Quantitative Differences In Expression Research Articles

Does the egg capsule protect against chronic UV-B radiation? A study based on encapsulated and decapsulated embryos of cuttlefish Sepia officinalis.

Although the egg capsule plays a crucial role in the embryonic development of cephalopods, its ability to protect embryos from Ultraviolet (UV) radiation is unknown. Our study evaluated the photoprotection mechanisms of S. officinalis to UV-B radiation and estimated the ability of the black capsule to act as a physical shield against it. Embryos with and without capsule and juveniles were exposed to four experimental UVB conditions for 55 days. The effects of different UVB doses were evaluated in terms of morphological abnormalities and differences in gene expression between each group. We observed that the development might be severely impaired in embryos exposed to UVB without capsule protection, and these effects were time- and UVB-dose-dependent. In addition, we found variations in gene expression levels (light-sensitive, stress response and DNA repair) in different tissues as a function of UVB doses. We suggest a relationship between morphological abnormalities and the limit of molecular regulation. These results suggest that the quantitative differences in expression are essential for defining the survivability of the embryo face to UVB. Thus, we demonstrated that the egg capsule could ensure successful embryonic development of the cuttlefish S. officinalis even at high doses of UVB.

Mapping the dynamics of insulin-responsive pathways in the blood–brain barrier endothelium using time-series transcriptomics data

Critical functions of the blood–brain barrier (BBB), including cerebral blood flow, energy metabolism, and immunomodulation, are regulated by insulin signaling pathways. Therefore, endothelial insulin resistance could lead to BBB dysfunction, which is associated with neurodegenerative diseases such as Alzheimer’s disease (AD). The current study aims to map the dynamics of insulin-responsive pathways in polarized human cerebral microvascular endothelial cell (hCMEC/D3) monolayers. RNA-Sequencing was performed on hCMEC/D3 monolayers with and without insulin treatment at various time points. The Short Time-series Expression Miner (STEM) method was used to identify gene clusters with distinct and representative expression patterns. Functional annotation and pathway analysis of genes from selected clusters were conducted using Webgestalt and Ingenuity Pathway Analysis (IPA) software. Quantitative expression differences of 16,570 genes between insulin-treated and control monolayers were determined at five-time points. The STEM software identified 12 significant clusters with 6880 genes that displayed distinct temporal patterns upon insulin exposure, and the clusters were further divided into three groups. Gene ontology (GO) enrichment analysis demonstrated that biological processes protecting BBB functions such as regulation of vascular development and actin cytoskeleton reorganization were upregulated after insulin treatment (Group 1 and 2). In contrast, GO pathways related to inflammation, such as response to interferon-gamma, were downregulated (Group 3). The IPA analyses further identified insulin-responsive cellular and molecular pathways that are associated with AD pathology. These findings unravel the dynamics of insulin action on the BBB endothelium and inform about downstream signaling cascades that are potentially disrupted due to brain insulin resistance prevalent in AD.

Investigations into SCAMP5, a candidate lupus risk gene expressed in plasmacytoid dendritic cells

ObjectiveWe have investigated the molecular function of SCAMP5, a candidate risk gene for SLE exclusively expressed in plasmacytoid dendritic cells (pDCs) among peripheral leucocytes.MethodsWe tested the independence of the association...

Variation of microRNA expression in the human placenta driven by population identity and sex of the newborn

