TaqMan RT-qPCR Research Articles

5156 Circulating MicroRNA-based Biomarker Combinations for Differentiating Uni- and Bilateral Primary Aldosteronism Using a Machine Learning Approach

Abstract Disclosure: B. Vekony: None. G. Nyiro: None. Z. Herold: None. J. Fekete: None. F. Ceccato: None. S. Gruber: None. L. Kurzinger: None. M. Parasiliti-Caprino: None. B. Szeredas: None. S.K. Nazri: None. V. Fell: None. M. Bassiony: None. E.A. Azizan: None. I. Bancos: None. F. Beuschlein: None. P. Igaz: None. Background: The differentiation of uni- and bilateral forms of primary aldosteronism (PA) is of pivotal clinical relevance. Whereas unilateral PA warrants surgical treatment, bilateral PA is treated by aldosterone antagonists. The most reliable way to differentiate these two entities is adrenal venous sampling (AVS) that is an invasive method with limited availability and requiring great expertise. Aim: To develop a circulating blood-borne microRNA-based approach using machine learning for the differentiation of uni- and bilateral PA. Methods: Circulating microRNA profiling on an Illumina MiSeq platform was performed on total RNA samples isolated from EDTA-anticoagulated blood specimens taken by AVS from 18 pairs of right and left suprarenal veins (10 uni-, 8 bilateral PA). Significantly differentially expressed microRNAs were analyzed by a neural network (90-10% learner-tester cross-validation simulation). From the top 29 significantly differentially expressed microRNAs, 9 were selected for validation by Taqman RT-qPCR on a separate cohort of 30 AVS-confirmed samples (15-15 uni- and bilateral forms) both on plasma samples from adrenal as well as from peripheral veins. The method was finally tested on another independent cohort of peripheral blood samples from 80 patients (40 uni- and 40 bilateral PA). Results: Regarding the 9 microRNAs selected for validation, microRNA abundance was non-significantly lower in peripheral samples compared to AVS samples. By analyzing peripheral plasma samples, ten combinations of 4-8 microRNAs from the pool of 9 microRNAs turned out to have maximal sensitivity and specificity values over 85 %. The best combination included 6 microRNAs with a sensitivity of 86.3 % and a specificity of 87.91 % (AUC = 0.871). The inclusion of further parameters such as imaging, potassium and renin levels did not improve the model’s performance. Conclusion: The analysis of circulating blood-borne microRNAs represents a minimally invasive and potentially reliable way of differentiating uni- and bilateral PA. If bilateral PA is confirmed by the model, no more localization efforts would be necessary thus simplifying the management of PA patients. This would be relevant both from the patient’s and a public health perspective. Presentation: 6/3/2024

Comparison of Different Reverse Transcriptase-Polymerase Chain Reaction-Based Methods for Wastewater Surveillance of SARS-CoV-2: Exploratory Study.

Many countries have applied the wastewater surveillance of the COVID-19 pandemic to their national public health monitoring measures. The most used methods for detecting SARS-CoV-2 in wastewater are quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and reverse transcriptase-droplet digital polymerase chain reaction (RT-ddPCR). Previous comparison studies have produced conflicting results, thus more research on the subject is required. This study aims to compare RT-qPCR and RT-ddPCR for detecting SARS-CoV-2 in wastewater. It also aimed to investigate the effect of changes in the analytical pipeline, including the RNA extraction kit, RT-PCR kit, and target gene assay, on the results. Another aim was to find a detection method for low-resource settings. We compared 2 RT-qPCR kits, TaqMan RT-qPCR and QuantiTect RT-qPCR, and RT-ddPCR based on sensitivity, positivity rates, variability, and correlation of SARS-CoV-2 gene copy numbers in wastewater to the incidence of COVID-19. Furthermore, we compared 2 RNA extraction methods, column- and magnetic-bead-based. In addition, we assessed 2 target gene assays for RT-qPCR, N1 and N2, and 2 target gene assays for ddPCR N1 and E. Reverse transcription strand invasion-based amplification (RT-SIBA) was used to detect SARS-CoV-2 from wastewater qualitatively. Our results indicated that the most sensitive method to detect SARS-CoV-2 in wastewater was RT-ddPCR. It had the highest positivity rate (26/30), and its limit of detection was the lowest (0.06 gene copies/µL). However, we obtained the best correlation between COVID-19 incidence and SARS-CoV-2 gene copy number in wastewater using TaqMan RT-qPCR (correlation coefficient [CC]=0.697, P<.001). We found a significant difference in sensitivity between the TaqMan RT-qPCR kit and the QuantiTect RT-qPCR kit, the first having a significantly lower limit of detection and a higher positivity rate than the latter. Furthermore, the N1 target gene assay was the most sensitive for both RT-qPCR kits, while no significant difference was found between the gene targets using RT-ddPCR. In addition, the use of different RNA extraction kits affected the result when the TaqMan RT-qPCR kit was used. RT-SIBA was able to detect SARS-CoV-2 RNA in wastewater. As our study, as well as most of the previous studies, has shown RT-ddPCR to be more sensitive than RT-qPCR, its use in the wastewater surveillance of SARS-CoV-2 should be considered, especially if the amount of SARS-CoV-2 circulating in the population was low. All the analysis steps must be optimized for wastewater surveillance as our study showed that all the analysis steps including the compatibility of the RNA extraction, the RT-PCR kit, and the target gene assay influence the results. In addition, our study showed that RT-SIBA could be used to detect SARS-CoV-2 in wastewater if a qualitative result is sufficient.

