Real-time Polymerase Chain Reaction Approach Research Articles

Evaluation of two detection methods of microorganisms in platelet concentrates

The performance of a bacterial 16S ribosomal DNA real-time polymerase chain reaction (PCR) assay was evaluated and validated with an automated culture system to determine its use for screening of platelet concentrates (PCs). PCs were spiked with suspensions of Escherichia coli, Serratia marcescens, Staphylococcus epidermidis and St. aureus at 1, 10, and 100 colony-forming units (CFUs) mL and stored for 5 days. DNA amplification was performed using real-time PCR. The BacT/ALERT was used as a reference method and samples were inoculated into an aerobic culture bottle; for the PCR assay, aliquots were drawn from all (spiked) PCs on days 0 to 5 of storage. Real-time PCR detected only the gram-positive bacteria in PCs spiked with low bacterial titres (1 CFU mL) after 48 h; however, it was able to detect all positive samples in PCs spiked with 10 CFU mL of either gram-positive or gram-negative bacteria after 48 h. In addition, real-time PCR detected all positive samples in PCs spiked with high gram-positive bacterial titres (100 CFU mL) after 24 h. On the other hand, the BacT/ALERT system showed positive results in all samples within 24 h. The BacT/ALERT method is more sensitive and should continue to be the gold standard for identifying bacterial contaminations in blood samples. The real-time PCR approach can be used for the screening of PCs for microbial detection before they are released from blood centres or shortly before they are used in blood transfusion, and thus allow an extended shelf life of the platelets.

A quantitative real‐time polymerase chain reaction assay for the seagrass pathogen Labyrinthula zosterae

The protist Labyrinthula zosterae (Phylum Bigyra, sensu Tsui et al. 2009) has been identified as a causative agent of wasting disease in eelgrass (Zostera marina), of which the most intense outbreak led to the destruction of 90% of eelgrass beds in eastern North America and western Europe in the 1930s. Outbreaks still occur today, albeit at a smaller scale. Traditionally, L. zosterae has been quantified by measuring the necrotic area of Z. marina leaf tissue. This indirect method can however only lead to a very rough estimate of pathogen load. Here, we present a quantitative real-time polymerase chain reaction (qPCR) approach to directly detect and quantify L. zosterae in eelgrass tissue. Based on the internal transcribed spacer (ITS) sequences of rRNA genes, species-specific primers were designed. Using our qPCR, we were able to quantify accurately and specifically L. zosterae load both from culture and eelgrass leaves using material from Europe and North America. Our detection limit was less than one L. zosterae cell. Our results demonstrate the potential of this qPCR assay to provide rapid, accurate and sensitive molecular identification and quantification of L. zosterae. In view of declining seagrass populations worldwide, this method will provide a valuable tool for seagrass ecologists and conservation projects.

Quantitative analyses of ammonia-oxidizing Archaea and bacteria in the sediments of four nitrogen-rich wetlands in China

With the rapid development of ammonia-synthesizing industry, the ammonia-nitrogen pollution in wetlands acting as the sink of point and diffuse pollution has been increased dramatically. Most of ammonia-nitrogen is oxidized at least once by ammonia-oxidizing prokaryotes to complete the nitrogen cycle. Current research findings have expanded the known ammonia-oxidizing prokaryotes from the domain Bacteria to Archaea. However, in the complex wetlands environment, it remains unclear whether ammonia oxidation is exclusively or predominantly linked to Archaea or Bacteria as implied by specific high abundance. In this research, the abundance and composition of Archaea and Bacteria in sediments of four kinds of wetlands with different nitrogen concentration were investigated by using quantitative real-time polymerase chain reaction, cloning, and sequencing approaches based on amoA genes. The results indicated that AOA distributed widely in wetland sediments, and the phylogenetic tree revealed that archaeal amoA functional gene sequences from wetlands sediments cluster as two major evolutionary branches: soil/sediment and sediment/water. The bacteria functionally dominated microbial ammonia oxidation in different wetlands sediments on the basis of molecule analysis, potential nitrification rate, and soil chemistry. Moreover, the factors influencing AOA and AOB abundances with environmental indicator were also analyzed, and the results addressed the copy numbers of archaeal and bacterial amoA functional gene having the higher correlation with pH and ammonia concentration. The pH had relatively great negative impact on the abundance of AOA and AOB, while ammonia concentration showed positive impact on AOB abundance only. These findings could be fundamental to improve understanding of the importance of AOB and AOA in nitrogen and other nutrients cycle in wetland ecosystems.

