Differences In Transcriptional Profiles Research Articles

P134: ROLE OF AIRE GENE (AUTOIMMUNE REGULATOR) IN TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL REGULATION OF HLA-G

Non-classical HLA-G and HLA-E molecules have well-recognized immunomodulatory effects on peripheral blood immune-competent cells; however, little is known regarding the effect of these molecules in central tolerance. Aire is responsible for promiscuous gene expression of tissue-related antigens by thymic cells during induction of central tolerance. We characterized the differential transcript profiles of murine MHC class Ib (Qa-1/HLA-E, Qa-2/HLA-G) and Aire genes during ontogeny of thymus, and identified that the transcriptional profile of the functional homolog of HLA-G, (H2-Q7/Qa2) was positively correlated with Aire during thymus fetal development, indicating that non-classical MHC class I genes could be under the transcriptional control of Aire. This study aimed to evaluate the transcriptional and post-transcriptional relation between Aire and HLA-G. Using previously validated Aire plasmid-based constructs, we transfected different cell-lines: HLA-G + Cells (JEG3), HLA-G- Cells (U251) and thymic cells (4D6 and LT-TEC2), and assessed HLA-G gene and protein expression. Using an in silico approach, we found 4 putative binding sites for Aire-complex at the 5’-promoter region of the HLA-G gene. We cloned, selected (enzyme digestion: EcoRI/HindIII) and validated (sequencing/alignment) the bacterial-vectors containing Aire. Initially, we observed that only JEG-3 cells were HLA-G + with transcript levels being 10.000-fold greater than the other cell lines and 50% of cell population presenting HLA-G in cell surface. After transient transfection, HLA-G transcript levels were increased only in 4D6 and LT-TEC2 cells (P = 0.03 and P = 0.007, respectively) compared to control (empty-vector). We did not observe any modification in the HLA-G cell surface expression in all cell lines. Concluding, Aire may play a role in the transcriptional regulation of HLA-G, which appears to be cell type-dependent, i. e., primarily inducted in thymic cell lines.

Differential transcriptional profile of Corynebacterium pseudotuberculosis in response to abiotic stresses

BackgroundThe completion of whole-genome sequencing for Corynebacterium pseudotuberculosis strain 1002 has contributed to major advances in research aimed at understanding the biology of this microorganism. This bacterium causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death. In the current study, we simulated the conditions experienced by the bacteria during host infection. By sequencing transcripts using the SOLiDTM 3 Plus platform, we identified new targets expected to potentiate the survival and replication of the pathogen in adverse environments. These results may also identify possible candidates useful for the development of vaccines, diagnostic kits or therapies aimed at the reduction of losses in agribusiness.ResultsUnder the 3 simulated conditions (acid, osmotic and thermal shock stresses), 474 differentially expressed genes exhibiting at least a 2-fold change in expression levels were identified. Important genes to the infection process were induced, such as those involved in virulence, defence against oxidative stress, adhesion and regulation, and many genes encoded hypothetical proteins, indicating that further investigation of the bacterium is necessary. The data will contribute to a better understanding of the biology of C. pseudotuberculosis and to studies investigating strategies to control the disease.ConclusionsDespite the veterinary importance of C. pseudotuberculosis, the bacterium is poorly characterised; therefore, effective treatments for caseous lymphadenitis have been difficult to establish. Through the use of RNAseq, these results provide a better biological understanding of this bacterium, shed light on the most likely survival mechanisms used by this microorganism in adverse environments and identify candidates that may help reduce or even eradicate the problems caused by this disease.

Differential transcript profiles of MHC class Ib(Qa-1, Qa-2, and Qa-10) and Aire genes during the ontogeny of thymus and other tissues.

Qa-2 and Qa-1 are murine nonclassical MHC class I molecules involved in the modulation of immune responses by interacting with T CD8+ and NK cell inhibitory receptors. During thymic education, the Aire gene imposes the expression of thousands of tissue-related antigens in the thymic medulla, permitting the negative selection events. Aiming to characterize the transcriptional profiles of nonclassical MHC class I genes in spatial-temporal association with the Aire expression, we evaluated the gene expression of H2-Q7(Qa-2), H2-T23(Qa-1), H2-Q10(Qa-10), and Aire during fetal and postnatal development of thymus and other tissues. In the thymus, H2-Q7(Qa-2) transcripts were detected at high levels throughout development and were positively correlated with Aire expression during fetal ages. H2-Q7(Qa-2) and H2-T23(Qa-1) showed distinct expression patterns with gradual increasing levels according to age in most tissues analyzed. H2-Q10(Qa-10) was preferentially expressed by the liver. The Aire transcriptional profile showed increased levels during the fetal period and was detectable in postnatal ages in the thymus. Overall, nonclassical MHC class I genes started to be expressed early during the ontogeny. Their levels varied according to age, tissue, and mouse strain analyzed. This differential expression may contribute to the distinct patterns of mouse susceptibility/resistance to infectious and noninfectious disorders.

