Quantitative Real-time Polymerase Chain Reaction Analysis Research Articles

Molecular cloning and characterization of genes involved in rosmarinic acid biosynthesis from Prunella vulgaris.

Prunella vulgaris L., commonly known as "self-heal" or "heal-all," is a perennial herb with a long history of medicinal use. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme-A (CoA) ligase (4CL) are important enzymes in the phenylpropanoid pathway and in the accumulation of rosmarinic acid (RA), which is a major secondary metabolite in P. vulgaris. In this study, we isolated cDNAs encoding PvPAL, PvC4H, and Pv4CL from P. vulgaris using rapid amplification of cDNA ends polymerase chain reaction (PCR). The amino acid sequence alignments of PvPAL, PvC4H, and Pv4CL showed high sequence identity to those of other plants. Quantitative real-time PCR analysis was used to determine the transcript levels of genes involved in RA biosynthesis in the flowers, leaves, stems, and roots of P. vulgaris. The transcript levels of PvPAL, PvC4H, and Pv4CL1 were the highest in flowers, whereas Pv4CL2 was the highest in roots. High-performance liquid chromatography analysis also showed the highest RA content in the flowers (3.71 mg/g dry weight). We suggest that the expression of the PvPAL, PvC4H, and Pv4CL1 genes is correlated with the accumulation of RA. Our results revealed that P. vulgaris flowers are appropriate for medicinal usage, and our findings provide support for increasing RA production in this plant.

Influence of BCL2 gene in major depression susceptibility and antidepressant treatment outcome

BackgroundOur recent work indicated that low-expression of the anti-apoptotic protein B-cell/lymphoma 2 (Bcl-2) mRNA was observed among untreated major depressive disorder (MDD) patients, and the subsequent altered level of Bcl-2 was found to be close to the antidepressant treatment outcome. The primary aim of this present study was to examine whether a particular gene, encoding Bcl-2 (BCL2) confers risk to MDD, and likewise to investigate whether this gene acts as an indicator of antidepressant treatment outcome. MethodsWe enrolled 178 treatment-resistant depression (TRD) and 612 non-treatment-resistant depression (NTRD) patients as well as 725 healthy controls. In total, three selected tagging SNPs (tagSNPs) of BCL2 (rs2279115, rs1801018 and rs1564483) were genotyped to test for possible association. Using TaqMan relative quantitative real-time polymerase chain reaction (PCR), we analyzed leukocytic expression of BCL2 mRNA in 47 healthy subjects. ResultsOf the three SNPs, we observed no significant differences in genotype and allele frequencies between the MDD and control groups as well as between the TRD and NTRD groups. However, we found a significant association between the rs2279115C allele and TRD in males (corrected P=0.048) but not in females. Further real-time quantitative PCR analysis in healthy subjects revealed that the rs2279115 polymorphism significantly influenced BCL2 mRNA expression (P=0.03). LimitationsThis is a preliminary investigation with relatively small sample size and cross-sectional design. ConclusionsThese initial findings strengthen the hypothesis that BCL2 may play an important role in mediating the outcome of antidepressant treatment, a result that may further be confirmed by future genetic studies from large-scale populations that can overcome the limited sample size of this preliminary finding.

Silencing in Apolygus lucorum of the olfactory coreceptor Orco gene by RNA interference induces EAG response declining to two putative semiochemicals

Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) is an agronomically important pest that causes severe economic damage to the cotton, fruit, and vegetable industries. Similar to other insects, A. lucorum can perceive and discriminate olfactory cues. A highly conserved and broadly expressed olfactory coreceptor (Orco) is crucial for insect olfaction, and Orco orthologs have been identified in several insect species. In this study, a homology-based polymerase chain reaction (PCR) method was utilized to identify AlucOrco, an Orco ortholog essential for olfaction in A. lucorum. AlucOrco shares significant sequence homology with known Orco proteins in other insects. Quantitative real-time PCR (qRT-PCR) analysis revealed that AlucOrco was abundantly expressed in adult A. lucorum. AlucOrco expression level was the highest in the antennae; by contrast, AlucOrco showed negligible expression level in other tissues. We injected AlucOrco siRNA into the conjunctivum between the prothorax and mesothorax of A. lucorum and evaluated its expression 36h after RNA interference. The results of qRT-PCR demonstrated that the level of mRNA expression was significantly reduced (>90%) in AlucOrco siRNA-treated A. lucorum than in water-injected and non-injected controls. The electroantennogram responses of A. lucorum to two putative semiochemicals, trans-2-hexenal and trans-2-hexenyl butyrate, were also reduced significantly (∼80%) in RNAi-treated A. lucorum than in the controls. These results suggest that AlucOrco is crucial in mediating odorant perception of A. lucorum, especially in perceiving trans-2-hexenal and trans-2-hexenyl butyrate.