Expression of nisin genes in cheese—A quantitative real-time polymerase chain reaction approach

The role of bacteriocins in different environments has not been thoroughly explained, mainly because of the difficulties related to the detection of their production. Nisin, an antimicrobial peptide produced by Lactococcus lactis has a long history of safe use in food products and has been studied from many aspects of genetics, biosynthesis, immunity, regulation, and mode of action. Still, some aspects concerning the dynamics of nisin gene expression remain unknown, especially in complex media like cheese. The main objective of the present study was to quantify in a cheese-like medium the expression of nisin genes in L. lactis M78, a well-characterized nisin A producer isolated from raw milk. The expression of all 11 genes involved in nisin biosynthesis was evaluated during cheese production by real-time reverse transcription-PCR. Total RNA was extracted from cheeses using a direct extraction method without prior separation of microbial cells. The M78 strain grew well in experimental cheeses, producing detectable amounts of nisin after 4h of fermentation. The presence of nisin as an activator modified both the expression of nisin genes and the accumulation of active nisin. Four groups could be distinguished based on gene expression as a function of time: nisA, nisFEG, nisRK and nisBTCIP. Based on nisin-producing strain growth, nisin activity, function of nisin genes, and their location, correlations were established that contribute to the explanation of regulation of nisin biosynthesis and immunity. This study is the first in which the evolution of bacteriocin gene transcripts has been quantified rigorously in a cheese-like medium.

RESEARCH ARTICLE: Early identification of Aspergillus carbonarius in artificially and naturally contaminated grape berries by real-time polymerase chain reaction

Objectives The contamination of agricultural commodities with mycotoxins can occur without visible fungal contamination. Therefore, it is very important to develop a method for detecting very low levels of Aspergillus carbonarius DNA, which is the main agent responsible for ochratoxin A contamination in grape and wine. Methods The aim of this study was to detect the presence of A. carbonarius in grapes early during different stages of fungal development by the SYBR s Green real-time polymerase chain reaction or quantitative polymerase chain reaction approach by designing species-specific primers (Acpks) on the basis of the polyketide synthase sequences. Results The real-time polymerase chain reaction amplification results show early detection of a specific A. carbonarius from nonsymptomatic grape berries harvested directly from the field. Moreover, the amplification of fungal DNA extracted from grape berries artificially inoculated with A. carbonarius has shown an amplification starting from 6h after inoculation, i.e. when the mycelium is not yet visible under stereomicroscope ( � 100) observation. Conclusion The results indicate that these highly specific and sensitive polymerase chain reaction-based methods are able to discriminate grape berries infected with A. carbonarius from uninfected ones. Thus, in a working day, it is possible to harvest the berries, extract high-quality DNA and perform A. carbonarius-specific real-time polymerase chain reaction amplification. The method proposed in this work could contribute toward food safety (e.g., wine and table grapes) because it could be used to predict the potential risk of contamination by potentially ochratoxigenic strains of A. carbonarius.

Differential oxidative stress gene expression profile in mouse brain after proton exposure

Radiation is known to potentially interfere with cellular functions at all levels of cell organization. The radiation-induced stress response is very complex and involves altered expression of many genes. Identification of specific genes may allow the determination of pathways important in radiation responses. Although several radiation-related research have been studied extensively, the molecular and cellular processes affected by proton exposure remain poorly understood. Our earlier reports have shown that proton radiation induces reactive oxygen species (ROS) formation and lipid peroxidation and inhibits antioxidants, superoxide dismutase, and glutathione. Therefore, in this present study, we used quantitative real-time reverse transcription polymerase chain reaction approach and showed the modulation of several genes including oxidative stress, antioxidants defense mechanism, ROS metabolism, and oxygen transporters related genes expression in 2-Gy proton-exposed mouse brain. Literature evidences suggest that change in oxidants and antioxidants levels induce DNA damage, followed by cell death. In conclusion, changes in the gene profile of mouse brain after proton irradiation are complex and the exposed cells might undergo programmed cell death through alteration of genes responsible for oxidative stress signaling mechanism.

