Semi-quantitative Real-time PCR Research Articles

Preclinical Assessment of the Effectiveness of Magnetic Resonance Molecular Imaging of Extradomain-B Fibronectin for Detection and Characterization of Oral Cancer.

Oral squamous cell carcinoma (OSCC) has not seen a substantial improvement in patient survival despite therapeutic advances, making accurate detection and characterization of the disease a clinical priority. Here, we aim to demonstrate the effectiveness of magnetic resonance imaging (MRI) with the targeted MRI contrast agent MT218 specific to extradomain-B fibronectin (EDB-FN) in the tumor microenvironment for detection and characterization of aggressive OSCC tumors. EDB-FN expression was evaluated in human normal tongue and OSCC specimens with immunohistochemistry. Invasiveness of human CAL27, HSC3, and SCC4 OSCC cells was analyzed with spheroid formation and transwell assays. EDB-FN expression in the cells was analyzed with semiquantitative real-time PCR, western blotting, and a peptide binding study with confocal microscopy. Contrast-enhanced MRI with MT218 was performed on subcutaneous OSCC mouse models at a dose of 0.04mmol/kg, using gadoteridol (0.1mmol/kg) as a control. Strong EDB-FN expression was observed in human untreated primary and metastatic OSCC, reduced expression in treated OSCC, and little expression in normal tongue tissue. SCC4 and HSC3 cell lines demonstrated high invasive potential with high and moderate-EDB-FN expression, respectively, while CAL27 showed little invasive potential and low-EDB-FN expression. In T1-weighted MRI, MT218 produced differential contrast enhancement in the subcutaneous tumor models in correlation with EDB-FN expression in the cancer cells. Enhancement in the high-EDB-FN tumors was greater with MT218 at 0.04mmol/kg than gadoteridol at 0.1mmol/kg. The results suggest EDB-FN has strong potential as an imageable biomarker for aggressive OSCC. MRMI results demonstrate the effectiveness of MT218 and the potential for differential diagnostic imaging of oral cancer for improving the management of the disease.

Bronchiolitis and recurrent wheezing are distinguished by type 2 innate lymphoid cells and immune response.

BackgroundRecurrent wheezing (RW) is frequently developed in infants that have suffered bronchiolitis (BCH) during first months of life, but the immune mechanism underlying is not clear. The goal was to analyze the innate immune response that characterizes BCH and RW.MethodsNinety‐eight and seventy hospitalized infants with BCH or RW diagnosis, respectively, were included. Nasopharyngeal aspirate (NPA) was processed. Cellular pellet was employed to evaluate type 2 innate lymphoid cells (ILC2) by flow cytometry and mRNA expression assays by semi‐quantitative real‐time PCR (qRT‐PCR). In supernatant, twenty‐seven pro‐inflammatory and immunomodulatory factors, as well as lipid mediators and nitrites, were evaluated by ELISA and Luminex.ResultsBronchiolitis patients showed higher ILC2 percentage compared with RW (P < .05). Also, ST2+/ILC2 percentage was higher in the BCH group than in the RW group (P < .01). TLR3, IL33, IFNG, IL10, and FLG mRNA levels were significantly increased in BCH vs RW (P < .05). In supernatant, no significant differences were reached, observing similar levels of parameters linked to vascular damage, monocyte activation, and fibroblast growth. Prostaglandin E2 and cysteinyl leukotrienes C4 were evaluated; a significant difference was only found in their ratio.ConclusionBronchiolitis is associated with elevated nasal percentage of ILC2. This cellular population could be the key element in the differential immune response between BCH and RW which share some mechanisms such us monocyte activation, vascular damage, and fibroblast repair. Lipid mediators could play a role in the evolution of the disease later in life through innate lymphoid cells.

Increasing the expression of programmed death ligand 2 (PD-L2) but not 4-1BB ligand in colorectal cancer cells.