BackgroundAnalysis of lymphocyte cell lines revealed substantial differences in the expression of mRNA and microRNA (miRNA) among human populations. The extent of such population-associated differences in actual human tissues remains largely unexplored. The placenta is one of the few solid human tissues that can be collected in substantial numbers in a controlled manner, enabling quantitative analysis of transient biomolecules such as RNA transcripts. Here, we analyzed microRNA (miRNA) expression in human placental samples derived from 36 individuals representing four genetically distinct human populations: African Americans, European Americans, South Asians, and East Asians. All samples were collected at the same hospital following a unified protocol, thus minimizing potential biases that might influence the results.ResultsSequence analysis of the miRNA fraction yielded 938 annotated and 70 novel miRNA transcripts expressed in the placenta. Of them, 82 (9%) of annotated and 11 (16%) of novel miRNAs displayed quantitative expression differences among populations, generally reflecting reported genetic and mRNA-expression-based distances. Several co-expressed miRNA clusters stood out from the rest of the population-associated differences in terms of miRNA evolutionary age, tissue-specificity, and disease-association characteristics. Among three non-environmental influenced demographic parameters, the second largest contributor to miRNA expression variation after population was the sex of the newborn, with 32 miRNAs (3% of detected) exhibiting significant expression differences depending on whether the newborn was male or female. Male-associated miRNAs were evolutionarily younger and correlated inversely with the expression of target mRNA involved in neuron-related functions. In contrast, both male and female-associated miRNAs appeared to mediate different types of hormonal responses. Demographic factors further affected reported imprinted expression of 66 placental miRNAs: the imprinting strength correlated with the mother’s weight, but not height.ConclusionsOur results showed that among 12 assessed demographic variables, population affiliation and fetal sex had a substantial influence on miRNA expression variation among human placental samples. The effect of newborn-sex-associated miRNA differences further led to expression inhibition of the target genes clustering in specific functional pathways. By contrast, population-driven miRNA differences might mainly represent neutral changes with minimal functional impacts.

1013 – CAPTURING THE TRANSCRIPTOME OF THE FIRST FUNCTIONAL HEMATOPOIETIC STEM CELLS

Cell fate and behavior are regulated by dynamic intrinsic and extrinsic processes. These processes affect single cells to produce a fully integrated organism with distinct functional cell types and tissues. Experimental hematology provides insight into the independent iterations of blood cell development (primitive, definitive progenitor and definitive stem) in the different embryonic sites/times prior to adult hematopoietic system formation. Hematopoietic stem cell (HSC) generation occurs in a short developmental time window from a natural transdifferentiation of aortic endothelial-to-hematopoietic cells (EHT). The Gata2 transcription factor is a dosage-dependent intrinsic regulator of EHT controlled by signals from the extrinsic developmental niche. Hematopoietic progenitor cells (HPC) are also generated by EHT, although at an earlier stage. So, what mechanistically determines HPC or HSC fate during EHT? Vital time-lapse imaging reveals highly dynamic Gata2 expression changes in cells emerging during EHT. We used Gata2 levels to examine the "inner" HSC transcriptional regulatory network. Single cell iterative RNAseq, index-sorting and in vivo/vitro hematopoietic analyses show that the transcriptome of the first aortic HSCs is unique compared to HPCs. The observed heterogeneity and quantitative expression differences of pivotal EHT/HSC genes suggest that establishment of functional hematopoietic fate is a stochastic process. Additionally, dynamic changes occur in the aortic niche. Transient cell-cell interactions occur between motile yolk sac HPC-derived macrophages and emerging aortic hematopoietic cells to affect hematopoietic output/function. The extrinsic signals from this new pro-inflammatory macrophage subset, together with intrinsic dynamic Gata2 expression changes in endothelial cells could assist strategies for ex vivo HSC generation.

Association of Selenoprotein S Expression and its Variants with Metabolic Syndrome in Subjects with Cardiovascular Disease

Selenoproteins S (SELS or VIMP) may regulate cytokine production, and thus play a key role in the control of the inflammatory response. This study consisted of 136 Iranian patients with cardiovascular disease (65 MetS-affected and 71 MetS un-affected individuals) in the selengene study. Expression of two variants of VIMP including VIMP I and II were analyzed in all subjects using Real-Time PCR and ELISA. The level of VIMP was lower in MetS+ compared to the MetS- subjects (p<0.05). We found no significant differences in quantitative expression of VIMP I and VIMP II in both groups. VIMP I reveal a reverse correlation with fasting blood sugar (FBS) (r=-0.45, p=0.009). Moreover, SELS in protein level has negative correlation with WC (r=-0.171, p=0.049) and positive correlation with HDL (r=0.176, p=0.046). Our study suggests that VIMP in protein level is significantly lower in MetS and shows a reverse correlation with WC and positive correlation with HDL. Therefore, with regard to the functional role of this protein, it is possible to deduce that its lower expression leads to the higher secretion of unfolded proteins into the cytosol and outside the cell, where they cannot play their exact roles in the different pathways. Moreover, the reverse correlation of VIMP I with FBS suggests further consideration of VIMP and its variant VIMP I expression in regards to potential development of major CVD risk factors.

