Reverse Transcriptase-polymerase Chain Reaction Data Research Articles

3-Bromopyruvate, a caloric restriction mimetic, exerts a mitohormetic effect to provide neuroprotection through activation of autophagy in rats during aging.

In the present study, attempts have been made to evaluate the potential role of 3 Bromopyruvate (3-BP) a glycolytic inhibitor and a caloric restriction mimetic (CRM), to exert neuroprotection in rats during aging through modulation of autophagy. Young male rats (4 months), and naturally aged (22 months) male rats were supplemented with 3-BP (30mg/kg b.w., orally) for 28 days. Our results demonstrate a significant increase in the antioxidant biomarkers (ferric reducing antioxidant potential level, total thiol, superoxide dismutase, and catalase activities) and a decrease in the level of pro-oxidant biomarkers such as protein carbonyl after 3-BP supplementation in brain tissues. A significant increase in reactive oxygen species (ROS) was observed due to the mitohormetic effect of 3-BP supplementation in the treated rats. Furthermore, the 3-BP treatment also enhanced the activities of electron transport chain complexes I and IV in aged brain mitochondria thus proving its antioxidant potential at the level of mitochondria. Gene expression analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the expression of autophagy, neuroprotective and aging marker genes. RT-PCR data revealed that 3-BP up-regulated the expression of autophagy markers genes (Beclin-1 and LC3 β), sirtuin-1, and neuronal marker gene (NSE), respectively in the aging brain. The results suggest that 3-BP induces a mitohormetic effect through the elevation of ROS which reinforces defensive mechanism(s) targeted at regulating autophagy. These findings suggest that consistently low-dose 3-BP may be beneficial for neuroprotection during aging and age-related disorders.

Temporal Modulation of Differential Alternative Splicing in HaCaT Human Keratinocyte Cell Line Chronically Exposed to Arsenic for up to 28 Wk.

Background:Chronic arsenic exposure via drinking water is associated with an increased risk of developing cancer and noncancer chronic diseases. Pre-mRNAs are often subject to alternative splicing, generating mRNA isoforms encoding functionally distinct protein isoforms. The resulting imbalance in isoform species can result in pathogenic changes in critical signaling pathways. Alternative splicing as a mechanism of arsenic-induced toxicity and carcinogenicity is understudied.Objective:This study aimed to accurately profile differential alternative splicing events in human keratinocytes induced by chronic arsenic exposure that might play a role in carcinogenesis.Methods:Independent quadruplicate cultures of immortalized human keratinocytes (HaCaT) were maintained continuously for 28 wk with 0 or sodium arsenite. RNA-sequencing (RNA-Seq) was performed with poly(A) RNA isolated from cells harvested at 7, 19, and 28 wk with subsequent replicate multivariate analysis of transcript splicing (rMATS) analysis to detect and quantify differential alternative splicing events. Reverse transcriptase-polymerase chain reaction (RT-PCR) for selected alternative splicing events was performed to validate RNA-Seq predictions. Functional enrichment was performed by gene ontology (GO) analysis of the differential alternative splicing event data set at each time point.Results:At least 600 differential alternative splicing events were detected at each time point tested, comprising all the five main types of alternative splicing and occurring in both open reading frames (ORFs) and untranslated regions (UTRs). Based on functional relevance ELK4, SHC1, and XRRA1 were selected for validation of predicted alternative splicing events at 7 wk by RT-PCR. Densitometric analysis of RT-PCR data corroborated the rMATS predicted alternative splicing for all three events. Protein expression validation of the selected alternative splicing events was challenging given that very few isoform-specific antibodies are available. GO analysis demonstrated that the enriched terms in differential alternatively spliced mRNAs changed dynamically with the time of exposure. Notably, RNA metabolism and splicing regulation pathways were enriched at the 7-wk time point, when the greatest number of differentially alternatively spliced mRNAs are detected. Our preliminary proteomic analysis demonstrated that the expression of the canonical isoforms of the splice regulators DDX42, RMB25, and SRRM2 were induced upon chronic arsenic exposure, corroborating the splicing predictions.Discussion:These results using cultures of HaCaT cells suggest that arsenic exposure disrupted an alternative splice factor network and induced time-dependent genome-wide differential alternative splicing that likely contributed to the changing proteomic landscape in arsenic-induced carcinogenesis. However, significant challenges remain in corroborating alternative splicing data at the proteomic level. https://doi.org/10.1289/EHP9676

Population-Based Estimates of SARS-CoV-2 Seroprevalence in Houston, Texas as of September 2020.