Immune checkpoint (ICP) molecules modulate the immune response by either inducing or preventing T cell activation. Over-expression of some ICPs on malignant cells has been shown to regulate anti-tumor immune responses. We aimed to investigate the expression levels of two immune checkpoint molecules which have not been studied extensively in patients with colorectal cancer (CRC). Programmed Death Ligand 2 (co-inhibitory) and 4-1BB ligand (co-stimulatory) were assessed in tumor tissues of CRC patients compared to the adjacent normal tissues. Following tissue excision during surgical operation from 21 CRC patients, RNA extraction, cDNA synthesis and semi-quantitative real-time PCR were done for measuring the expressions of PD-L2 and 4-1BBL genes. In protein level, indirect immunohistochemistery (IHC) was performed on tissue sections. We revealed that PD-L2 was expressed in about 81% CRCs and insignificantly correlated with the tumor differentiation grade. Although a 3.25-fold change in the gene expression of PD-L2 was found in tumor tissues compared to the adjacent normal tissues (P = 0.005), but decreased level of 4-1BBL in counterpart tissues was not significant. Our results were confirmed by IHC for PDL-2 (P = 0.02) and 4-1BBL, however it was not statistically significant for the latter one. Although not significant, we could find an association between the elevated expression of PD-L2 and the tumor differentiation grade. Increased expression of negative regulator of the anti-tumor immune responses like PD-L2, as a prominent way of tumor escape, can be considered for cancer immunotherapy approaches in CRC patients using blocking monoclonal antibodies.

Molecular Cloning and the Expression Pattern of a Phospholipid Hydroperoxide Glutathione Peroxidase in Kalidium foliatum under NaCl Treatment

The halophyte Kalidium foliatum (Pall.) Moq. exhibits strong tolerance to salinity. Salt stress leads to reactive oxygen species (ROS) production, and high contents of ROS are harmful to plants. The phospholipid hydroperoxide glutathione peroxidase (PHGPx) responds positively to damage induced by ROS. To understand the mechanism of tolerance to salt stress in halophytes, a PHGPx gene of K. foliatum was cloned, and its expression pattern was analysed under salt treatment. Treatments of whole plants were carried out with 0, 150, 200, 250, 300, 350 or 400 mM sodium chloride for 72 h. The extraction of total RNA from 4‑month-old seedlings was conducted using the leaves of both treated and untreated plants. The full-length cDNA of KfPHGPx was cloned with reverse transcription PCR (RT-PCR), the KfPHGPx expression patterns were analysed using semi-quantitative RT-PCR and real-time PCR, and the activities of phospholipid hydroperoxide glutathione peroxidase enzyme in K. foliatum were examined. The full-length cDNA clone encoding the full-length sequence consisted of 714 bp of nucleotides and included an open reading frame (ORF) that encoded a polypeptide of 237 amino acids. The deduced amino acid sequence of the KfPHGPx gene has significant similarity with Spinacia oleracea (XP_021837527.1) and Sesuvium portulacastrum (ADE07246.1). The semi-quantitative RT-PCR and real-time PCR analyse showed that KfPHGPx expression can be markedly induced by moderate salt treatment.

Uterine expression of smooth muscle alpha- and gamma-actin and smooth muscle myosin in bitches diagnosed with uterine inertia and obstructive dystocia

Primary uterine inertia (PUI) is the most common type of dystocia in dogs. We hypothesized that PUI develops because of lower than normal expression of the basic contractile elements in the uterus, i.e., smooth muscle (SM) α- and γ-actin and SM-myosin, and that the expression of these proteins is influenced by the number of fetuses present in utero. Full-thickness inter-placental uterine biopsies were collected during Cesarean sections from dogs with PUI (n = 11), and from bitches with obstructive dystocia (OD) still presenting strong labor contractions (designated as the control group, n = 7). Relative gene expression was determined by semi-quantitative real-time (TaqMan) PCR, and protein localization by immunohistochemistry. Gene expression between PUI and OD bitches, and between PUI bitches carrying small, large, or average number of fetuses according to their breed, were compared. Uterine SM-γ-actin and SM-myosin mRNA levels were significantly higher in PUI than in OD dogs, while SM-α-actin did not differ. PUI bitches carrying large litters had lower uterine SM-γ-actin gene expression than those with small litters (P = 0.008). Immunostaining for SM-actin isoforms and SM-myosin was present in the myometrium, and localization pattern and staining intensity appeared similar in the PUI and OD groups. All proteins stained in blood vessels, and SM-γ-actin was also present in endometrial luminal and glandular epithelium. In conclusion, higher uterine SM-γ-actin and SM-myosin gene expression in PUI bitches, compared with OD dogs, might be an indication of abnormal progression with labor. Whether this is the cause of PUI due to an intrinsic error of the myometrium not becoming committed to labor, or the consequence of inadequate endocrine or mechanical stimuli, is not clear. Litter size was previously shown to be one of the risk factors for the development of uterine inertia in dogs, and our findings suggest possible differing uterine pathophysiology of PUI with respect to litter size.

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome.

The importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 (Galnt3)-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. Using semiquantitative real-time PCR, we found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands. Additionally, 16S rRNA gene sequencing revealed that the loss of Galnt3 resulted in changes in the structure, composition, and stability of the oral microbiome. Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted.

Clinical significance of plasma MMP-2 and MMP-9 levels as biomarkers for tumor expression in breast cancer patients in Egypt.

Matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9) are involved in the breakdown of extracellular matrix in normal physiological processes as well as in disease processes, such as cancer metastasis. We conducted this work to study the role of MMP-2 and MMP-9 in breast cancer by measuring their plasma concentrations before and after surgery. Also, to examine if their levels can reflect the stage of disease and prognosis. Forty-eight breast cancer patients and 13 patients with benign breast diseases were included in the study. MMP-2 and MMP-9 levels were measured by ELISA and semi-quantitative real-time PCR. MMP-2 and MMP-9 levels in plasma were determined by ELISA immediately before surgery and during 6 to 12months after curative surgery. We observed a significant increase in the level of MMP-9 mRNA expression in breast cancer patients in comparison to their normal breast tissues and to tissues of benign breast disease. In all TNM tumor stages, the plasma levels of MMP-2 and MMP-9 were increased significantly before curative surgery in the studied patients with breast carcinoma and decreased significantly after surgery. Both MMP-2 and MMP-9 may be used as a possible marker for follow-up or as a marker that reflects the response of the disease to treatment.

A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus)

Berries and vegetables are potential transmission vehicles for eggs of pathogenic parasites, such as Echinococcus spp. We developed a SYBR Green based semi-quantitative real-time PCR (qPCR) method for detection of Echinococcus multilocularis and Echinococcus canadensis DNA from berry samples. A set of primers based on the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene was designed and evaluated. To assess the efficacy of the assay, we spiked bilberries (Vaccinium myrtillus) with a known amount of E. multilocularis eggs. The detection limit for the assay using the NAD1_88 primer set was 4.37 × 10−5 ng/μl of E. multilocularis DNA. Under artificial contamination of berries, 50 E. multilocularis eggs were reliably detected in 250 g of bilberries. Analytical sensitivity of the assay was determined to be 100% with three eggs. As an application of the assay, 21 bilberry samples from Finnish market places and 21 bilberry samples from Estonia were examined. Previously described sieving and DNA extraction methods were used, and the samples were analyzed for E. multilocularis and E. canadensis DNA using semi-quantitative real-time PCR and a melting curve analysis of the amplified products. Echinococcus DNA was not detected in any of the commercial berry samples. This easy and fast method can be used for an efficient detection of E. multilocularis and E. canadensis in bilberries or other berries, and it is applicable also for fruits and vegetables.

Comparison Of drrA And drrB Efflux Pump Genes Expression In Drug-Susceptible And -Resistant Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients In Iran.

