Gene Expression Ratio Research Articles

Towards Understanding Therapeutic Failures in Masquelet Surgery: First Evidence that Defective Induced Membrane Properties are Associated with Clinical Failures.

The two-stage Masquelet induced-membrane technique (IMT) consists of cement spacer-driven membrane induction followed by an autologous cancellous bone implantation in this membrane to promote large bone defect repairs. For the first time, this study aims at correlating IMT failures with physiological alterations of the induced membrane (IM) in patients. For this purpose, we compared various histological, immunohistochemical and gene expression parameters obtained from IM collected in patients categorized lately as successfully (Responders; n = 8) or unsuccessfully (Non-responders; n = 3) treated with the Masquelet technique (6 month clinical and radiologic post-surgery follow-up). While angiogenesis or macrophage distribution pattern remained unmodified in non-responder IM as compared to responder IM, we evidenced an absence of mesenchymal stem cells and reduced density of fibroblast-like cells in non-responder IM. Furthermore, non-responder IM exhibited altered extracellular matrix (ECM) remodeling parameters such as a lower expression ratio of metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinases (TIMP-1) mRNA as well as an important collagen overexpression as shown by picrosirius red staining. In summary, this study is the first to report evidence that IMT failure can be related to defective IM properties while underlining the importance of ECM remodeling parameters, particularly the MMP-9/TIMP-1 gene expression ratio, as early predictive biomarkers of the IMT outcome regardless of the type of bone, fracture or patient characteristics.

Apoptosis Induction with Enhancement of BAX/BCL2 Gene Expression Ratio via Combination Therapy in HT29 Colon Cancer Cells

Background and Aim: Combination therapy is one of the new strategies that minimize resistance to chemotherapy and reduces drug toxicity. Here, we investigated the effect of combination therapy with 5-Fluorouracil and Gamma Tocopherol on cell survival and BAX/BCL2 gene expression ratio in HT29 colon cancer cells. Methods: The proliferation of cancer cells was determined via colony formation assay. BAX/BCL2 ratio was evaluated after incubation with concentrations of 5-Fluorouracil and Gamma Tocopherol via real-time-PCR. Results: The average number of colonies in the cells treated with 5-Fluorouracil, Gamma Tocopherol and their combination of them was 63±4, 78±3, and 28±2, respectively which significantly decreased in the combination group. In contrast with the control group, the BAX/BCL2 ratio remarkably increased when the cell underwent combinational treatment (p<0.05). Conclusion: 5-Fluorouracil and Gamma Tocopherol reduced HT 29 cell proliferation. Our results suggest that combination therapy with 5- Fluorouracil and Gamma Tocopherol can be considered as a strategy for induction of apoptosis via increasing the BAX/BCL2 ratio. *Corresponding Author: Nadereh Rashtchizadeh; Email: rashtchizadeh@rocketmail.com Please cite this article as: Bazzaz R, Yaghmaei P, Dastmalchi S, Rashtchizadeh N. Apoptosis Induction with Enhancement of BAX/BCL2 Gene Expression Ratio via Combination Therapy in HT29 Colon Cancer Cells. Arch Med Lab Sci. 2020;6:1-7 (e21). https://doi.org/10.22037/amls.v6.33489

Pentagalloyl Glucose- and Ethyl Gallate-Rich Extract from Maprang Seeds Induce Apoptosis in MCF-7 Breast Cancer Cells through Mitochondria-Mediated Pathway.

