A novel system for large-scale gene expression analysis: bacterial colonies array

In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.

Gene Expression Profiles Stratified according to Type 1 Diabetes Mellitus Susceptibility Regions

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expression of genes related to apoptosis, cell cycle and signaling pathways are independent of TP53 status in urinary bladder cancer cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Protein expression upon desiccation and imbibition of Magnolia ovata A. St.-Hil seeds

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expression of genes related to quality of Longissimus dorsi muscle meat in Nellore (Bos indicus) and Canchim (5/8 Bos taurus×3/8 Bos indicus) cattle

This study was performed to compare CAPN1, CAPN2, CAST, TG, DGAT1 and LEP gene expressions and correlate them with meat quality traits in two genetic groups (Nellore and Canchim) in order to assess their expression profile and use their expression profile as genetic markers. We analyzed 30 young bulls (1. year old), 15 of each genetic group. Samples of the Longissimus dorsi muscle were collected for analysis of: total lipids (TL) and meat tenderness measured as Warner-Bratzler shear force (SF) and myofibrillar fragmentation (MFI) at day of slaughter and 7. days of aging. Gene expression profiles were obtained via RT-qPCR. TL and MFI showed differences between breeds, higher MFI in Canchim and higher TL in Nellore. Calpains showed no differential expression between groups, as did DGAT1, TG, and LEP. CAST was expressed more in the Nellore cattle. The only significant within-breed correlation (0.79) between gene expression and meat traits was found for DGAT1 and MFI in Canchim breed. Although the number of animals used in this study was small, the results indicate that the increased expression of CAST in Nellore may reflect tougher meat, but the lack of correlations with the meat traits indicates it is not a promising genetic marker. © 2013 Elsevier Ltd.

Effects of BST and high energy diet on gene expression in mammary parenchyma of dairy heifers

The objective of this study was to determine the effects of dietary energy and recombinant bovine somatotropin (bST) injection to identify genes that might control mammogenesis. Total RNA was extracted from the parenchymal tissue of 32 heifers randomly assigned to one of four treatments: two diets (a standard diet and a high energy, high protein diet), each with or without bST. To perform microarray experiments, RNA samples were pooled (2 animals/pool) before reverse transcription and labeling with Cy3 or Cy5. A 4-node loop design was used to examine the differential gene expression among treatments using a bovine-specific cDNA micro array (National Bovine Functional Genomics Consortium Library, NBFGC) containing 18,263 unique expressed sequence tags (EST). Significance levels of differential gene expression among treatments were assessed using a mixed model approach. Injection of bST altered the expression of 12 % of the genes on NBFGC slide related to tissue development, whereas 6% were altered by diet. Administration of bST increases the expression of genes positively related to cell proliferation and mammary parenchyma to a greater extent than a high energy diet. © 2013 Sociedade Brasileira de Zootecnia.

Global liver gene expression differences in Nelore steers with divergent residual feed intake phenotypes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of sex on basal and dickkopf-1 regulated gene expression in the bovine morula

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Flavonoids modify root growth and modulate expression of SHORT-ROOT and HD-ZIP III

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

ANXA1Ac₂₋₂₆ peptide reduces ID1 expression in cervical carcinoma cultures

Cervical cancer is the second most frequent cancer in women worldwide and is associated with genetic alterations, infection with human papilloma virus (HPV), angiogenesis and inflammatory processes. The idea that inflammation is involved in tumorigenesis is supported by the frequent appearance of cancer in areas of chronic inflammation. On the other hand, the inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1), a 37 kDa protein was detected as a modulator of inflammatory processes and is expressed by tumor cells. The study was carried out on the epithelial cancer cell line (SiHa) treated with the peptide of annexin A1 (ANXA1Ac2-26). We combined subtraction hybridization approach, Ingenuity Systems software and quantitative PCR, in order to evaluate gene expression influenced by ANXA1. We observed that ANXA1Ac2-26 inhibited proliferation in SiHa cells after 72h. In these cells, 55 genes exhibited changes in expression levels in response to peptide treatment. Six genes were selected and the expression results of 5 up-regulated genes (TPT1, LDHA, NCOA3, HIF1A, RAB13) and one down-regulated gene (ID1) were research by real time quantitative PCR. Four more genes (BMP4, BMPR1B, SMAD1 and SMAD4) of the ID1 pathway were investigated and only one (BMPR1B) shows the same down regulation. The data indicate the involvement of ANXA1Ac2-26 in the altered expression of genes involved in tumorigenic processes, which could potentially be applied as a therapeutic indicator of cervical cancer.