BackgroundAmong different resistance mechanisms in Mycobacterium tuberculosis (MTB), efflux pumps may have a role in drug-resistance property of MTB. So, the aim of this study was to compare the relative overexpression of two important efflux pump genes, drrA and drrB, among MTB isolates from TB patients.MethodsA total of 37 clinical isolates of confirmed MTB isolates were analyzed. Drug susceptibility testing (DST) was performed using the conventional proportional method. Real-time semiquantitative PCR profiling of the efflux pump genes of drrA and drrB was performed for clinical isolates. The receiver operating curve (ROC) analysis for differentiation of resistant from susceptible isolates on the basis of efflux pump expression fold changes was also performed.ResultsAccording to DST, 16 rifampin (RIF) monoresistant, 3 isoniazid (INH) monoresistant, 5 multidrug-resistant (MDR) and 13 pan-susceptible isolates of MTB were evaluated for gene expression. The highest values of drrA and drrB gene expression fold changes were seen in MDR isolates, which were significant in comparison with susceptible isolates and H37Rv reference strain. By using comparative ROC analysis, the obtained cutoff point for drrA and drrB gene overexpression was the folds of >1.6 and >2.3, respectively.ConclusionThe results of the present study confirm the role of DrrA-DrrB efflux pump in antibiotic resistance in clinical MTB isolates. As the large number of efflux pumps are located in the cell envelope of MTB, we cannot correlate a single efflux pump overexpression to the drug-resistance phenotype, unless all the pumps simultaneously investigated.

Branched chain fatty acid composition of yak milk and manure during full-lactation and half-lactation

BackgroundBranched chain fatty acids (BCFA) are bioactive food compounds and are well known to be essential components of human, cow and caprine milk. In Qinghai-Tibet plateau, yaks are domesticated in large numbers and their milk in addition to meat are commercially important to millions of Tibetans and Chinese. ObjectiveWe tested the hypotheses that concentrations of BCFA in yak milk and manure differ between lactation periods and evaluated gene expression levels of certain genes involved in the biosynthesis and elongation of fatty acids. DesignFresh milk and manure were collected from each yak and their fatty acid compositions compared with emphasis on BCFA. Participants/settingYak milk and manure samples from the full lactation (October, 2015) and half lactation periods (March, 2016) were collected and BCFA levels were analyzed in detail by GC-FID and structures verified by GC-EI-MS/MS. Gene expression studies were carried out by semi-quantitative real time PCR method. Statistical analyses performedThe difference between full lactation and half lactation was tested using student's t-test. Linear regression model was modelled in Excel and its significance was tested by ANOVA. Statistical significance was determined by performing student's t-test for gene expression studies. ResultsBCFA ranged from 3–6% of total fatty acids in yak milk samples. The half-lactation yak milk contained higher levels of BCFA (5.29 ± 0.53) than the full-lactation milk (4.00 ± 0.46). The total BCFA in yak manure was found to be 14.67 ± 1.21, high in anteiso-15:0 and anteiso-17:0. ELOVL1 enzyme involved in the elongation of saturated C18 to C26 acyl-CoA substrates and MCAT enzyme involved in the transfer of a malonyl group to the mitochondrial acyl carrier protein are significantly upregulated in full-lactation milk. ConclusionsBCFA in yak manure especially anteiso BCFA are positively correlated with yak milk from the same animal, indicating that these BCFA come from dietary sources. Yak milk delivers 777 mg BCFA compared to 158 mg per cup of whole U.S. dairy milk. QTP herders known to consume up to 2 kg of yak yogurt take in an estimated 3,500–5,000 mg BCFA per day. We conclude that BCFA intake for yak milk consumers is among the highest known in the world, higher when drawn from half lactating yaks.