Bouea macrophylla Griffith, locally known as maprang, has important economic value as a Thai fruit tree. The maprang seed extract (MPSE) has been shown to exhibit antibacterial and anticancer activities. However, the bioactive constituents in MPSE and the molecular mechanisms underlying these anticancer activities remain poorly understood. This study aims to identify the active compounds in MPSE and to investigate the mechanisms involved in MPSE-induced apoptosis in MCF-7 treated cancer cells. The cytotoxic effect was determined by MTT assay. The apoptosis induction of MPSE was evaluated in terms of ROS production, mitochondrial membrane potential depolarization, and apoptosis-related gene expression. The compounds identified by HPLC and LC/MS analysis were pentagalloyl glucose, ethyl gallate, and gallic acid. MPSE treatment decreased cell proliferation in MCF-7 cells, and MPSE was postulated to induce G2/M phase cell cycle arrest. MPSE was found to promote intracellular ROS production in MCF-7 treated cells and to also influence the depolarization of mitochondrial membrane potential. In addition, MPSE treatment can lead to increase in the Bax/Bcl-2 gene expression ratio, suggesting that MPSE-induced apoptosis is mitochondria-dependent pathway. Our results suggest that natural products obtained from maprang seeds have the potential to target the apoptosis pathway in breast cancer treatments.

Comparative Subtyping Of An Upfront Resected Cohort Of Pancreatic Adenocarcinoma Using Immunohistochemical And Rnaseq-Based Approaches To Identify Treatment Unresponsive Subgroups

Presenter: Emilio Alonso MD | Providence Portland Medical Center Background: Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the leading causes of cancer death in the Unites States, with an increasing incidence over the past decades. Despite the high mortality rate and dismal prognosis, the therapeutic responses are poor and heterogenous even to our current standards of treatment. Recently it has been shown that PDAC could be subdivided into clinically relevant subgroups using immunohistochemistry (IHC) for two protein markers, KRT81 and HNF1A. We aimed to validate their IHC-subtyping and evaluate the gene expression HNF1A and KRT81 in an upfront-resected PDAC cohort. Alternative PDAC subtyping using gene-expression profiles have been previously described by Collisson and Bailey, which we aim to see their overlap using our cohort. Methods: We performed IHC for HNF1A and KRT81 on 61 patients with upfront resected PDAC from 2010 to 2014. These were read by a board-certify pathologist confirming its presence or absence in the tumor cells. Of the 68 patients, 34 patients had RNAseq. Gene expression analysis for KRT81/HNF1A were generated, from which gene expression ratio were calculated. Expression of HNF1A/KRT81 in the The-Cancer-Genome-Atlas (TCGA) pancreatic cancer database, were similarly evaluated on 138 patients after the data was obtained from cBioPortal. Using RNAseq as a more sensitive test, the accuracy, sensitivity, and specificity of IHC-subtyping was accessed. Molecular subtyping was also performed as previously described by Collisson and Bailey. Results: Using IHC-subtyping, we were able to validate the Results of the previous study. The IHC HNF1A-subtype showed a better overall and disease-free survival than KRT81. Similarly, the HNF1A positive RNAseq subtype had a statistically better overall survival and disease-free survival over the KRT81 positive subtype (P < .016 and P < 0.023, respectively). When validated externally using the TCGA-database, the RNAseq HNF1A subtype had a statistically better disease-free survival than the KRT81 subtype (P < 0.0193). We were not able to recapitulate this on the overall survival given the early censorship of the TCGA cohort of patients. Using gene expression as a more sensitive tool, the accuracy (53%), sensitivity (54%) and specificity (75%) were calculated. Thus, IHC had a better positive than negative predictive value (84% vs 6%) in stratifying PDAC. Additionally, our gene profile subtypes did not match the previously described subtypes of Collisson and Bailey as they did not correlate with survival outcomes in our patients. Conclusion: RNAseq stratification was able to accurately recapitulate the IHC subtypes of PDAC using the gene expression of HNF1A and KRT81, which was associated with significant different outcomes in both our internal cohort of upfront resected PDAC and externally using the TCGA database. Unfortunately, we were not able to recapitulate the previous molecular subtyping defined by Collisson and Bailey. This study, introduces a more sensitive analytical tool, RNAseq, for the stratification of this PDAC subtypes, which may play a role in our future understanding of the biology of these two subgroups. With the growing development in price and workflow, RNAseq analysis on endosonographically acquired core needle biopsies may serve as a biomarker-based stratification stool for PDAC patients.