Development and evaluation of one-step RT-qPCR TaqMan multiplex panels applied to six viruses occurring in lily and tulip bulbs

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics.

Plasma microRNA Signature as Companion Diagnostic for Abiraterone Acetate Treatment in Metastatic Castration-Resistant Prostate Cancer: A Pilot Study.

Abiraterone acetate (AA) serves as a medication for managing persistent testosterone production in patients with metastatic castration-resistant prostate cancer (mCRPC). However, its efficacy varies among individuals; thus, the identification of biomarkers to predict and follow treatment response is required. In this pilot study, we explored the potential of circulating microRNAs (c-miRNAs) to stratify patients based on their responsiveness to AA. We conducted an analysis of plasma samples obtained from a cohort of 33 mCRPC patients before and after three, six, and nine months of AA treatment. Using miRNA RT-qPCR panels for candidate discovery and TaqMan RT-qPCR for validation, we identified promising miRNA signatures. Our investigation indicated that a signature based on miR-103a-3p and miR-378a-5p effectively discriminates between non-responder and responder patients, while also following the drug's efficacy over time. Additionally, through in silico analysis, we identified target genes and transcription factors of the two miRNAs, including PTEN and HOXB13, which are known to play roles in AA resistance in mCRPC. In summary, our study highlights two c-miRNAs as potential companion diagnostics of AA in mCRPC patients, offering novel insights for informed decision-making in the treatment of mCRPC.

Development of TaqMan RT-qPCR for the detection of regulated citrus viruses and viroids in Aotearoa New Zealand

The major citrus species include several economically important fruits, such as orange, mandarin, lemon, limes, grapefruit and pomelos. Since the 1980 s, total production and consumption of citrus has grown strongly with the current annual worldwide production at over 105 million tonnes. New Zealand's citrus exports, for instance, had an estimated worth of NZ$ 11.6 million (approx. US$ 7 million) in 2020. Citrus plants are prone to viral diseases, which can lead to substantial economic losses. In New Zealand, the citrus Import Health Standard (IHS) has identified 22 viruses and viroids that are subject to regulation and requires citrus nursery stock to be free of these pathogens. As such, there is a need for reliable, sensitive, and rapid detection methods to screen for these viruses and viroids during post entry quarantine. In this study, we developed TaqMan RT-qPCR assays for the detection of nine of these regulated viruses and viroids, namely citrus leaf rugose virus (CiLRV), citrus leprosis virus C (CiLV-C), citrus leprosis virus C2 (CiLV-C2), citrus leprosis virus N (CiLV-N), citrus psorosis virus (CPsV), citrus yellow mosaic virus (CYMV), citrus bent leaf viroid (CBLVd), citrus viroid V (CVd-V), and citrus viroid VI (CVd-VI). These assays have been validated and found to be highly sensitive, specific, and reliable. The implementation of these assays will facilitate the safe importation of citrus nursery stock, thus safeguarding the country's horticultural and economic interests.

