated humidity. Resulting blastocysts were collected in pools of 10 and stored at –80°C, pending analysis. Following total mRNA extraction, cDNA syntesis and RT-qPCR were performed according to manufacturers. Values were normalized to the internal control Ppia gene. Relative gene expression was calculated using the 2−ΔΔ Ct approach. Blastocyst rates after IVC were compared by the Chi-squared test (P &lt; 0.05), with relative Igf2 and Igf2r expression data and Igf:Igf2r ratio analysed by ANOVA, after log-transformation when needed, with pairwise comparisons done by the Tukey test (P &lt; 0.05). No differences in blastocyst rates after IVC were observed among groups (Control: 94.2%; P1: 95.4%; P2: 94.1%). However, the Igf2 mRNA relative abundances in blastocysts were 6.3- and 4.2-fold lower in P1 (P &lt; 0.01) and P2 (P = 0.07) than in the Control group, respectively. Likewise, the Igf2r relative transcription levels were 6.6- and 2.2-fold down-regulated in blastocysts from the P1 (P &lt; 0.001) and P2 (P &lt; 0.01) groups, respectively, when compared with controls. Although the relative expression for both genes followed a down-regulation pattern in blastocysts exposed to HGP at the 8-cell stage, the Igf2:Igf2r ratio was 1.9-fold lower in blastocysts in the P2 group (P &lt; 0.05) than Controls, which was similar to the P1 group, indicating a potential stress adaptation response for embryo growth and development after exposure to HGP in the P1 group. It is known that cells under certain conditions of stress may halter growth and development as a response to initiate cellular events to maintain viability. Results from this study appear to translate such response process to HGP in both experimental groups. However, as embryo development and the Igf2:Igf2r ratio in embryos were similar between the Control and the P1 group, exposure to 16 MPa HGP for 2 h at the 8-cell-stage embryo does not seem to affect cell signalling to growth and proliferation up to the blastocyst stage.

Ectopic expression of TWIST1 upregulates the stemness marker OCT4 in the esophageal squamous cell carcinoma cell line KYSE30

BackgroundThe transcription factor TWIST1 plays an important role in the epithelial–mesenchymal transition (EMT) process and in the migration, invasion and metastasis of cancer cells. OCT4, which is a homeobox transcription factor, has an important role in the self-renewal potential of cancer cells. Our aim here is to elucidate impact of ectopic expression of TWIST1 on OCT4 gene expression in esophageal squamous cell carcinoma (ESCC).MethodsThe ESCC line was KYSE30. GP293T cells were transfected with purf-IRES-GFP and pGP plasmids to produce recombinant viral particles. A semi-confluent KYSE30 culture was transduced with the prepared retroviral particles. mRNA extraction and cDNA synthesis were performed from normal KYSE30 cells and those ectopically expressing TWIST1. Expressional analysis of TWIST1 and OCT4 were performed with relative comparative real-time PCR.ResultsEctopic expression of TWIST1 in KYSE30 cells was related to its significant overexpression: nearly nine-fold higher in GFP-hTWIST1 KYSE-30 cells than in control GFP cells. This induced expression of TWIST1 caused significant upregulation of OCT4 in GFP-hTWIST1 KYSE-30 cells: nearly eight-fold higher. In silico analysis predicted the correlation of TWIST1 and OCT4 through ETS2.ConclusionsOverexpressed TWIST1 can be correlated with upregulation of the cancer stem cell marker OCT4 and the protein may play critical regulatory role in OCT4 gene expression. Since OCT4 is involved in the self-renewal process, the results may suggest a new linkage between TWIST1 and OCT4 in the cell biology of ESCC, highlighting the probable role of TWIST1 in inducing self-renewal.

Urinary mRNA and lupus disease flare.