Liver X Receptor Agonism Sensitizes a Subset of Hepatocellular Carcinoma to Sorafenib by Dual-Inhibiting MET and EGFR

Sorafenib is the first approved systemic therapy for advanced hepatocellular carcinoma (HCC) and is the first-line choice in clinic. Sustained activation of receptor tyrosine kinases (RTKs) is associated with low efficacy of sorafenib in HCC. Activation of liver X receptor (LXR) has been reported to inhibit some RTKs. In this study, we found that the LXR agonist enhanced the anti-tumor activity of sorafenib in a subset of HCC cells with high LXR-β/α gene expression ratio. Mechanically, the activation of LXR suppressed sorafenib dependent recruitment of MET and epidermal growth factor receptor (EGFR) in lipid rafts through cholesterol efflux. Our findings imply that LXR agonist can serve as a potential sensitizer to enhance the anti-tumor effect of sorafenib.

Predominance of M2 macrophages in gliomas leads to the suppression of local and systemic immunity.

Glioblastoma is a highly prevalent and aggressive form of primary brain tumor. It represents approximately 56% of all the newly diagnosed gliomas. Macrophages are one of the major constituents of tumor-infiltrating immune cells in the human gliomas. The role of immunosuppressive macrophages is very well documented in correlation with the poor prognosis of patients suffering from breast, prostate, bladder and cervical cancers. The current study highlights the correlation between the tumor-associated macrophage phenotypes and glioma progression. We observed an increase in the pool of M2 macrophages in high-grade gliomas, as confirmed by their CD68 and CD163 double-positive phenotype. In contrast, less M1 macrophages were noticed in high-grade gliomas, as evidenced by the down-regulation in the expression of CCL3 marker. In addition, we observed that higher gene expression ratio of CD163/CCL3 is associated with glioma progression. The Kaplan-Meier survival plots indicate that glioma patients with lower expression of M2c marker (CD163), and higher expression of M1 marker (CCL3) had better survival. Furthermore, we examined the systemic immune response in the peripheral blood and noted a predominance of M2 macrophages, myeloid-derived suppressor cells and PD-1+ CD4 T cells in glioma patients. Thus, the study indicates a high gene expression ratio of CD163/CCL3 in high-grade gliomas as compared to low-grade gliomas and significantly elevated frequency of M2 macrophages and PD-1+ CD4 T cells in the blood of tumor patients. These parameters could be used as an indicator of the early diagnosis and prognosis of the disease.

151P - Gene expression profiling for a better understanding of gastric cancer: From the perspective of metabolic rearrangement

BackgroundMetabolic rearrangement has been shown to be an important characteristic for stomach adenocarcinomas. Here we aimed to improve our understanding of the tumorigenesis of gastric cancer by means of gene and protein expression profile analysis with a focus on metabolic genes and pathways. MethodsArray-based gene expression profiling of fresh frozen cancer tissues and adjacent normal tissues were obtained from 8 patients with gastric cancer at early stage by using Affymetrix oligonucleotide microarray. Assays targeted 179 unique genes related to cancer metabolism. The raw expression data were normalized using nSolver Analysis Software 3.0 and a dataset of gene expression ratios for GC vs. controls was generated. The p values were calculated using a paired t-test, and the threshold for up- and down-regulated genes was set at p value < 0.05. The protein expression of the dysregualted genes were detected by immunohistochemistry (IHC) in the formalin fixed paraffin embedded tissue blocks. The signal was quantified by the Allred score system which represented the estimated intensity and proportion of positive-staining cells. ResultsOur microarray results showed increased expression of 20 metabolic genes and decreased expression of 6 metabolic genes in all cases of gastric cancer at early stage. Besides of the undetected NOX4, the protein levels of all the others dysregualted genes, detected by IHC, showed consistently results. Half of all dysregulated genes (AKT2, EGLN3, G6PD, GLS, HIF1A, HK2, HRAS, MAP2K1, NTRK3, PGK1, PLCG1, RET and RPS6KB1) are implicated in Carbon Metabolism, a pivotal metabolic approach involving in nucleic acid biosynthesis. Five genes (ARNT, EGLN1, EGLN3, HIF1A and NOX4) are molecules implicated in hypoxia signaling. Besides, the upregulated HIF1A is a cancer metabolism driver, which means that HIF1A may induce the oncogenesis of gastric cancer. ConclusionsThe gastric mucosa in gastric cancer at early stage is characterized by dysregulated expression of a limited repertoire of metabolic genes. The nature of the corresponding metabolic rearrangement and pathways may help guide further investigations into its etiology. Legal entity responsible for the studyFudan University Shanghai Cancer Center. FundingNational Human Genetic Resources Sharing Service Platform (2005DKA21300), National Natural Science Foundation of China (81602078),. DisclosureAll authors have declared no conflicts of interest.