Absolute dominance of hydrogenotrophic methanogens in full-scale anaerobic sewage sludge digesters

Anaerobic digestion (AD) is gaining increasing attention due to the ability to covert organic pollutants into energy-rich biogas and, accordingly, growing interest is paid to the microbial ecology of AD systems. Despite extensive efforts, AD microbial ecology is still limitedly understood, especially due to the lack of quantitative information on the structures and dynamics of AD microbial communities. Such knowledge gap is particularly pronounced in sewage sludge AD processes although treating sewage sludge is among the major practical applications of AD. Therefore, we examined the microbial communities in three full-scale sewage sludge digesters using qualitative and quantitative molecular techniques in combination: denaturing gradient gel electrophoresis (DGGE) and real-time polymerase chain reaction (PCR). Eight out of eleven bacterial sequences retrieved from the DGGE analysis were not affiliated to any known species while all eleven archaeal sequences were assigned to known methanogen species. Quantitative real-time PCR analysis revealed that, based on the 16S rRNA gene abundance, the hydrogenotrophic order Methanomicrobiales is the most dominant methanogen group (> 94% of the total methanogen population) in all digesters. This corresponds well to the prevailing occurrence of the DGGE bands related to Methanolinea and Methanospirillum, both belonging to the order Methanomicrobiales, in all sludge samples. It is therefore suggested that hydrogenotrophic methanogens, especially Methanomicrobiales strains, are likely the major players responsible for biogas production in the digesters studied. Our observation is contrary to the conventional understanding that aceticlastic methanogens generally dominate methanogen communities in stable AD environments, suggesting the need for further studies on the dominance relationship in various AD systems.

Downregulation of PTEN expression in psoriatic lesions

Considerable studies showed that tumor suppressor phosphatase and tensin homolog (PTEN) deleted on chromosome 10 is a major tumor suppressor, which inhibits cell proliferation through inactivation of the PI3 kinase (PI3K)/Akt signal pathway. In many human tumors, there is loss of function or mutations in PTEN. In our previous study, it was found that Akt activity in psoriatic lesions increased compared with that in normal controls. However, the expression of PTEN and the correlation between PTEN and PI3K/Akt have not been investigated in patients with psoriasis. Skin tissue samples were obtained from 18 patients with psoriasis and 19 healthy controls. The expressions of PTEN were measured with quantitative real-time polymerase chain reaction (RT-PCR) assay, Western blotting, and immunohistochemistry. Quantitative RT-PCR analysis demonstrated that the mRNA levels of PTEN in psoriatic lesions were decreased in comparison with that in normal skin. Western blotting and immunohistochemistry analysis of PTEN showed that PTEN protein was lowly expressed in psoriatic lesions compared with that in normal controls, which suggested that the reduction of PTEN mRNA expression leads to decreased PTEN protein levels. The downregulation of PTEN expressions may play a role in the overactivation of the PI3K/Akt pathway in psoriatic lesions and correlate with the hyperproliferation of psoriatic keratinocytes.

Real-Time Quantitative Polymerase Chain Reaction (RQ-PCR) on Peripheral Blood (PB) and Bone Marrow (BM) Samples for Monitoring Minimal Residual Disease (MRD) in Patients (pts) With Acute Promyelocytic Leukemia (APL) Treated With All-Trans-Retinoic Acid (ATRA) and Arsenic Trioxide (ATO)