Differential Transcriptional Profiles of Dormancy-Related Genes in Apple Buds

The production of temperate fruit crops depends on plant developmental processes, primarily the shift from the juvenile phase to the reproductive phase, dormancy transitions and flowering. Apple tree (Malus × domestica Borkh.) development is regulated by chilling temperatures, which are required for bud dormancy progression. The apple cultivar Castel Gala is a spontaneous mutation of "Gala Standard". "Castel Gala" is characterized by a 50 % decrease in the chilling requirement (CR) for dormancy release, which results in an earlier budbreak. This work explores the contrasting phenotypes of these cultivars using suppression subtractive hybridization (SSH). From 1,019 unigenes identified by SSH, we selected 28 candidate genes putatively associated with dormancy cycling. Reverse transcription-quantitative polymerase chain reaction was used to validate the differential expression profiles and to transcriptionally characterize these genes in three distinct apple cultivars ("Castel Gala", "Royal Gala" and "Fuji Standard") during a cycle comprising growth to dormancy. Of the 28 candidate genes analyzed, 17 confirmed the differences in expression predicted by SSH. Seasonal transcript accumulation during the winter was observed for several genes, with higher steady-state mRNA levels maintained longer in cultivars with a high CR. The transcription profiles suggest that these genes may be associated with dormancy establishment and maintenance. Of the 17 candidate genes, transcripts coding for dormancy-associated MADS-box (DAM), dehydrins, GAST1, LTI65, NAC, HTA8, HTA12 and RAP2.12-like proteins displayed major differences in gene expression between cultivars through the winter. These genes were therefore considered good candidates for key roles in the dormancy process in apple trees.

Cluster analysis reveals differential transcript profiles associated with resistance training-induced human skeletal muscle hypertrophy

Using genomic microarray analysis, we sought to identify and annotate differences in the pretraining skeletal muscle transcriptomes among human subjects clustered as nonresponders (Non), modest responders (Mod), and extreme responders (Xtr) based on differential magnitudes of myofiber hypertrophy in response to progressive resistance training (RT) (Non-6 μm², Mod 1,111 μm², or Xtr 2,475 μm²). In prior work, we noted differences among clusters in the prevalence of myogenic stem cells prior to and during RT (35), and in the translational signaling responses to the first bout of resistance exercise (30). Here we identified remarkable differences in the pretraining transcript profiles among clusters (8,026 gene transcripts differentially expressed between Xtr and Non, 2,463 between Xtr and Mod, and 1,294 between Mod and Non). Annotated functions and networks of differentially expressed genes suggest Xtr were "primed" to respond to RT through transcriptional regulation, along with a uniquely expressed network of genes involved in skeletal muscle development, while the failed response in Non may have been driven by excessive proinflammatory signaling. Protein follow-up analysis revealed higher basal levels of acetylated histone H3 (K36) in the two responder clusters (Mod, Xtr) compared with Non, and only the responders experienced alterations in the muscle content of select proteins (e.g., α-tubulin, p27(kip)) in response to the first resistance exercise stimulus. Overall, the widely disparate transcriptomes identified prior to RT among the three clusters support the notion that at least some of the interindividual heterogeneity in propensity for RT-induced myofiber hypertrophy is likely predetermined.

Transcriptional and metabolic insights into the differential physiological responses of arabidopsis to optimal and supraoptimal atmospheric CO2.