Specific plasma microRNA profiles could be potential non-invasive biomarkers for biochemical pregnancy loss following embryo transfer

BackgroundPlasma microRNAs act as biomarkers for predicting and diagnosing diseases. Reliable non-invasive biomarkers for biochemical pregnancy loss have not been established. We aim to analyze the dynamic microRNA profiles during the peri-implantation period and investigate if plasma microRNAs could be non-invasive biomarkers predicting BPL.MethodsIn this study, we collected plasma samples from patients undergoing embryo transfer (ET) on ET day (ET0), 11 days after ET (ET11), and 14 days after ET (ET14). Patients were divided into the NP (negative pregnancy), BPL (biochemical pregnancy loss), and CP (clinical pregnancy) groups according to serum hCG levels at day11~14 and ultrasound at day28~35 following ET. MicroRNA profiles at different time-points were detected by miRNA-sequencing. We analyzed plasma microRNA signatures for BPL at the peri-implantation stage, we characterized the dynamic microRNA changes during the implantation period, constructed a microRNA co-expression network, and established predictive models for BPL. Finally, the sequencing results were confirmed by Taqman RT-qPCR.ResultsBPL patients have distinct plasma microRNA profiles compared to CP patients at multiple time-points during the peri-implantation period. Machine learning models revealed that plasma microRNAs could predict BPL. RT-qPCR confirmed that miR-181a-2-3p, miR-9-5p, miR-150-3p, miR-150-5p, and miR-98-5p, miR-363-3p were significantly differentially expressed between patients with different reproductive outcomes.ConclusionOur study highlights the non-invasive value of plasma microRNAs in predicting BPL.

Investigating the Impact of Circulating miRNAs on Cardiovascular System Remodeling During Pregnancy

During pregnancy, women undergo significant remodeling of their cardiovascular system, a complex process involving various physiological adaptations to ensure an adequate blood supply to the developing fetus. An intriguing area of research focuses on the role of circulating microRNAs, particularly the primate-specific chromosome 19 miRNA cluster (C19MC) and the rodent-specific chromosome 2 miRNA cluster (C2MC), both of which exhibit high expression levels in the placenta. Extracellular vesicles, such as exosomes, play a crucial role in intercellular communication and may carry microRNAs that contribute to the physiological changes observed during pregnancy. However, the influence of circulating C19MC and C2MC microRNAs on maternal cardiovascular system remodeling remains unexplored. We hypothesized that the increased levels of circulating C2MC or C19MC microRNAs during pregnancy might play a role in the maternal cardiovascular system adaptations. To investigate this hypothesis, we conducted a study to determine whether circulating exosomes during pregnancy lead to elevated expression levels of C19MC or C2MC microRNAs in various organs, including the heart, kidney, liver, spleen, lungs, and brain. In our investigation, we utilized three distinct mouse models: wild-type (WT) mice expressing only C2MC, C2MC knock-out (KO) mice, and a novel humanized mouse model, which combined C2MC KO with either a single or double copy of the human C19MC. We extracted total RNA from the organs of these mice and performed cDNA synthesis. Subsequently, we measured the expression levels of two representative microRNAs, miR-517a and miR-466a, corresponding to C19MC and C2MC, respectively, in the organs of pregnant and non-pregnant mice using TaqMan RT-qPCR. Our results indicated no significant differences in the expression levels of miR-517a and miR-466a when normalized to the housekeeping gene snoRNA-202 across different organs in pregnant and non-pregnant mice. To comprehensively assess the expression patterns of all microRNAs within the C19MC and C2MC clusters in various organs of pregnant and non-pregnant mice, further investigation with a larger sample size and small RNA-Seq analysis is warranted. This approach will enable us to explore a broader spectrum of microRNAs and potentially identify specific candidates that may play a crucial role in the essential physiological process of maternal cardiovascular system remodeling. This study is supported by USF Cardiovascular Summer Undergraduate Research Fellowship (SURF) sponsored by the American Heart Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

First report of infections with isolates of the Asian genotype of Cucumber green mottle mosaic virus in cucumber crops in Belgium.