Abstract 2623 Background: Monitoring minimal residual disease (MRD) using reverse transcriptase polymerase chain reaction (RT-PCR) for the PML-RARA fusion transcript in bone marrow (BM) samples has been used to predict relapse in patients (pts) with acute promyelocytic leukemia (APL). Rigorous, serial real-time quantitative PCR (RQ-PCR) has been shown to be the strongest predictor of relapse-free survival in APL (Grimwade D, JCO, 2009), and BM was more sensitive than peripheral blood (PB) for detecting pending relapse. However, the clinical utility of MRD monitoring and use of PB as a sample source for MRD detection remains to be established for APL pts treated with ATRA and ATO without chemotherapy. Purpose: To evaluate the reliability of using PB real-time quantitative PCR (RQ-PCR) in monitoring MRD in patients with APL treated with ATRA and ATO ± gemtuzumab ozogamicin (GO). Methods: From June 2007 to August 2011, 78 pts with newly diagnosed APL were treated on a clinical trial (NCT00413166) combining ATRA and ATO at our institution. High risk pts (WBC > 10 × 109/L) and pts with rising WBC also received GO on the first day or when WBC rose above 10, respectively. RQ-PCR for PML-RARα fusion transcript was performed on BM and PB after induction, every 3 months throughout consolidation (8 months of ATRA and ATO) for the first 2 years and then PB and/or BM analysis was performed every 6 months with follow up. The Spearman correlation and Paired Wilcoxon test were used to analyze the relation, generate the correlation coefficient and analyze the differences between BM and PB RQ-PCR values. Results: A total of 432 PB and 618 BM samples were obtained for RQ-PCR analysis. The median number of PB RQ-PCR and BM RQ-PCR obtained per patient were 6 (range, 0–16) and 8 (range, 0–14), respectively. Two hundred thirty six samples were obtained concomitantly from PB and BM (defined as obtained within a day of each other) from 63 patients. The median time to CR was 30 days (range, 9–63). The median time to complete molecular response (CMR) was 130 days (range, 20–271). Two patients died, 1 during induction (before assessing his response to treatment), the other one during 4th consolidation while in CR of unknown cause. Two pts have relapsed, one of them with CNS disease only, the other one with systemic then CNS relapse. Both patients had previously achieved CMR. The patient with CNS relapse remained RQ-PCR-negative on BM at the time of relapse (PB not available at time of relapse). The other relapsed patient had a positive PB RQ-PCR at time of relapse. There was a strong correlation (Spearman correlation, r=0.8, p Conclusions: Among the available paired samples, there is a strong correlation between BM and PB RQ-PCR for PML-RARα fusion transcripts for patients treated with ATRA+ATO. PB RT-PCR is a reliable and practical method to monitor pts in CMR and for possible impending relapse. Disclosures: Off Label Use: Arsenic trioxide is used off label for the treatment of acute promyelocytic leukemia.

Saliva Polymerase Chain Reaction Assay for Detection and Follow-up of Herpesvirus Reactivation in Patients With Drug Reaction With Eosinophilia and Systemic Symptoms (DRESS)

Reactivations of human herpesviruses (HHVs) contribute to the development of drug reaction with eosinophilia and systemic symptoms (DRESS). Diagnosis of HHV reactivation is conventionally based on quantitative real-time polymerase chain reaction (PCR) analysis of blood samples, but these viruses are present in the oropharynx and are shed in saliva. To evaluate the use of a saliva PCR assay for demonstrating HHV shedding in patients with DRESS. Shedding of HHV in saliva was prospectively studied in patients with DRESS. Reactivations of HHV, including HHV-6, HHV-7, cytomegalovirus (CMV), and Epstein-Barr virus (EBV), were studied by performing quantitative real-time PCR analysis of blood samples obtained at admission) and serial samples of saliva obtained within the first 2 weeks of DRESS; saliva samples from controls were compared. The study included 5 patients who met definite criteria for DRESS and 15 controls (5 immunosuppressed patients and 10 healthy adults). DNA viral loads of HHV, including HHV-6, HHV-7, CMV, and EBV as measured with real-time PCR in blood and saliva samples from patients with DRESS and saliva samples from immunosuppressed and healthy controls. The PCR assay demonstrated shedding of HHV-7, EBV, HHV-6, and CMV, listed by order of magnitude. The DNA viral loads in blood and saliva samples, also measured with real-time PCR, were found to be close. In 1 patient, reactivations in saliva preceded clinical manifestations of CMV disease. Significant production of HHV-7 and EBV was demonstrated in saliva samples from the controls, but neither HHV-6 nor CMV were detected. The saliva PCR assay is a useful tool for demonstration and follow-up of HHV reactivation. The interpretation of HHV viral loads in saliva differs according to HHV type.

Effects of Jianpi Jiedu Recipe (健脾解毒方) on reversion of P-glycoprotein-mediated multidrug resistance through COX-2 pathway in colorectal cancer

To evaluate the underlying mechanism of Jianpi Jiedu Recipe (, JJR) in the reversion of multidrug resistance concerning colorectal cancer in vitro and in vivo. Mice were treated orally with JJR at a daily 4.25 g/(kg·day) or injected with vinblastine (VCR) 2.5 mg/(kg·day) for 3 weeks after having been inoculated with HCT8/V cells; tumor tissues were assayed by hematoxylin and eosin staining. Firstly, the effects of JJR on the expression of cyclooxygenase-2 (COX-2) were tested by real-time polymerase chain reaction (PCR) technique and COX-2 gene silenced by siRNA. Secondly, the variation of intracellular concentration of oxaliplatin (L-OHP) was evaluated by the inductively coupled plasma mass spectroscopy (ICPMS) in HCT8/V and its COX-2 siRNA cells; the concentration of JJR combined with chemotherapeutic drugs and the reverse effect of multidrug resistance (MDR) in HCT8/V cells was evaluated by the MTT assay. Thirdly, real-time quantitative PCR and Western blot analysis were used to detect the multidrug resistance gene 1 (MDR1) mRNA and P-gp expression. JJR had an inhibitory effect on the growth of tumors in vivo, and it, in combination with chemotherapeutic drugs, could reverse the drug-resistance of HCT8/V cells and increase the sensitivity of HCT8/V cells to VCR, DDP, 5-Fu, and THP. ICP-MS results showed that JJR could increase the concentration of drugs in HCT8/V cells (P<0.01). Furthermore, it was shown that JJR could reverse drug resistance of colorectal cancer cells by decreasing MDR1 expression and P-gp level via downregulation of COX-2, which has been represented as one of the major mechanisms that contributes to the MDR phenotype (P<0.01). JJR reversed multidrug resistance and enhanced the sensitivity to chemotherapy, which could be attributed to the down-regulation of COX-2 in MDR1/P-gp-mediated MDR colorectal cancer after chemotherapy.

Accuracy of quantitative real-time PCR analysis

When solving many problems in biology and med� icine, it is necessary to detect the presence of specific nucleic acid sequences in samples. For this purpose, polymerase chain reaction (PCR)—a chain enzy� matic reaction of multiplication of the analyzed DNA fragment, realized in the form of successive rounds of duplication of fragments present in the reaction—is commonly used. In addition to identification of a specific DNA sequence, it is often necessary to determine its con� centration in the sample. At present, the most com� mon method for quantitative analysis of nucleic acids

Real-Time Quantitative Polymerase Chain Reaction (RQ-PCR) On Peripheral Blood (PB) and Bone Marrow (BM) Samples for Monitoring Minimal Residual Disease (MRD) in Patients (pts) with Acute Promyelocytic Leukemia (APL) Treated with All-Trans-Retinoic Acid (ATRA) and Arsenic Trioxide (ATO).

Abstract 2623▪▪This icon denotes a clinically relevant abstract Background:Monitoring minimal residual disease (MRD) using reverse transcriptase polymerase chain reaction (RT-PCR) for the PML-RARA fusion transcript in bone marrow (BM) samples has been used to predict relapse in patients (pts) with acute promyelocytic leukemia (APL). Rigorous, serial real-time quantitative PCR (RQ-PCR) has been shown to be the strongest predictor of relapse-free survival in APL (Grimwade D, JCO, 2009), and BM was more sensitive than peripheral blood (PB) for detecting pending relapse. However, the clinical utility of MRD monitoring and use of PB as a sample source for MRD detection remains to be established for APL pts treated with ATRA and ATO without chemotherapy. Purpose:To evaluate the reliability of using PB real-time quantitative PCR (RQ-PCR) in monitoring MRD in patients with APL treated with ATRA and ATO ± gemtuzumab ozogamicin (GO). Methods:From June 2007 to August 2011, 78 pts with newly diagnosed APL were treated on a clinical trial (NCT00413166) combining ATRA and ATO at our institution. High risk pts (WBC > 10 × 109/L) and pts with rising WBC also received GO on the first day or when WBC rose above 10, respectively. RQ-PCR for PML-RARα fusion transcript was performed on BM and PB after induction, every 3 months throughout consolidation (8 months of ATRA and ATO) for the first 2 years and then PB and/or BM analysis was performed every 6 months with follow up. The Spearman correlation and Paired Wilcoxon test were used to analyze the relation, generate the correlation coefficient and analyze the differences between BM and PB RQ-PCR values. Results:A total of 432 PB and 618 BM samples were obtained for RQ-PCR analysis. The median number of PB RQ-PCR and BM RQ-PCR obtained per patient were 6 (range, 0–16) and 8 (range, 0–14), respectively. Two hundred thirty six samples were obtained concomitantly from PB and BM (defined as obtained within a day of each other) from 63 patients. The median time to CR was 30 days (range, 9–63). The median time to complete molecular response (CMR) was 130 days (range, 20–271). Two patients died, 1 during induction (before assessing his response to treatment), the other one during 4th consolidation while in CR of unknown cause. Two pts have relapsed, one of them with CNS disease only, the other one with systemic then CNS relapse. Both patients had previously achieved CMR. The patient with CNS relapse remained RQ-PCR-negative on BM at the time of relapse (PB not available at time of relapse). The other relapsed patient had a positive PB RQ-PCR at time of relapse. There was a strong correlation (Spearman correlation, r=0.8, p <0.05) between PB and BM RQ-PCR on the 236 concomitant samples (fig 1); no significant difference were found between quantitative PCR values for BM and PB by paired Wilcoxon test analysis (p = 0.30, paired Wilcoxon test). The patient with systemic relapse (who had secondary APL) was successfully retreated with ATRA+ATO, then with systemic and intrathecal (IT) chemotherapy prior to allogeneic stem cell transplant at second relapse. The patient with CNS relapse was successfully treated with IT chemotherapy. Conclusions:Among the available paired samples, there is a strong correlation between BM and PB RQ-PCR for PML-RARα fusion transcripts for patients treated with ATRA+ATO. PB RT-PCR is a reliable and practical method to monitor pts in CMR and for possible impending relapse. [Display omitted] Disclosures:Off Label Use: Arsenic trioxide is used off label for the treatment of acute promyelocytic leukemia.

Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis

Strongyle and trichostrongyle parasites are ubiquitous nematodes of grazing livestock. Several molecular diagnostic tests are based upon measuring and quantifying DNA obtained from parasite eggs. It is well known that such eggs undergo development during storage, but it remains unknown to which extent developmental stages can affect the variation of diagnostic test results. This study investigated the influence of developmental stages of strongyle eggs on the variation real-time polymerase chain reaction (PCR) results. Mixed species strongyle eggs were obtained from the faeces of a naturally infected horse. Eggs were isolated and placed in microtiter plates with demineralized water. A total of 25 wells containing 100 eggs each were set up and kept refrigerated for up to five days. Once daily, five wells were examined on an inverted microscope at 100× magnification, where the developmental stages of the eggs were noted, and then eggs harvested for DNA extraction. The protocol was repeated three times. Genomic DNA was extracted using a commercial kit previously validated for strongyle type eggs. PCR reactions were performed with a primer set specific for the ribosomal DNA region for all strongyle type parasites (NC1, NC2). SYBR Green Real-Time PCRs were performed in triplicates. Results revealed a statistically significant increase in PCR yield after three days, which was statistically associated with beginning embryonation of the eggs. In conclusion, storage time and developmental stage of strongyle eggs are significant sources of error in studies based on quantitative real-time PCR analysis. This study suggests that for refrigerated storage of more than three days, eggs should be inactivated and preserved for further analysis.

Biomarkers of Organic Contamination in the South American Fish Poecilia vivipara and Jenynsia multidentata

South American cyprinodontiform fish are potential candidates to be used as model biomarker species of exposure in environmental toxicology. The aim of this study was to identify molecular and biochemical biomarkers of pollution using Poecilia vivipara (Poecilidae) and Jenynsia multidentata (Anablepidae). Partial nucleotide sequences for cytochrome P-450 1A (CYP1A), a classical biomarker of exposure to organic contaminants in fish, were identified in P. vivipara and J. multidentata (approximately 650 nucleotides) using degenerated primers and polymerase chain reaction (PCR). These sequences shared approximately 90% identity in the predicted amino acid sequence with the corresponding CYP1A region of Fundulus heteroclitus. Real-time quantitative PCR (RT-qPCR) analysis confirmed that CYP1A transcription was markedly induced in the liver and gills of J. multidentata (approximately185-fold and 20-fold, respectively) and P. vivipara (122-fold and 739-fold, respectively) 24 h after exposure to 1 μM synthetic CYP1A inducer β-naphthoflavone (BNF). At 24 h after injection with 1 μg/g environmental carcinogenic contaminant benzo[a]pyrene (BaP), a decreased total antioxidant capacity against peroxyl radicals was observed both in liver of J. multidentata and gills of P. vivipara. BaP injection in both fish did not produce changes in lipid peroxide (thiobarbituric acid-reactive substances, TBARS) levels, suggesting an absence of an oxidative stress condition. The newly identified CYP1A may thus serve as general biomarker of exposure to organic contaminant in future studies using P. vivipara and J. multidentata. Data also indicate the importance of species-specific differences in biomarker responses in these South American cyprinodontiform fish, suggesting distinct resistance/susceptibility properties to polycyclic aromatic hydrocarbons.

Involvement of Corticotrophin‐Releasing Hormone and Corticosteroid Receptors in the Brain–Pituitary–Gill of Tilapia During the Course of Seawater Acclimation

The mRNA expression of genes for corticotrophin-releasing hormone (CRH) and the hormone receptors CRH-receptor/CRH-R, glucocorticoid receptor 1/2 (GR1/2) and mineralocorticoid receptor (MR) was studied in the brain, pituitary and gill of tilapia (Oreochromis mossambibus) during salinity and handling stress by real-time quantitative-polymerase chain reaction analysis. The results indicated that the transcripts of CRH and CRH-R were increased in the forebrain, midbrain and gill, whereas elevated hypothalamic CRH mRNA suppressed the CRH-R mRNA in the pituitary in seawater (SW) fish. The levels of plasma osmolality and cortisol were significantly increased in SW compared to freshwater fish. The up-regulation of GR1, GR2, MR and α-NKA (Na(+) /K(+) -ATPase) transcripts in SW fish provided evidence that cortisol responds to stress and involves ion-base regulation via the GR1, GR2 and MR receptors in the gill. These data suggest that GR1, GR2 and MR have a pivotal role in the brain and gill. GR1, GR2 and MR expression may be dependent on CRH and cortisol expression in the brain and gill. In addition, we performed in situ hybridisation analysis to localise and differentiate the CRH, CRH-R, GR1, GR2 and MR transcripts in the brain of FW- and SW-acute acclimated tilapia during salinity stress. In almost all transcripts, the hybridisation signal was significantly abundant in the SW-acute acclimated tilapia brain, especially in the dorsal ventral cephalon, dorsal nucleus preopticus pars magnocellularis and dorsal nucleus preopticus pars parvocellularis. Salinity stress induced differential and specific responses in the gill and brain compared to handling stress.

Selection of Suitable Reference Genes for RT-qPCR Analyses in Cyanobacteria

Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR) is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works.

High Levels of Nuclear MYC Protein Predict the Presence of MYC Rearrangement in Diffuse Large B-cell Lymphoma

Determining the presence of MYC gene rearrangements is becoming an increasingly important part of the diagnostic workup in aggressive lymphoma. Cytogenetic MYC alterations aid in differentiating diffuse large B-cell lymphoma (DLBCL) from Burkitt lymphoma. In addition, MYC aberrations are associated with poor prognosis in DLBCL. Fluorescence in situ hybridization and karyotyping are standard tests for detecting MYC aberrations, but these techniques are laborious and expensive. Here, we studied MYC status of 219 DLBCLs and Burkitt lymphomas using fluorescence in situ hybridization, immunohistochemistry, and quantitative real-time polymerase chain reaction (QRT-PCR). Overall, 15% of the cases had an MYC break. QRT-PCR analysis of MYC expression showed that 72% of DLBCLs with an MYC break had aberrantly high or low levels of MYC transcript. Excluding the cases with aberrantly low MYC expression, we found a significant positive correlation between levels of MYC transcripts and MYC tumor cells; however, QRT-PCR is not readily applicable as a screening tool. Immunohistochemically, all tumors showed a nuclear staining pattern that was simple to evaluate. The percentage of MYC lymphoma cells correlated closely with MYC rearrangement status. In all, 93% of cases with an MYC break had ≥80% MYC cells, in contrast to 3% of nonrearranged cases (P<0.0001). Receiver operating characteristic curve analysis showed ≥70% MYC tumor cells to be the optimal cutoff (sensitivity=100%, specificity=93%). Area under the receiver operating characteristic curve was 0.992, indicating that immunostaining for Myc protein is an excellent screening test to predict whether an MYC rearrangement is present.

TCF/Lef1-Mediated Control of Lipid Metabolism Regulates Skin Barrier Function

Defects in the function of the skin barrier are associated with a wide variety of skin diseases, many of which are not well characterized at the molecular level. Using Lef1 (lymphoid enhancer-binding factor 1) dominant-negative mutant mice, we demonstrate here that altered epidermal TCF (T cell factor)/Lef1 signaling results in severe impairment of the stratum corneum skin barrier and early postnatal death. Barrier defects were accompanied by major changes in lipid composition and ultrastructural abnormalities in assembly and extrusion of lipid lamellae of the interfollicular epidermis, as well as abnormal processing of profilaggrin. In contrast, tight-junction formation and stratified organization of the interfollicular epidermis was not obviously disturbed in Lef1 mutant mice. Molecular analysis revealed that TCF/Lef1 signaling regulates expression of lipid-modifying enzymes, such as Elovl3 and stearoyl coenzyme A desaturase 1 (SCD1), which are key regulators of barrier function. Promoter analysis and chromatin immunoprecipitation experiments indeed showed that SCD1 is a direct target of Lef1. Together, our data demonstrate that functional TCF/Lef1 signaling governs important aspects of epidermal differentiation and lipid metabolism, thereby regulating skin barrier function.

RNA Stability in Human Liver: Comparison of Different Processing Times, Temperatures and Methods

The accuracy of information garnered by real-time quantitative polymerase chain reaction (RT-qPCR), an important technology for elucidating molecular mechanisms of disease, is dependent on tissue quality. Thus, this study aimed to determine the effects of intra-operative manipulation, extended processing times, different temperatures or storage in RNAlater on RNA quality in liver samples for tissue banking. Liver samples, flash-frozen or in RNAlater, were collected over a time course (during surgery before blood arrest up to 1 day after surgery) with samples kept either at room temperature (RT) or on ice. This study showed that at the longest time-point at RT, the RNA quality decreased significantly by 20%. However, relative gene expressions of FOS, GUSB, MYC, HIF1α and GFER were in general not significantly different when the time-points were compared. In conclusion, samples should be kept on ice during processing, and either RNAlater or snap-freezing should be utilised for storage. Further, intra-operative manipulation and extended postoperative processing time generally does not change relative gene expression levels for the 5 genes studied, making such sampling suitable for RT-qPCR analysis. Thus, if relative gene expression of a gene of interest is stable, these guidelines will lead to increased accrual of samples to the tissue bank.

Correlation between mitochondrial TRAP-1 expression and lymph node metastasis in colorectal cancer

To evaluate the effect of mitochondrial tumor necrosis factor receptor-associated protein-1 (TRAP-1) on the lymph node metastasis (LNM) in Chinese colorectal cancer (CRC) patients, and develop potential LNM-associated biomarkers for CRC using quantitative real-time polymerase chain reaction (RT-PCR) analysis. Differences in mitochondrial TRAP-1 gene expression between primary CRC with LNM (LNM CRC) and without LNM (non-LNM CRC) were assessed in 96 Chinese colorectal carcinoma samples using quantitative RT-PCR analysis, Western blotting, and confirmed with immunohistochemical assay. The relationship between clinicopathological parameters and potential diagnostic biomarkers was also examined. TRAP-1 was significantly upregulated in LNM CRC compared with non-LNM CRC, which was confirmed by RT-PCR, Western blotting and immunohistochemical assay. The expression of TRAP-1 in two different metastatic potential human colorectal cancer cell lines, LoVo and HT29, was analyzed with Western blotting. The expression level of TRAP-1 was dramatically higher in LoVo than in HT29. Overexpression of TRAP-1 was significantly associated with LNM (90.2% in LNM group vs 22% in non-LNM group, P < 0.001), the advanced tumor node metastasis stage (89.1% in LNM group vs 26.9% in non-LNM group, P < 0.001), the increased 5-year recurrence rate (82.7% in LNM group vs 22.6% in non-LNM group, P < 0.001) and the decreased 5-year overall survival rate (48.4% in LNM vs 83.2% in non-LNM group, P < 0.001). Univariate and multivariate analyses indicated that TRAP-1 expression was an independent prognostic factor for recurrence and survival of CRC patients (Hazard ratio of 2.445 in recurrence, P = 0.017; 2.867 in survival, P = 0.028). Mitochondria TRAP-1 affects the lymph node metastasis in CRC, and may be a potential biomarker for LNM and a prognostic factor in CRC. Over-expression of TRAP-1 is a predictive factor for the poor outcome of colorectal cancer patients.

Comparison Between RT-PCR and RQ-PCR for Minimal Residual Disease Detection in Acute Promyelocytic Leukemia: The International Consortium on Acute Promyelocytic Leukemia (IC-APL) Experience,