Altered Cell Surface N-Glycosylation of Resting and Activated T Cells in Systemic Lupus Erythematosus.

Altered cell surface glycosylation in congenital and acquired diseases has been shown to affect cell differentiation and cellular responses to external signals. Hence, it may have an important role in immune regulation; however, T cell surface glycosylation has not been studied in systemic lupus erythematosus (SLE), a prototype of autoimmune diseases. Analysis of the glycosylation of T cells from patients suffering from SLE was performed by lectin-binding assay, flow cytometry, and quantitative real-time PCR. The results showed that resting SLE T cells presented an activated-like phenotype in terms of their glycosylation pattern. Additionally, activated SLE T cells bound significantly less galectin-1 (Gal-1), an important immunoregulatory lectin, while other lectins bound similarly to the controls. Differential lectin binding, specifically Gal-1, to SLE T cells was explained by the increased gene expression ratio of sialyltransferases and neuraminidase 1 (NEU1), particularly by elevated ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1)/NEU1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6)/NEU1 ratios. These findings indicated an increased terminal sialylation. Indeed, neuraminidase treatment of cells resulted in the increase of Gal-1 binding. Altered T cell surface glycosylation may predispose the cells to resistance to the immunoregulatory effects of Gal-1, and may thus contribute to the pathomechanism of SLE.

Increased Chondrocytic Gene Expression Is Associated With Improved Repair Tissue Quality and Graft Survival in Patients After Autologous Chondrocyte Implantation

Background: Assays to quantitate the quality of autologous chondrocyte implants have recently become available. However, the correlation of the assay score with radiological and clinical outcomes has not been established. Purpose/Hypothesis: The purpose was to assess the influence of cell identity (chondrocyte/synoviocyte gene expression ratio) and viability on patient-reported outcome measures, graft survival, and repair tissue quality. It was hypothesized that greater cell product quality as assessed through an identity assay and cell viability is associated with superior outcomes after autologous chondrocyte implantation (ACI) for symptomatic cartilage defects. Study Design: Cohort study; Level of evidence, 3. Methods: Seventy-nine patients with a minimum follow-up of 2 years were included in this study. Of these, 67 patients were available for imaging assessment utilizing the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scoring system. Patients were assigned to groups either below or above the cohort’s mean based on their individual cell identity score and viability percentage. Results: Patients were predominantly female (57.7%) with a mean age of 30.0 ± 9.3 years. No differences were seen between Knee injury and Osteoarthritis Outcome Score, Lysholm, Tegner, or International Knee Documentation Committee Subjective Knee Evaluation Form within the viability and cell identity groups at a final follow-up of 3.8 ± 1.4 years after ACI (P > .05). In a subset of patients, the mean MOCART score was 68.3 ± 15.6 at an average magnetic resonance imaging follow-up of 17.7 ± 9.56 months. Low cell identity was significantly associated with the degree of defect filling (P = .025), integration of border zone (P = .01), effusion (P = .024), and ACI graft failure (P = .002). Patients with above-average cell identity scores had a significantly higher survival rate at 5-year follow-up compared with patients with below-average scores (95.8% vs 64.7%; P = .013). Cell viability did not influence MOCART subscales or graft failure (all P > .05). Cell viability and identity showed no significant correlation with each other (r = −0.045; P = .694). Conclusion: Cell identity was significantly correlated with structural repair quality and graft survival after second-generation ACI for symptomatic chondral lesions in the knee. While improved imaging outcome and higher graft survivorship were associated with a higher individual cell identity score indicating a higher chondrocyte/synoviocyte gene expression ratio in the final cell product, clinical outcome did not correlate with the identity score.