BackgroundIn tightly closed human habitats such as space stations, locations near volcano vents and closed culture vessels, atmospheric CO2 concentration may be 10 to 20 times greater than Earth’s current ambient levels. It is known that super-elevated (SE) CO2 (>1,200 µmol mol−1) induces physiological responses different from that of moderately elevated CO2 (up to 1,200 µmol mol−1), but little is known about the molecular responses of plants to supra-optimal [CO2].Methodology/Principal FindingsTo understand the underlying molecular causes for differential physiological responses, metabolite and transcript profiles were analyzed in aerial tissue of Arabidopsis plants, which were grown under ambient atmospheric CO2 (400 µmol mol−1), elevated CO2 (1,200 µmol mol−1) and SE CO2 (4,000 µmol mol−1), at two developmental stages early and late vegetative stage. Transcript and metabolite profiling revealed very different responses to elevated versus SE [CO2]. The transcript profiles of SE CO2 treated plants were closer to that of the control. Development stage had a clear effect on plant molecular response to elevated and SE [CO2]. Photosynthetic acclimation in terms of down-regulation of photosynthetic gene expression was observed in response to elevated [CO2], but not that of SE [CO2] providing the first molecular evidence that there appears to be a fundamental disparity in the way plants respond to elevated and SE [CO2]. Although starch accumulation was induced by both elevated and SE [CO2], the increase was less at the late vegetative stage and accompanied by higher soluble sugar content suggesting an increased starch breakdown to meet sink strength resulting from the rapid growth demand. Furthermore, many of the elevated and SE CO2-responsive genes found in the present study are also regulated by plant hormone and stress.Conclusions/SignificanceThis study provides new insights into plant acclimation to elevated and SE [CO2] during development and how this relates to stress, sugar and hormone signaling.

Differential transcript profiling through cDNA-AFLP showed complexity of rutin biosynthesis and accumulation in seeds of a nutraceutical food crop (Fagopyrum spp.)

BackgroundBuckwheat, consisting of two cultivated species Fagopyrum tataricum and F. esculentum, is the richest source of flavonoid rutin. Vegetative tissues of both the Fagopyrum species contain almost similar amount of rutin; however, rutin content in seed of F. tataricum are ~50 folds of that in seed of F. esculentum. In order to understand the molecular basis of high rutin content in F. tataricum, differential transcript profiling through cDNA-AFLP has been utilized to decipher what genetic factors in addition to flavonoid structural genes contribute to high rutin content of F. tataricum compared to F. esculentum.ResultsDifferential transcript profiling through cDNA-AFLP in seed maturing stages (inflorescence to seed maturation) with 32 primer combinations generated total of 509 transcript fragments (TDFs). 167 TDFs were then eluted, cloned and sequenced from F. tataricum and F. esculentum. Categorization of TDFs on the basis of their presence/absence (qualitative variation) or differences in the amount of expression (quantitative variation) between both the Fagopyrum species showed that majority of variants are quantitative (64%). The TDFs represented genes controlling different biological processes such as basic and secondary metabolism (33%), regulation (18%), signal transduction (14%), transportation (13%), cellular organization (10%), and photosynthesis & energy (4%). Most of the TDFs except belonging to cellular metabolism showed relatively higher transcript abundance in F. tataricum over F. esculentum. Quantitative RT-PCR analysis of nine TDFs representing genes involved in regulation, metabolism, signaling and transport of secondary metabolites showed that all the tested nine TDFs (Ubiquitin protein ligase, ABC transporter, sugar transporter) except MYB 118 showed significantly higher expression in early seed formation stage (S7) of F. tataricum compared to F. esculentum. qRT-PCR results were found to be consistent with the cDNA-AFLP results.ConclusionsThe present study concludes that in addition to structural genes, other classes of genes such as regulators, modifiers and transporters are also important in biosynthesis and accumulation of flavonoid content in plants. cDNA-AFLP technology was successfully utilized to capture genes that are contributing to differences in rutin content in seed maturing stages of Fagopyrum species. Increased transcript abundance of TDFs during transition from flowers to seed maturation suggests their involvement not only in the higher rutin content of F. tataricum over F. esculentum but also in nutritional superiority of the former.