Identification of stably expressed genes for normalization of gene expression data in oral tumors: a preliminary analysis

We sought to identify stably expressed genes in tumors of gingivo-buccal region and tongue from untreated as well as treated patients. The study was undertaken in view of the ambiguity with regards to the choice of reference genes for normalization of gene expression data from gingivo-buccal region and tongue. This aspect was also examined in tumors from treated patients since it could provide clues for such analyses in the assessment of treatment modalities in the future. Expression of ten candidate housekeeping genes, identified in array-based studies, was tested using TaqMan based semi-quantitative real-time PCR. Thirty-five buccal mucosa derived (18 from treated patients) and 15 tongue tumors (8 from treated patients) were studied. Most stable genes were identified based on the consensus between the results of the three methods, Comparative δCt, BestKeeper and NormFinder, used for data analysis. CHMP2A and VPS29 were identified as the most stably expressed genes suitable for normalization of data from buccal-mucosa tumors, whereas RPS13 and PSMB2 were indicated for similar specimens from treated patients. The same criteria identified stable expression of PSMB2 and PUM1 in tumors from tongue and OAZ1 and RPS13 for the post-treatment tongue tumors. We have identified stably expressed genes in common oral cancers which can be used for normalization of the gene expression data. Results also established differences in tumors arising at different sites of the oral cavity and highlighted further changes following exposure to therapy.

CoCl2 simulated hypoxia induce cell proliferation and alter the expression pattern of hypoxia associated genes involved in angiogenesis and apoptosis

Background/aimsHypoxia microenvironment plays a crucial role during tumor progression and it tends to exhibit poor prognosis and make resistant to various conventional therapies. HIF-1α acts as an important transcriptional regulator directly or indirectly associated with genes involved in cell proliferation, angiogenesis, apoptosis and energy metabolism during tumor progression in hypoxic microenvironment. This study was aimed to investigate the expression pattern of the hypoxia associated genes and their association during breast cancer progression under hypoxic microenvironment in breast cancer cells.MethodsCell proliferation in MCF-7 and MDA-MB-231 cell lines treated with different concentration of CoCl2 was analyzed by MTT assay. Flow cytometry was performed to check cell cycle distribution, whereas cell morphology was examined by phase contrast microscopy in both the cells during hypoxia induction. Expression of hypoxia associated genes HIF-1α, VEGF, p53 and BAX were determined by semiquantitative RT-PCR and real-time PCR. Western blotting was performed to detect the expression at protein level.ResultsOur study revealed that cell proliferation in CoCl2 treated breast cancer cells were concentration dependent and varies with different cell types, further increase in CoCl2 concentration leads to apoptotic cell death. Further, accumulation of p53 protein in response to hypoxia as compare to normoxia showed that induction of p53 in breast cancer cells is HIF-1α dependent. HIF-1α dependent BAX expression during hypoxia revealed that after certain extent of hypoxia induction, over expression of BAX conquers the effect of anti-apoptotic proteins and ultimately leads to apoptosis in breast cancer cells.ConclusionIn conclusion our results clearly indicate that CoCl2 simulated hypoxia induce the accumulation of HIF-1α protein and alter the expression of hypoxia associated genes involved in angiogenesis and apoptosis.

Isolation of primary odontoblasts: Expectations and limitations

The purpose of this study was to evaluate different protocols of enzymatic treatment (collagenase with either protease, trypsin or hyaluronidase) to isolate mature odontoblasts. Primary odontoblasts were obtained from human molars, which was confirmed by histology and scanning electron microscopy. The combination of collagenase with protease appeared most suitable and resulted in higher cell numbers and better integrity of the odontoblast processes, whereas combination with hyaluronidase or trypsin led to truncated processes and detachment of cell patches instead of single cells. However, trypan blue staining after 24h showed that odontoblasts in culture did not remain viable. Gene expression analysis was possible after mRNA extraction from tissues exvivo and real-time semi-quantitative PCR revealed increased expression of collagen, nestin, bone sialoprotein and dentin matrix acidic phosphoprotein1 in the odontoblast layer. Though primary odontoblasts could not be cultivated after isolation, characteristic genes were identified to differentiate odontoblasts from pulp fibroblasts.