Multivariate gene expression analysis reveals functional connectivity changes between normal/tumoral prostates

Abstract Background Prostate cancer is a leading cause of death in the male population, therefore, a comprehensive study about the genes and the molecular networks involved in the tumoral prostate process becomes necessary. In order to understand the biological process behind potential biomarkers, we have analyzed a set of 57 cDNA microarrays containing ~25,000 genes. Results Principal Component Analysis (PCA) combined with the Maximum-entropy Linear Discriminant Analysis (MLDA) were applied in order to identify genes with the most discriminative information between normal and tumoral prostatic tissues. Data analysis was carried out using three different approaches, namely: (i) differences in gene expression levels between normal and tumoral conditions from an univariate point of view; (ii) in a multivariate fashion using MLDA; and (iii) with a dependence network approach. Our results show that malignant transformation in the prostatic tissue is more related to functional connectivity changes in their dependence networks than to differential gene expression. The MYLK, KLK2, KLK3, HAN11, LTF, CSRP1 and TGM4 genes presented significant changes in their functional connectivity between normal and tumoral conditions and were also classified as the top seven most informative genes for the prostate cancer genesis process by our discriminant analysis. Moreover, among the identified genes we found classically known biomarkers and genes which are closely related to tumoral prostate, such as KLK3 and KLK2 and several other potential ones. Conclusion We have demonstrated that changes in functional connectivity may be implicit in the biological process which renders some genes more informative to discriminate between normal and tumoral conditions. Using the proposed method, namely, MLDA, in order to analyze the multivariate characteristic of genes, it was possible to capture the changes in dependence networks which are related to cell transformation.

Identification of protein-coding and non-coding RNA expression profiles in CD34+ and in stromal cells in refractory anemia with ringed sideroblasts

Abstract Background Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods In this study, gene expression profiles of CD34+ cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ≤ 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ≤ 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34+ cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

Life stage differences in mammary gland gene expression profile in non-human primates

Breast cancer (BC) is the most common malignancy of women in the developed world. To better understand its pathogenesis, knowledge of normal breast development is crucial, as BC is the result of disregulation of physiologic processes. The aim of this study was to investigate the impact of reproductive life stages on the transcriptional profile of the mammary gland in a primate model. Comparative transcriptomic analyses were carried out using breast tissues from 28 female cynomolgus macaques (Macaca fascicularis) at the following life stages: prepubertal (n = 5), adolescent (n = 4), adult luteal (n = 5), pregnant (n = 6), lactating (n = 3), and postmenopausal (n = 5). Mammary gland RNA was hybridized to Affymetrix GeneChip(®) Rhesus Macaque Genome Arrays. Differential gene expression was analyzed using ANOVA and cluster analysis. Hierarchical cluster analysis revealed distinct separation of life stage groups. More than 2,225 differentially expressed mRNAs were identified. Gene families or pathways that changed across life stages included those related to estrogen and androgen (ESR1, PGR, TFF1, GREB1, AR, 17HSDB2, 17HSDB7, STS, HSD11B1, AKR1C4), prolactin (PRLR, ELF5, STAT5, CSN1S1), insulin-like growth factor signaling (IGF1, IGFBP1, IGFBP5), extracellular matrix (POSTN, TGFB1, COL5A2, COL12A1, FOXC1, LAMC1, PDGFRA, TGFB2), and differentiation (CD24, CD29, CD44, CD61, ALDH1, BRCA1, FOXA1, POSTN, DICER1, LIG4, KLF4, NOTCH2, RIF1, BMPR1A, TGFB2). Pregnancy and lactation displayed distinct patterns of gene expression. ESR1 and IGF1 were significantly higher in the adolescent compared to the adult animals, whereas differentiation pathways were overrepresented in adult animals and pregnancy-associated life stages. Few individual genes were distinctly different in postmenopausal animals. Our data demonstrate characteristic patterns of gene expression during breast development. Several of the pathways activated during pubertal development have been implicated in cancer development and metastasis, supporting the idea that other developmental markers may have application as biomarkers for BC.

Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.

Bioremediation with White-Rot Fungi at Fisherville Mill: Analyses of Gene Expression and Number 6 Fuel Oil Degradation

Extracellular enzymes that white-rot fungi secrete during lignin decay have been proposed as promising agents for oxidizing pollutants. We investigated the abilities of the white-rot fungi Punctularia strigosozonata, Irpex lacteus, Trichaptum biforme, Phlebia radiata, Trametes versicolor, and Pleurotus ostreatus to degrade Number 6 fuel oil in wood sawdust cultures. Our goals are to advise bioremediation efforts at a brownfield redevelopment site on the Blackstone River in Grafton, Massachusetts and to contribute to the understanding of decay mechanisms in white-rot fungi. All species tested degraded a C10 alkane. When cultivated for 6 months, Irpex lacteus, T. biforme, P. radiata, T. versicolor and P. ostreatus also degraded a C14 alkane and the polycyclic aromatic hydrocarbon phenanthrene. Gene expression analyses of P. strigosozonata indicate differential gene expression in the presence of Number 6 oil and on pine and aspen sawdust.