Differential transcriptional profiles mediated by exposure to the cannabinoids cannabidiol and Δ9‐tetrahydrocannabinol in BV‐2 microglial cells

Apart from their effects on mood and reward, cannabinoids exert beneficial actions such as neuroprotection and attenuation of inflammation. The immunosuppressive activity of cannabinoids has been well established. However, the underlying mechanisms are largely unknown. We previously showed that the psychoactive cannabinoid Δ(9) -tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signalling pathways. To characterize the transcriptional effects of CBD and THC, we treated BV-2 microglial cells with these compounds and performed comparative microarray analysis using the Illumina MouseRef-8 BeadChip platform. Ingenuity Pathway Analysis was performed to identify functional subsets of genes and networks regulated by CBD and/or THC. Overall, CBD altered the expression of many more genes; from the 1298 transcripts found to be differentially regulated by the treatments, 680 gene probe sets were up-regulated by CBD and 58 by THC, and 524 gene products were down-regulated by CBD and only 36 by THC. CBD-specific gene expression profile showed changes associated with oxidative stress and glutathione depletion, normally occurring under nutrient limiting conditions or proteasome inhibition and involving the GCN2/eIF2α/p8/ATF4/CHOP-TRIB3 pathway. Furthermore, CBD-stimulated genes were shown to be controlled by nuclear factors known to be involved in the regulation of stress response and inflammation, mainly via the (EpRE/ARE)-Nrf2/ATF4 system and the Nrf2/Hmox1 axis. These observations indicated that CBD, but much less than THC, induced a cellular stress response in microglial cells and suggested that this effect could underlie its anti-inflammatory activity. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Analysis of differential transcriptional profiling in wheat infected by Blumeria graminis f. sp. tritici using GeneChip

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating disease of wheat. The use of wheat cultivars resistant to powdery mildew provides an effective, economical, and environmentally friendly method to control the disease. Previously, we identified a dominant resistance gene, temporarily named Pmhym, from the wheat cultivar Hongyoumai. In order to screen differential transcripts related to Pmhym-mediated resistance, four F3 homozygous resistant and four susceptible progenies derived from the Hongyoumai/Yumai13 cross were selected to construct two different pools, respectively, representing an incompatible and compatible interaction with Bgt. Pre-inoculated control and the pathogen-inoculated treatments at 24 h post inoculation (hpi) were used. Three groups of differential genes were categorized from three comparisons as pre- and post-induced, respectively, in two interactions, and post-induced between incompatible and compatible interaction. It was found that salicylic acid (SA), jasmonate (JA), and ethylene (ET) signaling-related genes were differentially expressed, thus suggesting that they are involved in the defensive response against Bgt infection. In compatible interactions, the genes involved in the abscisic acid (ABA) signaling pathway might be inhibitory to the above-mentioned three pathways, resulting in a susceptible reaction. Genes involved in disease/defense, signal transduction, and reactive oxygen species (ROS) metabolism were up-regulated in incompatible interactions, implying a role in resistant response. The results of qRT-PCR analysis on several candidate genes were consistent in their expression patterns as revealed by microarray analysis. The differential expression analyses in the present study are good candidates for further elucidation of wheat defensive response to powdery mildew.

Overexpression of an apoplastic peroxidase gene CrPrx in transgenic hairy root lines of Catharanthus roseus

Peroxidases are a family of isoenzymes found in all higher plants and are known to be involved in a broad range of physiological processes. However, very little information is available concerning their role in Catharanthus roseus. The present study describes the impact of both overexpression and suppression of a peroxidase gene, CrPrx in C. roseus transgenic hairy root lines. Real-time PCR analysis in 35S-CrPrx and CrPrx-RNAi transgenic lines indicated differential transcript profile for peroxidases as well as for genes and regulators involved in MIA (monoterpenoid indole alkaloid) pathway of C. roseus. Comparative analysis revealed that MIA pathway genes showing elevated levels of expression in 35S-CrPrx transgenic lines showed a significant reduction in their transcript level in CrPrx-RNAi transgenic lines. Metabolite analysis detected higher levels of ajmalicine and serpentine accumulation in overexpressed lines. It was observed that all overexpressed transgenic lines produced more amount of H(2)O(2). These results indicate a role of CrPrx gene in the regulation of MIA pathway genes and regulators, thus affecting the production of specific alkaloids.

MAPT Isoforms: Differential Transcriptional Profiles Related to 3R and 4R Splice Variants

