Reverse Transcriptase PCR Method Research Articles

Comparative efficacy of S-ELISA, N & F gene based reverse transcriptase PCR and cell culture methods for detection of PPR virus in clinical specimens

Comparative efficacy of S-ELISA, N & F gene based reverse transcriptase PCR and cell culture methods for detection of PPR virus in clinical specimens

Investigating expression of IGF-1 candidate gene for growth-associated microsatellite genotypes in a resource population of RIR chicken

Present study was undertaken to analyze relative expression of IGF-1 gene in liver and breast muscle, and to investigate probable association of chicken growth-linked microsatellite genotypes with IGF-1 gene expression in a resource population of RIR chicken. In this experiment, 114 birds of combined sexes were screened for growth-associated microsatellite genotypes and 25 alleles were separated through 3.4% MetaPhore agarose gel electrophoresis with their sizes ranging from 97 bp at MCW0010 to 349 bp at LEI0071. Twelve birds of either sexes comprising equally from these birds were selected at the age of 12 weeks for gene expression study by quantitative reverse transcriptase PCR method. Higher 40-ΔCt value was considered as higher expression. Least squares analysis of variance revealed that birds’ hatch factor had significant effect on relative mRNA expression of IGF-1 gene in liver tissue other than breast muscle. Birds from the second hatch showed higher expression (35.86±1.120) in liver tissue in comparison to the chicks from first hatch (32.58±0.792). The results also evidenced that the liver tissue had higher expression (34.22±0.690) than that in the breast muscle (33.923±0.186) tissue. The males had numerically higher expressions in both the tissues as compared to female birds. The microsatellites could not demonstrate significant genotypic differences in IGF-1 gene expression in liver and breast muscle tissues of the birds. It is suggested to study a large sample to investigate significant variation in IGF-1 gene expression in the birds under different sexes and growth-linked microsatellite genotypes to draw a valid conclusion.

Biosynthesis of silver nanoparticles using Citrus hystrix leaf extract and evaluation of its anticancer efficacy against HeLa cell line

Background Cervical cancer continues to be the leading cause of death worldwide despite the availability of many therapeutic options. Biogenic synthesis of metal nanoparticles paves a new way for the development of targeted drug delivery modalities of cancer therapeutics. Objective In this study, we demonstrate the efficacy of biosynthesized silver nanoparticles from methanolic leaf extract of Citrus hystrix as an anticancer agent used against cervical cancer cell line HeLa. Methods The addition of 1 mM silver nitrate to methanolic leaf extract of Citrus hystrix resulted in the biosynthesis of silver nanoparticles during the reaction mixture and was incubated in the dark for 1 h at pH 9 with gentle stirring. Characterization of synthesized NPs was carried out using various analyses. MTT assay, DAPI, AO/EB double staining, and reverse transcriptase-PCR (RT-PCR) analysis were carried out to evaluate the cytotoxic activity of Citrus hystrix mediated green synthesized silver nanoparticles (ChAgNPs). Results and conclusion The absorption band at 430 nm, as shown by UV–vis spectroscopy revealed the formation of AgNPs. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis shows that most of the ChAgNPs were spherical in shape and X-ray diffraction (XRD) patterns revealed the crystalline nature of the particles. Moreover, its potent cytotoxic effect on the HeLa cell line was analyzed using MTT assay with an IC50 value of 56 µg/ml by decreasing the cell viability in a dose and time-dependent manner. The induced apoptotic activity was confirmed by DAPI and double staining methods. Autophagic and apoptotic mediated cell death in ChAgNPs treated HeLa cell line was confirmed by staining procedures and RT-PCR methods.

An isothermal lab-on-phone test for easy molecular diagnosis of SARS-CoV-2 near patients and in less than 1 hour

