cDNA Levels Research Articles

Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data

Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.

Dystrophin Dp71 Isoforms Are Differentially Expressed in the Mouse Brain and Retina: Report of New Alternative Splicing and a Novel Nomenclature for Dp71 Isoforms.

Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

Effects of Fertilization on Soil Microbial Abundance and Community Structure at DNA and cDNA Levels in Paddy Soils

Fertilizer applications have important effects on soil microbial abundance and community structure. In this study, total soil microbial DNA and RNA were directly extracted from paddy soils of N0 (control treatment, no nitrogen fertilizer), NPK (balanced fertilization), NPK+LS (balanced fertilization with additional 3.0 t·hm-2 rice straw incorporation) and NPK+HS (balanced fertilization with additional 6.0 t·hm-2 rice straw incorporation) treatments in a long-term fertilization experiment of double rice cropping system in Changsha County, Hunan Province. Soil bacteria community structures were evaluated by analyzing the 16S rRNA gene fragments at DNA and cDNA levels with Terminal Restriction Fragment Length Polymorphism (T-RFLP) and quantitative PCR techniques. Balancing fertilization with chemical fertilizers and rice straw incorporation significantly changed the composition of bulk (DNA-based) and potentially active (mRNA-based) soil bacterial community as shown in T-RFLP profiles, and also reduced the bulk soil microbial diversity, but not the potentially active ones, as compared with the control treatment. The DNA-based abundance of 16S rRNA gene was on average 377 times as many as the m-RNA based population size. Compared to N0,balanced fertilization with rice straw incorporation (NPK+LS and NPK+HS) increased the bulk and active copy numbers of 16S rRNA gene, but not for balanced fertilization (NPK). The abundance and microbial community structure were not significantly different between the NPK+LS and NPK+HS treatments. Redundancy analysis (RDA) showed that soil ammonium was the key environmental factor determining the bulk and active soil microbial community structure among the treatments. In conclusion, the effect of fertilization on soil microbial abundance and community structure could be indicated at both DNA and cDNA levels; the cDNA information could better reflect the adaptability of bacterial community to the environmental stress.

Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αβγ and αβδ Expression Using Flow Cytometry and Electrophysiology: EVIDENCE FOR ALTERNATIVE SUBUNIT STOICHIOMETRIES AND ARRANGEMENTS

The subunit stoichiometry and arrangement of synaptic αβγ GABAA receptors are generally accepted as 2α:2β:1γ with a β-α-γ-β-α counterclockwise configuration, respectively. Whether extrasynaptic αβδ receptors adopt the analogous β-α-δ-β-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or β2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and β2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of β2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1β2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αβγ and αβδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αβγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical β-α-γ/δ-β-α receptors, including β-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and β-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies.

Effect of the addition of phytosterols and tocopherols on Streptococcus thermophilus robustness during industrial manufacture and ripening of a functional cheese as evaluated by qPCR and RT-qPCR

The quality of functional food products designed for the prevention of degenerative diseases can be affected by the incorporation of bioactive compounds. In many types of cheese, the performance of starter microorganisms is critical for optimal elaboration and for providing potential probiotic benefits. Phytosterols are plant lipophilic triterpenes that have been used for the design of functional dairy products because of their ability to lower serum cholesterol levels in humans. However, their effect on the starter culture behavior during cheesemaking has not yet been studied. Here, we followed DNA and RNA kinetics of the bacterium Streptococcus thermophilus, an extensively used dairy starter with probiotic potential, during industrial production of a functional, semi-soft, reduced-fat cheese containing phytosterol esters and alpha-tocopherol as bioactive compounds. For this purpose, real-time quantitative PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) assays were optimized and applied to samples obtained during the manufacture and ripening of functional and control cheeses. An experimental set-up was used to evaluate the detection threshold of free nucleic acids for extraction protocols based on pelleted microorganisms. To our knowledge, this straight-forward approach provides the first experimental evidence indicating that DNA is not a reliable marker of cell integrity, whereas RNA may constitute a more accurate molecular signature to estimate both bacterial viability and metabolic activity. Compositional analysis revealed that the bioactive molecules were effectively incorporated into the cheese matrix, at levels considered optimal to exert their biological action. The starter S. thermophilus was detected by qPCR and RT-qPCR during cheese production at the industrial level, from at least 30min after its inoculation until 81days of ripening, supporting the possible role of this species in shaping organoleptic profiles. We also showed for the first time that the addition of phytosterols at functional concentrations, not only did not affect starter performance but also correlated with a significant increase in target DNA and cDNA levels in most of the time points evaluated throughout cheesemaking. Therefore, these findings suggest that the growth and metabolism of S. thermophilus may be enhanced by the incorporation of these biologically active molecules during cheese production, providing important information for the industrial design of novel fermented foods.