Tau aggregation in neurofibrillary tangles is a pathological hallmark in tauopathies including Alzheimer's disease (AD). The predominant aggregation of certain MAPT (tau gene) isoforms, either the 4-repeat (4R tau) or the 3-repeat (3R tau) isoform has been widely described in tauopathies. Alterations of the 4R tau to 3R tau ratio may be a key for tau-related neurodegeneration. To study the biological consequences in expression between tau splicing isoforms 4R and 3R, we analyzed the main neurobiological effects of inclusion of the repeat region coded by exon 10 in MAPT. We compared the transcriptional profiles of the 4R tau isoforms to 3R tau isoforms using whole-genome gene expression profiling microarrays using human neuroblastoma SH-SY5Y cell lines overexpressing either human 4R tau or 3R tau isoforms. We identified 68 transcripts that differed significantly (at p < 0.001) between 4R and 3R isoforms as conditioned on a second variant, the so-called 2N inclusion. We extended these findings in a 2 × 2 ANOVA to examine interaction effects of these variants. Transcripts involved in embryonic development were downregulated when exon 10 was present, while transcripts related to outgrowth of neurites were generally upregulated. An important pathway implicated in AD also differed between the 3R and 4R cell lines, Wnt signaling. These studies demonstrate expression differences between MAPT isoforms 4R tau and 3R tau due to the inclusion/exclusion of the repeat region coded for by exon 10. Our data add to complex findings on the role of 3R/4R in normal and abnormal neuronal function and highlight several molecular mechanisms that might drive neurodegeneration, or perhaps, set the stage for it.

Differential transcriptional profiles induced by amphotericin B formulations on human monocytes during response to hyphae ofAspergillus fumigatus

Amphotericin B formulations possess diverse immunomodulatory properties that may contribute to the activity of phagocytes against invasive aspergillosis. In this work we provide a novel set of data on different gene transcriptional profiles of monocytes exposed to the combination of Aspergillus fumigatus and amphotericin B formulations. We used pathway-specific microarray analysis, RT-PCR analysis and enzyme-linked immunosorbent assays to compare the effects of amphotericin B deoxycholate (DAMB) at 1 μg/ml and amphotericin B lipid complex (ABLC) at 5 μg/ml to assess gene expression of immune molecules of THP-1 cells exposed to A. fumigatus hyphae (AF) for 4 h. A. fumigatus hyphae at effector/target ratio 10/1 induced mostly chemotactic factors for monocyte recruitment. DAMB at 1 μg/ml in the presence or absence of AF induced the most pronounced changes in pro-inflammatory and chemokine gene expression, while ABLC under the same conditions caused less dramatic effect. There was a reciprocal response of increased expression of the genes encoding IL-1β and IL-20 and decreased expression of IL-10, IL-2 and IL-3 in response of monocytes to both the hyphae and antifungal agents. These results demonstrate that amphotericin B formulations exert differential effects on genes encoding pro-inflammatory molecules, immunoregulatory molecules and chemokines by human monocytes during response to A. fumigatus and that these molecules may affect antifungal activity.

Involvement of multiple molecular events in pyrogallol-induced hepatotoxicity and silymarin-mediated protection: Evidence from gene expression profiles

In this study, the involvement of various molecular events in pyrogallol-mediated hepatotoxicity was deciphered by differential mRNA transcription profiles of control and pyrogallol treated mice liver. The modulatory effects of silymarin on pyrogallol-induced differentially expressed transcripts were also looked into. Swiss albino mice were treated with or without pyrogallol. In some set of experiments, mice were also treated with silymarin 2h prior to pyrogallol. Total RNA was isolated from liver and polyadenylated RNA was reverse-transcribed into Cye 3 or Cye 5 labelled cDNA. Equal amounts of labelled cDNA from two different groups were mixed and hybridized with mouse 15k array. The hybridized arrays were scanned, analyzed and the expression level of each transcript was calculated. The differential expression was validated by quantitative real time polymerase chain reaction. Comparative transcription pattern showed an alteration in the expression of 183 transcripts (150 up-regulated and 33 down-regulated) associated with oxidative stress, cell cycle, cytoskeletal network, cell–cell adhesion, extra-cellular matrix, inflammation, apoptosis, cell-signaling and intermediary metabolism in pyrogallol-exposed liver and silymarin pre-treatment modulated the expression of many of these transcripts. Results obtained thus suggest that pyrogallol induces multiple molecular events leading to hepatotoxicity and silymarin effectively counteracts pyrogallol-mediated alterations.