ObjectivesThe performance of a new point-of-care CE-IVD-marked isothermal lab-on-phone COVID-19 assay was assessed in comparison to a gold standard real-time reverse transcriptase–PCR method. MethodsThe study was conducted following a nonprobability sampling of ≥16-year-old volunteers from three different laboratories, using direct mouthwash (N = 24) or nasopharyngeal (N = 191) clinical samples. ResultsThe assay demonstrated 95.19% sensitivity and 100% specificity for detection of SARS-CoV-2 in direct nasopharyngeal crude samples and 78.95% sensitivity and 100% specificity in direct mouthwash crude samples. It also successfully detected currently predominant SARS-CoV-2 variants of concern (Beta B.1.351, Delta B.1.617.2, and Omicron B.1.1.529) and demonstrated to be inert against potential cross-reactions of other common respiratory pathogens that cause infections that present similar symptoms to COVID-19. ConclusionThis lab-on-phone pocket-sized assay relies on an isothermal amplification of SARS-CoV-2’s N and E genes, taking just 50 minutes from sample to result, with only 2 minutes of hands-on time. It presents good performance when using direct nasopharyngeal crude samples, enabling a low-cost, real-time, rapid, and accurate identification of SARS-CoV-2 infections at the point of care, which is important for both clinical management and population screening, as a tool to break the chain of transmission of COVID-19 pandemic, especially in low-resources environments.

Studying the potential of upregulated PTGS2 and VEGF-C besides hyper-methylation of PTGS2 promoter as biomarkers of Acute myeloid leukemia.

Hereby, we aimed to investigate the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and Vascular Endothelial Factor-C (VEGF-C) besides the methylation of PTGS2 in AML patients. VEGF-C and PTGS2 expression analysis were evaluated in newly diagnosed AML patients and healthy controls by quantitative Reverse Transcriptase PCR method. Also, PTGS2 methylation status was evaluated by Methylation-Sensitive High-Resolution Melting Curve Analysis (MS-HRM). While 34% of patients were female, the mean age of the patients was 43.41 ± 17.60 years suffering mostly from M4 (48.21%) type of AML. Although methylation level between patients and controls was not significantly different, none of the normal controls showed methylation in the PTGS2 promoter. PTGS2 and VEGF-C levels were elevated in AML cases and correlated with WBC, Platelet, and Hemoglobin levels. The survival of patients with overexpressed VEGF-C and PTGS2 was poorer than others. It can be concluded that PTGS2 and especially VEGF-C expression but not PTGS2 methylation can be considered as diagnostic biomarkers for AML.

Development and validation of methodology to study the genetic polymorphism of organic cation transporter 1 (OCT1) - rs622342: A Prospective open-label study

The main objective is to develop and validate the methodology to study the genetic polymorphism of Organic cation transporter (OCT1) rs622342. The Reverse Transcriptase-PCR method for identifying the OCT1 rs622342 genetic polymorphism was developed and validated. This study is a prospective open-label study conducted over a period of six months with 61 South Indian healthy male volunteers. Phenol chloroform method was used for the DNA isolation, which was subjected to PCR for identifying the polymorphism. The method was successfully developed. After the addition of restriction enzyme (PsiI) in the ten samples, none of the samples showed fragmentation, which shows that there is a lack of rs622342 genetic polymorphism in the selected ten samples. Keywords: Genetic polymorphism, Organic cation transporter, Methodology & Validation, Healthy volunteers.

DOCKING STUDIES AND SYNTHESIS OF NOVEL 4- THIAZOLIDINONE DERIVATIVES BEARING 1, 3, 4- OXADIAZOLE MOIETY AS SIRT-3 ACTIVATORS TARGETING PARKINSON’S DISEASE

Seven protein families known as class III histone deacetylase for their characteristic features have been reported in mammals. Sirtuin3 (SIRT3) has gained considerable attention among the sirtuin members for its role in neurodegenerative disease. A new series of 4-thiazolidinones fused 1, 3, 4-oxadiazole derivatives have been designed in silico by using Schrodinger software. Molecules showing high affinity with the target protein (4JSR) were selected for the synthesis and analytical methods were used to determine the structures. The synthesized compounds III (a-h) were taken for in vitro cytotoxicity studies by MTT assay technique using SHSY5Y neuroblastoma cell lines. The synthesized compounds III (a-h) were taken for in vitro cytotoxicity studies by MTT assay technique using SHSY5Y neuroblastoma cell lines. Gene expression study was then performed on SIRT-3 gene by reverse transcriptase PCR method. Compound III b and III g were effective with IC50 values of 126.70 and 157.42 respectively and were found to possess good expression on the SIRT-3 gene.