Common pathogenic mechanism in patients with dropped head syndrome caused by different mutations in the MYH7 gene

Mutations in the MYH7 gene are the source of an allelic series of diseases, including various cardiomyopathies and skeletal myopathies that usually manifest in adulthood. We observed a 1.5 y.o. male patient with congenital weaknesses of the axial muscles, “dropped head” syndrome, and dilated cardiomyopathy. The clinical evaluation included medical history, an echocardiogram, electromyography, and a histopathological study. The genetic evaluation included whole exome sequencing. Muscle biopsy samples from the proband were used for mRNA extraction. We revealed a novel genetic variant c.5655 + 5G > C in the MYH7 gene. The analysis of the cDNA showed an in-frame skipping of exon 38 (p.1854_1885del). This variant and two previously published mutations (c.5655G > A and c.5655 + 1G > A), also presumably leading to exon 38 skipping, were studied by expression analysis in the HEK293T cell line transfected with 4 plasmids containing the MYH7 minigene (wt, c.5655G > C, c.5655 + 1G > A and c.5655 + 5G > A). A quantitative difference in expression was shown for cell lines with each of the three mutant plasmids. All mutation carriers had a similar phenotype and included congenital axial myopathy and variable cardiac involvement. Prominent dropped head syndrome was mentioned in all patients. Early-onset axial myopathy with a dropped head syndrome is a distinct clinical entity within MYH7-related disorders. We suggest that mutations in the MYH7 gene affecting the C-terminal domain of beta-myosin heavy chain should also be considered as a possible cause in cases of early-onset myopathy with “dropped head” syndrome.

Mining for heat-stress responsive genes by large scale gene expression data from Betula luminifera

Global average temperature has been predicted to rise by 1.8–4.0 °C within this century, accompanied by an increase in the magnitude and frequency of high-temperature events. Developing new cultivars better adapted to high temperature is essential for forestry ecosystems. Analyzing smooth-bark birch (Betula luminifera) gene expression profiles in response to heat stress is critical to understanding how birch manages heat stress, and provides vital information to improve thermoresistance for stable wood production. In this study, the leaf transcriptomics of B. luminifera were evaluated, thus up- and down-regulated genes were identified under heat stress through digital gene expression analysis. Expression patterns of 17 related genes of two genotypes following exposure to heat stress were compared. A total of 867 differentially expressed genes were identified, including 447 upregulated genes and 420 downregulated genes under heat stress, while 10 genes and 58 genes were detected only in control and heat-stress leaves, respectively. Among them, Hsfs, HSPs, DREB2, Cpn60-β2, ATP9 and other genes involved in the response to heat stress displayed quantitative differences in expression between the genotypes. The expression levels of the transcription factors MybC2-L1 and MYC1 were repressed significantly under heat stress. These results showed that a panoply of genes were induced by heat stress, while the expression of many genes were reduced or off in the presence of excessively high temperatures. It can be speculated that the distinct expression levels between genotypes in response to heat stress underlie their different capacities to resist high temperatures.

Enhancer Divergence and cis-Regulatory Evolution in the Human and Chimp Neural Crest

cis-regulatory changes play a central role in morphological divergence, yet the regulatory principles underlying emergence of human traits remain poorly understood. Here, we use epigenomic profiling from human and chimpanzee cranial neural crest cells tosystematically and quantitatively annotate divergence of craniofacial cis-regulatory landscapes. Epigenomic divergence is often attributable to genetic variation within TF motifs at orthologous enhancers, with a novel motif being most predictive of activity biases. We explore properties of this cis-regulatory change, revealing the role of particular retroelements, uncovering broad clusters of species-biased enhancers near genes associated with human facial variation, and demonstrating that cis-regulatory divergence is linked to quantitative expression differences of crucial neural crest regulators. Our work provides a wealth of candidates for future evolutionary studies and demonstrates the value of "cellular anthropology," a strategy of using in-vitro-derived embryonic cell types to elucidate both fundamental and evolving mechanisms underlying morphological variation in higher primates.