BackgroundIn contrast to studies that relied on volunteers or convenience sampling, there are few population-based SARS-CoV-2 seroprevalence investigations and most were conducted early in the pandemic. The health department of the fourth largest city in the U.S. recognized that sound estimates of viral impact were needed to inform decision-making.MethodsAdapting standardized disaster research methodology in September 2020, the city was divided into high and low strata based on RT-PCR positivity rates, and census block groups within each stratum were randomly selected with probability proportional to size, followed by random selection of households within each group. Using two immunoassays, the proportion of infected individuals was estimated for the city, as well as by positivity rate and by sociodemographic and other characteristics. The degree of under ascertainment of seroprevalence was estimated based on RT-PCR positive cases.ResultsSeroprevalence was estimated to be 14% with a near two-fold difference in areas with high (18%) versus low (10%) RT-PCR positivity rates and was four times higher compared to case-based surveillance data.ConclusionsSeroprevalence was higher than previously reported and is greater than that estimated from RT-PCR data. Results will be used to inform public health decisions about testing, outreach, and vaccine rollout.

Spermidine, a caloric restriction mimetic, provides neuroprotection against normal and D-galactose-induced oxidative stress and apoptosis through activation of autophagy in male rats during aging.

Spermidine (SPD) is a natural polyamine present in all living organisms and is involved in the maintenance of cellular homeostasis by inducing autophagy in different model organisms. Its role as a caloric restriction mimetic (CRM) is still being investigated. We have undertaken this study to investigate whether SPD, acting as a CRM, can confer neuroprotection in D-galactose induced accelerated senescence model rat and naturally aged rats through modulation of autophagy and inflammation. Young male rats (4months), D-gal induced (500mg/kg b.w., subcutaneously) aging and naturally aged (22months) male rats were supplemented with SPD (10mg/kg b.w., orally) for 6weeks. Standard protocols were employed to measure prooxidants, antioxidants, apoptotic cell death and electron transport chain complexes in brain tissues. Gene expression analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the expression of autophagy and inflammatory marker genes. Our data demonstrate that SPD significantly (p ≤ 0.05) decreased the level of pro-oxidants and increased the level of antioxidants. SPD supplementation also augmented the activities of electron transport chain complexes in aged brain mitochondria thus proving its antioxidant potential at the level of mitochondria. RT-PCR data revealed that SPD up-regulated the expression of autophagy genes (ATG-3, Beclin-1, ULK-1 and LC3B) and down-regulated the expression of the inflammatory gene (IL-6) in aging brain. Our results provide first line of evidence that SPD provides neuroprotection against aging-induced oxidative stress by regulating autophagy, antioxidants level and also reduces neuroinflammation. These results suggest that SPD may be beneficial for neuroprotection during aging and age-related disorders.

Read trimming is not required for mapping and quantification of RNA-seq reads at the gene level.

RNA sequencing (RNA-seq) is currently the standard method for genome-wide expression profiling. RNA-seq reads often need to be mapped to a reference genome before read counts can be produced for genes. Read trimming methods have been developed to assist read mapping by removing adapter sequences and low-sequencing-quality bases. It is however unclear what is the impact of read trimming on the quantification of RNA-seq data, an important task in RNA-seq data analysis. In this study, we used a benchmark RNA-seq dataset and simulation data to assess the impact of read trimming on mapping and quantification of RNA-seq reads. We found that adapter sequences can be effectively removed by read aligner via ’soft-clipping’ and that many low-sequencing-quality bases, which would be removed by read trimming tools, were rescued by the aligner. Accuracy of gene expression quantification from using untrimmed reads was found to be comparable to or slightly better than that from using trimmed reads, based on Pearson correlation with reverse transcriptase-polymerase chain reaction data and simulation truth. Total data analysis time was reduced by up to an order of magnitude when read trimming was not performed. Our study suggests that read trimming is a redundant process in the quantification of RNA-seq expression data.