In July 2019, four rows of cucumber plants (Cucumis sativus) in a commercial glasshouse in the north of Belgium showed severe mosaic, blistering and distortion of the leaves, with symptoms resembling those caused by Cucumber green mottle mosaic virus (CGMMV). CGMMV is a Tobamovirus that mainly affects cucurbit crops worldwide (Dombrovsky et al., 2017). Phylogenetic analyses in previous studies have shown two major clades, one including isolates that were initially identified in Europe and Russia (European genotype) and the second one with isolates initially identified in Asia and Israel (Asian genotype) (Dombrovsky et al., 2017; Pitman et al., 2022; Mackie et al., 2023). A symptomatic leaf sample was collected and total RNA was isolated from 100 mg of leaf tissue (Spectrum™ Plant Total RNA kit, Sigma-Aldrich). CGMMV was detected using a one-step TaqMan RT-qPCR (Hongyun et al., 2008). High-throughput sequencing (HTS) confirmed the presence of CGMMV. The sample was prepared using the Novel enrichment technique of viromes protocol (NETOVIR protocol, Conceição-Neto et al., 2015). The leaf material was homogenized, enriched for virus-like particles and the RNA was extracted (QIAamp Viral RNA mini kit, QIAGEN). The extract was randomly amplified (Whole Transcriptome Amplification kit, Sigma Aldrich), used for library preparation (Nextera XT DNA library preparation kit, Illumina) and sequenced on a NovaSeq platform. HTS data analysis was performed using Geneious Prime software (Biomatters, Auckland, New Zealand, version 2023.2). After quality filtering and trimming, 26.7M reads were obtained (132 nt mean length). In total, 20.6M reads were mapped to two genomes KP772568 and GQ411361 (considered as reference for the Asian and European genotypes respectively) with Geneious. This revealed 100% coverage of the full sequences (6422 nt) with 99.4% and 90% nucleotide identities to the reference genomes of Asian and European genotypes, respectively. Phylogenetic analyses confirmed that isolate 2019-26A-BE, with GenBank ID OR724740, relates to the Asian genotype. The HTS data were additionally processed using the ViPER pipeline (De Coninck, 2021). The raw reads were quality filtered and trimmed, (Trimmomatic) and then used to perform de novo assembly (metaSPAdes). The produced contigs were classified using DIAMOND and visualized with KronaTools. The results showed that no other virus was detected in the sample. Finally, cucumber seedlings were inoculated using the original symptomatic sample and were grown in a research glasshouse. After 3 weeks, severe CGMMV symptoms, similar to the original symptoms observed in the commercial glasshouse, were observed in the inoculated plants. Infection with CGMMV was verified via RT-qPCR, and the isolate present in the inoculated plants was confirmed to belong to the Asian genotype via RT-PCR-RFLP (Crespo et al., 2017). Later samplings of symptomatic leaves confirmed the presence of isolates belonging to the Asian genotype of CGMMV at four other commercial glasshouse locations, specializing in cucumber crop, in Belgium in 2020, 2021 and 2023 by RT-PCR-RFLP. While the Asian genotype was previously found elsewhere in Europe (Pitman et al., 2022), to our knowledge, this is the first report of infections with isolates of this genotype in glasshouse cucumber crops in Belgium. Further investigation is required to determine the spread and impact of infections with isolates of the Asian genotype in cucumber crops in Belgium.

Abstract 3665: Stratifying prostate cancer patients through circulating genes related to prostate cell subtypes, drug targets, and therapeutic resistance

Abstract Stratification remains an obstacle for the optimal treatment of prostate cancer (PCa) patients throughout the treatment trajectory. Targeting the androgen receptor (AR) kills the majority of luminal-like cells, which are AR-positive and express Prostate Specific Antigen (PSA). However, a subset of surviving AR-positive cells develop resistance to treatments while AR-negative cells of the neuroendocrine (NE) or stem-like phenotypes emerge through selection. We reported on the clinical relevance of cell-subtype genes in whole blood RNA of advanced PCa patients. Here, we studied an expanded panel of genes to include drug targets and therapeutic resistance as predictive biomarkers to stratify patients at diagnosis and at the advanced stage of disease.Genes were chosen based on literature review, results of clinical trials in PCa, and PCa transcriptomic data. Technical validation of TaqMan RT-qPCR assays was carried out for each gene, including rigorous testing of reproducibility. Whole blood RNA was extracted from 26 healthy controls with no prostatic disease, 16 patients prior to prostatectomy, and 43 blood samples from 28 metastatic cases. Gene overexpression was defined as the 99.5% confidence interval of expression in controls. Clinical data were retrieved from patients’ charts.A panel of 64 genes was built, showing overexpression in advanced PCa but low or no expression in normal blood (including whole blood, white and red blood cell populations and platelets). Testing in control blood showed no correlation with age. The proportions of patients’ white blood cells did not correlate with gene expression in their blood. Overall, up to 44/64 genes were overexpressed in at least one patient sample. Patients with prostatic intraductal carcinoma at prostatectomy showed more circulating genes, including more NE and stemness genes, and more PCa cell subtypes represented. Intermediate and high-risk patients showed more circulating NE genes. In metastatic patients, signatures of luminal, NE, stemness, and resistance to AR inhibitors or taxanes were associated with progression. PCa-specific luminal genes were associated with shorter overall survival. Treatment resistance genes correlated with lines of treatment and current taxanes. Two targetable NE genes were overexpressed in distinct categories of patients, suggesting that they may benefit from more specific treatments.In conclusion, phenotypic and functional differences in circulating gene patterns of PCa patients correlate with pathological features, treatments, progression. They may be clinically meaningful to stratify patients and predict therapeutic response. Circulating genes encoding drug targets may justify clinical trials to offer personalized treatments and impact on lethal PCa. Citation Format: Seta Derderian, Edouard Jarry, Arynne Santos, Mohanachary Amaravadi, Quentin Vesval, Lucie Hamel, Raphael Sanchez-Salas, Alexis Rompré-Brodeur, Wassim Kassouf, Raghu Rajan, Marie Duclos, Fadi Brimo, Armen Aprikian, Simone Chevalier. Stratifying prostate cancer patients through circulating genes related to prostate cell subtypes, drug targets, and therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3665.

Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese.

This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3-57.2%) and 41.4% (95% CI 29.1-53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4-24.9%, and 12.3% GII, 95% CI 7.0-17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.

Alcohol and e-cigarette damage alveolar-epithelial barrier by activation of P2X7r and provoke brain endothelial injury via extracellular vesicles

BackgroundUse of nicotine containing products like electronic cigarettes (e-Cig) and alcohol are associated with mitochondrial membrane depolarization, resulting in the extracellular release of ATP, and mitochondrial DNA (mtDNA), mediating inflammatory responses. While nicotine effects on lungs is well-known, chronic alcohol (ETH) exposure also weakens lung immune responses and cause inflammation. Extracellular ATP (eATP) released by inflammatory/stressed cells stimulate purinergic P2X7 receptors (P2X7r) activation in adjacent cells. We hypothesized that injury caused by alcohol and e-Cig to pulmonary alveolar epithelial cells (hPAEpiC) promote the release of eATP, mtDNA and P2X7r in circulation. This induces a paracrine signaling communication either directly or via EVs to affect brain cells (human brain endothelial cells - hBMVEC).MethodsWe used a model of primary human pulmonary alveolar epithelial cells (hPAEpiC) and exposed the cells to 100 mM ethanol (ETH), 100 µM acetaldehyde (ALD), or e-Cig (1.75 µg/mL of 1.8% or 0% nicotine) conditioned media, and measured the mitochondrial efficiency using Agilent Seahorse machine. Gene expression was measured by Taqman RT-qPCR and digital PCR. hPAEpiC-EVs were extracted from culture supernatant and characterized by flow cytometric analysis. Calcium (Ca2+) and eATP levels were quantified using commercial kits. To study intercellular communication via paracrine signaling or by EVs, we stimulated hBMVECs with hPAEpiC cell culture medium conditioned with ETH, ALD or e-cig or hPAEpiC-EVs and measured Ca2+ levels.ResultsETH, ALD, or e-Cig (1.8% nicotine) stimulation depleted the mitochondrial spare respiration capacity in hPAEpiC. We observed increased expression of P2X7r and TRPV1 genes (3-6-fold) and increased intracellular Ca2+ accumulation (20-30-fold increase) in hPAEpiC, resulting in greater expression of endoplasmic reticulum (ER) stress markers. hPAEpiC stimulated by ETH, ALD, and e-Cig conditioned media shed more EVs with larger particle sizes, carrying higher amounts of eATP and mtDNA. ETH, ALD and e-Cig (1.8% nicotine) exposure also increased the P2X7r shedding in media and via EVs. hPAEpiC-EVs carrying P2X7r and eATP cargo triggered paracrine signaling in human brain microvascular endothelial cells (BMVECs) and increased Ca2+ levels. P2X7r inhibition by A804598 compound normalized mitochondrial spare respiration, reduced ER stress and diminished EV release, thus protecting the BBB function.ConclusionAbusive drugs like ETH and e-Cig promote mitochondrial and endoplasmic reticulum stress in hPAEpiC and disrupts the cell functions via P2X7 receptor signaling. EVs released by lung epithelial cells against ETH/e-cig insults, carry a cargo of secondary messengers that stimulate brain cells via paracrine signals.

Viabilidade da substituição da técnica de isolamento viral em camundongos pela RT-qPCR para o diagnóstico da raiva

ABSTRACT: Rabies is a viral encephalitis that affects mammals, including humans. Rapid and effective laboratory diagnosis of the rabies virus is critical for public health. This study evaluated the operational, technical, and financial viability of the RT-qPCR in replacement of inoculation in mice for diagnosing rabies in the laboratory routine. A total of 316 samples of mammalian brains were analyzed, 121 positives and 195 negatives, previously diagnosed by direct immunofluorescence (DIF) and mouse inoculation test (MIT). The samples were submitted to the duplex TaqMan RT-qPCR technique. The accuracy, sensitivity, and specificity of RT-qPCR were analyzed. We analyzed the costs for performing the RT-qPCR technique and compared it with the cost of MIT. The results showed 99.37% accuracy, 99.17% sensitivity, and 99.49% specificity by RT-qPCR when related to DIF and MIT results, which proved to be a robust and repeatable technique. The minimum time for a positive diagnosis was reduced in RT-qPCR (1 day) if compared to MIT (9.64 days), with a 17.7% reduction in the cost of the molecular technique. The present study demonstrated that the molecular biology technique is an efficient tool to diagnose rabies in the laboratory routine, being able to replace MIT.

Liposome-encapsulated zoledronate increases inflammatory macrophage population in TNBC tumours

BackgroundTumour associated macrophages (TAMs) are important players in breast tumour progression and metastasis. Clinical and preclinical evidence suggests a role for zoledronate (ZOL) in breast cancer metastasis prevention. Further, zoledronate is able to induce inflammatory activation of monocytes and macrophages, which can be favourable in cancer treatments. The inherent bone tropism of zoledronate limits its availability in soft tissues and tumours. In this study we utilised an orthotopic murine breast cancer model to evaluate the possibility to use liposomes (EMP-LIP) to target zoledronate to tumours to modify TAM activation. MethodsTriple-negative breast cancer 4T1 cells were inoculated in the 4th mammary fat pad of female Balb/c mice. Animals were divided according to the treatment: vehicle, ZOL, EMP-LIP and liposome encapsulated zoledronate (ZOL-LIP). Treatment was done intravenously (with tumour resection) and intraperitoneally (without tumour resection). Tumour growth was followed by bioluminescence in vivo imaging (IVIS) and calliper measurements. Tumour-infiltrating macrophages were assessed by immunohistochemical and immunofluorescence staining. Protein and RNA expression levels of inflammatory transcription factors and cytokines were measured by Western Blotting and Taqman RT-qPCR. ResultsLiposome encapsulated zoledronate (ZOL-LIP) treatment suppressed migration of 4T1 cell in vitro. Tumour growth and expression of the angiogenic marker CD34 were reduced upon both ZOL and ZOL-LIP treatment in vivo. Long-term ZOL-LIP treatment resulted in shift towards M1-type macrophage polarization, increased CD4 T cell infiltration and activation of NF-κB indicating changes in intratumoural inflammation, whereas ZOL treatment showed similar but non-significant trends. Moreover, ZOL-LIP had a lower bisphosphonate accumulation in bone compared to free ZOL. ConclusionResults show that the decreased bisphosphonate accumulation in bone promotes the systemic anti-tumour effect of ZOL-LIP by increasing inflammatory response in TNBC tumours via M1-type macrophage activation.

MOY 2 MicroRNAs as Bile Based Biomarkers for Pancreaticobiliary Cancers (MIRABILE)

Abstract Aims MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. They are commonly deregulated in cancer and are incredibly stable molecules in tissues and biofluids. Accurate identification of a malignant biliary stricture can sometimes be challenging. We aimed to identify miRNAs in the bile that are able to differentiate between malignant (pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA)) and benign pancreaticobiliary disease. Methods There were 111 patients recruited prospectively at initial endoscopic retrograde cholangiopancreatography (ERCP) for obstructive jaundice and bile aspirated for analysis. Total cell-free RNA was extracted from the bile from a discovery cohort of patients (n=78: 27 PDAC, 14 CCA, and 37 benign disease) and small RNA sequencing was performed. LASSO regression analysis was used to define bile miRNA signatures, and NormFinder used to identify endogenous controls. A validation cohort was used (n=87: 34 PDAC, 14 CCA, and 39 benign disease) to confirm candidate miRNAs using TaqMan RT-qPCR. Results We identified 6 potential bile miRNAs, of which 4 were suitable for RT-qPCR validation. We found miR-340 and miR-182 were significantly differentially expressed (p=0.0268 and p=0.0004 respectively) in malignant vs. benign bile, with combined AUC of 0.80 (sensitivity 64.6%; specificity 82.1%) for predicting malignant pancreaticobiliary disease. Adding serum CA 19-9 to this bile miRNA model improved the diagnostic potential (AUC 0.85: sensitivity of 86.4%; specificity of 71.4%). Conclusions Bile obtained at ERCP contains miRNAs able to differentiate benign from malignant pancreaticobiliary disease. Our novel bile miRNAs can be used with serum CA 19-9 to rapidly detect CCA and PDAC in patients presenting with obstructive jaundice.

Differential Expression of miR-146 and miR-155 in Active and Latent Tuberculosis Infection.

Tuberculosis (TB) is one of the leading causes of death worldwide. Besides, one-third of the world population is infected with Mycobacterium tuberculosis (MTB) while staying clinically asymptomatic; the situation is called latent TB infection (LTBI). MiR-21, miR-31, miR-146a, and miR-155 play an important role in many immune and inflammatory pathways. In the present study the expression levels of MiR-21, miR-31, miR-146a, and miR-155 in peripheral blood mononuclear cells (PBMCs) from patients with active TB, latently infected individuals (LTBI), and healthy controls (HC) were investigated. Participants were recruited at the Bouali Hospital, Zahedan University of Medical Sciences, Zahedan, Iran from 2010 to 2011. PBMCs were stimulated with PPD before RNA extraction. TaqMan RT-qPCR assay was used to analyze the expression levels of miRNAs. The results indicated no significant differences in the expression of miR-21 and miR-31 between different groups; however, in patients with active TB, the expression of miR-21 (P=0.03) and miR-31 (P=0.04) were significantly increased after stimulation with PPD compared to the unstimulated condition. The expression of miR-146 in response to PPD in both LTBI (P=0.02) and TB (P=0.03) groups compared to the HC group was increased. No significant differences were found in the expression level of miR-155 in response to PPD between LTBI and HC groups. However, the fold change was significantly higher in the TB group in comparison with the HC (P=0.03) and LTBI (P=0.05) groups. The results confirm the main role of miR-146 and miR-155 in TB infection and suggest a role for miR-146 and miR-155 as infection and activation markers in tuberculosis infection, respectively.

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR

The natural host range for brassica yellows virus (BrYV) is generally limited to Cruciferae. However, we found that BrYV can naturally infect strawberry. The full-length genome sequences of BrYV-MB (accession No. MZ666129) and BrYV-HY (accession No. ON060762) identified in strawberry from Yantai and Beijing, China, were obtained by high-throughput sequencing (HTS) combined with the RT-PCR and RACE techniques. The complete genome sequences of BrYV-MB and BrYV-HY are 5666 nt and contain six open reading frames (ORFs). The two isolates have the highest nucleotide (nt) sequence identity of 99.0%. The infectious cDNA clone of BrYV-HY was constructed through homologous recombination and used to agroinfiltrate Nicotiana benthamiana and Arabidopsis thaliana. The inoculated leaves of N. benthamiana showed necrotic symptoms after 4 days of inoculation (dpi), and the systematic leaves of A. thaliana exhibited purple symptoms at 14 dpi. To develop a rapid and high-sensitive method for the detection of BrYV, a TaqMan real-time fluorescence quantitative RT-PCR method (TaqMan RT-qPCR) was established. Under optimum reaction conditions, the sensitivity of the detection was as low as 100 fg and approximately 100-fold more sensitive than the conventional RT-PCR, so it can be used in large-scale testing.

A TaqMan RT-qPCR assay for absolute quantification of citrus leprosis virus C lineage SJP: disclosing the subgenomic/genomic ratio in plant and mite vector, plant organ-specific viral loads, and the kinetics of viral accumulation in plants

A TaqMan RT-qPCR assay for absolute quantification of citrus leprosis virus C lineage SJP: disclosing the subgenomic/genomic ratio in plant and mite vector, plant organ-specific viral loads, and the kinetics of viral accumulation in plants

Hypoxia-inducible factor pathway genes predict survival in metastatic clear cell renal cell carcinoma.