In the past 20 years, extraction and quantification of mRNA from urinary sediment has emerged as a novel laboratory technique. The potential application of urinary mRNA quantification to the clinical care of patients with SLE has become an active field of research. However, published studies in this area are limited to small-scale ones. mRNA levels of several of cytokine and transcript factor genes have been found to have the potential for the diagnosis or monitoring of disease activity in patients with lupus nephritis. Our previous studies showed that a high urinary mRNA level of T-bet, the key transcription factor of type 1 T helper cells (Th1), was an independent predictor of lupus flare and probably reflects an intrinsic characteristic of patients with predisposition to Th1 activation. Other studies suggest that monitoring of monocyte chemoattractant protein-1(MCP-1), interleukin (IL)-17, GATA-3 (the key transcription factor of type 2 T helper cells) and forkhead box P3 (FOXP3, the key transcription factor of regulatory T cells) mRNA level in urinary sediment may provide an early clue for detecting disease flare in lupus patients. As a simple and non-invasive method, urinary mRNA level deserves further studies to validate its role in risk stratification and monitoring of patients with lupus nephritis.

Selective and Efficient RNA Analysis by Solid-Phase Microextraction.

In this study, a solid-phase microextraction (SPME) method was developed for the purification of mRNA (mRNA) from complex biological samples using a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for quantification. The chemical composition of the polymeric ionic liquid (PIL) and a polyacrylate (PA) SPME sorbent coating was optimized to enhance the extraction performance. Of the studied SPME sorbent coatings, the PIL containing carboxylic acid moieties in the monomer and halide-based anions extracted the highest amount of mRNA from aqueous solutions, whereas the native PA fiber showed the lowest extraction efficiency. On the basis of RT-qPCR data, electrostatic interactions and an ion-exchange mechanism between the negatively charged phosphate backbone of RNA and the PIL cation framework were the major driving forces for mRNA extraction. The optimized PIL-based SPME method purified a high quantity of mRNA from crude yeast cell lysate compared to a phenol/chloroform extraction method. The reusability and robustness of PIL-based SPME for RNA analysis represents a significant advantage over conventional silica-based solid-phase RNA extraction kits. The selectivity of the SPME method toward mRNA was enhanced by functionalizing the PA sorbent with oligo dT20 using carbodiimide-based amide linker chemistry. The oligo dT20-modified PA sorbent coating demonstrated superior extraction performance than the native PA sorbent coating with quantification cycle (Cq) values 33.74 ± 0.24 and 39, respectively. The modified PA sorbent extracted sufficient mRNA from total RNA at concentrations as low as 5 ng μL-1 in aqueous solutions without the use of organic solvents and time-consuming multiple centrifugation steps that are required in traditional RNA extraction methods.

Abstract 112: Myeloid Mineralocorticoid Receptor Controls Inflammatory and Fibrotic Responses After Renal Ischemic Injury via Macrophage Interleukin-4 Receptor

Introduction: Patients who survive an episode of acute kidney injury (AKI) are at high risk of de novo chronic kidney disease (CKD) development. Pharmacological mineralocorticoid receptor (MR) antagonism is useful to prevent CKD after a single episode of ischemic AKI in the rat. Objective: Test the involvement of myeloid MR in the development of kidney fibrosis after an ischemic AKI episode. Methods: We included 18 male C57/B6 mice that were divided in: sham, renal ischemia for 22.5 min and IR plus treatment with the non-steroidal MR antagonist finerenone (10 mg/kg) at -48, -24 and -1 h before IR. MR inactivation in myeloid cells (MR MyKO ) was achieved by crossing mice with the MR alleles flanked by loxP sites (MR f/f ) with mice expressing the Cre recombinase under the LysM promoter activity. In MR f/f and MR MyKO mice we induced renal IR of 22.5 min or sham surgery. The mice were followed-up during 4 weeks to test for AKI to CKD transition. In another set of mice, the macrophages were sorted from kidneys after 24 h of reperfusion and flow cytometry characterization or mRNA extraction was performed. Thyoglycolate elicited peritoneal macrophages were used for in vitro studies. Results: The progression of AKI to CKD after 4 weeks of renal ischemia in the untreated C57/B6 and MR f/f mice was characterized by a 50% increase in plasma creatinine, a 2-fold increase in the mRNA levels of TGF-β and fibronectin as well as by severe tubule-interstitial fibrosis. The mice that received finerenone or MR MyKO mice were protected against these alterations. Increased expression of M2-anti-inflamatory markers in kidney-isolated macrophages from finerenone-treated or MR MyKO mice was observed. The inflammatory population of Ly6C high macrophages was reduced by 50%. In peritoneal macrophages in culture, MR inhibition promoted increased IL-4 receptor expression and activation, facilitating macrophage polarization to an M2 phenotype. Conclusion: MR antagonism or myeloid MR deficiency facilitates macrophage polarization to a M2, anti-inflammatory phenotype after kidney IR, preventing maladaptive repair and chronic kidney fibrosis and dysfunction. MR inhibition acts through the modulation of IL-4 receptor signaling to facilitate macrophage phenotype switching.