Clinical and Molecular Characterization of NF1 Patients: Single-Center Experience of 32 Patients From China.

Neurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, and the presence of pseudogenes.Our goal was to establish a sensitive, feasible, and comparatively economical protocol to detect NF1 mutations using blood samples.We developed a method to screen patients for mutations. Thirty-two NF1 patients from 32 unrelated families and 120 unrelated population-match controls were investigated in this study. Specific primers were designed for NF1 to avoid pseudogenes. NF1 mutations were detected by sequencing at the deoxyribonucleic acid (DNA) and complementary DNA (cDNA) levels, and multiplex ligation-dependent probe amplification (MLPA) and familial segregation analyses were used.Forty-four specific primers designed according to the NF1 structure were successfully used for polymerase chain reaction (PCR) and DNA sequencing, which was more feasible and useful than cDNA sequencing. Thirty distinct NF1 mutations were identified in 32 patients. Thirteen mutations were novel and most were frameshift mutations (33.3%). Mutations were detected at a rate of 93.8%.Our study suggests that this sensitive, feasible, and comparatively economical protocol is effective for the detection of NF1 mutations.

Effects of simvastatin and taurine on delayed cerebral vasospasm following subarachnoid hemorrhage in rabbits.

The aim of the current study was to observe the effects of simvastatin and taurine on delayed cerebral vasospasm (DCVS) following experimental subarachnoid hemorrhage (SAH) in rabbits. A total of 48 New Zealand white rabbits were allocated at random into four groups (control, SAH, SAH + simvastatin and SAH + taurine groups; n=12 each). The rabbit model of DCVS was established using a double hemorrhage method, which involved injecting autologous arterial blood into the cisterna magna in the SAH groups. The SAH + simvastatin group was administered oral simvastatin (5 mg/kg) daily between days 0-6. The SAH + taurine group was administered oral taurine (50 mg/kg) daily between days 0-6. Starch (50 mg/kg) was administered orally to the animals in the other two groups (control and SAH groups). The control group were not subjected to any other injections or treatment. The internal diameter and internal diameter/wall thickness of the basilar artery (BA) were measured. The expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were determined using immunohistochemical and quantitative polymerase chain reaction methods following the sacrifice of all animals on day 7. The activity of nuclear factor (NF)-κB in the BA was also measured using an electrophoretic mobility shift assay. The BA walls in the SAH + simvastatin and SAH + taurine groups exhibited reduced narrowing and corrugation of the tunica elastica interna compared with the SAH group. At the protein and cDNA levels, it was found that cerebral vasospasm of the BA in the SAH + simvastatin and SAH + taurine groups was alleviated, as indicated by the reduced expression of TNF-α, IL-1β, IL-6 and NF-κB compared with the SAH group (P<0.05). In conclusion, simvastatin and taurine reduced DCVS following SAH in rabbits, which suggests that these compounds may exert anti-inflammatory effects.

Clinical and Biological Characterization of Children with FLT3ITD Mutated Acute Myeloid Leukemia (AML): A Report from the AIEOP AML-2002 Study Group