Altered immune parameters associated with Koala Retrovirus (KoRV) and Chlamydial infection in free ranging Victorian koalas (Phascolarctos cinereus)

Koala Retrovirus (KoRV) has been widely speculated to cause immune suppression in koalas (Phascolarctos cinereus) and to underlie the koala’s susceptibility to infectious disease, however evidence for immunomodulation is limited. The aim of this study is to determine whether immunophenotypic changes are associated with KoRV infection in free ranging Victorian koalas. qPCR was used to examine mRNA expression for Th1 (IFNγ), Th2-promoting (IL6, IL10) and Th17 (IL17A) cytokines, along with CD4 and CD8 in whole blood of koalas (n = 74) from Mt Eccles and Raymond Island in Victoria, Australia, with and without natural chlamydial infection. KoRV positive koalas had significantly lower levels of IL17A (p`0.023) and IFNγ (p = 0.044) gene expression along with a decreased CD4:CD8 gene expression ratio (p = 0.025) compared to negative koalas. No effect of chlamydial infection or combined effect of KoRV and chlamydial infection was detected in these populations. The decreased expression of IFNγ could make KoRV infected koalas more susceptible to persistent chlamydial infection, and a decrease in IL17A could make them more susceptible to gram negative bacterial, fungal and mycobacterial infection; but more tolerant of chlamydial infection.

Phenotype-based drug screening reveals association between venetoclax response and differentiation stage in acute myeloid leukemia.

Ex vivo drug testing is a promising approach to identify novel treatment strategies for acute myeloid leukemia. However, accurate blast-specific drug responses cannot be measured with homogeneous "add-mix-measure" cell viability assays. In this study, we implemented a flow cytometry-based approach to simultaneously evaluate the ex vivo sensitivity of different cell populations in 34 primary acute myeloid leukemia samples to seven drugs and 27 rational drug combinations. Our data demonstrate that different cell populations present in acute myeloid leukemia samples have distinct sensitivity to targeted therapies. Particularly, blast cells of FAB M0/1 acute myeloid leukemia showed high sensitivity to venetoclax. In contrast, differentiated monocytic cells abundantly present in M4/5 subtypes showed resistance to Bcl-2 inhibition, whereas immature blasts in the same samples were sensitive, highlighting the importance of blast-specific readouts. Accordingly, in the total mononuclear cell fraction the highest BCL2/MCL1 gene expression ratio was observed in M0/1 and the lowest in M4/5 acute myeloid leukemia. Of the seven tested drugs, venetoclax had the highest blast-specific toxicity, and combining venetoclax with either MEK inhibitor trametinib or JAK inhibitor ruxolitinib effectively targeted all venetoclax-resistant blasts. In conclusion, we show that ex vivo efficacy of targeted agents and particularly Bcl-2 inhibitor venetoclax is influenced by the cell type, and accurate blast-specific drug responses can be assessed with a flow cytometry-based approach.

The Application of Ultrasonic Vibration in Human Sperm Cryopreservation as a Novel Method for the Modification of Physicochemical Characteristics of Freezing Media

The application of ultrasonic vibration was performed to modify the water molecules as the main compositions of the freezing medium used for human sperm cryopreservation. Different time periods of ultrasonic vibration (ULV) at the frequency of 28 kHz were applied for the evaluation of physicochemical properties of the water molecules. The most significant bubble size, zeta potential, and pH were obtained for the water molecules exposed to ultrasonic vibrations for 18 minutes and this time period was selected for further experiments due to the optimum results. In the next stage, semen samples were diluted with freezing medium containing ULV-exposed water and then cryopreserved. All the semen parameters were significantly reduced in cryopreserved groups as compared with the fresh control group. The highest percentage of total and progressive motility, viability, membrane and DNA integrity, and mitochondrial membrane potential were observed in frozen ULV compared with the frozen control. The rate of apoptosis in frozen ULV was significantly lower than that of in the frozen control. Furthermore, the gene expression ratios of α- and β-tubulins were significantly increased during cryopreservation, while the expression ratio of the tubulin polymerization promoting protein (TPPP) gene was decreased. Similar results were also observed when the protein levels of the genes mentioned earlier were evaluated by the ELISA method. Therefore, the changes in physicochemical properties of the freezing medium of human sperm cryopreservation using ULV can improve the quality of frozen products.