Ethanolic extract of Salacca zalacca peel reduce IL-1β and apoptosis in high glucose induced zebrafish embryo

Gestational diabetes is a glucose intolerance level first identified during pregnancy. This condition is caused by hormone secretion that hinders insulin action from the placenta which caused insulin resistance that will cause hyperglycemia. This condition will increase oxidative stress, and in consequence increasing inflammation and cell death which can hinder fetal growth and development. Salacca zalacca peel is a food waste that contains antioxidants and anti-inflammatory properties. This research aimed to study the effect of SZ peel ethanolic extract in the expression of IL-1β and cell death marker (BAX and Apaf-1) in glucose 3% induced zebrafish embryo. Glucose metabolism is measured by the level of Phosphoenolpyruvate carboxykinase (PEPCK) at the age of 3 dpf. The expression of PEPCK, IL-1β, Bax, and Apaf-1 is measured using reverse transcriptase PCR (RT-PCR) method. Salacca zalacca peel extract is given in the concentration of 0,1; 0,2; and 0,4 mg/mL. The result of this research proves that PEPCK decreases in the group which was given glucose 3%. Salacca zalacca peel extract significantly decreases the expression of IL-1β and Bax in the concentration of 0,4 mg/mL, and also able to decrease Apaf-1 expression with the result that most closely resemble the negative control in the concentration of 0,4 mg/mL. In conclusion, Salacca zalacca peel extract protects from gestational diabetes condition through the decrease of IL-1β, Bax, and Apaf-1.

Development of a rapid viability RT-PCR (RV-RT-PCR) method to detect infectious SARS-CoV-2 from swabs

Since the rapid onset of the COVID-19 pandemic, its causative virus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), continues to spread and increase the number of fatalities. To expedite studies on understanding potential surface transmission of the virus and to aid environmental epidemiological investigations, we developed a rapid viability reverse transcriptase PCR (RV-RT-PCR) method that detects viable (infectious) SARS-CoV-2 from swab samples in <1 day compared to several days required by current gold-standard cell-culture-based methods. The method integrates cell-culture-based viral enrichment in a 96-well plate format with gene-specific RT-PCR-based analysis before and after sample incubation to determine the cycle threshold (CT) difference (ΔCT). An algorithm based on ΔCT ≥ 6 representing ∼ 2-log or more increase in SARS-CoV-2 RNA following enrichment determines the presence of infectious virus. The RV-RT-PCR method with 2-hr viral infection and 9-hr post-infection incubation periods includes ultrafiltration to concentrate virions, resulting in detection of <50 SARS-CoV-2 virions in swab samples in 17 h (for a batch of 12 swabs), compared to days typically required by the cell-culture-based method. The SARS-CoV-2 RV-RT-PCR method may also be useful in clinical sample analysis and antiviral drug testing, and could serve as a model for developing rapid methods for other viruses of concern.

Facility-Based Surveillance Activities for COVID-19 Infection and Outcomes among Healthcare Workers in a Nigerian Tertiary Hospital.

ABSTRACTCOVID-19 in healthcare workers (HCWs) can result in nosocomial transmission, depletion in available workforce, and enhanced community transmission. This article describes surveillance for COVID-19 in HCWs at a tertiary healthcare facility, and documents the outcomes. A descriptive cross-sectional study of all HCWs identified from surveillance for COVID-19 from March 31 to August 31, 2020 was conducted. Healthcare workers were categorized as high risk and low risk using an adapted WHO Risk Assessment tool. Nasopharyngeal and oropharyngeal swab specimens obtained from high-risk subjects were tested by a reverse transcriptase PCR method. Data were analyzed with IBM SPSS version 25.0 software (IBM SPSS Statistics for Windows, Version 25.0, Armonk, NY), and results were presented as frequencies and percentages. The level of significance was set at P < 0.05. During 5 months of surveillance, 1,466 HCWs with a mean age of 38.1 ± 9.7 years were identified as contacts. On risk assessment, 328 (22.4%) were adjudged high risk. High risk was associated with increasing age (P < 0.001), male gender (P = 0.001), and nonclinical staff (P = 0.002). Following testing, 78 (5.3%) in the high-risk category were confirmed to have COVID-19. There was no record of COVID-19 in HCWs adjudged low risk. Forty-four (56.4%) cases were epidemiologically linked to the community, 20 (25.7%) to patients, and 14 (17.9%) to another HCW. Surveillance and risk assessment are crucial to COVID-19 response in healthcare facilities and revealed HCW infections with predominantly nonoccupational epidemiological links in this study.