Soil type determines the abundance and community structure of ammonia-oxidizing bacteria and archaea in flooded paddy soils

Ammonia oxidation plays an important role in global nitrogen cycle. However, little information is available on ammonia oxidizers in paddy soils. This study aimed to understand the controlling factors of ammonia oxidizers in the paddy soils. Three types of paddy soils were collected from southwest [Chongqing City (CQ)], central [Honghu City (HH)], and northeast [Panjin City (PJ)] of China, respectively. The soils were cultured with unplanted and rice-planted pot experiments for 10 weeks. The abundance and composition of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) of the paddy soil samples were investigated by real-time polymerase chain reaction and denaturing gradient gel electrophoresis approaches based on amoA genes. In this study, minor changes in the abundance and community structure of both AOB and AOA were observed among the treatments of unplanted and rhizosphere and bulk of rice-planted soil samples in the same type of paddy soil. The AOB population sizes in CQ were lower than those in HH and PJ, while the AOA in CQ and HH were lower than those in PJ. The amoA gene copy numbers of AOA were more abundant than those of AOB in all treatments of the three paddy soils. The community structure of both AOB and AOA in all paddy soils was different from each other. The population size and community structure of AOB and AOA in the flooded paddy soils were mainly determined by the soil types, irrespective of rice-planted or unplanted.

Multicolor Real‐Time PCR Genotyping of ABO System Using Displacing Probes

Rapid and informative ABO genotyping has become increasingly popular in forensic use. We developed a multiplex real-time polymerase chain reaction (PCR) approach to genotype ABO major groups and subgroups. Seven differently fluorophor-labeled displacing probes for O(1)(261delG), A(261G), A(796C/803C), B(796A/803C), O(2) (802G>A), A(2) (1059delC), and A(2) (1009A>G) were combined in one or two PCRs to determine either ABO major groups or subgroups. The method correctly detected 13 reference DNA samples. A blind test of 237 samples resulted in complete agreement with their phenotypes, and 110 of these 237 samples as well as with PCR-SSP method. The whole analysis could be finished in less than 100 min at substantially low material cost and the template DNA ranging from 0.16 to 500 ng per reaction could be quantitatively detected. Despite the limited informativeness of ABO genotyping, the developed methods could find application in rapid and inexpensive screening of forensic settings.

Biomolecular identification of allergenic pollen: a new perspective for aerobiological monitoring?

Accurate and updated information on airborne pollen in specific areas can help allergic patients. Current monitoring systems are based on a morphologic identification approach, a time-consuming method that may represent a limiting factor for sampling network enhancement. To verify the feasibility of developing a real-time polymerase chain reaction (PCR) approach, an alternative to optical analysis, as a rapid, accurate, and automated tool for the detection and quantification of airborne allergenic pollen taxa. The traditional cetyl trimethyl ammonium bromide-based method was modified for DNA isolation from pollen. Taxon-specific DNA sequences were identified via bioinformatics or literature searches and were PCR amplified from the matching allergenic taxa; based on the sequences of PCR products, complementary or degenerate TaqMan probes were developed. The accuracy of the quantitative real-time PCR assay was tested on 3 plant species. The setup of a modified DNA extraction protocol allowed us to achieve good-quality pollen DNA. Taxon-specific nuclear gene fragments were identified and sequenced. Designed primer pairs and probes identified selected pollen taxa, mostly at the required classification level. Pollen was properly identified even when collected on routine aerobiological tape. Preliminary quantification assays on pollen grains were successfully performed on test species and in mixes. The real-time PCR approach revealed promising results in pollen identification and quantification, even when analyzing pollen mixes. Future perspectives could concern the development of multiplex real-time PCR for the simultaneous detection of different taxa in the same reaction tube and the application of high-throughput molecular methods.