Impact of interplay between autophagy and interferon-alpha in HCV and HCV/HIV infection.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are among the most dangerous pathogens globally. Infection with HCV has been reported in a high percentage of HIV patients. Viruses are obligate intracellular pathogens and their survival is associated with their capability to subvert antiviral defenses of cells and to improve cellular processes required for their replication. The aim of this study was to compare the expression rate of the key gene for autophagy process, Beclin-1, as a cellular response to viral infections, and its effect on IFN-α expression in both HCV and HCV/HIV patient groups. In this study, a total number of 40 samples of peripheral blood mononuclear cells (PBMCs) including 20 HCV and 20 HCV/HIV patients before treatment were evaluated. The HCV viral load in both groups was evaluated by semi quantitative real-time PCR. The level of Beclin-1 and IFN-α gene expression was examined in all samples by semi quantitative real-time PCR assay. The median viral load was 8.3×105 copies/ml in HCV group and 2.1×106 copies/ml in HCV/HIV patients. While the expression level of Beclin-1 gene in HCV group was significantly higher, the level of IFN-α expression was lower compared to the HCV/HIV group (P Keywords: autophagy; Beclin gene; HCV; HIV; IFN gene.

The novel sugar transporter SLC50A1 as a potential serum-based diagnostic and prognostic biomarker for breast cancer.

BackgroundThe novel sugar transporter and membrane protein SLC50A1 has been identified as a potential candidate biomarker for breast cancer; however, its potential as a serum biomarker for breast cancer detection and prognosis is unclear. The aim of this study was to investigate the serum expression profile of SLC50A1 and to determine its diagnostic and prognostic significance in breast cancer.Materials and methodsBioinformatics analysis was conducted, and data for SLC50A1 expression in human breast cancer were collected. Semi-quantitative real-time PCR and ELISA were performed to compare SLC50A1 expression in several breast cancer cell lines, one paired tissue cohort (n=20) and two independent cohorts of human breast cancer patients (n=85) and healthy individuals (n=30). The results were analyzed statistically, and associations between clinicopathological and survival data were evaluated by multivariate Cox regression analysis.ResultsSLC50A1 was confirmed as a candidate breast cancer gene by bioinformatics analysis. SLC50A1 mRNA expression levels were significantly upregulated in breast cancer (P<0.001). Serum SLC50A1 levels were able to discriminate between women with breast cancer and healthy women with a sensitivity of 75.3% and a specificity of 100.0% (P<0.001; area under the curve=0.915). Interestingly, SLC50A1 protein expression was associated with estrogen receptor (P=0.016) and HER2 status (P=0.037). Furthermore, SLC50A1 levels were positively related to unfavorable 3-year outcomes in patients with high-grade breast cancer (HR =1.823, P=0.01), indicating its potential use as an independent prognostic factor.ConclusionSLC50A1 can be used as a serum-based diagnostic and prognostic biomarker in breast cancer. However, further studies are needed to clarify its potential role as a therapeutic target.

Transforming Growth Factor-β2 and Its Receptor Type II Messenger RNA Levels in Mice Endometrium and Their Regulation by Sex Steroids During Estrous Cycle

Despite increasing evidence pointing to the involvement of transforming growth factor-β superfamily in the regulation of reproduction, a definitive role of TGF-β2 and its receptor type II in the uterus is currently unclear. In this study, using semi-quantitative reverse transcriptase PCR (RT-PCR) and real-time PCR techniques (qPCR), TGF-β2 and its receptor type II (TGF-βRII) mRNA levels were examined in mouse endometrium during estrous cycle and in response to an acute exposure of 17β-estradiol (E2) and progesterone (P4). We found that TGF-βRII mRNA was expressed at a very low level during diestrus, and remained comparatively higher at proestrus, estrus and metestrus stages with no significant differences between them. On the contrary, the average TGF-β2 mRNA levels abruptly increased from estrus to diestrus stages and then briskly decreased at proestrus, the proliferative stage of the cycle. These results suggest that both TGF-β2 and its receptor type II are functional during proliferation as well as apoptosis of mice endometrial epithelial and stromal cells. A single injection of E2 or P4 at diestrus stage mice caused significant attenuation of the expression of TGF-β2 mRNA after 8 h of treatment compared to sesame oil control. Inhibition induced by P4 was comparatively stronger than that induced by E2 and results further showed that combined injection of E2 and P4 had similar inhibitory effects as that of P4 alone on TGF-β2 expression. In contrast, expression of TGF-βRII mRNA was not significantly altered by ovarian steroids administered either alone or in combination. Results further demonstrated that in short-term endometrial tissue incubation, p38 MAPK pathway was involved in the steroid-mediated inhibition of TGF-β2 mRNA with no effect on TGF-βRII mRNA. This result suggests that p38 MAPK pathway may be an important regulator of TGF-β2 and TGF-βRII mRNA expression in vitro in diestrus endometrium, in cooperation with ovarian steroids.

Tissue Morphology and Gene Expression Characterisation of Transplantable Adenocarcinoma Bearing Mice Exposed to Fluorodeoxyglucose-Conjugated Magnetic Nanoparticles.

Fluorodeoxyglucose-conjugated magnetic nanoparticles, designed to target cancer cells with high specificity when heated by an alternating magnetic field, could provide a low-cost, non-toxic treatment for cancer. However, it is essential that the in vivo impacts of such technologies on both tumour and healthy tissues are characterised fully. Profiling tissue gene expression by semi-quantitative reverse transcriptase real-time PCR can provide a sensitive measurement of tissue response to treatment. However, the accuracy of such analyses is dependent on the selection of stable reference genes. In this study, we determined the impact of fluorodeoxyglucose-conjugated magnetic nanoparticles on tumour and non-tumour tissue gene expression and morphology in MAC16 adenocarcinoma established male NMRI mice. Mice received an injection of 8 mg/kg body weight fluorodeoxyglucose-conjugated magnetic nanoparticles either intravenously in to the tail vein, directly into the tumour or subcutaneously directly overlying the tumour. Tissues from mice were sampled between 70 minutes and 12 hours post injection. Using the bioinformatic geNorm tool, we established the stability of six candidate reference genes (Hprt, Pgk1, Ppib, Sdha, Tbp and Tuba); we observed Pgk1 and Ppib to be the most stable. We then characterised the expression profiles of several apoptosis genes of interest in our adenocarcinoma samples, observing differential expression in response to mode of administration and exposure duration. Using histological assessment and fluorescent TUNNEL staining, we observed no detrimental impact on either tumour or non-tumour tissue morphology or levels of apoptosis. These observations define the underlying efficacy of fluorodeoxyglucose-conjugated magnetic nanoparticles on tumour and non-tumour tissue morphology and gene expression, setting the basis for future studies.

Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow

BackgroundIn recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.MethodsA total of 3 QCs were implemented and processed through the whole mNGS workflow: a no-template-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).ResultsThe optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6 to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).ConclusionsAlthough the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

Cytoplasmic location of NR4A1 in aggressive lymphomas is associated with a favourable cancer specific survival

The nuclear orphan receptor NR4A1 functions as tumour suppressor in aggressive lymphomas by pro-apoptotic genomic and non-genomic effects. Here, we immunohistochemically studied the clinico-pathological relevance of NR4A1 protein expression patterns in a cohort of 60 diffuse large B cell lymphoma (DLBCL) patients and non-neoplastic lymph nodes. We observed a significant association between high cytoplasmic NR4A1 and favourable cancer-specific survival and the germinal centre B cell-like subtype, respectively. Moreover, the percentage of lymphoma cells exhibiting cytoplasmic NR4A1 significantly correlated to those showing cleaved caspase 3. Complementary, functional profiling using gene set enrichment of Reactome pathways based on publicly available microarray data was applied to determine pathways potentially implicated in cytoplasmic localization of NR4A1 and validated by means of semi quantitative real-time PCR. The pathway analysis revealed changes in the ERK1/2 pathway, and this was corroborated by the finding that high cytoplasmic NR4A1 was associated with higher expression of ERK1/2 targets in our cohort. These data indicate that high cytoplasmic NR4A1 is associated with a favourable lymphoma-specific survival and highlights the importance of NR4A1 expression patterns as potential prognostic marker for risk assessment in aggressive lymphomas.