Synthesis, characterization and biological evaluation of some novel sulfonylthiosemicarbazides

A series of thiosemicarbazides were synthesized and structurally characterized by spectroscopic techniques (NMR, FT-IR) besides elemental analysis. These compounds were evaluated for their cytotoxicity against human breast cancer cell line MCF7 and prostate cancer cell line PC3 and nonmalignant fibroblast L929 cell line by MTT assay. Among the compounds, N-[2-(4-chlorophenyl)ethyl]-2-[(4-methylphenyl)sulfonyl]hydrazinecarbothioamide (3d) and 2-[(4-methylphenyl)sulfonyl]-N-[4-(trifluoromethoxy)phenyl]hydrazinecarbothioamide (3f) were found to display significant cytotoxicity with IC50 of 13.87 μM (against PC3 cell line) and 1.47 μM (against MCF7 cell line), respectively. These compounds were non-cytotoxic to normal cell line with IC50>100 μM. Western blotting studies demonstrated that compound 3f induced apoptosis and caused cell death in the MCF7 and PC3 cell lines via an increase in Bax protein expression and a slight decrease in Bcl-2 protein expression. The gene expression ratio Bax/Bcl-2 showed the induction of mitochondrial apoptosis in cancer cell lines. All of synthesized compounds have also been tested for antioxidant activity and all compounds achieved strong inhibition of the DPPH radical. These findings showed that compound 3f, displays potential to be further explored in the development of new anticancer agents.

Targeted transcriptional profiling of the tumor microenvironment reveals lymphocyte exclusion and vascular dysfunction in metastatic osteosarcoma

ABSTRACTOsteosarcoma (OS) is the most common bone tumor in pediatric and adolescent/young adult patients yet little is known about the microenvironment that supports this aggressive disease. We have used targeted gene expression profiling and immunohistochemistry to characterize the microenvironment of metastatic and non-metastatic OS specimens from pediatric patients exhibiting poor histologic response to chemotherapy. Our results indicate that metastatic specimens exhibit lymphocyte exclusion as T cells are confined to the periphery of the pulmonary lesions. Furthermore, our data provides evidence of vascular dysfunction in metastatic OS indicated by increased expression of VEGFA, an increased ANGPT2:ANGPT1 gene expression ratio, and decreased expression of SELE, the gene encoding the adhesion molecule E-selectin. Moreover, correlation analyses show an inverse relationship between lymphocyte abundance and markers of vascular dysfunction exclusively in the metastatic specimens. Together, our data shows that the non-metastatic OS specimens demonstrate increased expression of various immunotherapeutic targets in comparison metastatic specimens and identifies vascular dysfunction and lymphocyte exclusion as important processes for therapeutic intervention in metastatic disease.

Genes expression and phenotypic differences in corpus luteum and cumulus cells of commercial line and piau breed gilts