Purpose. While most of the recurrent molecular marker of AML have been already discovered and used for risk stratification in clinical protocols, post-treatment monitoring of these abnormalities can be useful in the clinical management of patients. In particular, monitoring of minimal residual disease (MRD), largely used in acute lymphoblastic leukemia, is gaining popularity also in AML, in the perspective of optimizing risk stratification of patients in terms of subsequent clinical relapse. While MRD monitoring in AML is mainly based on flow-cytometry approaches, molecular MRD measurements are not currently routinely used for taking clinical decision. Here we examine if the molecular MRD and Allelic Ratio (AR) levels monitoring may influence the survival of FLT3 internal tandem duplication (ITD)-mutated AML patients.Patients and methods. We retrospectively analyzed 507 children with de novo AML for FLT3ITD mutation by RT-PCR. Mutation was sequenced and the AR was calculated by Genescan. Bone marrow samples after induction treatment were analyzed for MRD levels by Real-Time PCR. We correlated these parameters with both patient event-free survival (EFS) and gene expression profile (GEP) findings.Results. 54/507 patients (10.6%) harboured FLT3ITD mutation. AR was calculated both at cDNA and DNA levels showing a reliable correlation (R=0.68), even if only AR measured on cDNA was found to be a significant poor prognostic feature. This latter observation supports the concept that expression of the mutation is more important than genetic bulk architecture at diagnosis. Patients with high AR showed a significant worse EFS as compared to those with low AR (19.2% for AR>0.51, vs 63.5% for AR<0.51), mainly because of a failure to achieve complete remission. Molecular monitoring of MRD showed that a low MRD reduction (<2 logs) after I induction course was an independent prognostic factor predicting both a high risk of incidence of relapse (cumulative incidence of relapse, CIR=68.7% vs 24.3, p <0.05), and a lower EFS (22.2% vs 59.4%, p<0.05). After the second induction course, a low MRD reduction also predicted a worse EFS. Through GEP analysis, we showed that high ITD-AR patients had a different transcriptional profile that those with low ITD-AR. Gene ontology revealed that patients with high AR had a negative regulation of genes involved in H3K27 tri-methylation tightly associated with inactivation of gene promoters as compared to those with low ITD-AR, suggesting that methylation might play a crucial role in this subgroup of FLT3ITD patients. Furthermore, we found that the cyclinA1 was significantly overexpressed. We hypothesised that cyclinA1-CDK2 complex is driving B-MYB phosphorylation, which, in turn, over-activates its own expression and sustains the B-MYB transcriptional oncogenic program in FLT3ITD patients with high AR. Support to this hypothesis is given by the observation that B-MYB signature was found to discriminate patients with low and high ITD-AR. GEP was also performed by comparing poor or good responders patients, showing that the former have a significant enrichment in stemness, in resistance to drugs and histone acetylation pathways.Conclusion. Our results show that ITD-AR and MRD status are important independent prognostic factors for the management of of FLT3ITD patients, to be taken into consideration in planning the post-induction treatment. The high expression of the FLT3ITD mutation and the persistence of the mutated clone confer an aberrant hyperactivation of the downstream oncogenic FLT3 pathway influencing the outcome within these AML. Transcriptional profiles opens for further consideration of epigenetic targeting for FLT3ITD patients. DisclosuresNo relevant conflicts of interest to declare.

Defects in RNase H2 Stimulate DNA Break Repair by RNA Reverse Transcribed into cDNA.

Eukaryotic ribonucleases (RNase) H1 and H2 are endonucleases that cleave RNA in a double- stranded RNA-DNA molecule. RNase H2 can also cleave a single ribonucleotide embedded in DNA duplex. While the activity of RNase H1 and H2 has been extensively characterized in vitro, still much is unclear about the specific targets of these enzymes in vivo. We recently demonstrated that yeast cells can repair a double-strand break (DSB) in DNA by homologous recombination (HR) using antisense (non-coding) RNA, either directly, or indirectly after converting RNA into cDNA. In wildtype RNase H1 and/or H2 cells, repair by cDNA dominates, whereas in the absence of RNase H1 and H2 functions cDNA and, in particular, direct transcript-RNA repair mechanisms are markedly stimulated. Here we found that null alleles of any of the three RNase H2 subunits stimulate DSB repair by cDNA significantly more than a null allele of RNase H1. These results show that RNase H2 is the preferred RNase H enzyme to target cDNA in yeast. Targeting of cDNA by RNase H2 does not require RNase H2 interaction with the DNA clamp proliferating cell nuclear antigen (PCNA). Moreover, yeast RNase H2 orthologous mutants of two common RNase H2 defects associated with Aicardi Goutières syndrome (AGS) in humans, displayed elevated cDNA-driven repair of a DSB when combined with each other or with RNase H1 null mutation. Our findings support the hypothesis that defective RNase H2 alleles have higher level of cDNA derived from either coding or non-coding RNA in the form of RNA-cDNA hybrids.

Methylation of KvDMR1 involved in regulating the imprinting of CDKN1C gene in cattle.

The CDKN1C gene encodes a cyclin-dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith-Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele-specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting.