The suitability of using a modified real-time reverse transcriptase PCR method in the detection of Enterovirus in respiratory samples from Iraq

The suitability of using a modified real-time reverse transcriptase PCR method in the detection of Enterovirus in respiratory samples from Iraq

443. Comparison of Clinical Characteristics of Endemic vs. SARS-CoV-2 Coronavirus Infection in Patients Admitted to a Community Teaching Hospital

BackgroundEndemic coronaviruses [EC] (NL63, 229E, OC43, HKU1) have been identified in humans since the 1960s. Most are associated with mild upper respiratory tract infections (RTIs) but can be associated serious disease. More recently, novel coronaviruses associated with SARS, MERS, and COVID-19 caused by SARS-CoV-2 have emerged and are often associated with serious disease and death. With this report, we compare presenting clinical characteristics and some outcomes of patients hospitalized with RTIs caused by ECs or by SARS-CoV-2 (COVID-19).MethodsPatients admitted to the Summa Health System with RTIs associated with EC were identified via multiplex PCR method (BioFire™). Patients admitted with COVID-19 were identified by real-time, reverse transcriptase-PCR method using one of several platforms. Patients were selected from two existing quality improvement registries encompassing EC and COVID-19 cases. We compared clinical characteristics and outcomes of EC patients to those of COVID-19 patients using chi-squared tests for proportions and independent samples t-tests for means.ResultsShown in Table 1. Significant differences in patient characteristics with EC vs. COVID-19 included: older age, primarily Caucasian, smoking history, requiring O2 supplementation on admission, and having chronic heart, lung, or renal disease. Significant differences in patient characteristics with COVID-19 vs. EC included: admission from extended care facility, obesity, presenting with fever, gastrointestinal symptoms and/or myalgia, presence of radiographic infiltrates, abnormal liver tests, and longer length of stay (LOS). A non-significant difference was noted in 30 day mortality rates. Coronavirus OC43 accounted for 54% of EC strains. ConclusionThere were significant differences in clinical characteristics between the two groups with EC patients more likely to have lung disease (often COPD) and requiring admission for need of O2 supplementation. COVID-19 patients were more likely to present with a febrile illness associated with pneumonia and require longer LOS (often requiring O2 supplementation later in hospitalization). Patients admitted with COVID-19 present with different clinical characteristics than those with EC with numerically higher mortality rate.Disclosures All Authors: No reported disclosures

Differential Gene Expression Pattern of Drought Responsive Transcription Factors in Chickpea: An Expressional Analysis

Chickpea (Cicer arietinum L.) is an important grain crop mainly grown in arid and semi-arid regions of the world. Drought is the major factor limiting chickpea growth and productivity. Transcription factors (TFs) genes have been reported as key regulators of drought tolerance in plants. In this study, we determined the relative gene expression of transcription factors of WRKY, DREB2A, and CarNAC3 in three weeks seedlings of tolerant (MCC537) and susceptible (MCC674) genotypes under progressive water deficit through semi-quantitative reverse transcriptase PCR (SqRT-PCR) method. Furthermore, results of SqRT-PCR were more confirmed by quantitative PCR (qPCR). Relative gene expression analysis of the selected genes using qPCR revealed different dehydration-responsive expression patterns. The expression of WRKY gene was significantly induced in both tolerant and sensitive genotypes under severe drought stress (at the last time point). Also, in tolerant genotype, DREB2A gene was approximately expressed three, five and fourfold higher than control plants at three times points, respectively. Moreover, the expression level of CarNAC3 gene in this genotype increased four and twofold higher compared to the relative control at 2 and 4 days after stress, respectively. However, the expression of these genes in the susceptible genotype was constant or decreased relative to the control. Furthermore, the CarNAC3 and DREB2A genes showed higher and more effective expression than the WRKY gene. Overall, the results demonstrated that these TFs may play an important role to improve drought tolerance and have a potential to facilitate molecular breeding and development of drought-tolerant chickpea varieties.

Orthogonal analysis of dystrophin protein and mRNA as a surrogate outcome for drug development.