Hypoxia inducible factor (HIF) pathway alterations drive progression of clear cell renal cell carcinoma (ccRCC). We aim to evaluate genes within the canonical and non-canonical HIF pathways as predictors of survival in metastatic ccRCC. Gene expression was determined from 324 archival pretreatment nephrectomy specimens from CALGB90206, a phase III trial of patients treated with interferon alpha (INF-α) vs. INF-α plus bevacizumab. TaqMan RT-qPCR was performed using RNA from tumors macrodissected based on review by genitourinary pathology. A total of 35 HIF-related genes were assessed by Cox regression analysis. After adjusting for sex and Memorial Sloan Kettering Cancer Center risk score (MSKCC-RS), 11 genes predicted OS: HIF2A (HR 1.059, P = 0.012), EGLN3 (HR 1.089, P = 0.012), VEGFC (HR 0.904, P = 0.039), VEGFD (HR 1.085, P = 0.016), FLT4 (HR 1.093, P = 0.038), CCND1 (HR 1.077, P = 0.026), TGFA (HR 1.127, P = 0.003), EGFR (HR 1.151, P = 0.028), VHL (HR 0.764, P = 0.002), HSP90AA1 (HR 0.845, P = 0.002), and PTEN (HR 1.163, P = 0.050); 7 genes predicted PFS: HIF2A (HR 1.060, P = 0.011), CCND1 (HR 1.082, P = 0.016), TGFA (HR 1.096, P = 0.026), EP300 (HR 1.171, P = 0.031), VHL (HR 0.775, P = 0.007), HSP90AA1 (HR 0.871, P = 0.015), and TP53 (HR 1.119, P = 0.050). Most of these genes validated as significant predictors of survival in the external, TCGA dataset. In multivariate analysis of all externally validated genes, VEGFC (HR 0.906, P = 0.043), TGFA (HR 1.122, P = 0.003), CITED2 (HR 1.113, P = 0.035) and EP300 (HR 1.136, P = 0.049) predicted OS; and HIF2A (HR 1.049, P = 0.036) and EP300 (HR 1.199, P = 0.010) predicted PFS. EGLN3 (HR 1.156, P = 0.045) and BNIP3 (HR 1.254, P = 0.049) significantly interacted with treatment status and predicted PFS in patients treated with IFN-α and IFN-α+bevacizumab, respectively. We identified specific gene isoforms in both the canonical and non-canonical HIF pathways associated with metastatic RCC survival. EGLN3 and BNIP3 showed significant interaction with treatment arm and may be predictive of treatment response. We have identified genes for future prospective investigation as predictive biomarkers and novel drug targets.

INVERSE EXPRESSION BETWEEN TGF-β1 AND TWIST IN ORAL SQUAMOUS CELL CARCINOMA CELL LINE AFTER CHOLESTEROL DEPLETION

Objectives To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). Study Design SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. Results The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Conclusions Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology. To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology.

Investigation on Natural Infection of Covert Mortality Nodavirus in Farmed Giant Freshwater Prawn (Macrobrachium rosenbergii).

Simple SummaryCovert mortality nodavirus (CMNV) is a newly discovered aquatic animal virus in recent years. Here, we detected CMNV positive in farmed giant freshwater prawn (Macrobrachium rosenbergii) from Jiangsu, China by TaqMan RT-qPCR. Meanwhile, in situ hybridization and histological analysis indicated that the intestine, gill, hepatopancreas and ovary of giant freshwater prawn were the target organs of CMNV. In addition, a large number of CMNV-like particles were observed in the hepatopancreas and gill tissues under transmission electron microscopy. Overall, our study confirms that giant freshwater prawn is a susceptible host of CMNV, further expands the known host range of CMNV, and provided a new direction for further investigation and exploration of multiple pathogenic factors of giant freshwater prawn disease. Covert mortality nodavirus (CMNV), from the Nodaviridae family, is characterized by its unique cross-species transmission and wide epidemic distribution features. In this study, Macrobrachium rosenbergii was proved to be infected naturally by CMNV, which further expand the known host range of CMNV. Here, 61.9% (70/113) of the M. rosenbergii samples collected from Jiangsu Province were CMNV positive in the TaqMan RT-qPCR assay, which indicated the high prevalence of CMNV in M. rosenbergii. Meanwhile, the sequences of CMNV RdRp gene cloned from M. rosenbergii were highly identical to that of the original CMNV isolate from Penaeus vannamei. In situ hybridization (ISH) and histology analysis indicated that the intestine, gill, hepatopancreas and ovary were the targeted organs of CMNV infection in M. rosenbergii, and obvious histopathological damage including vacuolation and karyopyknosis were occurred in the above organs. Notably, the presence of CMNV in gonad alerted its potential risk of vertical transmission in M. rosenbergii. Additionally, numerous CMNV-like particles could be observed in tissues of hepatopancreas and gill under transmission electron microscopy. Collectively, our results call for concern of the potential negative impact of the spread and prevalence of CMNV in M. rosenbergii on its aquaculture, as well as providing a renewed orientation for further investigation and exploration of the diverse pathogenic factors causing M. rosenbergii diseases.