Characterization and expression analyses of five interferon regulatory factor transcripts (Irf4a, Irf4b, Irf7, Irf8, Irf10) in Atlantic cod (Gadus morhua)

The interferon regulatory factor (IRF) family of genes encodes a group of transcription factors that have important roles not only in regulating the expression of Type I interferons (IFNs) and other genes in the IFN pathway, but also in growth, development and the regulation of oncogenesis. In this study, several IRF family members (Irf4a, Irf4b, Irf7, Irf8, Irf10) in Atlantic cod (Gadus morhua) were characterized at the cDNA and putative amino acid levels, allowing for phylogenetic analysis of these proteins in teleost fish, as well as the development of gene-specific primers used in RT-PCR and quantitative PCR (QPCR) analyses. Two Atlantic cod Irf10 splice variants were identified and their presence confirmed by sequencing of the Irf10 genomic region. RT-PCR showed that Irf7, Irf8 and both Irf10 transcripts were expressed in all 15 cod tissues tested, while Irf4a and Irf4b were absent in some tissues. QPCR analysis of spleen expression expanded upon this, and upon previous work. All IRF transcripts in the study were responsive to stimulation by the viral mimic poly(I:C), and all except Irf4a were responsive to exposure to formalin-killed Aeromonas salmonicida (ASAL). These IRF genes, thus, are likely important in the cod immune response to both viral and bacterial infections. Increased temperature (10 °C to 16 °C) was also observed to modulate the antibacterial responses of all IRF transcripts, and the antiviral responses of Irf4b and Irf10-v2. This research supports earlier studies which reported that elevated temperature modulates the expression of many immune genes in Atlantic cod.

Elevated mRNA-levels of distinct mitochondrial and plasma membrane Ca(2+) transporters in individual hypoglossal motor neurons of endstage SOD1 transgenic mice.

Disturbances in Ca2+ homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial and sporadic forms of Amyotrophic Lateral Sclerosis (ALS), a fatal degenerative motor neuron disease. However, the distinct molecular ALS-pathology remains unclear. Recently, an activity-dependent Ca2+ homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs) from a common ALS mouse model, the endstage superoxide dismutase SOD1G93A transgenic mouse, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca2+ uptake capacity and elevated Ca2+ extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca2+ transporters in individual, choline-acetyltransferase (ChAT) positive hMNs from wildtype (WT) and endstage SOD1G93A mice, by combining UV laser microdissection with RT-qPCR techniques, and specific data normalization. As ChAT cDNA levels as well as cDNA and genomic DNA levels of the mitochondrially encoded NADH dehydrogenase ND1 were not different between hMNs from WT and endstage SOD1G93A mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca2+ transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca2+ transporters MCU/MICU1, Letm1, and UCP2 in remaining hMNs from endstage SOD1G93A mice. These higher expression-levels of mitochondrial Ca2+ transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification. Normalized mRNA-levels for the plasma membrane Na+/Ca2+ exchanger NCX1 were also about 2-fold higher in hMNs from SOD1G93A mice. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a neuroprotective strategy for ALS.

Postoperative immunosuppression markers and the occurrence of sepsis in patients with benign and malignant disease

To investigate associations between the postoperative immune response and the levels of extracellular circulating DNA (cDNA), C-reactive protein (CRP), neutrophil/lymphocyte (N/L) ratio, and regulatory T (Treg) cells in the peripheral blood and their role as potential predictors of postoperative septic complications. This was a prospective observational study involving 115 adult patients who underwent elective surgery. Patients were divided into three groups: with benign disease, with malignant disease, and with malignant disease and administration of dexamethasone. Serum CRP levels, N/L ratio, monocyte human leukocyte antigen-DR (HLA-DR) expression, proportion of Treg cells, and cDNA levels were measured at different time points before and after surgery. All patients had increased CRP levels after surgery. Septic patients had higher serum CRP levels at baseline. Compared with the other groups, the dexamethasone group had significantly higher CRP levels before and after surgery, a significantly higher N/L ratio before surgery, a significantly lower rise in the N/L ratio after surgery, and a significantly lower HLA-DR expression at baseline, which remained stable after surgery. In the malignant-disease group, we observed a significant postoperative decrease in the HLA-DR expression. Our results suggest that the immunosuppressive effect of surgery and the presence of a malignant disease may contribute to a higher risk of postoperative sepsis. Preoperative CRP levels may be a reliable predictor of sepsis in oncological patients.