Fisetin as a caloric restriction mimetic protects rat brain against aging induced oxidative stress, apoptosis and neurodegeneration

AimIn the present study, attempts have been made to evaluate the potential role of fisetin, a caloric restriction mimetic (CRM), for neuroprotection in D-galactose (D-gal) induced accelerated and natural aging models of rat. Main methodsFisetin was supplemented (15mg/kg b.w., orally) to young, D-gal induced aged (D-gal 500mg/kg b.w subcutaneously) and naturally aged rats for 6weeks. Standard protocols were employed to measure pro-oxidants, antioxidants and mitochondrial membrane potential in brain tissues. Gene expression analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the expression of autophagy, neuronal, aging as well as inflammatory marker genes. We have also evaluated apoptotic cell death and synaptosomal membrane-bound ion transporter activities in brain tissues. Key findingsOur data demonstrated that fisetin significantly decreased the level of pro-oxidants and increased the level of antioxidants. Furthermore, fisetin also ameliorated mitochondrial membrane depolarization, apoptotic cell death and impairments in the activities of synaptosomal membrane-bound ion transporters in aging rat brain. RT-PCR data revealed that fisetin up-regulated the expression of autophagy genes (Atg-3 and Beclin-1), sirtuin-1 and neuronal markers (NSE and Ngb), and down-regulated the expression of inflammatory (IL-1β and TNF-α) and Sirt-2 genes respectively in aging brain. SignificanceThe present study suggests that fisetin supplementation may provide neuroprotection against aging-induced oxidative stress, apoptotic cell death, neuro-inflammation, and neurodegeneration in rat brain.

EpCAM expression in breast cancer cells is associated with enhanced bone metastasis formation.

Epithelial cell adhesion molecule (EpCAM) has been implicated in multiple cellular functions including cell adhesion. EpCAM has also recently been identified as a marker for cancer stem cells (CSCs). Here, we examined the roles of EpCAM in the development of bone metastasis of breast cancer by using well-characterized animal models. Morphological and real-time reverse transcriptase-polymerase chain reaction data showed that the EpCAM-negative and -positive (EpCAM(neg) and EpCAM(pos) ) cell populations isolated from breast cancer cell lines exhibited mesenchymal and epithelial phenotypes, respectively. Flow cytometric analysis revealed that EpCAM(pos) , but not EpCAM(neg) , cells possessed self-renewal and differentiation potentials. Tumorsphere formation in suspension cultures and tumorigenicity in the orthotopic mammary fat pad of mice were significantly greater in EpCAM(pos) cells than in EpCAM(neg) cells. The development of bone metastases induced by an intracardiac injection was markedly increased in mice inoculated with EpCAM(pos) cells. Furthermore, intracardiac inoculations of parental cells demonstrated that the EpCAM(pos) population in cancer cells that colonized in bone was significantly higher than that in parental cells. However, stable transduction of EpCAM into EpCAM(neg) cells failed to reproduce the phenotypes of EpCAM(pos) cells. These results suggest that the expression of EpCAM in breast cancer cells is associated with CSC-like phenotypes, which contribute to the promotion of bone metastases by enhancing tumorigenicity. Our results also support the possibility that the epithelial phenotypes of EpCAM-expressing cells confer advantageous properties for the development of bone metastases, at least after entering the circulation, while EpCAM is likely not solely responsible for the phenotypes of EpCAM(pos) cells.

Expression of tissue levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in renal cell carcinoma

BackgroundMatrix metalloproteinases (MMPs) are one of the major classes of proteolytic enzymes involved in tumor invasion and metastasis and are inhibited by naturally occurring tissue inhibitors of metalloproteinases (TIMPs). {AU Query: Please verify that corrections made to previous sentence did not alter intended meaning}. In this study, we examined the expression of MMP-2, MMP-9, membrane-type 1 (MT1)-MMP, TIMP-1, and TIMP-2 in renal tissue samples of renal cell cancer and examined the correlation between their expression and clinicopathological parameters.MethodsRenal tissue samples from 76 patients with renal cell carcinoma were available for this study. To determine the expression of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out on tumor and normal tissues.ResultsMean MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 mRNA expression in the renal cell carcinomas was significantly higher than in the normal renal tissue (P <0.05). The RT-PCR data of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 did not show any significant correlation with tumor type or pathologic grade of renal cell carcinoma. MMP-2, MMP-9 and MT1-MMP mRNA expression increased significantly with the TNM stage of the tumor.ConclusionsMean MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 mRNA expression in the renal cell carcinomas was significantly higher than in the normal renal tissue.