Proteomic analysis of plasma in adult active pulmonary tuberculosis patients with diabetes mellitus

Abstract:There is a high burden of both diabetes and tuberculosis in China. Althoug both diabetes and tuberculosis have been the objects of intensive study the association between the two diseases is not fully understood. The aim of this study was to determine whether thetqualitative and quantitative differences in expression of some protein expression in plasma could be related to active pulmonary tuberculosis complicated with diabetes. Using two‐dimensional gel electrophoresis and mass spectrometry we analyzed expression of the proteins associated with active pulmonary tuberculosis complicated with diabetes. Results showed that under the baseline condition, the levels of a‐1 antitrypsin precursor, vitamin D‐binding protein precursor, CD5 antigen like precursor, clusterin precursor, apolipoprotein A‐I precursor, haptoglobin and fibrinogen g‐chain differed between patients with active pulmonary tuberculosis and active pulmonary tuberculosis complicated with diabetes. Western blott results confirmed differential expression of clusterin. These results suggest that active pulmonary tuberculosis complicated with diabetes may be associated with specific protein expression pattern in plasma. C‐terminal haptoglobin is a possible candidate protein of interest, which might be a link between active pulmonary tuberculosis and diabetes. The dynamics of protein expression during disease progression may improve our understanding of the pathogenesis of active pulmonary tuberculosis complicated with diabetes.Supported by NSFC‐81172662

Insulin-like growth factor-1 mRNA isoforms and insulin-like growth factor-1 receptor mRNA expression in chronic hepatitis C.

To evaluate the expression of different insulin-like growth factor (IGF)-1 mRNA isoforms and IGF-1 receptor (IGF-1R) mRNA in hepatitis C virus (HCV)-infected livers. Thirty-four liver biopsy specimens from chronic hepatitis C (CH-C) patients were obtained before anti-viral therapy. Inflammatory activity (grading) and advancement of fibrosis (staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturer's instruction. Expression levels of IGF-1 mRNA isoforms (IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms (P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2 (r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA (r = 0.891; r = 0.821, respectively), with IGF-1B mRNA (r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA (r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA (r = 0.956; r = 0.869, respectively), and B with C isoforms (r = 0.868) (P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading (G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data (e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile.

Differential protein expression profiling of Arabidopsis thaliana callus under microgravity on board the Chinese SZ-8 spacecraft.

Exposure of Arabidopsis callus to microgravity has a significant impact on the expression of proteins involved in stress responses, carbohydrate metabolism, protein synthesis, intracellular trafficking, signaling, and cell wall biosynthesis. Microgravity is among the main environmental stress factors that affect plant growth and development in space. Understanding how plants acclimate to space microgravity is important to develop bioregenerative life-support systems for long-term space missions. To evaluate the spaceflight-associated stress and identify molecular events important for acquired microgravity tolerance, we compared proteomic profiles of Arabidopsis thaliana callus grown under microgravity on board the Chinese spacecraft SZ-8 with callus grown under 1g centrifugation (1g control) in space. Alterations in the proteome induced by microgravity were analyzed by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry with isobaric tags for relative and absolute quantitation labeling. Forty-five proteins showed significant (p < 0.05) and reproducible quantitative differences in expression between the microgravity and 1g control conditions. Of these proteins, the expression level of 24 proteins was significantly up-regulated and that of 21 proteins was significantly down-regulated. The functions of these proteins were involved in a wide range of cellular processes, including general stress responses, carbohydrate metabolism, protein synthesis/degradation, intracellular trafficking/transportation, signaling, and cell wall biosynthesis. Several proteins not previously known to be involved in the response to microgravity or gravitational stimuli, such as pathogenesis-related thaumatin-like protein, leucine-rich repeat extension-like protein, and temperature-induce lipocalin, were significantly up- or down-regulated by microgravity. The results imply that either the normal gravity-response signaling is affected by microgravity exposure or that microgravity might inappropriately induce altered responses to other environmental stresses.

Expression of p53, Ki67, EcPV2- and EcPV3 DNA, and viral genes in relation to metastasis and outcome in equine penile and preputial squamous cell carcinoma.

Equine penile and preputial squamous cell carcinoma (SCC) is a potentially lethal disease of which little is known regarding the relationship between tumour characteristics and prognosis. To assess the relationship between tumour differentiation grade (tumour subtype), presence of papillomaviruses, expression of viral genes (E2, E6, L1), nuclear proteins p53 and Ki67 and metastasis in equine penile and preputial SCC and to assess the relationship of tumour subtype, presence of papillomavirus type 2, p53 and Ki67 with survival. Retrospective case-control study using archived material. Samples (n = 103) from 87 horses with penile and/or preputial intraepithelial neoplasia (PIN), papilloma or SCC and corresponding case files were evaluated. Tumours were graded microscopically and p53 and Ki67 expression evaluated immunohistochemically. Equine papillomavirus (EcPV) types 2 and 3 DNA was detected by conventional PCR. Real-time PCR was used for quantification of E2, E6 and L1 mRNA. Equine papillomavirus type 2 DNA was detected in 89.4% and EcPV3 in 1.5% of horses. No differences in quantitative expression of E2, E6 and L1 oncogenes between subtypes were found. Expression of p53 and occurrence of metastasis were positively correlated to a less differentiated subtype (r = 0.429, P<0.001 and r = 0.769, P = 0.001, respectively). Differences in survival between subtypes were significant (log Rank P<0.001); horses with less differentiated tumours were more likely to die of the disease (papilloma 8.3%; G1 26.1%; G2 26.3%; G3 63.3%). In equine penile and preputial SCC, tumour grading is an important prognosticator for survival and a predictor for presence of metastases. Expression of p53 and Ki67 and presence or expression of EcPV2 and EcPV3 do not appear to be important prognosticators.

Intraocular Biomarker Identification in Uveitis Associated With Juvenile Idiopathic Arthritis

To investigate the presence of biomarkers in aqueous humor (AH) from patients with uveitis associated with juvenile idiopathic arthritis (JIA). AH (N = 73) AND SERUM (N = 105) SAMPLES FROM 116 CHILDREN WERE ANALYZED USING SURFACE ENHANCED LASER DESORPTION/IONIZATION TIME OF FLIGHT MASS SPECTROMETRY (SELDI-TOF MS). JIA, silent chronic anterior uveitis (AU), other uveitis entities, and noninflammatory controls. Statistical biomarker identification was performed using the SELDI-ToF Biomarker Analysis Cluster Wizard followed by multivariate statistical analysis. Biochemical identification of biomarkers was performed by polyacrylamide gel protein separation, followed by liquid chromatography tandem mass spectrometry. ELISA was performed in a number of AH samples representing all four study groups. In the JIA group, one AH protein peak at mass/charge (m/z) 13,762 had qualitative and quantitative differences in expression compared with the other uveitis entities and the controls, but not to the group of silent chronic AU. Its quantitative expression in AH of patients with JIA and other silent chronic AU was positively associated with uveitis activity. The protein at m/z 13,762 in AH was identified as transthyretin (TTR). The TTR concentration in AH differed significantly between the study groups (P = 0.006) with considerably higher TTR concentrations in JIA and silent chronic AU samples positive for m/z 13,762 than those of the other uveitis and control groups. TTR is a potential intraocular biomarker of JIA- associated uveitis. Its role in the pathogenesis of silent chronic AU with and without arthritis needs further investigation.

Protein and Glycoprotein Patterns Related to Morphogenesis in Horseradish Tissue Culture

In this study cellular proteins of in vitro grown horseradish (Armoracia lapathifolia Gilib.) tissues of different morphologies, leaves (L), teratoma (TM) and tumor (TN), were separated by one-dimensional (1-DE) and two-dimensional polyacrylamide gel electrophoresis (2-DE) in order to reveal developmentally specific proteins. Moreover, proteins were subjected to analyses by lectin assays to reveal whether the morphological differences were reflected in protein glycosylation. 1-DE glycoprotein pattern showed substantial distinctions between horseradish tissues of different morphologies, which was in contrast to silver stained protein profiles, where only slightly qualitative changes were observed. 2-DE revealed more differences in expression of cellular proteins between examined tissues in comparison to 1-DE, while 2-DE blots confirmed quantitative difference in expression of 43 kDa glycoprotein between investigated tissues. Mass spectrometry analysis identified this protein as a typical horseradish peroxidase. Obtained results imply that the oligosaccharide structures could be important determinants of specificities of horseradish tissue morphology.

An increase in milk IgA correlates with both pIgR expression and IgA plasma cell accumulation in the lactating mammary gland of PRM/Alf mice

In mice, during late pregnancy and lactation, maternal precursors of IgA-containing cells (cIgA-cells) are primed in the gut and home to the mammary gland where they secrete IgA. In turn, the ensuing increase in milk IgA mediates immune protection of the newborn gastrointestinal tract. PRM/Alf is an inbred mouse strain which exhibits a substantial post-natal intestinal lengthening which develops throughout the neonatal suckling period, suggesting that the availability of cIg-A cells and the level of protective IgA in milk might also be increased. We confirmed that PRM/Alf milk contains higher amounts of IgA than C57BL/6J throughout lactation, concomitantly with an increase of pIgR on epithelial cells and a higher density of cIgA-cells in the PRM/Alf mammary gland. Furthermore, a search for variations in cellular and humoral factors implicated in regulating cIgA-cell migration towards the mammary gland, including the vascular addressins MAdCAM-1 (mucosal addressin cell adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) as well as the mucosal epithelial chemokine CCL28, did not reveal any quantitative differences in expression between PRM/Alf and C57BL/6J mice strains. Thus our results indicate that these factors are not limiting in the recruitment of cIgA-cells released from the elongated gut of PRM/Alf mice. In the context of intestinal lengthening, these findings strengthen the notion of an entero-mammary gland link, where the neonatal gut is protected by the maternal gut through the immune function of the mammary gland.

Smoking-Related Gene Expression in Laser Capture–Microdissected Human Lung

PURPOSE: Interindividual differences in quantitative expression could underlie a propensity for lung cancer. To determine precise individual gene expression signatures on a lung compartment-specific basis, we investigated the expression of carcinogen metabolism genes encoding cytochromes P450 (CYP) 1B1, 2A13, GSTP1, and a tumor suppressor gene p16 in laser capture-microdissected samples of human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissue from 62 smokers and nonsmokers. EXPERIMENTAL DESIGN: Tobacco exposure was determined by plasma nicotine, cotinine, and smoking history. Precise mRNA expression was determined using our RNA-specific qRT-PCR strategy, and correlated with detailed demographic and clinical characteristics. RESULTS: Several correlations of mRNA expression included (a) CYP1B1 in AC (positively with plasma nicotine level, P = 0.008; plasma cotinine level, P = 0.001), (b) GSTP1 in AC (positively with plasma cotinine level, P = 0.003), and (c) GSTP1 in BEC (negatively with smoke dose, P = 0.043; occupational risk, P = 0.019). CYP2A13 was rarely expressed in AC and not expressed in BEC. p16 expression was not correlated with any measured factor. For each gene, subjects showed expression that was individually concordant between these compartments. No clear association of mRNA expression with lung cancer risk was observed in this pilot analysis. CONCLUSIONS: The association between lung mRNA expression and tobacco exposure implies that gene-tobacco interaction is a measurable quantitative trait, albeit with wide interindividual variation. Gene expression tends to be concordant for alveolar and bronchial compartments for these genes in an individual, controlling for proximate tobacco exposure. (Clin Cancer Res 2009;15(24):7562-70).

A newly-developed community microarray resource for transcriptome profiling in Brassica species enables the confirmation of Brassica-specific expressed sequences