Contactin-2 Expression in the Cardiac Purkinje Fiber Network

Purkinje cells (PCs) comprise the most distal component of the cardiac conduction system, and their unique electrophysiological properties and the anatomic complexity of the Purkinje fiber network may account for the prominent role these cells play in the genesis of various arrhythmic syndromes. Differential transcriptional profiling of murine Purkinje fibers and working ventricular myocytes was performed to identify novel genes expressed in PCs. The most highly enriched transcript in Purkinje fibers encoded Contactin-2 (Cntn2), a cell adhesion molecule critical for neuronal patterning and ion channel clustering. Endogenous expression of Cntn2 in the murine ventricle was restricted to a subendocardial network of myocytes that also express beta-galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein. Both Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic reporter mice confirmed expression of Cntn2 in the Purkinje fiber network, as did immunohistochemical staining of single canine Purkinje fibers. Whole-cell patch-clamp recordings and measurements of Ca(2+) transients in Cntn2-EGFP(+) cells revealed electrophysiological properties indicative of PCs and distinctive from those of cardiac myocytes, including prolonged action potentials and frequent afterdepolarizations. Cntn2 is a novel marker of the specialized cardiac conduction system. Endogenous expression of Cntn2 as well as Cntn2-dependent transcriptional reporters provides a new tool through which Purkinje cell biology and pathophysiology can now more readily be deciphered. Expression of a contactin family member within the CCS may provide a mechanistic basis for patterning of the conduction system network and the organization of ion channels within Purkinje cells.

Abscisic acid regulated gene expression in bread wheat (Triticum aestivum L.)

The studies about the elucidation of molecular components of drought response in major crops have become an urgent priority. Since abscisic acid (ABA) is one of the major plant hormones influencing main stress responses including drought, cold, and heat, there has been overwhelming interest in investigation of ABA-associated genes. Therefore, the identification and characterization of ABAassociated genes from bread wheat is significant for gaining better insights about the multiple stress-response mechanisms. Due to the pivotal role of ABA in stress response mechanism, we tried to determine spatiotemporal expressional characteristics of ABA-related genes in wheat. The comparative differential transcript profiling experiments revealed a total of 60 differentially-regulated cDNA fragments, thirty of which were sequenced to obtain more details about their functional identities. Some of the sequenced fragments showed significant similarity with the genes known to be associated with ABA-induced signaling networks; e.g., Germin-Like Proteins, Tonoplast Intrinsic Proteins, MAP kinases and leucine-rich repeat (LRR) receptor-like proteins. To get more details about ABAassociated genes in wheat, the expressional characteristics of the transcripts from the initial screening was further investigated by using quantitative Real Time PCR. For example, the amount of transcripts MAPK4 and TIP1 homologs in leaf tissue of bread wheat is gradually increased and eventually peaked in about 2 hours after ABA treatment, and subsequently declined with the prolonged exposure. On the other hand, induction in the GLP homolog expression was much faster than the aforementioned transcripts; reaching to the apex in roughly an hour after the treatment. This study provided insights about the expressional characteristics of some ABA-associated genes from bread wheat. This is the first study investigating the early response genes under ABA-treatment in hexaploid bread wheat.

Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease

The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of 111In-labeled neutrophils at these sites and clearance of 32P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

Identification of differential transcript profiles between mutual crossbred embryos of zebrafish ( Danio rerio) and Chinese rare minnow ( Gobiocypris rarus) by cDNA-AFLP

The crosstalk between naive nucleus and maternal factors deposited in egg cytoplasm before zygotic genome activation is crucial for early development. In this study, we utilized two laboratory fishes, zebrafish ( Danio rerio) and Chinese rare minnow ( Gobiocypris rarus), to obtain mutual crossbred embryos and examine the interaction between nucleus and egg cytoplasm from different species. Although these two types of crossbred embryos originated from common nuclei, various developmental capacities were gained due to different origins of the egg cytoplasm. Using cDNA amplified fragment length polymorphism (cDNA-AFLP), we compared transcript profiles between the mutual crossbred embryos at two developmental stages (50%- and 90%-epiboly). Three thousand cDNA fragments were generated in four cDNA pools with 64 primer combinations. All differentially displayed transcript-derived fragments (TDFs) were screened by dot blot hybridization, and the selected sequences were further analyzed by semi-quantitative RT-PCR and quantitative real-time RT-PCR. Compared with ZR embryos, 12 genes were up-regulated and 12 were down-regulated in RZ embryos. The gene fragments were sequenced and subjected to BLASTN analysis. The sequences encoded various proteins which functioned at various levels of proliferation, growth, and development. One gene (ZR6), dramatically down-regulated in RZ embryos, was chosen for loss-of-function study; the knockdown of ZR6 gave rise to the phenotype resembling that of RZ embryos.