Aim: Detection of drug-induced dystrophin in patient muscle biopsy is a surrogate outcome measure for Duchenne muscular dystrophy. We sought to establish and validate an orthogonal approach to measurement of dystrophin protein and RNA in muscle biopsies. Materials & methods: Validated methods were developed for dystrophin western blotting, mass spectrometry, immunostaining and reverse transcriptase PCR of biopsy mRNA using muscle biopsy standards. Results: Both western blotting and mass spectrometry validated methods demonstrated good linearity, and acceptable precision and accuracy with a lower limit of quantitation at 1%. Immunostaining and reverse transcriptase PCR methods were shown to be reliable. Conclusion: The described orthogonal approach is sufficient to support measures of dystrophin as a surrogate outcome in a clinical trial.

Risk of otitis media with effusion (OME) in children by Pseudomonas aeruginosa

ObjectiveTwo third of children in the world experience otitis media with effusion (OME) at least once in their life. According to the importance of knowing OME reason in pediatrics, beside introduced probable bacterial and viral causal agents, Pseudomonas aeruginosa was evaluated either. MethodsIn this study, 42 middle ear fluid (MEF) samples were collected from pediatrics who confirmed OME by an ears, nose and throat (ENT) specialist. Samples were cultured on bacteriological media for bacterial growth and were either extracted for total DNA and RNA to be tested for introduced bacterial and viral agents with simple and Reverse Transcriptase PCR method within specific primers. ResultsTotal results from culture and molecular methods showed that the most prevalent infections were Pseudomonas aeruginosa and streptococcus pneumonia with 33.33% and 14.29% respectively. In total, 66.67% of patients were infected with bacteria, 11.9% with test viruses while in 21.42% of patients no infectious agents were detected. Influenza type A was the only virus was detected. ConclusionPseudomonas aeruginosa was the most prevalent agent while mostly detected in patients who were referred from the tropical and humid region. According to these results, it is highly recommended to know the pattern of OME infection in each area separately for more successful treatment.

Expression Profile of Cellular Retinol- Binding Protein IV (CRBP-IV) Gene in Rhode Island Red Chicken

The investigation was aimed to study expression profiling of Cellular Retinol- binding protein IV (CRBP-IV) in Rhode Island Red (RIR) chicken. Cellular retinol binding protein IV (CRBP-IV) belongs to the family of cellular retinol binding proteins and plays a major role in absorption, transport, and metabolism of vitamin A. Vitamin A is correlated with reproductive performance, so measured relative mRNA expression of CRBP IV gene in kidney, liver and oviduct tissues collected from ten numbers of birds belonging to different ages viz., 12, 32 and 40 weeks, by quantitative reverse transcriptase PCR (qRT-PCR) method and data was analyzed using JMP of SAS (2010). Analysis revealed that he expression of CRBP IV gene differed significantly among three age groups in kidney (P≤0.05) and oviduct (P≤0.1) in RIR chicken. Highest expression of CRBP IV gene was observed at 40 weeks in kidney with a 40-∆Ct value as 33.96±0.7 which was significantly more than those at 32 weeks and 12 weeks, where the estimates were 29.09±0.70 and 27.81±0.60, respectively. These data may help to understand the role of CRBP IV gene on the laying period of Rhode Island Red Chicken.

The Serum Exosome Derived MicroRNA−135a, −193b, and −384 Were Potential Alzheimer's Disease Biomarkers

ObjectiveMicroRNAs (miRs) are attractive molecules to be considered as one of the blood-based biomarkers for neurodegenerative disorders such as Alzheimer's disease (AD). The goal of this study was to explore their potential value as biomarkers for the diagnosis of AD. MethodsThe expression levels of exosomal miR−135a, −193b, and −384 in the serum from mild cognitive impairment (MCI), dementia of Alzheimer-type (DAT), Parkinson's disease with dementia (PDD), and vascular dementia (VaD) patients were measured with a real-time quantitative reverse transcriptase PCR (qRT-PCR) method. ResultsBoth serum exosome miR-135a and miR-384 were up-regulated while miR-193b was down-regulated in serum of AD patients compared with that of normal controls. Exosome miR-384 was the best among the three miRs to discriminate AD, VaD, and PDD. Using the cut-off value could better interpret these laboratory test results than reference intervals in the AD diagnosis. ROC curve showed that the combination of miR−135a, −193b, and −384 was proved to be better than a particular one for early AD diagnosis. ConclusionOur results indicated that the exosomal miRs in the serum were not only potential biomarker of AD early diagnosis, but might also provide novel insights into the screen and prevention of the disease.