Diminazene aceturate prevents nephropathy by increasing glomerular ACE2 and AT2 receptor expression in a rat model of type1 diabetes.

One of the protective actions of angiotensin converting enzyme-2 (ACE2) is the inactivation of angiotensin II. Expression and activity of ACE2 was reduced in glomeruli of diabetic patients and in animal models of diabetes. Recently the potential role of recombinant ACE2 administration in preventing diabetic nephropathy (DN) has been shown. Here we have tested the effects of the ACE2 activator, diminazene aceturate (DIZE), in a model of DN. Male Wistar rats were rendered diabetic using a single dose of streptozotocin (55mg·kg-1 , i.p.). After 4weeks, diabetic animals were divided into experimental groups and treated with DIZE, at a low dose (5mg·kg-1 ·day-1 ), a high dose (15mg·kg-1 ·day-1 ) and the high dose with of the AT2 receptor antagonist PD123319 (10mg·kg-1 ·day-1 ). At the end of the treatment , kidneys from all the groups were collected and processed separately for glomerular isolation, protein isolation, mRNA extraction and for immunohistochemical studies. Treatment with DIZE restored ACE2 expression in glomeruli and increased expression of AT2 receptors in whole kidney and isolated glomeruli of diabetic animals. DIZE administration reduced angiotensin II levels and increased angiotensin-(1-7) levels in diabetic kidney. However, PD123319 treatment reversed all these actions of DIZE. DIZE treatment reduced diabetes-induced renal damage as shown by reduction of fibrosis and apoptosis. These protective actions of DIZE were blocked by the AT2 receptor antagonist. Taken together, these results suggest that DIZE protected against DN through the ACE2/angiotensin-(1-7)/ AT2 receptor axis.

Abstract 5615: Genomic differential expression analysis of fusion proteins incorporating the pro-apoptotic molecule Granzyme B reveals new potential targets for treatment of breast cancer

Abstract Granzyme B (GrB) is a member of the serine protease family of enzymes that play a critical role in the body’s defense against viral infection and tumor development. Cytotoxic T lymphocyctes (CTL) and natural killer (NK) cells directly deliver granzymes to target cells, which induces apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. Anti-tumor efficacy studies have suggested that the targeted delivery of human Granzyme B to tumor cells has a significant potential for cancer treatment. We had previously developed a novel fusion construct composed of the VEGF121 growth factor and human GrB. The GrB/VEGF121 construct was found to be highly cytotoxic to vascular endothelial cells expressing the R2 (KDR) receptor for VEGF. In this study, we examined the mechanism of GrB/VEGF121-induced cytotoxicity against cells in culture at the genomic level. The TNBC cell line MDA-MB-231 was treated with an IC50 dose of GrB/VEGF121 for 24 hours; the cells were then harvested and the effect of GrB/VEGF121 on intracellular events was examined by extraction of mRNA followed by microarray analysis. Gene-level differential expression analysis revealed that a total of twenty genes were upregulated by over 3-fold, while 25 were downregulated 3-fold or more. These included genes involved in signal transduction, stress response, cell cycle control, hypoxia and metastasis. Particularly impacted were the Nuclear Receptors Meta pathway, NRF2 pathway, nuclear receptors in lipid metabolism and toxicity, matrix metalloproteinases, and the notch signaling pathway. Validated data will be reported following complete analysis of gene level differential expression and alternative splicing. Our data suggests that GrB/VEGF121 induces expression of genes known to be induced by VEGF alone, as well as molecules previously not associated with either Granzyme B or VEGF.This data suggests a previously unsuspected impact of the serine protease GrB on various molecular pathways within the cell and may lead to a new understanding of how these agents operate at the molecular level. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Michael G. Rosenblum. Genomic differential expression analysis of fusion proteins incorporating the pro-apoptotic molecule Granzyme B reveals new potential targets for treatment of breast cancer [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 5615. doi:10.1158/1538-7445.AM2017-5615

Abstract 3784: Expression of estrogen receptor specific signaling transcriptome in epithelial cell adhesion molecule (EpCAM) captured circulating tumor cells from patients with breast cancer

Abstract Background: Breast cancer (BC) is the second leading cause of cancer related death in American women. There is a critical need for new biomarkers to evaluate treatment response and emerging mechanisms of resistance. The identification and characterization of new biomarkers such as Circulating Tumor Cells (CTCs) may be helpful tools for these purposes as well as understanding the biology of metastatic BC. Methods: Blood samples were processed with a Ficoll gradient to isolate nucleated cells. CTCs were isolated from these samples using an exclusion-based sample preparation technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. EpCAM positive CTCs were captured in the VERSA followed by mRNA extraction for gene expression profiling for the estrogen receptor (ER) and other breast cancer relevant targets. CTCs were defined as Hoechst positive, CD45/CD11b/CD14 negative, and cytokeratin positive. Captured CTCs were evaluated for ER protein staining intensity and localization. Results: The VERSA platform was validated to capture and enumerate CTCs from patients with breast cancer using EpCAM capture and immunofluorescent staining for intact nucleus, ER, and cytokeratin while negative for multiple immune markers (CD45, CD11b, CD14). The VERSA can capture cells with &amp;gt;90% capture efficiency with EpCAM antibody in MCF7 and T47D cell lines. ER specific transcriptome analysis in BC patient CTCs and cell lines was performed. We evaluated expression of ER signaling pathway, prognostic, metastatic/EMT; stemness; epithelial markers with concordant expression of RPLPO and HPRT1. We identified expression of ER in 6 out of 7 BC samples. Expression of PR and HER2 in a subset of samples confirms capture of breast cancer cells for further molecular interrogation. Conclusion: In conclusion, we report the expression profiles of ER signaling genes in EpCAM captured CTCs from BC patients. These tools will be used to compare subpopulations of CTCs from patients with BC as predictive and pharmacodynamic biomarkers of response to targeted therapies or chemotherapies. Citation Format: Ashok Singh, Jamie M. Sperger, Jennifer Schehr, Tessa Witkowski, Beth A. Weaver, Mark E. Burkard, Joshua M. Lang. Expression of estrogen receptor specific signaling transcriptome in epithelial cell adhesion molecule (EpCAM) captured circulating tumor cells from patients with breast cancer [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 3784. doi:10.1158/1538-7445.AM2017-3784

Abstract 1722: Novel multiplex liquid biopsy detection of ALK and ROS1 rearrangements and intratumoral heterogeneity in lung cancer using circulating tumor cells (CTCs) isolated from NSCLC patient bloods and TR-CytoTrapNanoTM technology

Abstract Introduction: ALK and ROS1 rearrangements are common gene alterations found in subsets of patients with NSCLC. Identification of ALK and ROS1 rearrangement in NSCLC is critical for highly active targeted therapies aiming for these two main tumorigenic drivers. Due to the similarity of tyrosine kinase domains between ALK and ROS1, many ALK inhibitors are also functional in treatment of ROS1 subtype NSCLC. For example, crizotinib, which is a standard therapy for advanced ALK-rearranged NSCLC is highly effective in advanced ROS1-rearranged genotype NSCLC patients. The mutational analysis of these genes requires serial tumor biopsying from primary lesions, which requires high risk clinical procedures. Further, the current tissue-based diagnostics, such as fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are known to be challenging technically with limited sensitivity, subjective interpretation and inaccuracy in monitoring the current tumor status. The TR-CytoTrapNanoTM CTC technology is a blood-based assay to utilize CTCs as a biosource for mRNA extraction to detect genetic alteration or gene expression profiles including ALK and ROS1 gene rearrangements. In this study, we determined the variant specific expression of ALK fusion transcripts (EML4-ALK, KIF-ALK, and TGF-ALK) and ROS1 fusion transcripts (EZR-ROS1, SDC4-ROS1, TPM3-ROS1, LRIG3-ROS1, GOPC-ROS1, CD74-ROS1, and SLC34A2-ROS1) using multiplex digital droplet polymerase chain reaction(ddPCR) technology. Method: The Peripheral Blood Mononuclear Cells isolated from NSCLC patient bloods run through the TR-CytoTrapNanoTM microfluidic system. The CTCs are enriched and captured on the chip surface conjugated with Anti-EpCAM targeting antibody, which specifically binds to CTCs of epithelial origin. Following lysis step facilitate total RNA release from trapped CTCs and then pure mRNAs are isolated using oligo-dT conjugated magnetic beads. The mRNAs are used for following ALK and ROS1 specific reverse transcription reaction to make cDNAs for either ALK or ROS1 fusion transcripts. The specific subtyping of ALK and ROS1 rearrangement are then validated using ddPCR. Results and Conclusion: We tested several liquid biopsies from ALK and ROS1 rearrangement positive patients and corresponding positive data to FISH assays using solid tumor biopsies. Interestingly, some of the CTCs harboring ALK and ROS1 rearrangement has shown high intratumor heterogeneity in the subtypes of fusion partners. we envision that our novel TR-CytoTrapNanoTM CTC ALK/ROS1 rearrangement Assay to monitor treatment response and recurrence in NSCLC patients will be used to monitor treatment response and disease recurrence following treatment with Tyrosine Kinase Inhibitors (TKIs). Note: This abstract was not presented at the meeting. Citation Format: sangjun lee. Novel multiplex liquid biopsy detection of ALK and ROS1 rearrangements and intratumoral heterogeneity in lung cancer using circulating tumor cells (CTCs) isolated from NSCLC patient bloods and TR-CytoTrapNanoTM technology [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 1722. doi:10.1158/1538-7445.AM2017-1722

Effects of FGF2 administration for different time periods on chondrocytes

Objective To study the effects of fibroblast growth factor 2 (FGF2) on the proliferation and gene expression profiles of rabbit articular chondrocytes in vitro after different time periods of stimulation. Methods The chondrocytes were isolated and cultured in vitro, and the 3rd generation cells were harvested. Cells were divided into three groups. In the group 1 (FGF2 short-time action group), chondrocytes were cultured in medium with FGF2 for one day, and then transferred to fresh culture medium without FGF2 and cultured for another 6 days. In the group 2 (FGF2 long-time action group), chondrocytes were cultured in medium with FGF2 for 7 days. In the Group 3 (control group), chondrocytes were cultured in culture medium without FGF2 for 7 days. After culture for 1, 3, and 7 days, the proliferation of chondrocytes in the all groups was detected respectively. Following extraction of mRNA, the gene expressions of BMP2, BMP4, SOX9 and COL2A1 of the chondrocytes in the all groups were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The content of type II collagen was measured via immunofluorescence staining. Results Compared with the control group, FGF2 promoted the proliferation of chondrocytes in the short-and long-time action groups and there was no significant difference between the two FGF2-treated groups. The results of qRT-PCR indicated that different treatment induced different gene expression profile. Particularly, compared with the control group and the FGF2 long-time action group, the expression of BMP2, BMP4, SOX9 and COL2A1 in the short-time action group were significantly upregulated at the 7th day. Immunofluorescence intensity of type II collagen in the group 1 was stronger than that in the control group and group 2. Conclusions Different administration of FGF2 for different time periods induced different responses of chondrocytes. Short-term FGF2 stimulation was more beneficial to maintain the phenotype of chondrocytes and the synthesis of extracellular matrix. Key words: Fibroblast growth factor 2; Chondrocytes; Long-time action; Short-time action

Interferon-γ Released by Activated CD8+ T Lymphocytes Impairs the Calcium Resorption Potential of Osteoclasts in Calcified Human Aortic Valves

In calcific aortic valve disease (CAVD), activated T lymphocytes localize with osteoclast regions; however, the functional consequences of this association remain unknown. We hypothesized that CD8+ T cells modulate calcification in CAVD. CAVD valves (n=52) dissected into noncalcified and calcified portions were subjected to mRNA extraction, real-time quantitative PCR, enzyme-linked immunosorbent assay, and immunohistochemical analyses. Compared with noncalcified portions, calcified regions exhibited elevated transcripts for CD8, interferon (IFN)-γ, CXCL9, Perforin 1, Granzyme B, and heat shock protein 60. Osteoclast-associated receptor activator of NK-κB ligand (RANKL), tartrate-resistant acid phosphatase (TRAP), and osteoclast-associated receptor increased significantly. The stimulation of tissue with phorbol-12-myristate-13-acetate and ionomycin, recapitulating CAVD microenvironment, resulted in IFN-γ release. Real-time quantitative PCR detected mRNAs for CD8+ T-cell activation (Perforin 1, Granzyme B). In stimulated versus unstimulated organoid cultures, elevated IFN-γ reduced the mRNAs encoding for RANKL, TRAP, and Cathepsin K. Molecular imaging showed increased calcium signal intensity in stimulated versus unstimulated parts. CD14+ monocytes treated either with recombinant human IFN-γ or with conditioned media-derived IFN-γ exhibited low levels of Cathepsin K, TRAP, RANK, and tumor necrosis factor receptor-associated factor 6 mRNAs, whereas concentrations of the T-cell co-activators CD80 and CD86 increased in parallel with reduced osteoclast resorptive function, effects abrogated by neutralizing anti-IFN-γ antibodies. CD8+ cell-derived IFN-γ suppresses osteoclast function and may thus favor calcification in CAVD.

Gene Expression of the GIP and GLP‐1 Receptors in the Horse

IntroductionThe Glucagon‐like peptide‐1 (GLP‐1R) and gastric insulinotropic polypeptide (GIPR) receptors are members of the secretin family of G‐protein coupled receptors that regulate insulin secretion from pancreatic beta cells. Both receptors are well studied in rodents and humans, but there are limited data available for horses. In horses, GIP and GLP‐1 contribute to hyperinsulinaemia, which causes a painful foot disease called laminitis.AimThis study aimed to determine whether the GIPR and GLP‐1R genes are expressed in equine tissues.MethodsArchived tissue samples (pancreas, skeletal muscle, heart, liver, kidney, digital lamellae (which is the organ affected by laminitis), tongue and small intestine) from healthy horses were subject to mRNA extraction and cDNA synthesis prior to primers design and PCR. Sequencing of the PCR product was undertaken to confirm amplification of the target gene.ResultsBoth the GIPR and GLP‐1R were expressed in the pancreas as expected, and this tissue was used as a positive control for tissue distribution studies. The GIPR was expressed in the heart, liver, kidney and small intestine. The GLP‐1R gene was expressed in all tissues except the tongue and small intestine. Sequence identity to the Equus caballus mRNA sequence available from GenBank was 98% for GIPR and 100% for GLP‐1R.DiscussionThese data indicate that GIPR and GLP‐Rs may be potential therapeutic targets for the treatment/prevention of hyperinsulinaemia and laminitis in horses. The expression of both receptors in tissues other than the pancreas suggests that they may have other physiological functions and this requires investigation.Support or Funding InformationAustralian Research Council (ARC)

The immune mechanism of intestinal tract Toll-like receptor in mediating EV71 virus type severe hand-foot-and-mouth disease and the MAPK pathway.