Modulation of Liver l-γ-Glutamyl-l-cysteinylglycine Homeostasis By N-Acetyl-Glucosamine-thiazolidine-4(R)-carboxylic Acid in Mice

The properties of modulating liver l-γ-glutamyl-l-cysteinylglycine (GSH) homeostasis by thiazolidine derivative N-acetyl-glucosamine-thiazolidine-4(R)-carboxylic acid (GlcNAcCys) and the underlying mechanisms were investigated in l-buthionine-[S,R]-sulfoximine (BSO)-induced mice liver GSH depletion model. The data show that BSO (6 mmol/kg body weight; intraperitoneally) significantly decreased liver total sulfhydryl and GSH concentrations when compared with control. When mice were treated with different doses of GlcNAcCys (200, 400, 900 mg/kg body weight; intraperitoneally, respectively), total sulfhydryl and GSH concentrations were significantly increased when measured 6 hours after treatment. The activities of GSH-associated enzymes were also measured. Liver glutathione S-transferase (GST) activities were significantly decreased by BSO compared with the control, and GlcNAcCys significantly increased GST activity. Moreover, reverse-transcriptase polymerase chain reaction data indicated that GlcNAcCys could significantly induce glutamylcysteine ligase catalytic subunit c mRNA transcription. The mRNA levels of transcription factors c-jun and c-fos were increased by BSO administration but were decreased back to normal after the administration of GlcNAcCys. In a conclusion, GlcNAcCys can modulate liver GSH homeostasis, which may be related to its ability to induce glutamylcysteine ligase catalytic subunit transcription. GlcNAcCys has potential hepatoprotective properties by increasing GSH content, increasing GST activity.

Evaluating the effect of spastin splice mutations by quantitative allele‐specific expression assay

mutations in the SPG4/SPAST gene are the most common cause for hereditary spastic paraplegia (HSP). The splice-site mutations make a significant contribution to HSP and account for 17.4% of all types of mutations and 30.8% of point mutations in the SPAST gene. However, only few studies with limited molecular approach were conducted to investigate and decipher the role of SPAST splice-site mutations in HSP. a reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative allele-specific expression assay were performed. we have characterized the consequence of two novel splice-site mutations (c.1493 + 1G>A and c.1414-1G>A) in the SPAST gene in two different families with pure HSP. The RT-PCR analysis revealed that both spastin mutations are indeed splice-site mutations and cause skipping of exon 12. Furthermore, RT-PCR data suggested that these splice-site mutations may cause leaky splicing. By means of a quantitative allele-specific expression assay, we could confirm that both splice-site mutations cause leaky splicing, as the relative expression of the exon 12-skipped transcript was reduced (21.1 ± 3.6 compared to expected 50%). our finding supports a "threshold-effect-model" for functional spastin in HSP. A higher level (78.8 ± 3.9%) of functional spastin than the expected ratio of 50% owing to leaky splicing might cause late age at onset of HSP. Remarkably, we could show that a quantitative allele-specific expression assay is a simple and effective tool to evaluate the role of most types of spastin splice-site mutations in HSP.

Cyclodextrin-Containing Polymer Delivery System for Light-Directed siRNA Gene Silencing

In this study, we have investigated the possibility of combining a cyclodextrin-containing polymer (CDP) with siRNA molecules to modulate gene expression in a light-directed manner through photochemical internalization (PCI) technology. We utilized S100A4 as a model gene to evaluate the efficacy of gene silencing. After optimization of carrier/cargo ratio and illumination dose, real-time reverse transcriptase-polymerase chain reaction data showed between 80% and 90% silencing in the siRNA samples treated with PCI compared with untreated control. In contrast, only a 0%-10% silencing effect was detected in the siRNA samples without PCI treatment, demonstrating the potency of light-specific delivery of siRNA molecules. Light-directed siRNA delivery was shown in 2 different cell lines with corresponding potency. Further, time-lapse results demonstrated maximum gene silencing only at 5 hours after endosomal release, implying, for example, rapid carrier decondensation when using the CDP. This work represents a first success in using a CDP delivery agent, without endosomolytic properties for siRNA gene silencing in a light-directed manner, opening the opportunity to use CDPs for light-directed siRNA gene silencing in vivo.

Encapsulated plant extract (Gelsemium sempervirens) poly (lactide-co-glycolide) nanoparticles enhance cellular uptake and increase bioactivity in vitro

Ethanolic extract of Gelsemium sempervirens (family: Loganiaceae), henceforth to be called EEGS, is used in various traditional systems of medicine. In homeopathy, EEGS is known as mother tincture of G. sempervirens, which is generally used to treat pain and respiratory ailments. We demonstrated earlier anticancer activity of crude EEGS by in vitro studies on human HeLa cells. To test the hypothesis if nanoparticle-encapsulated extract (now onwards to be called NEEGS) could enhance cellular uptake and thereby improve bioactivity, we formulated nanoparticle encapsulation based on poly (lactide-co-glycolide) (PLGA) and confirmed encapsulation by scanning electron microscopy (SEM) and atomic force microscopy. EEGS was encapsulated with 81.6% efficiency in PLGA biodegradable nanoparticle formulation and F68 (polyoxyethylene-polyoxypropylene) was used as a stabilizer. Dynamic laser light scattering and SEM indicated a particle diameter of 122.6 nm. The zeta potential of the drug-loaded nanoparticles was -14.8 mV. NEEGS was characterized for their biological activities in a skin cancer cell line A375 in vitro. NEEGS exhibited relatively rapid (30 min) and more efficient cellular uptake than their un-encapsulated counterpart (45 min). Analysis of data of apoptosis study using Annexin V-FITC, terminal transferase dUTP nick end labeling assay and DNA ladder revealed that encapsulated EEGS was more potent than their un-encapsulated counterpart in inducing apoptosis of A375 cells. Reverse transcriptase-polymerase chain reaction data of survivin, cyclin-D1, caspase-3, PCNA and p53 also corroborated well to suggest greater potentials of NEEGS as anticancer agents.

Radiation-Induced Interleukin-6 Expression Through MAPK/p38/NF-κB Signaling Pathway and the Resultant Antiapoptotic Effect on Endothelial Cells Through Mcl-1 Expression With sIL6-Rα

To investigate the mechanism of interleukin-6 (IL-6) activity induced by ionizing radiation. Human umbilical vascular endothelial cells (HUVECs) were irradiated with different doses to induce IL-6. The IL-6 promoter assay and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to examine transcriptional regulation. Specific chemical inhibitors, decoy double-stranded oligodeoxynucleotides, and Western blotting were conducted to investigate the signal transduction pathway. Recombinant soluble human IL-6 receptor alpha-chain (sIL6-Ralpha) and specific small interfering RNA were used to evaluate the biologic function of radiation-induced IL-6. Four grays of radiation induced the highest level of IL-6 protein. The promoter assay and RT-PCR data revealed that the induction of IL-6 was mediated through transcriptional regulation. The p38 inhibitor SB203580, by blocking nuclear factor-kappaB (NF-kappaB) activation, prevented both the transcriptional and translational regulation of radiation-induced IL-6 expression. The addition of sIL6-Ralpha rescued HUVECs from radiation-induced death in an IL-6 concentratio-dependent manner. The antiapoptotic effect of combined sIL6-Ralpha and radiation-induced IL-6 was inhibited by mcl-1-specific small interfering RNA. Radiation transcriptionally induces IL-6 expression in endothelial cells through mitogen-activated protein kinase/p38-mediated NF-kappaB/IkappaB (inhibitor of NF-kappaB) complex activation. In the presence of sIL6-Ralpha, radiation-induced IL-6 expression acts through Mcl-1 expression to rescue endothelial cells from radiation-induced death.

Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies

BackgroundThe reliability of gene expression profiling-based technologies to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. It strictly depends upon the technique that has been employed. Hence, the present study aimed at systematically comparing silica-gel column (SGC) and guanidine isothiocyanate (GTC) techniques of RNA isolation to answer the question which technique is preferable when frozen, long-term stored or fresh lung tissues have to be evaluated for the downstream molecular analysis.MethodsFrozen lungs (n = 3) were prepared by long-term storage (2.5 yrs) in -80°C while fresh lungs (n = 3) were harvested and processed immediately. The purity and quantification of RNA was determined with a spectrophotometer whereas the total amounted copy numbers of target sequences were determined with iCycler detection system for assessment of RNA intactness (28S and 18S) and fragment sizes, i.e. short (GAPDH-3' UTR), medium (GAPDH), and long (PBGD) with 200 bp, 700 bp, and 1400 bp distance to the 3'ends of mRNA motif, respectively.ResultsTotal yield of RNA was higher with GTC than SGC technique in frozen as well as fresh tissues while the purity of RNA remained comparable. The quantitative reverse transcriptase-polymerase chain reaction data revealed that higher mean copy numbers of 28S and a longer fragment (1400 bp) were obtained from RNA isolated with SGC than GTC technique using fresh as well as frozen tissues. Additionally, a high mean copy number of 18S and medium fragment (700 bp) were obtained in RNA isolated with SGC technique from fresh tissues, only. For the shorter fragment, no significant differences between both techniques were noticed.ConclusionOur data demonstrated that although the GTC technique has yielded a higher amount of RNA, the SGC technique was much more superior with respect to the reliable generation of an intact RNA and effectively amplified longer products in fresh as well as in frozen tissues.

Erythropoietin Attenuates Renal Injury in an Experimental Model of Rat Unilateral Ureteral Obstruction via Anti-Inflammatory and Anti-Apoptotic Effects

Erythropoietin was recently shown to exert important cytoprotective and anti-apoptotic effects in injury models of the brain, heart and kidney. We examined whether erythropoietin also attenuates renal injury in a rat model of unilateral ureteral obstruction via anti-apoptotic and anti-inflammatory actions. We divided Sprague-Dawley rats (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea) into 4 groups, including 1-vehicle treated with sham operation, 2-vehicle treated with unilateral ureteral obstruction for 3 days, 3-erythropoietin treatment with sham operation and 4-erythropoietin treatment for unilateral ureteral obstruction for 3 days. The erythropoietin treatment dose was 3,000 IU/kg per day intraperitoneally, administered daily. We compared competitive reverse transcriptase-polymerase chain reaction data on transforming growth factor-beta, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, osteopontin, Fas and Bcl-2. Furthermore, we examined Western blots for caspase-3 and light microscopy findings with hematoxylin and eosin staining. We applied immunohistochemistry for transforming growth factor-beta, ED-1 and caspase-3, and TUNEL in each group. Transforming growth factor-beta, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, osteopontin and Fas mRNA levels in the erythropoietin treated, unilateral ureteral obstruction group were significantly lower than in the obstruction only group. The Bcl-2 mRNA level in the erythropoietin treated obstruction group was significantly higher than in the obstruction only group. Caspase-3 activity in the erythropoietin treated obstruction group was significantly lower than in the obstruction only group. On light microscopy interstitially infiltrated inflammatory cells were significantly decreased in the erythropoietin treated obstruction group compared to the obstruction only group. On immunohistochemistry the erythropoietin treated obstruction group showed significantly fewer reactions for transforming growth factor-beta, ED-1 and caspase-3 compared to the obstruction only group. Erythropoietin treatment in rats with unilateral ureteral obstruction significantly decreased the number of TUNEL positive cells. Erythropoietin exerts renoprotective effects in an experimental unilateral ureteral obstruction rat model via anti-apoptotic and anti-inflammatory actions.

Hyaluronan-mediated motility: a target in oral squamous cell carcinoma

To characterize cancer-related gene expression changes in oral squamous cell carcinomas (OSCCs), we compared the gene expression profiles in OSCC-derived cell lines with human normal oral keratinocytes (HNOKs). Microarray analysis identified 166 genes that were up-regulated in OSCC-derived cell lines. Gene ontology analysis showed that cancer-related function had the highest significance. Among the genes mapped to the cancer-related network with the highest significance, the receptor for hyaluronan-mediated motility (RHAMM) was evaluated further for mRNA and protein expression in the OSCC cell lines, primary OSCCs. Overexpression of RHAMM protein was observed in all cell lines compared to HNOKs. Immunohistochemical analysis showed highly expressed RHAMM in primary OSCCs, whereas most corresponding normal tissues had no or significant down-regulation of protein immunoreactivity. Real-time quantitative reverse transcriptase-polymerase chain reaction data agreed with the protein expression. Moreover, the RHAMM expression status was correlated with the TNM stage (P<0.001). The results suggested that RHAMM expression may be correlated with tumor aggressiveness and offer clues to the development of new treatments for human OSCCs.

Optimization of comparative expressed sequence hybridization for genome-wide expression profiling at chromosome level

Comparative expressed sequence hybridization (CESH) has recently been developed for global expression profiling at chromosome level. To improve its specificity and sensitivity, we examined the effects of cDNA amplification and labeling methods on CESH profiles, using a gastric cancer cell line, Kato III, and compared the CESH profiles to cDNA microarray and reverse transcriptase–polymerase chain reaction (RT-PCR) data. CESH results were scarcely affected by the amplification process, either at the RNA level with T7 polymerase or at the cDNA level with degenerate oligonucleotide-primed PCR (DOP-PCR). The labeling method, however, did remarkably affect the CESH results; false positive shifts of the test/reference ratio (T/R) were not detected in self-matched CESH with pre-cDNA labeling and random priming labeling of cDNA but were consistently seen with DOP-PCR labeling in 11 chromosomes. The use of cDNA deriving from mRNA either with pre-cDNA or random priming labeling gave results of higher detection sensitivity for regions of up- or downregulated expression and higher concordance with the microarray and RT-PCR data in the corresponding regions than with conventional CESH. This modification of CESH with random priming labeling was found feasible by its application to Kato III cells with and without 5-aza-2′-deoxycytidine treatment; the regions identified as epigenetically silenced included genes that were reportedly methylated in Kato III.

Validation of a Mathematical Model of Gene Transcription in Aggregated Cellular Systems: Application to L1 Retrotransposition

We present a methodology aimed at partial validation and accuracy-precision assessment of a mathematical model of gene transcription at the cellular level. The method is based on the analysis of time-series measurements aggregated over a large number of cells. Such measurements are typically obtained via reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. The validation procedure presented herein uses as an example data on L1 retrotransposon gene in HeLa cells. The procedure compares model predicted values with the RT-PCR data for L1 by means of the standard Bayesian statistical techniques with the help of modern Markov-Chain Monte-Carlo methodology.

Clinical significance of tissue levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in gastric cancer

Matrix metalloproteinases (MMPs) are one of the major classes of proteolytic enzymes involved in tumor invasion and metastasis, being inhibited by naturally occurring tissue inhibitors of metalloproteinases (TIMPs). In this study, we examined the expression of MMP-2, MMP-9, membrane-type 1 (MT1)-MMP, TIMP-1, and TIMP-2 in biopsy tissues of gastric cancer, and the correlation between their expression and clinicopathological parameters. Biopsy specimens from 66 patients with gastric carcinoma were available for this study. To determine the expression of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out on tumor and normal tissues, respectively, sampled during diagnostic gastroscopic examination. Immunohistochemical staining of representative samples using monoclonal antibody directed against MT1-MMP was done, and the clinicopathological variables were reviewed retrospectively. The expression level of MMPs and TIMPs was evaluated using the tumor : normal (T/N) ratios of MMPs and TIMPs. The T/N ratio of MT1-MMP mRNA showed a significant correlation with lymph node metastasis and tumor stage (P < 0.05). The other RT-PCR data of MMP-2, MMP-9, TIMP-1, and TIMP-2 did not show any significant correlation with clinicopathological parameters. Immunohistochemistry for MT1-MMP showed a positive immunoreaction in gastric adenocarcinoma and negative staining in normal mucosa. The correlation between the increased expression of MT1-MMP and clinicopathological variables reflects a role in predicting the aggressive behavior of gastric cancer. Because an RT-PCR assay can be performed on biopsy specimens obtained before surgery, an evaluation of MT1-MMP expression in biopsy specimens by RT-PCR may provide useful preoperative information on tumor aggressiveness.

Transcriptional Profiling of the Nuclear Factor-κB Pathway Identifies a Subgroup of Primary Lymphoma of the Central Nervous System With Low BCL10 Expression

Recent studies point to a role of nuclear factor (NF)-kappaB signaling in a subset of diffuse large B cell lymphomas. We have analyzed the expression of 21 genes encoding NF-kappaB family members, upstream modulators, and targets in 32 primary central nervous system lymphomas (PCNSLs) by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Compared with nonmalignant germinal center centroblasts, expression of BCL10, REL, IAP1, and TRAF1 was significantly lower in PCNSLs, whereas that of BAX, BCLXL, BCL2, MALT1, CARD9, CARD10, CARD11, CARD14, CCND2, cFLIP, RELA, RELB, NFKB1, NFKB2, and IRF4 was higher. Hierarchical clustering of gene expression data revealed two distinct subgroups of PCNSLs, which were characterized by significantly different transcriptional levels, predominantly of BCL10, but also of REL and IAP1. Thus, these quantitative RT-PCR data with expression of genes of the NF-kappaB family as well as NF-kappaB-regulated genes together with immunohistochemical detection of nuclear RELA and REL indicate activation of the NF-kappaB pathway in PCNSLs, which may contribute to their high proliferative activity and the low level of apoptosis.