We aimed to characterize the expression of angiogenesis-related genes in corpus luteum (CL) and cumulus cells (CC) during estrous cycle in gilts of different genetic groups, as well as to study the relation between gene expression and phenotypic data. Forty five gilts were used as follows: L1, Commercial Line 1 (Large White x Landrace x Duroc) (n = 15); L2, Commercial Line 2 (Large White x Landrace x Pietrain) (n = 15); and Piau, Piau breed gilts (n = 15). Estrus observation started from 120 days of age. After the second observed estrus females were slaughtered (n = 3) on days 3, 5, 10, 14 and 18 of estrous cycle (first day of estrous cycle as Day 0). CL sampling was performed on days 3, 5, 10 and 14 and collection of CC and follicular fluid on days 14 and 18. Follicular fluid was used for analysis of estradiol levels and CC and CL samples for analysis of angiogenesis-related genes expression, ANGPT-1/-2 and TEK in CC and MMP-2, VEGFA, VEGFR-1/-2, ANGPT-1/-2 and TEK in CL. Piau gilts showed lower ovulation rate than both L1 and L2 gilts (P < 0.05), lower number of large antral follicles (>6 mm) at 18 days than L2 gilts (P < 0.05), and smaller diameter of the largest follicles at 14 days than L1 gilts (P < 0.05). Piau and L2 gilts showed higher estradiol levels in follicular fluid on day 18. Expression of ANGPT-1 and -2 genes in CC did not differ among genetic groups neither among days of the estrous cycle, but TEK gene expression was higher in L1 than L2 gilts on day 18. Expression of VEGFA, VEGFR-2 and MMP-2 genes in CL did not differ among genetic groups and days of cycle, but VEGFR-1 expression was higher in Piau than L2 gilts on days 10 and 14, and it was higher in L1 than L2 gilts on day 14. The ANGPT-1/-2 and TEK genes expression in CL were significantly higher in Piau than L1 gilts on day 10. The ANGPT-2/ANGPT-1 gene expression ratio in CL was higher in L1 than Piau and L2 gilts at 14 days, suggesting a shorter luteal phase for L1 gilts. Results indicated differences among genetic groups for the pattern of the angiogenesis-related genes expression in CL along estrous cycles, which may be reflected in phenotypic traits such as ovulation rate, estradiol levels in follicular fluid and number and diameter of antral follicles.

Pairwise efficiency: a new mathematical approach to qPCR data analysis increases the precision of the calibration curve assay

BackgroundThe real-time quantitative polymerase chain reaction (qPCR) is routinely used for quantification of nucleic acids and is considered the gold standard in the field of relative nucleic acid measurements. The efficiency of the qPCR reaction is one of the most important parameters in data analysis in qPCR experiments. The Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines recommends the calibration curve as the method of choice for estimation of qPCR efficiency. The precision of this method has been reported to be between SD = 0.007 (three replicates) and SD = 0.022 (no replicates).ResultsIn this article, we present a novel approach to the analysis of qPCR data which has been obtained by running a dilution series. Unlike previously developed methods, our method, Pairwise Efficiency, involves a new formula that describes pairwise relationships between data points on separate amplification curves and thus enables extensive statistics. The comparison of Pairwise Efficiency with the calibration curve by Monte Carlo simulation shows the two-folds improvement in the precision of estimations of efficiency and gene expression ratios on the same dataset.ConclusionsThe Pairwise Efficiency nearly doubles the precision in qPCR efficiency determinations compared to standard calibration curve method. This paper demonstrates that applications of combinatorial treatment of data provide the improvement of the determination.

IMTBGO: An Algorithm for Integrating Metabolic Networks with Transcriptomes Based on Gene Ontology Analysis.

Background: Constraint-based metabolic network models have been widely used in pheno-typic prediction and metabolic engineering design. In recent years, researchers have attempted to im-prove prediction accuracy by integrating regulatory information and multiple types of “omics” data into this constraint-based model. The transcriptome is the most commonly used data type in integration, and a large number of FBA (flux balance analysis)-based integrated algorithms have been developed.Methods and Results: We mapped the Kcat values to the tree structure of GO terms and found that the Kcat values under the same GO term have a higher similarity. Based on this observation, we developed a new method, called iMTBGO, to predict metabolic flux distributions by constraining reaction bounda-ries based on gene expression ratios normalized by marker genes under the same GO term. We applied this method to previously published data and compared the prediction results with other metabolic flux analysis methods which also utilize gene expression data. The prediction errors of iMTBGO for both growth rates and fluxes in the central metabolic pathways were smaller than those of earlier published methods.Conclusion: Considering the fact that reaction rates are not only determined by genes/expression levels, but also by the specific activities of enzymes, the iMTBGO method allows us to make more precise pre-dictions of metabolic fluxes by using expression values normalized based on GO.