Abstract 4648: Development of a rapid, precise, and sensitive molecular assay for ALK fusion detection

Abstract Introduction In non-small cell lung cancer (NSCLC), chromosomal translocation events that result in overactivation of the anaplastic lymphoma kinase (ALK) have been identified and shown to play a key role in tumorigenesis. Inhibitors of the ALK kinase, such as the FDA-approved crizotinib, are effective in treating ALK-fusion-positive non-small cell lung cancer. Molecular studies have identified many ALK fusion variants in various cancer types, encompassing a combination of 22 different gene partners as well as various break points within the partner genes. The most common fusion events in non-small cell lung cancer are between the echinoderm microtubule associated protein like 4 (EML4) gene and the ALK gene. Fusion events between the Kinesin Family Member 5B (KIF5B) gene and ALK have also been identified. Existing assays for ALK fusion detection include the time-consuming, costly, and challenging fluorescence in situ hybridization (FISH) method, which is the current gold standard. Next generation sequencing approaches are also available, which are also costly to implement, difficult to interpret, and have lengthy workflows. Reverse-transcriptase PCR methods are ideal, as they are cost-effective, sensitive, rapid, and require little interpretation expertise. Methodology We designed a multiplexed real-time RT-PCR method to enable indiscriminate detection of ten EML4-ALK and three KIF5B-ALK fusion variants (encompassing approximately 95% of EML4-ALK and 90% of KIF5B-ALK characterized fusion variants, based on the Catalogue of Somatic Mutations in Cancer (COSMIC) database). The assay uses standard real-time PCR instrumentation and was characterized using the Bio-Rad CFX384™, Roche LightCycler® 96, and Roche LightCycler® 480 II instruments. Assay preparation and run time requires 2 hours or less. Sensitivity testing showed that for the Roche LightCycler® 480 II and Bio-Rad CFX384™, 100% detection was achieved for 50 copies of fusion template for each of the 13 targeted variants. The Roche LightCycler® 96 showed slightly poorer sensitivity, with 100% detection observed at 50 copies for 9 fusion variants and 100 copies for the remaining four variants. Inter- and intra-run precision testing demonstrated good reproducibility and repeatability, with a coefficient of variation of less than 10% for all targeted fusion variants. Using ALK-positive and ALK-negative FFPE reference materials, it was demonstrated that 50 ng of total FFPE RNA was sufficient and up to approximately 200 ng showed good performance. Conclusion In summary, we have developed a rapid, precise, and sensitive assay for the detection of thirteen EML4-ALK and KIF5B-ALK fusion events to enable molecular characterization of non-small cell lung cancer tumors. Citation Format: Mona D. Shahbazian, Yulei Shang, Maidar Jamba, Michael J. Powell. Development of a rapid, precise, and sensitive molecular assay for ALK fusion detection [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 4648. doi:10.1158/1538-7445.AM2017-4648

Diagnostic and Prognostic Implications of Prohibitin Overexpression in Normal Karyotype Acute Myeloid Leukemia

Abstract Background: Prohibitin (PHB) is a highly conserved protein, widely expressed in multiple cellular compartments such as mitochondria and nucleus. Recent studies have demonstrated that PHB played an important role in the regulation of mitochondrial function, cell proliferation and development. Our proteome analysis has revealed PHB was highly expressed in primary AML cells compared with normal cells. This finding prompted us to investigate whether PHB could serve as a potential diagnostic, prognostic, and therapeutic target in AML. Patients and Methods: PHB2 protein expression was assessed in 133 newly diagnosed normal karyotype AML (NK-AML) patients by using immunohistochemical staining (IHCS) on paraffin-embedded bone marrow sections. The degree of PHB2 protein expression was scored as 0-8 as a sum of a diffuseness score (0-5) and an intensity score (0-3). Overall survival (OS) and event-free survival (EFS) was analyzed according to PHB2 expression score. Also, we developed quantitative reverse transcriptase PCR (qRT-PCR) method to measure mRNA expression of PHB1 and PHB2 genes for the optimal algorithms for the discrimination of AML cells from the normal blood or hematopoietic cells. Because accumulation of beta-catenin in the nucleus has been identified in leukemic cells, we investigated the effect of beta-catenin accumulation on PHB expression. We monitored the expression levels of PHB with lithium chloride treatment, which blocks GSK-3beta activity and ultimately affects beta-catenin accumulation. For the development of anti-leukemic drug candidates targeting PHB in AML cells, we synthesized two potent chemical substances that can alkylate PHB: cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU). We evaluated their therapeutic effects in the primary AML and AML cell lines. Results: In the PHB2 IHCS, using the cutoff as positivity score 4.0, PHB2 positive patients showed inferior OS (23.7% vs 50.0%; P = 0.026) and high hazard ratio (1.808; P = 0.028) compared to PHB2 negative patients (figure 1). Using the same cutoff, PHB2 positive patients showed inferior EFS (20.7% vs 47.1%; P= 0.024) and high hazard ratio (1.795; P = 0.026) (figure 2). In the qRT-PCR assay of PHB1 and PHB2, quantitative expression (ddCt) of PHB1 gene was higher in the control group: 0.31-28.32 (4.40+/-5.80; Mean+/-SD) versus 0.22-6.52 (1.83+/-1.56) in patient group. Meanwhile, PHB2 gene expression was higher in the NK-AML patient group: 0.15-3.49 (0.80+/-0.47) versus 0.00-0.46 (0.16+/-0.13) in control group. Using linear discrimination analysis, we derived a linear equation for discrimination between normal and AML cells. Estimating the prevalence of AML as 57.2/1,000,000, the sensitivity of newly developed algorithm was 93.6%, specificity was 76.9%, negative predictive value was 99.9%, and positive predictive value was 2.28% (figure 3). PHB protein expression was increased by inhibition of the MAPK pathway and decreased by activation of EGF signal. We identified the TCF-4/LEF-1 binding motif, CATCTG, in the promoter region of PHB by site-directed mutagenesis and ChIP assay. This beta-catenin-mediated activation of PHB expression was independent of c-MYC activation, a product of Wnt signaling. These data indicated that PHB was a direct target of beta-catenin and the increased level of PHB in leukemic cells can be regulated by Wnt signaling. Using CCEU and ICEU, time and dose dependent manner of proliferation suppression was observed in primary AML and AML cell lines. Notable morphological transformation of AML cells was observed when treated with 10-100 umol of CCEU and ICEU for 24 hours. The half maximal inhibitory concentration (IC50) was 25 umol for most AML cells. Conclusions: Overexpression of PHB2 was associated with poor prognosis in NK-AML. Newly developed discrimination algorithm using qRT-PCR showed the possibility as a rapid diagnostic tool for leukemic cell identification. Our functional study suggested that the elevated level of PHB in leukemic cells was the result of Wnt signals which were up-regulated in AML cells. Finally, this study developed novel alkylating chemotherapeutics targeting PHB for selective killing of leukemic cells. PHB2 expression status can provide clinically valuable information for the diagnosis, prediction of the prognosis, detection of minimal residual disease, and therapeutic targeting of NK-AML. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

Evaluation and validation of reference genes for qPCR analysis to study climate change-induced stresses in Sinularia cf. cruciata (Octocorallia: Alcyonidae)

Coral reef organisms, including octocorals, are facing the consequences of anthropogenic activities, such as increasing oceanic pH and sea surface temperature, threatening their long-term survival and well-being. Gene expression studies based on quantitative PCR (qPCR) are important tools to provide insight into the molecular basis of octocoral stress responses and their potential resilience mechanisms. Nevertheless, a lack of experimentally validated, stably expressed reference genes for the normalization of gene expression using quantitative reverse transcriptase PCR (qPCR) method limit such investigations among octocorals. Here, assessment of the expression stability of seven candidate reference genes was performed using a palette of statistical tools for valid qPCR-based gene expression studies on the octocoral Sinularia cf. cruciata during thermal (34°C) and low-pH (pH7.5) stress and the most suitable set of reference genes was determined for such experiments. The reliability of the selected reference genes was confirmed in a qPCR assay that targeted the heat shock protein 70 (HSP70) gene. The HSP70 gene was found to be significantly upregulated during thermal stress, whereas during low-pH stress the expression level of this gene decreased. This study provides experimentally validated stress-specific sets of stably expressed reference genes during climate change-induced stresses, which will benefit future gene expression studies on Sinularia cf. cruciata as well as other octocorals. These results also highlight potentially different acclimation strategies of octocorals to different sources of abiotic stresses, contributing to our understanding of the potential for the adaptation of coral reef organisms to a changing world.