Cdcs1 a major colitis susceptibility locus in mice; Subcongenic analysis reveals genetic complexity

The cytokine-deficiency-induced colitis susceptibility (Cdcs)1 locus is a major modifier of murine inflammatory bowel disease (IBD) and was originally identified in experimental crosses of interleukin-10-deficient (Il10(-/-)) mice. Congenic mice, in which this locus was reciprocally transferred between IBD-susceptible C3H/HeJBir-Il10(-/-) and resistant C57BL/6J-Il10(-/-) mice, revealed that this locus likely acts by inducing innate hypo- and adaptive hyperresponsiveness, associated with impaired NF-kappaB responses of macrophages. The aim of the present study was to dissect the complexity of Cdcs1 by further development and characterization of reciprocal Cdcs1 congenic strains and to identify potential candidate genes in the congenic interval. In total, 15 reciprocal congenic strains were generated from Il10(-/-) mice of either C3H/HeJBir or C57BL/6J genetic backgrounds by 10 cycles of backcrossing. Colitis activity was monitored by histological grading. Candidate genes were identified by fine mapping of congenic intervals, sequencing, microarray analysis, and a high-throughput real-time reverse-transcription polymerase chain reaction (RT-PCR) approach using bone marrow-derived macrophages. Within the originally identified Cdcs1-interval, 3 independent regions were detected that likely contain susceptibility-determining genetic factors (Cdcs1.1, Cdcs1.2, and Cdcs1.3). Combining results of candidate gene approaches revealed Fcgr1, Cnn3, Larp7, and Alpk1 as highly attractive candidate genes with polymorphisms in coding or regulatory regions and expression differences between susceptible and resistant mouse strains. Subcongenic analysis of the major susceptibility locus Cdcs1 on mouse chromosome 3 revealed a complex genetic structure. Candidate gene approaches revealed attractive genes within the identified regions.

Charles River altered Schaedler flora (CRASF®) remained stable for four years in a mouse colony housed in individually ventilated cages

As recommendations for specific pathogen-free housing change, mouse facilities need to re-derive their colonies repeatedly in order to eliminate specified bacteria or viruses. This paper describes the establishment of a new mouse facility using as starting point a small colony of CD-1 mice colonized with the Charles River altered Schaedler flora (CRASF) housed in individually ventilated cages (IVCs). The import of new strains was performed exclusively via embryo transfer using CD-1 mice as recipients. The integrity of the CRASF in caecum samples of the original CD-1 colony and of three inbred mouse lines imported into the colony was proven by a quantitative realtime polymerase chain reaction approach. Furthermore, we searched for bacterial contaminants in the gut flora using non-specific 16S rRNA primers. The bacterial sequences found were closely related to but not exclusively sequences of altered Schaedler flora (ASF) members, suggesting that the ASF is heterogeneous rather than restricted to the eight defined bacteria. Moreover, no pathogens were found, neither using the non-specific 16S rRNA primers nor in routine quarterly health monitoring. As one effect of this defined gut flora, interleukin-10 knockout mice are devoid of colitis in our facility. In conclusion, our approach building up a mouse facility using foster mothers and embryo transfer as well as a strict barrier system and IVCs is suitable to maintain a colony free from contaminating bacteria over the long term. CRASF remained stable for seven mouse generations and was efficiently transferred to the imported mouse strains.

Decreased EGFR mRNA expression in response to antipsoriatic drug dithranol in vitro

Dithranol is enormously effective in the treatment of psoriasis; however its molecular mode of action should be further elucidated. Since epidermal growth factor receptor (EGFR) is involved in the pathogenesis of psoriasis, the objective of this study was to investigate the transcriptional effect of dithranol on EGFR gene expression in the HaCaT keratinocyte cell line, which is commonly employed as a model system to study psoriasis including experiments examining the effects of therapeutic drugs and cellular regulators on keratinocytes. Cultured HaCaT cells were treated with 0.1-0.5 ig/ml dithranol for 30 min. After 4 h, total cellular RNA isolated from HaCaT cells was reverse transcribed to cDNA which was subjected to polymerase chain reaction (PCR) with specific primer pair for EGFR. We found that dithranol treatment down-regulated the EGFR mRNA of HaCaT cells in a concentration-dependent manner. Our result was further substantiated using a quantitative real-time PCR approach. Taken together, the dithranol-induced down-regulation of the EGFR in cultured human keratinocytes might help to disclose the molecular therapeutic action of the drug.