Protective and modulatory effects of royal jelly used against the induced changes in silver nanoparticles on the hippocampus of male rats

Objective (s): Silver nanoparticles (NPs) have attracted considerable attention owing to their important properties, including antimicrobial and anti-oxidative stress effects. However, high concentrations of silver NPs have been reported to have toxic effects. The present study aimed to evaluate the modulatory and protective effects of royal jelly (RJ) against the harmful effects of silver NPs on hippocampal functions, such as learning and memory. Materials and Methods: This experimental study was conducted on 40 male Wistar rats. The animals were divided into four groups of 10, including the control group (no silver NPs and RJ), RJ group, silver NPs plus RJ, and silver NPs. Some functions of the hippocampus (e.g., learning and memory) were evaluated using Morris memory function tests for four consecutive days. In addition, the relative expression of TRPV1 was assessed using real-time polymerase chain reaction (RT-PCR). At the final stage, hippocampal tissues were collected for histological studies.Results: Levels of learning and memory, relative gene expression ratio of TRPV1, and the histological changes in the hippocampus were significantly different in the groups receiving silver NPs compared to the groups administered with RJ. Conclusion: According to the results, RJ may be the effective in the protection against the adverse effects of silver NPs and improve the function of the hippocampus.

Effect of paleopolyploidy and allopolyploidy on gene expression in banana

BackgroundBananas (Musa spp.) are an important crop worldwide. Most modern cultivars resulted from a complex polyploidization history that comprised three whole genome duplications (WGDs) shaping the haploid Musa genome, followed by inter- and intra-specific crosses between Musa acuminata and M. balbisiana (A and B genome, respectively). Unresolved hybridizations finally led to banana diversification into several autotriploid (AAA) and allotriploid cultivars (AAB and ABB). Using transcriptomic data, we investigated the impact of the genome structure on gene expression patterns in roots of 12 different triploid genotypes covering AAA, AAB and ABB subgenome constitutions.ResultsWe demonstrate that (i) there are different genome structures, (ii) expression patterns go beyond the predicted genomic groups, and (iii) the proportion of the B genome influences the gene expression.The presence of the B genome is associated with a higher expression of genes involved in flavonoid biosynthesis, fatty acid metabolism, amino sugar and nucleotide sugar metabolism and oxidative phosphorylation. There are cultivar-specific chromosome regions with biased B:A gene expression ratios that demonstrate homoeologous exchanges (HE) between A and B sub-genomes. In two cultivars, aneuploidy was detected. We identified 3674 genes with a different expression level between allotriploid and autotriploid with ~ 57% having recently duplicated copies (paralogous). We propose a Paralog Inclusive Expression (PIE) analysis that appears to be suitable for genomes still in a downsizing and fractionation process following whole genome duplications. Our approach allows highlighting the genes with a maximum likelihood to affect the plant phenotype.ConclusionsThis study on banana is a good case to investigate the effects of alloploidy in crops. We conclude that allopolyploidy triggered changes in the genome structure of a crop and it clearly influences the gene.

A likelihood ratio test for changes in homeolog expression bias

BackgroundGene duplications are a major source of raw material for evolution and a likely contributor to the diversity of life on earth. Duplicate genes (i.e., homeologs, in the case of a whole genome duplication) may retain their ancestral function, sub- or neofunctionalize, or be lost entirely. A primary way that duplicate genes evolve new functions is by altering their expression patterns. Comparing the expression patterns of duplicate genes gives clues as to whether any of these evolutionary processes have occurred.ResultsWe develop a likelihood ratio test for the analysis of the expression ratios of duplicate genes across two conditions (e.g., tissues). We demonstrate an application of this test by comparing homeolog expression patterns of 1448 homeologous gene pairs using RNA-seq data generated from leaves and petals of an allotetraploid monkeyflower (Mimulus luteus). We assess the sensitivity of this test to different levels of homeolog expression bias and compare the method to several alternatives.ConclusionsThe likelihood ratio test derived here is a direct, transparent, and easily implemented method for detecting changes in homeolog expression bias that outperforms alternative approaches. While our method was derived with homeolog analysis in mind, this method can be used to analyze changes in the ratio of expression levels between any two genes in any two conditions.