Abstract 3552The International Consortium on Acute Promyelocytic Leukemia (IC-APL) is an initiative of the International Members Committee of the ASH that aims to improve the treatment outcome of acute promyelocytic leukemia (APL) patients in developing countries, which was launched in Mexico, Brazil, Chile and Uruguay. The protocol is identical to the PETHEMA-LPA2005, except for the replacement of idarubicin by daunorubicin. In our interim analysis, estimated 2-year overall and disease-free survivals are 80% and 90%, respectively. The 2-year cumulative incidence of relapse was 5.6%. The median follow-up among survivors was 23 months (range: 1 – 56 months). A secondary aim of IC-APL was to establish molecular monitoring for minimal residual disease (MRD) as a standard practice for APL patients in these countries and to use the results obtained to guide therapy. According to the IC-APL protocol, testing for the PML-RARA fusion transcript was to be performed at diagnosis, end of induction (optional), after the third cycle of consolidation, and every 3 months during maintenance. Considering that real-time quantitative polymerase chain reaction (RQ-PCR) provides a number of advantages compared to conventional non-quantitative reverse transcriptase PCR (RT-PCR), we retrospectively compared the results obtained by both techniques. We analyzed 400 bone marrow (BM) samples from 97 patients with de novo APL enrolled in the IC-APL protocol in Brazil. Of the 97 patients, 78 were considered eligible. The mean age was of 35.8 years with 46 males. Among eligible patients, 49 corresponded to bcr1 ; one to bcr2 and 28 to bcr3 subtype of PML breakpoint. To quantify the fusion transcript PML-RARA we used standardized assays developed in the Europe Against Cancer (EAC) program, normalized to the expression of the ABL gene. The results were compared to plasmid standards (Ipsogen, Marseille) and expressed as Normalized Copy Numbers (NCN). Follow-up samples were considered PCR positive when PML-RARA transcripts amplified with Cycle Threshold (Ct) values of ≤40 in at least 2 out of 3 replicates, according to EAC criteria. A total of 71 samples at diagnosis, 50 at the end of induction, 47 after the third consolidation, 202 during maintenance phase and 30 samples after completion of treatment were analyzed. The median NCN of PML-RARA transcripts at diagnosis was 0.5151 and 0.5092 for the bcr1 and bcr3 subtypes, respectively. At the end of induction there was a reduction of about 3 logs (0.0004 for bcr1 and 0.0005 for bcr3). In this phase, six discrepant cases were observed, all presenting positivity by RQ-PCR. None of these cases relapsed and presented consecutive negative results. Considering samples obtained at the end of consolidation, we detected one case of molecular persistence detected by both methods, and two discrepant results, one positive by RT and another by RQ-PCR. Both cases did not relapse. Among samples collected during maintenance and after the end of treatment, two patients (2.5%) relapsed. Both of them were molecular relapses, defined as the detection (in the context of morphological remission) of the PML-RARA transcript by RT-PCR in two consecutive samples collected 15 days apart. RQ-PCR analysis provided much earlier warning of recurring disease, testing positive 5 and 6 months, respectively, before documentation of molecular relapse by conventional RT-PCR assay. Figure 1 show the kinetics of NCN in these two cases. Our results reinforce that the PML-RARA transcript may be detected after induction but this finding was not of prognostic value. However, our study underlines the importance of sequential monitoring to distinguish patients likely to be cured following front-line therapy from those destined to relapse. The RQ-PCR technique was shown to be more sensitive than RT-PCR, providing earlier warning of impending relapse, thereby allowing greater opportunity for successful delivery of pre-emptive therapy. Finally, our results demonstrate that the implementation of the IC-APL allowed the improvement of laboratory standards in parallel to advances in clinical management. [Display omitted] Disclosures:Pasquini:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Pagnano:Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau.

Deletion of estrogen receptor beta accelerates early stage of bone healing in a mouse osteotomy model

This study examined the role of estrogen receptor (ER) beta during mouse femoral fracture healing by employing ER knockout (KO) mice. The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification. This study was conducted to examine the role of ER beta during fracture healing. Female ERbeta knockout (KO) mice (18 weeks old) and age-matched female wild-type (WT) mice underwent open osteotomy on the right femur. They were sacrificed at 1, 2, 4 and 6 weeks post-fracture. The sera and callus samples were subjected to the following analyses: micro-computed tomography (CT)-based angiography, micro-CT evaluation, histological examination, histomorphometry examination, real-time polymerase chain reaction (PCR) analysis, biochemical marker, and mechanical testing. Micro-CT-based angiography showed that the total vessel volume at the fracture site was larger in the KO group than the WT group at 1 and 2 weeks post-fracture. Micro-CT analysis revealed that the callus volume was significantly higher in the KO group from week 2 to week 4 post-fracture when compared with the WT group consistent with the histological data. Analysis of biochemical markers indicated that circulating P1NP levels in the KO mice were significantly higher than in the WT mice from week 2 to week 4 and that temporal expression of circulating C-terminal telopeptide of type I collagen (CTX) levels was also higher in the KO mice than in the WT mice. These results were consistent with quantitative real-time PCR analysis. The ultimate load, stiffness, and energy to failure were significantly higher in the KO mice than in the WT mice at week 4. The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification, but not by the end of healing. Blockade of ERbeta can be considered as another therapeutic strategy for osteoporotic fracture and non-union fracture.