Rapid detection of Staphylococcus aureus bacteremia and methicillin resistance by real-time PCR in whole blood samples

We prospectively evaluated a real-time polymerase chain reaction (PCR) approach for the rapid diagnosis of Staphylococcus aureus bacteremia and presence of the mecA gene in 902 blood samples from 468 infectious episodes of 384 patients. Eight of 12 blood culture (BC)-confirmed samples were positive by the S. aureus-specific PCR. In addition, the mecA gene PCR correctly detected all cases of BC-confirmed methicillin-resistant Staphylococcus aureus (MRSA) infection. A positive PCR result was also obtained in ten of 462 BC-negative infectious episodes, including three patients with culture-confirmed S. aureus infection at other body sites.

Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction

This paper reports a duplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of members of the Aspergillus niger aggregate and A. carbonarius, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the β-ketosynthase and the acyl transferase domains of the poliketide synthase of A. carbonarius and the A. niger aggregate, respectively. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic T m-values demonstrating the specific, efficient and balanced amplification of the two PCR fragments. Subsequently, a TaqMan real-time PCR approach was settled, using 6-carboxy-fluorescein group (FAM) and VIC®-labelled specific probes for automated detection. Results indicated no differences in sensitivity when using either the two sets of primers and probes in separate or in the same reaction. However, when both targets are in very different amounts, there is a preferential amplification of the target which is in more concentration. CT-values obtained in the presence of grape DNA were very similar to those observed when only fungal purified DNA was present, indicating that the grape DNA does not interfere in the real-time PCR reaction. This procedure provides a fast and accurate tool to monitor, in a single reaction, the presence of OTA-producing species in grapes which, to some extent, will facilitate OTA contamination surveys to guarantee food safety in the wine industry.

PCR-coupled electrochemical sensing of Legionella pneumophila

Human infections with Legionella pneumophila represent a public health problem. Current culture assays for surveillance and control of L. pneumophila in water are time-consuming and limited by the sensitivity, especially when samples also contain microorganisms that inhibit Legionella growth. In this work, an electrochemical method, different from real-time polymerase chain reaction (PCR) approaches, for semiquantitative evaluation of L. pneumophila is presented. A PCR assay targeting the 16S-rRNA gene of L. pneumophila giving rise to a 95-mer amplicon was established. Amplicons were hybridized to a biotin-labeled reporter sequence and then to a thiolated stem-loop structure immobilized onto gold electrodes as a reporter molecule with 1-naphthyl phosphate as a substrate. 1-Naphthol enzymatically generated was determined by differential pulse voltammetry (DPV). For a constant number of amplification cycles, results show that the voltammetric signal is related to the number of copies in the sample thus achieving a useful semiquantitative estimation of L. pneumophila. After 40 cycles of PCR amplification this methodology has a limit of detection of 10 genomes, allowing the reliable detection of 102 genomes of L. pneumophila as well as distinguishing 103 and 104 genomes of the pathogen, values related to corrective actions in water systems in buildings, in accordance with the legislation currently in force.

Use of Real-Time PCR Technique in Studying Rumen Cellulolytic Bacteria Population as Affected by Level of Roughage in Swamp Buffalo

A real-time polymerase chain reaction approach was used in this study to determine the population of major ruminal bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) in digesta and rumen fluid of swamp buffalo (Bubalus bubalis). Four rumen-fistulated, male swamp buffalo were randomly assigned according to a 4 x 4 Latin square design to evaluate the effect of the urea-treated rice straw (roughage source)-to-concentrate ratio on cellulolytic bacterial distribution. Animals were fed roughage-to-concentrate (R:C) ratios of 100:0, 75:25, 50:50, and 25:75, respectively. At the end of each period, rumen fluid and digesta were collected at 0 h and 4 h post-morning-feeding. It was found that feeding urea-treated rice straw solely increased these three cellulolytic bacteria numbers up to 2.65 x 10(9) and 3.54 x 10(9) copies per milliliter for F. succinogenes, 5.10 x 10(7) and 7.40 x 10(7) copies per milliliter for R. flavefaciens, and 4.00 x 10(6) and 6.00 x 10(6) copies per milliliter for R. albus in rumen fluid and digesta, respectively. The distribution of the three cellulolytic bacteria species in digesta were highest at 3.21 x 10(9), 4.55 x 10(7), and 4.56 x 10(6) copies per milliliter for F. succinogenes, R. flavefaciens, and R. albus, respectively. Moreover, at 4 h post-morning-feeding, the populations of the three cellulolytic bacteria were higher than found at 0 h post-morning-feeding. It is most notable that F. succinogenes were the highest in population in the rumen of swamp buffalo and cellulolytic bacteria mostly adhered to feed digesta in the rumen.