Expression levels of vitellogenin receptor (Vtgr) during ovarian development and association between its single nucleotide polymorphisms (SNPs) and reproduction-related parameters of the giant tiger shrimp Penaeus monodon

Abstract Identification of molecular markers linked to phenotypic parameters of reproduction is important and can be applied to domestication and selective breeding programs of the giant tiger shrimp ( Penaeus monodon ). In this study, the full-length cDNA and expression levels of P. monodon vitellogenin receptor ( PmVtgr ) during ovarian development in wild shrimp and association between its single nucleotide polymorphisms (SNPs) and reproduction-related parameters in domesticated shrimp were examined. PmVtgr was 5990 bp in length composing of an open reading frame (ORF) of 5832 bp deducing to a polypeptide of 1943 amino acids. PmVtgr was only expressed in ovaries but not in other tissues of female broodstock. The expression level of PmVtgr in premature ovaries of juveniles was significantly lower than in those of broodstock ( P PmVtgr mRNA significantly increased in stage IV (mature) relative to stage I (previtellogenic) ovaries ( P PmVtgr was comparably expressed at all stages of ovarian development in eyestalk-ablated shrimp ( P > 0.05). In addition, SNPs in the PmVtgr gene segment of 14-month-old P. monodon ( N = 64) were indirectly examined by PCR-SSCP. Significant relationships between different SSCP patterns of PmVtgr and important reproduction-related phenotypes (i.e. gonadosomatic index, GSI and ovarian weight) of P. monodon were found. Results were consistent when further tested against non-related 19-month-old shrimp ( N = 54, P PmVtgr were cloned and sequenced ( N = 10 for each SSCP pattern). SNP positions 126 (C → T), 226 (T → A), 441 (T → A), 477 (T → A), 499 (A → C), 500 (T → C), 519 (A → G), 540 (A → G), 545 (G → A), 614 (T → C) and 678 (A → C) of PmVtgr were significantly associated with GSI values of examined shrimp ( N = 40).

The differential expression of alternatively spliced transcripts and imprinting status of MEG9 gene in cows.

Meg9/Mirg (maternally expressed gene 9/microRNA containing gene), a non-coding RNA (ncRNA) comprising many alternatively splicing isoforms, has been identified as maternally expressed in mouse and sheep, but its imprinting status and splicing variants are still unknown in cattle. In this study, we found three splicing variants of the cattle MEG9 gene expressed in a tissue-specific manner. A single nucleotide polymorphism site (SNP c.1354C>G) was identified in exon 3 of cattle MEG9 and used to distinguish between monoallelic and biallelic expression. Our results showed that MEG9 exhibited monoallelic expression in all examined cattle tissues by comparing sequencing results between genomic DNA and cDNA levels at the c.1354C>G SNP site, suggesting that MEG9 is imprinted in cattle.

Analysis of indoleamine 2,3-dioxygenase 1 (IDO1) expression of cultured cord blood adherent mononuclear cells as an indicator of atopic risk

* Correspondence: 8yz7@queensu.ca Department of Biomedical and Molecular Sciences/Medicine, Queen’s University, Kingston, ON, K7L 3N6, Canada Figure 1 IDO1 gene expression fold changes relative to plain media control. IDO1 expression levels were normalized to HPRT1 expression. The error bars represent the standard error of the mean. Numbers per stimulation group are as indicated beneath the graph. Cultures of atopic and non-atopic AMNCs were plated at 7.5x10 cells per condition. Following 5.5 hours incubation with either plain media, 1 μg/ml IFN-g, or 1 μg/ml IFN-g and 10 ng/ml CSE, cells were lysed for RNA extraction. RNA was reverse transcribed and cDNA levels were analyzed. Zhu et al. Allergy, Asthma & Clinical Immunology 2014, 10(Suppl 1):A72 http://www.aacijournal.com/content/10/S1/A72 ALLERGY, ASTHMA & CLINICAL IMMUNOLOGY

The potential of the human immune system to develop broadly neutralizing HIV-1 antibodies

Objectives and design:Developing an effective HIV-1 vaccine that elicits broadly neutralizing HIV-1 human antibodies (bnAbs) remains a challenging goal. Extensive studies on HIV-1 have revealed various strategies employed by the virus to escape host immune surveillance. Here, we investigated the human antibody gene repertoires of uninfected and HIV-1-infected individuals at genomic DNA (gDNA) and cDNA levels by deep sequencing followed by high-throughput sequence analysis to determine the frequencies of putative germline antibody genes of known HIV-1 monoclonal bnAbs (bnmAbs).Methods:Combinatorial gDNA and cDNA antibody libraries were constructed using the gDNAs and mRNAs isolated from uninfected and HIV-1-infected human peripheral blood mononuclear cells (PBMCs). All libraries were deep sequenced and sequences analysed using IMGT/HighV-QUEST software (http://imgt.org/HighV-QUEST/index.action). The frequencies of putative germline antibodies of known bnmAbs in the gDNA and cDNA libraries were determined.Results and conclusion:The human gDNA antibody libraries were more diverse in heavy and light chain V-gene lineage usage than the cDNA libraries, indicating that the human gDNA antibody gene repertoires may have more potential than the cDNA repertoires to develop HIV-1 bnAbs. The frequencies of the heavy and kappa and lambda light chain variable regions with identical V(D)J recombinations to known HIV-1 bnmAbs were extremely low in human antibody gene repertoires. However, we found relatively high frequencies of the heavy and kappa and lambda light chain variable regions that used the same V-genes and had the same CDR3 lengths as known HIV-1 bnmAbs regardless of (D)J-gene usage. B-cells bearing B-cell receptors of such heavy and kappa and lambda light chain variable regions may be stimulated to induce HIV-1 bnAbs.

Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop

Aims Previous studieshaveshownthatelephantgrassis colonized by nitrogen-fixing bacterial species; however, these results were based on culture-dependent methods, an approach that introduces bias due to an incomplete assessment of the microbial community. In this study, we used culture-independent methods to survey the diversity of endophytes and plant-associated bacterial communities in five elephant grass genotypes used in bioenergy production. Methods The plants of five genotypes of elephant grass were harvested from the experimental area of Embrapa Agrobiologia and divided into stem and root tissues. Total DNA and RNAwere extracted from plant tissues and the bacterial communities were analyzed by DGGE and clone library of the 16S rRNA and nifH genes at both the cDNA and DNA levels. Results Overall, the patterns based on DNA- and RNAderivedDGGE-profilesdiffered,especiallywithintissue samples. DNA-based DGGE indicated that both total bacterial and diazotrophic communities associated with roots (rhizoplane+endophytes) differed clearly from those obtained from stems (endophytes). These results were confirmed by the phylogenetic analyses of RNAderived sequences of 16S rRNA (total bacteria; 586 sequences), but not for nifH (186). In fact, rarefaction analyses showed a higher diversity of diazotrophic organisms associated with stems than roots. Based on 16S rRNA sequences, the clone libraries were dominated by sequences affiliated to members of Leptotrix (12.8 %) followed by Burkholderia (9 %) and Bradyrhizobium (6.5 %), while most of the nifH clones were closely related to the genus Bradyrhizobium (26 %). Conclusions Our results revealed an unexpectedly large diversity of metabolically active bacteria, providing new insights into the bacterial species predominantly found in

Neurotoxic effect of subacute benzo(a)pyrene exposure on gene and protein expression in Sprague-Dawley rats

Benzo(a)pyrene (B[a]P) is an environmental carcinogen that induces tumors in many animal species, but the neurotoxic effects of B[a]P have not been well studied. In the present study, we investigated the effects of subacute exposure to B[a]P in Sprague-Dawley (SD) rats. Male rats received daily injections of either B[a]P (0, 1, 2.5, or 6.25mg/kg, i.p.) or vehicle for 45 days. Exposure to B[a]P affected the behavior of rats in the Morris water maze test. Gene microarray and real-time PCR analyses revealed that exposure to B[a]P affected signal transduction in the rat hippocampus. Protein microarray analysis revealed that altered protein expression played a role in cell death in the functional annotation cluster analysis. Finally, major vault protein was found to display low cDNA and protein expression levels. The present study explored some of the possible mechanisms underlying B[a]P neurotoxicity and provided evidence that B[a]P plays a neurotoxic role in rats.

Preliminary evaluation of Prodigiosa herbal tea: cytotoxicity and GLUT2 expression in HepG2 cells

In this study, we characterized the potential cytotoxicity of Prodigiosa herbal tea on hepatocellular carcinoma (HepG2) cells in vitro. HepG2 were submitted to both concentration and time-dependent toxicity evaluation of the herbal tea of Prodigiosa (0–200 mg·L−1) following exposures of 24 and 48 h. Cell viability was determined with the (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the data reported relative to the non-treated cells. Cellular toxicity was observed at concentrations greater than 100 mg·L−1 of Prodigiosa with a clear indication of time-dependent decrease in cell viability. In contrast the 100 mg·L−1 significantly stimulated cell growth over the non-treated cells. The expression data indicated that Prodigiosa alters glucose transporter (GLUT2) protein and cDNA levels in HeG2 cells. The preliminary data from this study suggest that the effects from Prodigiosa herbal tea negatively influence HepG2 cell viability, GLUT2 protein, and mRNA expression at concentrations greater than 100 mg·L−1. Ongoing work in our laboratory will characterize the specific bioactive compound that is associated with the regulatory mechanisms of Prodigiosa on GLUT2 expression and the effects of Prodigiosa in normal liver cells.