BackgroundThe Brassica species include an important group of crops and provide opportunities for studying the evolutionary consequences of polyploidy. They are related to Arabidopsis thaliana, for which the first complete plant genome sequence was obtained and their genomes show extensive, although imperfect, conserved synteny with that of A. thaliana. A large number of EST sequences, derived from a range of different Brassica species, are available in the public database, but no public microarray resource has so far been developed for these species.ResultsWe assembled unigenes using ~800,000 EST sequences, mainly from three species: B. napus, B. rapa and B. oleracea. The assembly was conducted with the aim of co-assembling ESTs of orthologous genes (including homoeologous pairs of genes in B. napus from each of the A and C genomes), but resolving assemblies of paralogous, or paleo-homoeologous, genes (i.e. the genes related by the ancestral genome triplication observed in diploid Brassica species). 90,864 unique sequence assemblies were developed. These were incorporated into the BAC sequence annotation for the Brassica rapa Genome Sequencing Project, enabling the identification of cognate genomic sequences for a proportion of them. A 60-mer oligo microarray comprising 94,558 probes was developed using the unigene sequences. Gene expression was analysed in reciprocal resynthesised B. napus lines and the B. oleracea and B. rapa lines used to produce them. The analysis showed that significant expression could consistently be detected in leaf tissue for 35,386 unigenes. Expression was detected across all four genotypes for 27,355 unigenes, genome-specific expression patterns were observed for 7,851 unigenes and 180 unigenes displayed other classes of expression pattern. Principal component analysis (PCA) clearly resolved the individual microarray datasets for B. rapa, B. oleracea and resynthesised B. napus. Quantitative differences in expression were observed between the resynthesised B. napus lines for 98 unigenes, most of which could be classified into non-additive expression patterns, including 17 that showed cytoplasm-specific patterns. We further characterized the unigenes for which A genome-specific expression was observed and cognate genomic sequences could be identified. Ten of these unigenes were found to be Brassica-specific sequences, including two that originate from complex loci comprising gene clusters.ConclusionWe succeeded in developing a Brassica community microarray resource. Although expression can be measured for the majority of unigenes across species, there were numerous probes that reported in a genome-specific manner. We anticipate that some proportion of these will represent species-specific transcripts and the remainder will be the consequence of variation of sequences within the regions represented by the array probes. Our studies demonstrated that the datasets obtained from the arrays can be used for typical analyses, including PCA and the analysis of differential expression. We have also demonstrated that Brassica-specific transcripts identified in silico in the sequence assembly of public EST database accessions are indeed reported by the array. These would not be detectable using arrays designed using A. thaliana sequences.

Proteome analysis of muscadine grape leaves

Muscadine grapes are native to the southeastern United States and are used for making wine and consumed as fresh fruit. Grape berries, as ‘sink organs,’ rely on the use of available carbohydrate resources produced by photosynthesis to support their development and composition. A high throughput two-dimensional gel electrophoresis (2-DE) was conducted on muscadine (Vitis rotundifolia) grape leaf proteins to document complexity in their composition and to determine protein identity and function for enhancing photosynthetic efficiency of muscadine grape. 2-DE resolved muscadine leaf proteins into >258 polypeptides with pIs between 3.5 and 8.0 and molecular weight between 12,000 to 15,0000 Daltons. The consistently expressed proteins were excised and subjected to sequencing. Homology search of protein sequences showed 84% identity with Viridi plantae database. Identity of some of these proteins included RuBisCO, glutamine synthetase, pathogenesis-related protein, glyoxisomal malate dehydrogenase, ribonucleoprotein, chloroplast precursor, oxygen evolving enhancer protein. Comparative analysis of 10 muscadine cultivars showed quantitative differences in expression of 39 polypeptides among these genotypes. The results suggested that the polypeptide composition of muscadine grape leaf is complex, and polypeptide number and amount vary widely among muscadine genotypes, and these variations may be responsible for differences in their physiology, berry and stress tolerance characteristics.

Two-Dimensional Electrophoretic Analysis of Nuclear Matrix Proteins in Human Colon Adenocarcinoma

The aim of the present study was to observe possible qualitative and quantitative expression differences between nuclear matrix proteins (NMPs) of human colon adenocarcinoma and their mirror biopsies, using the technique of two-dimensional gel electrophoresis, in order to identify the existence of specific NMP fingerprints for colon cancer. Colon tissues were examined ultrastructurally and NMPs were isolated biochemically, by serial extraction of lipids, soluble proteins, DNA, RNA, and intermediate filaments and were separated according to their isoelectric point (pI) and their molecular weight (MW) by high-resolution two-dimensional electrophoresis (2D). By comparing the 2D electropherograms of colon cancer tissues and mirror biopsy tissues we observed qualitative and quantitative expression differences between their NMPs but also a differentiation of NMP composition between the stages of malignancy. Moreover, despite the similarities between mirror biopsy samples, a highlight percentage of exception was observed. Electrophoretic results provided in this study demonstrated that the examined NMPs could be further investigated as potential markers for detection of colorectal cancer in an early stage, for the assessment of the disease progression, as well as useful tools for individual therapy and for preventing a possible recurrence of cancer and metastasis.