High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA

MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of cellular processes. Research in this field is accelerating, and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods. Here we present the successful evaluation of the Megaplex reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. The Megaplex reaction provides simultaneous reverse transcription of 450 mature miRNAs, ensuring high-throughput detection. Further, the introduction of a complementary DNA pre-amplification step significantly reduces the amount of input RNA needed, even down to single-cell level. To evaluate possible pre-amplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional pre-amplification step. The normalized Cq values of all three sample pairs showed a good correlation with maintenance of differential miRNA expression between the cell lines. Moreover, pre-amplification using 10 ng of input RNA enabled the detection of miRNAs that were undetectable when using Megaplex alone with 400 ng of input RNA. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling.

Sirtuin gene expression in human mononuclear cells is modulated by caloric restriction

Sirtuins may provide novel targets for treating some diseases associated with oxidative stress, such as obesity and its comorbidities. However, there are a few in vivo studies in humans about the potential role of sirtuins as therapeutic targets among obese patients undergoing caloric restriction. Therefore, the aim of this study was to assess if the gene expression of sirtuins is modulated in peripheral blood mononuclear cells (PBMC) by a hypocaloric diet devised to lose weight in humans. Gene expression of two sirtuins (SIRT1 and SIRT2) in the PBMC of obese subjects (32.3 +/- 5.5 kg m(-2)) before and following an 8-week hypocaloric diet was investigated. NADH-coenzyme Q reductase (NDUFS2) and cytochrome c oxidase assembly protein (COX15) gene expression was selected together with plasma antioxidant power and nitric oxide as markers of antioxidant status. A quantitative real-time polymerase chain reaction approach was performed to assess the nutrigenomics outcome. Moreover, 2-keto[1-(13)C]isocaproate breath test (KICA-BT) parameters were evaluated to study mitochondrial oxidation in vivo. The intervention up-regulated the expression of both sirtuins, being inversely associated with total antioxidant capacity and directly related to nitric oxide, mitochondrial oxidation assessed by the KICA-BT and the expression of the mitochondrial proteins COX15 and NDUFS2. SIRT1 and SIRT2 may serve as key regulators for some obesity comorbidities related to antioxidant status, while PBMC could be a model to study the effect of the sirtuin response in obesity therapy.

Translational Machinery of Senegalese Sole (Solea senegalensis Kaup) and Atlantic Halibut (Hippoglossus hippoglossus L.): Comparative Sequence Analysis of the Complete Set of 60S Ribosomal Proteins and their Expression

Ribosomal proteins (RPs) comprise a large set of highly evolutionarily conserved proteins that are often over-represented in complementary DNA libraries. They have become very useful markers in comparative genomics, genome evolution, and phylogenetic studies across taxa. In this study, we report the sequences of the complete set of 60S RPs in Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species. Amino-acid sequence comparisons of the encoded proteins showed a high similarity both between these two flatfish species and with respect to other fish and human counterparts. Expressed sequence tag analysis revealed the existence of paralogous genes for RPL3, RPL7, RPL41, and RPLP2 in Atlantic halibut and RPL13a in Senegalese sole as well as RPL19 and RPL22 in both species. Phylogenetic analysis of paralogs revealed distinct evolutionary histories for each RP in agreement with three rounds of genome duplications and lineage-specific duplications during flatfish evolution. Steady-state transcript levels for RPL19 and RPL22 RPs were quantitated during larval development and in different tissues of sole and halibut using a real-time polymerase chain reaction approach. All paralogs were expressed ubiquitously although at different levels in different tissues. Most RP transcripts increased coordinately after larval first-feeding in both species but decreased progressively during the metamorphic process. In all cases, expression profiles and transcript levels of orthologous genes in Senegalese sole and Atlantic halibut were highly congruent. The genomic resources and knowledge developed in this survey will be useful for the study of Pleuronectiformes evolution.

Nucleophosmin ( NPM1) Mutations in Acute Myeloid Leukemia: An Ongoing (Cytoplasmic) Tale of Dueling Mutations and Duality of Molecular Genetic Testing Methodologies

This Commentary highlights two articles that focus on molecular techniques to identify mutations in nucleophosmin (NPM1), which is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML)