Statistical Difference In Expression Research Articles

Multiomics Analysis of Genetics and Epigenetics Reveals Pathogenesis and Therapeutic Targets for Atrial Fibrillation.

Objective This study is aimed at understanding the molecular mechanisms and exploring potential therapeutic targets for atrial fibrillation (AF) by multiomics analysis. Methods Transcriptomics and methylation data of AF patients were retrieved from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) and differentially methylated sites between AF and normal samples were screened. Then, highly expressed and hypomethylated and lowly expressed and hypermethylated genes were identified for AF. Weighted gene coexpression network analysis (WGCNA) was presented to construct AF-related coexpression networks. 52 AF blood samples were used for whole exome sequence. The mutation was visualized by the maftools package in R. Key genes were validated in AF using independent datasets. Results DEGs were identified between AF and controls, which were enriched in neutrophil activation and regulation of actin cytoskeleton. RHOA, CCR2, CASP8, and SYNPO2L exhibited abnormal expression and methylation, which have been confirmed to be related to AF. PCDHA family genes had high methylation and low expression in AF. We constructed two AF-related coexpression modules. Single-nucleotide polymorphism (SNP) was the most common mutation type in AF, especially T > C. MUC4 was the most frequent mutation gene, followed by PHLDA1, AHNAK2, and MAML3. There was no statistical difference in expression of AHNAK2 and MAML3, for AF. PHLDA1 and MUC4 were confirmed to be abnormally expressed in AF. Conclusion Our findings identified DEGs related to DNA methylation and mutation for AF, which may offer possible therapeutic targets and a new insight into the pathogenesis of AF from a multiomics perspective.

Molecular phylogenetic and in silico analysis of glyceraldeyde-3-phosphate dehydrogenase (GAPDH) gene from northern bobwhite quail (Colinus virginianus).

Many recent studies have been focused on prevalence and impact of two helminth parasites, eyeworm Oxyspirura petrowi and caecal worm Aulonocephalus pennula, in the northern bobwhite quail (Colinus virginianus). However, few studies have attempted to examine the effect of these parasites on the bobwhite immune system. This is likely due to the lack of proper reference genes for relative gene expression studies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that is often utilized as a reference gene, and in this preliminary study, we evaluated the similarity of bobwhite GAPDH to GAPDH in other avian species to evaluate its potential as a reference gene in bobwhite. GAPDH was identified in the bobwhite full genome sequence and multiple sets of PCR primers were designed to generate overlapping PCR products. These products were then sequenced and then aligned to generate the sequence for the full-length open reading frame (ORF) of bobwhite GAPDH. Utilizing this sequence, phylogenetic analyses and comparative analysis of the exon-intron pattern were conducted that revealed high similarity of GAPDH encoding sequences among bobwhite and other Galliformes. Additionally, This ORF sequence was also used to predict the encoded protein and its three-dimensional structure which like the phylogenetic analyses reveal that bobwhite GAPDH is similar to GAPDH in other Galliformes. Finally, GAPDH qPCR primers were designed, standardized, and tested with bobwhite both uninfected and infected with O. petrowi, and this preliminary test showed no statistical difference in expression of GAPDH between the two groups. These analyses are the first to investigate GAPDH in bobwhite. These efforts in phylogeny, sequence analysis, and protein structure suggest that there is > 97% conservation of GADPH among Galliformes. Furthermore, the results of these in silico tests and the preliminary qPCR indicate that GAPDH is a prospective candidate for use in gene expression analyses in bobwhite.

Relationship between hepatocellular carcinoma and depression via online database analysis

ABSTRACT There may be a mutually reinforcing relationship between hepatocellular carcinoma (HCC) and depression, but the mechanism is unknown. This study used bioinformatics to evaluate the relationship between HCC and depression at the genetic level. Genes associated with HCC and depression were obtained from pubmed2ensemble. Overlapping genes were annotated by gene ontology (GO) function and enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway. The cluster-1 genes obtained by Cytoscape were analyzed by GEPIA for expression and overall survival in HCC and, finally, introduced target genes to DGIdb to get associated drugs. A total of 199 genes were found to be in common between HCC and depression. GO term enrichment analysis on DAVID found the top-6 biological processes to be mainly associated with cell death and apoptosis. The top-6 cellular component terms are extracellular. The top-6 of molecular function terms are mainly associated with receptor binding. The top-6 pathways enriched by KEGG are mainly related to inflammatory response. IGF1, VEGFA, and SERPINE1 had statistical differences in expression and 10-year survival rate. There are total 45 drugs that act on VEGFA and SERPINE1. Based on our findings, we hypothesize that the mechanism of the interaction between HCC and depression may be related to cell death or apoptosis. Further studies are needed to verify this hypothesis.

Involvement of Src kinase in the down-regulation of ultra-rapid delayed rectifier K(+)current induced by tumor necrosis factor-α in cardiomyocytes

Objective: To investigate whether inflammatory factor tumor necrosis factor-α (TNF-α) is involved in the electrical remodeling of cardiomyocytes by regulating ultra-rapid delayed rectifier K(+) current (I(kur)) and the role of Src kinase. Methods: H9c2 cells, embryonic cardiomyocytes of rat, were cultured in Dulbecco's modified Eagle's medium (DMEM) and atrium-derived HL-1 cells were cultured in Claycomb medium. Both H9c2 and HL-1 cells were cultured at 37 ℃ with 5% CO(2). Cells cultured in normal conditions without additional treatment served as control group. Experimental groups were treated with different concentration of TNF-α (25 or 50 or 100 ng/ml) for 24 hours. To study whether Src specific inhibitor PP1 could abrogate the effect of TNF-α, cells were pre-treated with 10 μmol/L PP1 for 1 hour, followed by TNF-α (100 ng/ml) for 24 hours. Western blot and the whole cell patch clamp technique were used to detect the protein expression of Kv1.5 and Src and I(kur) in each group. Results: (1) In H9c2 cells, high concentration of TNF-α treatment (100 ng/ml) significantly reduced the Kv1.5 protein expression compared with control group and TNF-α 25 ng/ml group (both P<0.05). Compared with control group, the expression of p-Src protein was higher in 25 ng/ml, 50 ng/ml, 100 ng/ml TNF-α group (all P<0.05), but there was no statistical difference in the expression of Src protein among groups (P>0.05). In addition, the current density of I(kur) was decreased in 50 ng/ml, 100 ng/ml TNF-α group (both P<0.05). Furthermore, the expression of Kv1.5 protein and the current density of I(kur) were increased in PP1+TNF-α group compared with TNF-α 100 ng/ml group (both P<0.05). There was no statistical difference in the expression of Kv1.5 protein and the current density of I(kur) between the control group and PP1+TNF-α group (both P>0.05). (2) In atrium-derived HL-1 cells, the expression of Kv1.5 protein was reduced in 100 ng/ml TNF-α group compared with control group and TNF-α 25 ng/ml group (both P<0.01). In addition, the expression of p-Src protein was increased in TNF-α 100 ng/ml group compared with control group (P<0.05), but there was no statistical difference in the protein expression of Src among groups (P>0.05). The expression of Kv1.5 protein was increased in PP1+TNF-α group compared with TNF-α 100 ng/ml group (P<0.05). Conclusion: TNF-α is involved in the pathogenesis of atrial fibrillation, probably via decreasing I(kur) current density in atrium-derived myocytes through the activation of Src kinase.

Clock gene expression is altered in veterans with sleep apnea.

Clock gene dysregulation has been shown to underlie various sleep disorders and may lead to negative cardio-metabolic outcomes. However, the association between sleep apnea (SA) and core clock gene expression is unclear. We performed a cross-sectional analysis of 49 Veterans enrolled in a study of SA outcomes in veterans with chronic kidney disease, not selected for SA or sleep complaints. All participants underwent full polysomnography and next morning whole blood collection for clock gene expression. We defined SA as an apnea-hypopnea index ≥15 events/h; nocturnal hypoxemia(NH) was defined as ≥10% of total sleep time spent at <90% oxygen saturation. We used quantitative real-time PCR to compare the relative gene expression of clock genes between those with and without SA or NH. Clock genes studied were Bmal1, Ck1δ, Ck1ε, Clock, Cry1, Cry2, NPAS2, Per1, Per2, Per3, Rev-Erb-α, RORα, and Timeless. Our cohort was 90% male, mean age was 71 yr (SD 11), mean body mass index was 30 kg/m2 (SD 5); 41% had SA, and 27% had NH. Compared with those without SA, Per3 expression was reduced by 35% in SA ( P = 0.027). Compared with those without NH, NPAS2, Per1, and Rev-Erb-α expression was reduced in NH (50.4%, P = 0.027; 28.7%, P = 0.014; 31%, P = 0.040, respectively). There was no statistical difference in expression of the remaining clock genes by SA or NH status. Our findings suggest that SA or related NH and clock gene expression may be interrelated. Future study of 24 h clock gene expression in SA is needed to establish the role of clock gene regulation on the pathway between SA and cardio-metabolic outcomes.

Expression and cellular distribution of estrogen and progesterone receptors and the real-time proliferation of porcine cumulus cells

Although the expression of estrogen and progesterone receptors within porcine ovary and cumulus-oocyte complexes (COCs) is well recognized, still little information is known regarding expression of the progesterone receptor (PGR), PGR membrane component 1 (PGRMC1) and of estrogen-related receptors (ERRγ and ERRβ/γ) in separated cumulus cells in relation to real-time proliferation. In this study, a model of oocytes-separated cumulus cells was used to analyze the cell proliferation index and the expression PGR, PGRMC1 and of ERRγ and ERRβ/γ during 96-h cultivation in vitro using real-time quantitative PCR (qRT-PCR) and confocal microscopic observation. We found that PGR protein expression was increased at 0 h, compared with PGR protein expression after 96 h of culture (P < 0.001). The expression of PGRMC1, ERRγ and ERRβ/γ was unchanged. After using qRT-PCR we did not found statistical differences in expression of PGR, PGRMC1, ERRγ and ERRβ/γ during 96 h of cumulus cells in vitro culture (IVC). We supposed that the differential expression of the PGR protein at 0 h and after 96 h is related to a time-dependent down-regulation, which may activate a negative feedback. The distribution of PGR, PGRMC1 proteins may be linked with the translocation of receptors to the cytoplasm after the membrane binding of respective agonists and intra-cytoplasmic signal transduction. Furthermore, cumulus cells analyzed at 0 h were characterized by decreased proliferation index, whereas those after 96 h of culture revealed a significant increase of proliferation index, which may be associated with differentiation/luteinization of these cells during real-time proliferation.

Acute Pyelonephritis in Renal Allografts–A New Role for MicroRNAs?

Acute pyelonephritis (APN) versus acute rejection (AR) is a frequently encountered diagnostic and therapeutic dilemma in kidney transplants. Variable culture results, overlapping histologic features, and persistent graft dysfunction despite antibiotics are frequently encountered. Therefore, we explored the utility of intragraft microRNA profiles to distinguish between allograft APN and AR. Between 2003 and 2011, we identified 49 patients with biopsy features of APN, within the first 2 years posttransplant. MicroRNA profiling was performed on 20 biopsies (normal kidney, n=4; unequivocal AR, n=5; features of APN, n=11). Only 32% (16/49) of the patients had concomitant positive urine cultures at biopsy, and in 8 of 16 patients, colony count was less than 10 CFU/mL. In 14 of 49 patients, positive urine culture did not coincide with the biopsy, and in 19 of 49 patients, urine cultures were negative. On microRNA profiling, good clustering was seen among the normal kidneys and among AR biopsies. Among the 11 biopsies with features of APN, 4 biopsies showed good clustering with a pattern distinct from AR; (these patients recovered graft function with antibiotics); 7 of 11 biopsies showed heterogeneity in microRNA profiles and variable outcomes with antibiotic treatment. We identified a panel of 25 microRNAs showing statistical difference in expression between AR and APN. MiR-99b, miR-23b let-7b-5p, miR-30a, and miR-145 were validated using qPCR. Allograft pyelonephritis can be a diagnostic and therapeutic challenge. A gestalt approach is required. In addition to histology and cultures, differential intragraft microRNA expression may prove helpful to distinguish APN from AR in renal allograft biopsies.

GW24-e2389 Delivery of AAV9 cyclin-A2 via hyaluronic acid hydrogel induces cardiac regeneration as well as improves cardiac function in vivo post mycardial infarction

ObjectivesTo assess the effects of exogenous AAV9 Cyclin-A2 with hyaluronic acid in vivo post MI.MethodsForty-eight male Sprague Dawley rats were randomly divided into four groups: MI + PBS (n =...

Abstract 4173: MicroRNA expression of non-terminal respiratory unit type lung adenocarcinoma.

Abstract Background: Lung adenocarcinomas can be classified into two major subtypes, terminal respiratory unit (TRU) type and non-TRU type. We previously reported that non-TRU type adenocarcinoma has unique clinical and morphological features compared with TRU type adenocarcinoma. In brief, among 36 cases of non-terminal respiratory unit type adenocarcinoma, 24 cases revealed mucous columnar cell changes that were continuous with bronchial ciliated columnar cells. The mucous columnar cells became dysplastic showing loss of cilia, disorientation, and enlarged nuclei. Adenocarcinoma arose from these dysplastic mucous columnar cells and, characteristically, this type of adenocarcinoma showed acute inflammation, and honeycombing changes in the background. TTF1 immunostaining was consistently negative. In a case study with 14 males and 10 females, including 12 smokers or ex-smokers, EGFR and KRAS mutations were detected in 3 and 6 patients, respectively. We think that this kind of adenocarcinoma arising through mucous columnar cell change belongs to non-terminal respiratory unit type adenocarcinoma, and mucous columnar cell change is a precursor lesion of pulmonary adenocarcinoma. Objectives: We investigated whether microRNA (miRNA) expression profiles can differentiate between non-TRU type and TRU type lung adenocarcinoma. Methods: MiRNA expression in lung adenocarcinoma with non-TRU and TRU type was analyzed by microarray analysis with 1205 human and 144 human viral miRNAs. Results: Eight pairs of lung adenocarcinomas and corresponding noncancerous lung tissues (4 for non-TRU type and 4 for TRU type) were analyzed by microarray analysis. Initially, 44 miRNAs showed statistical differences in expression between lung cancer tissues and corresponding noncancerous tissues. Comparison analyses between non-TRU type and TRU type revealed 30 miRNAs with statistically different expression. Particularly, hsa-mir-494 and ebv-mir-BART19 were upregulated with more than 5-fold difference as compared with TRU type adenocarcinoma. Similarly, hsa-mir-551b was downregulated at more than 5-fold as compared with TRU type adenocarcinoma. Only 2 miRNAs (hsa-mir-1 and hsa-mir-133b) were shared in both non-TRU and TRU type of adenocarcinoma. Conclusion: We demonstrated that non-TRU type adenocarcinoma showed different miRNAs expression patterns compared with TRU type adenocarcinoma, suggesting different carcinogenic pathways for these tumors. Further studies are required for validation the results from microarray analysis. Citation Format: Mi Hyun Kim, Min Ki Lee, Dong Hoon Shin, Chang Hun Lee. MicroRNA expression of non-terminal respiratory unit type lung adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2013-4173

Expression of TAP1 and TNF-α in HBV-related hepatocellular carcinoma and its clinical significance

Objective To explore the association of expression of transporter associated with antigen processing（TAP1 ）, tumor necrosis factor-alpha （TNF-ot） with occurrence and development of HBV-related hepatocellular carcinoma（HCC）. Methods The expression of TAP1 and TNF-a in 35 liver carcinoma tissues, and 10 normal liver tissues were detected with immunohistochemical EnVision method,The expression and clinical significance of TAP1, TNF-a in HBV-related HCC were analyzed. Furthermore, the association of respective expression quantity with pathological typing of HCC was analyzed. Results The expression of TAP1 ,TNF-a was negative in hepatocytes of normal liver tissues. 91.4% of HCC tissues expressed TAP1. There was significantly statistical difference in expression of TAP1 between HCC tissues and the normal hepatic tissues（P 〈0.05） ;60% of HCC tissues expressed TNF-a. There was statistical difference in expression of TNF-a between HCC and the normal hepatic tissues （ P 〈 0. 05 ）, and the positive expression rate of TNF-a in well-differenciated HCC tissues was significantly higher than that in poorly-differenciated ones （P 〈 0. 05 ）. Conclusion TAPl-related MHC- I restricted antigen processing pathway was possibly normal in HBV-related HCC. The expression quantity of TNF-a was associated with the occurrence and degree of pathological differentiation of HBV-related HCC. Key words: Hepatitis B virus ; Hepatocellular carcinoma; Expression

Influence of fracture fragment displacement distance on fracture healing

Objective To study the influence of the distance of displaced fragment on the union of diaphysis fracture. Methods A wedge-shaped bone fragment was taken from central radial of the right forelimb of 120 New Zealand white rabbits for estabhshment of experimental animal model. The bone fragment was fixed to the main bone with two Kirschner wires, with certain space between bone fragment and the main bone. Then, the rabbits were divided into five groups, ie, Group A (in situ fixation),Group B (the space was 1/5 diameter of the radial shaft), Group C (the space was 2/5 diameter of the radial shaft), Group D (the space was 3/5 diameter of the radial shaft), Group E (the space was 4/5 diameter of the radial shaft). The animals were killed at 2, 4, 6, 8 weeks after operation. X-ray photos were taken to observe the fracture healing and the improved Gary X-ray used for scoring. HE staining after tissue section was employed to observe the histomorphological changes of fracture healing. Immunohistochemical method was used to determine expression of BMP-2. Results X-ray findings showed insignificant statistical difference between Group A and Group B, delayed union in Groups C and D and nonunion of bone absorption in Group E. Morphological observation showed same change in fracture site in Groups A and B lout significant late in emergence, formation and remodeling of the callus in the other groups compared with Group A, mainly with delayed fracture union or nonunion. There was no statistical difference in expression of BMP-2 between Group B and Group A (P ＞ 0. 05), but there was statistical significance in Groups C, D and E compared with Group A at 2 weeks (P ＜0.01). There was statistical difference between Group E and Group A at 4 weeks (P ＜0. 01) but no statistical difference at 6 and 8 weeks between either two groups (P ＞ 0. 05). Conclusions The distance of displaced fragment will influence fracture healing. The larger distance of the displaced fragment will beget more obvious influence on fracture healing. When the distance is more than 2/5 diameter of the bone shaft, the fracture will present union disorder. Key words: Fracture healing; Bone morphogenetic protien; Delayed union

W1816 Loss of TLR 2 and 4 on Ileal Plasmacytoid Dendritic Cells in Post-Operative Crohn's Disease Patients

Introduction Aberrant responses to bacterial flora are implicated in post-operative recurrence of Crohn9s disease (CD) after ileo-ceacal resection. Response to gut flora is conducted by dendritic cells (DC) which can induce a tolerogenic or active immune response. DC express Toll like receptors (TLR) which recognise bacterial products and are important in immune tolerance in health and highly expressed in IBD colonic tissue. Two subsets of DC are plasmacytoid (PDC) and myeloid cells (MDC). These DC show functional differences in their response to bacteria and are able to cross-talk. Expression of TLR on DC in the small intestine of post-operative CD patients is still largely unknown. We aimed to assess expression of TLR on DC from lamina propria mononuclear cells (LPMC) in post-operative CD patients as compared with normal controls. Methods Small intestine biopsies were obtained from 18 post-operative CD patients and 5 controls by colonoscopy. LPMC were collected using collagenase digestion, labelled with monoclonal antibodies to TLR2 and 4 and assessed by multicolour flow cytometry. Result A high percentage of DC from normal ileum expressed TLR2 and 4 with no significant differences in expression between PDC and MDC. However, expression of TLR 2 and 4 was significantly lower in PDC from post op CD patients compared with PDC in normal controls (mean 22.30% vs 60%, p=0.0003 and 38.55% vs 65.16%, p=0.02), respectively. Furthermore, PDC and MDC differed in expression of TLR2 and 4 in tissues from post-op CD patients; PDC expressed significantly less TLR2 and 4 compared with MDC (22.30% vs 38.11%, p=0.002 and 38.55% vs 50.89%, p=0.01), respectively. Conclusion Reduction of TLR2 and 4 expression in PDC compared with their counterparts in normal controls is likely to be the reason for the reduced expression of both TLRs on PDC compared with MDC in CD tissues. The lack of TLR2 and 4 expression on PDC maybe related to the disease activity as the normal controls did not show any statistical differences in expression of either TLR between the DC subsets. Other groups have suggested that functional differences exist between PDC and MDC in their activation by bacterial products and ability to produce innate and acquired inflammatory responses. In particular, blood PDC require the presence of MDC to respond to bacterial products to activate T cells rather than driving innate immunity. Our results indicate that PDC response to intestinal microflora may differ from that in MDC and provide initial background to the DC function and diversity in post-operative CD recurrence.

Comparison of Gene Expression Profiles in Tubulocystic Carcinoma and Collecting Duct Carcinoma of the Kidney

The relationship between tubulocystic carcinoma and collecting duct carcinoma of the kidney remains controversial. Some experts are of the opinion that the tumors are related, considering tubulocystic carcinoma to be synonymous with low-grade collecting duct carcinoma. However, others maintain that the 2 are distinct, unrelated entities on the basis of morphologic features and clinical outcome. To explore the relationship between tubulocystic carcinoma and collecting duct carcinoma, we compared the expression of several gene products at the mRNA level in cohorts of each tumor subtype. Seven cases of tubulocystic carcinoma and 8 cases of collecting duct carcinoma were identified. Total RNA was isolated from formalin-fixed paraffin-embedded tissue from each case. Relative expression levels of vimentin, alpha methylacyl CoA racemase, E-cadherin, p53, CD10 antigen, parvalbumin, cytokeratin 7, and cytokeratin 19 were assessed by quantitative reverse transcription polymerase chain reaction. Tubulocystic carcinoma was characterized by relative overexpression of vimentin, p53, and alpha methylacyl CoA racemase, compared with collecting duct carcinoma (P<0.05 for each gene, t test). In general, tubulocystic carcinoma expressed higher levels of E-cadherin and CD10, whereas collecting duct carcinoma expressed higher levels of cytokeratin 19; however, these trends did not reach statistical significance in this study cohort. Tubulocystic carcinoma and collecting duct carcinoma did not express cytokeratin 7 differentially. Case-to-case variability of gene expression limited the effectiveness of any one marker to distinguish the tumor types. Our study demonstrates that tubulocystic carcinoma and collecting duct carcinoma have different expression profiles of selected genes, including vimentin, p53, and alpha methylacyl CoA racemase. Further analysis of additional cases, using quantitative reverse transcription polymerase chain reaction and immunohistochemistry, will be useful to test the reproducibility of these findings. In addition, larger studies may establish statistical differences in expression of other genes analyzed in this study. Overall, these findings support the view that tubulocystic carcinoma and collecting duct carcinoma should be considered as 2 distinct entities at the molecular level.

Characterization of the Community Structure of a Dechlorinating Mixed Culture and Comparisons of Gene Expression in Planktonic and Biofloc-Associated “Dehalococcoides” andMethanospirillumSpecies

This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming "Dehalococcoides ethenogenes" strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of "Dehalococcoides" and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture.

Ontogeny of various solute carrier family (SLC) transporter expression in human pediatric liver biopsies

This study investigates developmental changes in mRNA expression of seven SLC transporters including SLCO1B1, SLCO1B3, SLCO2B1, SLCO4A1, SLC22A7, SLC22A1, and SLC10A1 in human pediatric liver. It is hypothesized that expression of these genes undergoes developmental changes during the early years of life. Sixty-one human pediatric liver samples were collected from living donors aged 0.3 to 12 years through liver biopsy. Real-time RT-PCR was used to quantify mRNA expression relative to cyclophilin for all target transporters. One-way ANOVA with post hoc pairwise comparisons was performed to assess statistical differences in expression amongst different age groups (A: 0.3–0.7, B: 0.7–2, C: 2–5, D: 5–12 yrs). Mean normalized relative mRNA expression is presented in the table (bold figures indicate statistical significance (p<0.05)). It is concluded that in human pediatric liver, there are significant age-dependent differences in mRNA expression of SLCO1B3 (OATP-8) and SLCO4A1 (OATP-E). Clinical Pharmacology & Therapeutics (2005) 77, P95–P95; doi: 10.1016/j.clpt.2004.12.258 Table 1. A(n = 9) B (n = 19) C (n = 12) D (n = 21) SLCO1B1 1928 7203 5370 3771 SLCO1B3 20.7 74.1 68.8 58.8 SLCO2B1 0.16 0.40 0.45 0.52 SLCO4A1 0.003 0.009 0.014 0.015 SLC22A7 1.65 4.23 4.17 3.31 SLC22A1 6.17 10.9 12.0 16.0 SLC10A1 28.0 35.7 20.9 25.4

Right- and left-sided colorectal cancers display distinct expression profiles and the anatomical stratification allows a high accuracy prediction of lymph node metastasis

Accurate preoperative prediction of lymph node metastasis and degree of tumor invasion would facilitate an appropriate decision of the extent of surgical resection of cancers to reduce unnecessary complication or to minimize the risk of recurrence in patients. We analyzed gene expression profiles characteristic of the invasiveness of colorectal carcinoma in a total of 89 cases, using a cDNA array and pattern classification algorithms. We set binary classes for a panel of clinicopathologic parameters, each of which was divided at different levels for categories (discrete) or values (continuous). We searched an optimal combination of genes to discriminate the classes by using of a feature subset selection algorithm, which was applied to a set of genes preselected on the basis of statistical difference in expression (two-sided t test, P ≤ 0.05). We used a sequential forward feature selection which additively searched a combination of genes, giving a minimal leave-one-out classification error rate of a k-nearest neighbor classifier. In the process of gene preselection, we found a remarkable difference in the expression pattern of genes according to the anatomical location of cancers. The difference was most prominent when the classes were set for cecum, ascending colon, transverse colon, and descending colon (CATD) versus sigmoid colon and rectum (SR). By stratifying these two locations, we were able to extract gene expression profiles characteristic of the classes of the presence versus absence of lymph node metastasis, lymphatic invasion, vascular invasion and degree of mural invasion, and pathological stages, with an accuracy of more than 90%. These results suggest that colorectal cancers harbor distinct molecular pathophysiological statuses according to their right-to-left locations, of which stratification is important for pattern classification of cDNA array data. Accurate preoperative prediction of lymph node metastasis and degree of tumor invasion would facilitate an appropriate decision of the extent of surgical resection of cancers to reduce unnecessary complication or to minimize the risk of recurrence in patients. We analyzed gene expression profiles characteristic of the invasiveness of colorectal carcinoma in a total of 89 cases, using a cDNA array and pattern classification algorithms. We set binary classes for a panel of clinicopathologic parameters, each of which was divided at different levels for categories (discrete) or values (continuous). We searched an optimal combination of genes to discriminate the classes by using of a feature subset selection algorithm, which was applied to a set of genes preselected on the basis of statistical difference in expression (two-sided t test, P ≤ 0.05). We used a sequential forward feature selection which additively searched a combination of genes, giving a minimal leave-one-out classification error rate of a k-nearest neighbor classifier. In the process of gene preselection, we found a remarkable difference in the expression pattern of genes according to the anatomical location of cancers. The difference was most prominent when the classes were set for cecum, ascending colon, transverse colon, and descending colon (CATD) versus sigmoid colon and rectum (SR). By stratifying these two locations, we were able to extract gene expression profiles characteristic of the classes of the presence versus absence of lymph node metastasis, lymphatic invasion, vascular invasion and degree of mural invasion, and pathological stages, with an accuracy of more than 90%. These results suggest that colorectal cancers harbor distinct molecular pathophysiological statuses according to their right-to-left locations, of which stratification is important for pattern classification of cDNA array data.

Prediction of lymphatic invasion/lymph node metastasis, recurrence, and survival in patients with gastric cancer by cDNA array-based expression profiling 1

We assessed the predictability of various classes of gastric carcinoma defined by clinicopathological parameters, such as invasiveness and clinical outcomes, using cDNA array data obtained from 54 cases. We searched an optimal combination of genes to discriminate the classes defined with the clinicopathological parameters by using a feature subset selection algorithm, which was applied to a set of genes preselected on the basis of statistical difference in expression (two-sided t test, P < or = 0.05). With the selected features (gene set), we evaluated the predictability of each parameter in a leave-one-out cross-validation test. We successfully selected sets of genes for which the classifier predicted better versus worse overall survival (tumor-specific death) and tumor-free survival (recurrence), with respective classification rates of 94 and 92%. A contingency table analysis (chi2 test) and Cox proportional hazard model analysis revealed that lymph node metastasis is the most important factor (confounding factor) in patients' prognoses and risks of recurrence. The feature subset selection procedure successfully extracted expression patterns characteristic of lymph node metastasis and lymphatic vessel invasion, yielding 92 and 98% prediction accuracies for these respective factors. We conclude that expression profiling using feature subset selection provides a powerful means of stratification of gastric cancer patients in regard to the prognostic factors. Further studies should be warranted to apply this method to personalization of the treatment options.

Right- and left-sided colorectal cancers display distinct expression profiles and the anatomical stratification allows a high accuracy prediction of lymph node metastasis

Accurate preoperative prediction of lymph node metastasis and degree of tumor invasion would facilitate an appropriate decision of the extent of surgical resection of cancers to reduce unnecessary complication or to minimize the risk of recurrence in patients. We analyzed gene expression profiles characteristic of the invasiveness of colorectal carcinoma in a total of 89 cases, using a cDNA array and pattern classification algorithms. We set binary classes for a panel of clinicopathologic parameters, each of which was divided at different levels for categories (discrete) or values (continuous). We searched an optimal combination of genes to discriminate the classes by using of a feature subset selection algorithm, which was applied to a set of genes preselected on the basis of statistical difference in expression (two-sided t test, P < or = 0.05). We used a sequential forward feature selection which additively searched a combination of genes, giving a minimal leave-one-out classification error rate of a k-nearest neighbor classifier. In the process of gene preselection, we found a remarkable difference in the expression pattern of genes according to the anatomical location of cancers. The difference was most prominent when the classes were set for cecum, ascending colon, transverse colon, and descending colon (CATD) versus sigmoid colon and rectum (SR). By stratifying these two locations, we were able to extract gene expression profiles characteristic of the classes of the presence versus absence of lymph node metastasis, lymphatic invasion, vascular invasion and degree of mural invasion, and pathological stages, with an accuracy of more than 90%. These results suggest that colorectal cancers harbor distinct molecular pathophysiological statuses according to their right-to-left locations, of which stratification is important for pattern classification of cDNA array data.

Comparative Study of Apoptotic Antitumor Effect between Angiogenesis Inhibitor AGM-1470 and MVAC Chemotherapy on Rat Urinary Bladder Cancer

Objectives: To investigate the antitumor apoptotic effect of AGM-1470 by comparing it to that induced by methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) therapy, currently the standard chemotherapy for bladder cancer, in a rat bladder cancer model. Materials and Methods: A total of 45 six-week-old female rats were divided into 3 equal groups: those receiving AGM-1470 treatment; those receiving MVAC treatment, and controls. All rats were cystectomized to evaluate the therapeutic effect with regard to macroscopic tumor findings, hematoxylin and eosin pathology, apoptosis detection, and immunohistochemistry (IHC) for bcl-2. Results: Our experimental protocol succeeded in producing invasive bladder tumors in the majority of rats. Tumor volume was significantly reduced in the AGM-1470 and MVAC treatment groups compared with that in the control group. The apoptotic indices of tumor cells was significantly higher in the AGM-1470 and MVAC treatment groups than in the control group. IHC for bcl-2 demonstrated no statistical difference in expression among the groups. However, the apoptotic indices of high-level bcl-2 expression were significantly lower than the indices of low-level expression in the AGM-1470 group. Conclusions: AGM-1470 and MVAC appear to exert a prominent mass reduction effect via tumor cell apoptosis in cases of invasive bladder tumor, although these therapies did not demonstrate any obvious modulation of bcl-2 protein expression status. Bcl-2 overexpression might be an obstacle to AGM-1470 therapy because of its significant inhibitory effect on apoptosis.

Evaluation of proliferative activity in middle ear cholesteatoma using proliferating cell nuclear antigen.

Middle ear cholesteatoma has a remarkable invasive activity accompanied by destruction of ossicles and temporal bone. Its aggressive growth and high tendency to recur have impact on the postoperative care of the patients. Proliferating cell nuclear antigen (PCNA) is a 36 KDa DNA-delta-polymerase-associated protein whose level of synthesis has been found to correlate directly with rates of cellular proliferation. In this present study, we used ABC (avidin-biotin complex) technique and monoclonal antibody to PCNA to evaluate the expression of PCNA in 37 cases of cholesteatoma epithelium and 21 cases of normal postauricular skin. The rate of PCNA-positive cells in basal, parabasal, and upper layer of cholesteatoma epithelium tissue is 78% (29 cases), 68% (25 cases), and 41% (15 cases). In each layer of the postauricular skin tissue is 71% (15 cases), 67% (14 cases) and 34% (7 cases). No statistical difference of expression of PCNA-positive cells exists between each layer of cholesteatoma epithelium and normal postauricular skin; however, a tendency of higher PCNA-positive cells in cholesteatoma epithelium was observed. Immunohistochemical method of PCNA has the advantages of spatial architecture preservation, the relative simplicity of the methodology and the rapid acquisition of results. Although the etiology and histopathology of the growth pattern and osteolytic activity of cholesteatoma are unclear, information on cell kinetics may assist in cholesteatoma classification and may help predict the risk of recurrence and bone destruction. The results of this report indicate that cholesteatoma has a similar proliferative activity to the normal postauricular skin, and cholesteatoma itself is not a real tumor, despite its clinical behavior, which is similar to neoplastic cells. It is necessary to further study whether the cell kinetic information we obtained from the PCNA immunohistochemical analysis provides a valuable tool in accessing the prognosis of the cholesteatoma.