Immunological response is thought to play a crucial role in the development of a severe hand-foot-and-mouth disease (HFMD) infection in children, but the mechanisms remain largely unknown. This study was designed to help in elucidating the immunopathological pathways involved in the disease by quantifying Toll-like receptor (TLR) mRNAs, MAPK factors and cytokine levels in children experiencing the disease. A total of 86 enterovirus 71 (EV71)-infected HFMD children (49 with mild and 27 with severe disease), along with 30 healthy children were involved in the study. Peripheral vein blood samples were collected from each individual, and used to isolate peripheral blood mononuclear cells (PBMCs) for mRNA extraction and sera for measuring levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-6 and IL-10. The average expression levels of TLR3, TLR4, TLR7 and TLR8 mRNA in PBMCs of children with severe HFMD were significantly higher than those in the other children, the lowest values were found in the healthy control group (P<0.05). The expression levels of TLR2 and TLR9 mRNA were not significantly different (P>0.05) among the groups. Additionally, the expression levels of TNF-α, IFN-γ, IL-6 and IL-10 in the serum of the children in the severe group were significantly higher than those in the other two groups, and the lowest values were again found in the control group (P<0.05). Pearson correlation analysis showed that the TLR3, TLR4, TLR7 and TLR8 mRNA levels in PBMCs were positively correlated with the TNF-α, IFN-γ, IL-6 and IL-10 levels in the serum (P<0.05). Furthermore, the expression levels of the ERK, JNK and p38 mRNA in PBMCs of children in the severe group were significantly higher than those in the other two groups, with the lowest values being in the control group (P<0.05). Pearson correlation analysis showed that the TLR3, TLR4, TLR7 and TLR8 mRNA levels in PBMCs were positively correlated with ERK, JNK and p38 mRNA levels (P<0.05). The results of our study seem to indicate that the high expression levels of TLR3, TLR4, TLR7 and TLR8 induced in severe EV71 HFMD regulate the expression of cytokines by MAPK signaling pathway and negatively affect the ability of the organism to resolve the infection. Further studies are needed to test the hypothesis that immuno-modulation would be an effective treatment approach in pediatric cases of severe HFMD.

High-yield extraction of Escherichia coli RNA from human whole blood.

Studies of bacterial transcriptomics during bloodstream infections are limited to-date because unbiased extraction of bacterial mRNA from whole blood in sufficient quantity and quality has proved challenging. Problems include the high excess of human cells, the presence of PCR inhibitors and the short intrinsic half-life of bacterial mRNA. This study aims to provide a framework for the choice of the most suitable sample preparation method. Escherichia coli cells were spiked into human whole blood and the bacterial gene expression was stabilized with RNAprotect either immediately or after lysis of the red blood cells with Triton X-100, saponin, ammonium chloride or the commercial MolYsis buffer CM. RNA yield, purity and integrity were assessed by absorbance measurements at 260 and 280 nm, real-time PCR and capillary electrophoresis. For low cell numbers, the best mRNA yields were obtained by adding the commercial RNAprotect reagent directly to the sample without prior lyses of the human blood cells. Using this protocol, significant amounts of human RNA were co-purified, however, this had a beneficial impact on the yields of bacterial mRNA. Among the tested lysis agents, Triton X-100 was the most effective and reduced the human RNA background by three to four orders of magnitude. For most applications, lysis of the human blood cells is not required. However, co-purified human RNA may interfere with some downstream processes such as RNA sequencing. In this case, blood cell lysis with Triton X-100 is desirable.