Differential transcriptional profile of the Wnt pathway in sporadic colorectal cancers with and without microsatellite instability

Patients with epidermal growth factor receptor (EGFR) activation mutation-positive non-small-cell lung cancer (NSCLC) respond well to EGFR tyrosine kinase inhibitors (EGFR-TKIs), but eventually become resistant in most cases. The hepatocyte growth factor/c-Met (HGF/c-Met) pathway is reported as a poor prognostic factor in various cancers. As c-Met is involved in EGFR-TKI resistance, a c-Met inhibitor and EGFR-TKI combination may reverse the resistance. This study evaluated the efficacy and safety of a c-Met selective inhibitor, tivantinib (ARQ 197), in combination with erlotinib, in Japanese EGFR mutation-positive patients with NSCLC who progressed while on EGFR-TKIs.This study enrolled 45 patients with NSCLC with acquired resistance to EGFR-TKIs, who were orally administered a daily combination of tivantinib/erlotinib. The primary end point was the overall response rate (ORR) and secondary end points included disease control rate, progression-free survival (PFS) and overall survival (OS). The patients underwent a mandatory second biopsy just after progression on EGFR-TKIs. The predictive biomarkers were extensively analysed using tumour and blood samples.The ORR was 6.7% (95% CI 1.4% to 18.3%), and the lower limit of 95% CI did not exceed the target of 5%. The median PFS (mPFS) and median OS (mOS) were 2.7 months (95% CI 1.4 to 4.2) and 18.0 months (95% CI 13.4 to 22.2), respectively. Both were longer in c-Met high patients (c-Met high vs low: mPFS 4.1 vs 1.4 months; mOS 20.7 vs 13.9 months). Partial response was observed in three patients, all of whom were c-Met and HGF high. The common adverse events and their frequencies were similar to those known to occur with tivantinib or erlotinib alone.Although this study did not prove clinical benefit of tivantinib in patients with acquired resistance to EGFR-TKIs, activated HGF/c-Met signalling, a poor prognostic factor, may define a patient subset associated with longer survival by the tivantinib/erlotinib combination.NCT01580735.

Transcriptome Analysis of Bull Semen with Extreme Nonreturn Rate: Use of Suppression-Subtractive Hybridization to Identify Functional Markers for Fertility1

Spermatozoa are terminally differentiated cells produced during the complex process of spermatogenesis. Although the role of their residual RNA content is still being debated, this transcriptome may represent a fingerprint of spermatogenesis quality. In the present study, we undertook differential transcript profiling of spermatozoa from fertile bulls with extreme nonreturn rates (NRRs): a low-fertile group, and a high-fertile group. Using the suppression-subtractive hybridization technique in combination with macroarray analysis, we also identified novel genes. Both extreme NRR index groups retained redundant identity, such as ribosomal and mitochondrial sequences, at a statistically significant level. An elevated number of 12S, 18S, and Large Chain R rRNA gene copies were found in low-fertile bulls and validated in spermatozoa by quantitative RT-PCR for a small cohort of bulls with known fertility index. Whereas the high-NRR library exhibited a large proportion (29%) of transcripts associated with known functions (e.g., metabolism, signal transduction, translation, glycosylation, and protein degradation), only 10% of the low-NRR sequences did. This difference is also conveyed by two other categories: 17% Bovine Genome and 48% unknown in the high-NRR library, compared with 3% and 80%, respectively, in the low-NRR library. Some of the unknown transcripts are similar to expressed sequence tags detected in the male reproductive organ of certain plants and retain homology to a putative human protein. Whereas the individual transcriptome profiles may be useful in fertility assessment, these findings also suggest cross-species conservation, could contribute to a better understanding of spermatogenesis, and provide new insights regarding idiopathic infertility.

Gender-enriched transcripts in Haemonchus contortus – predicted functions and genetic interactions based on comparative analyses with Caenorhabditis elegans

In the present study, a bioinformatic-microarray approach was employed for the analysis of selected expressed sequence tags (ESTs) from Haemonchus contortus, a key parasitic nematode of small ruminants. Following a bioinformatic analysis of EST data using a semiautomated pipeline, 1885 representative ESTs (rESTs) were selected, to which oligonucleotides (three per EST) were designed and spotted on to a microarray. This microarray was hybridized with cyanine-dye labelled cRNA probes synthesized from RNA from female or male adults of H. contortus. Differential hybridisation was displayed for 301 of the 1885 rESTs (∼16%). Of these, 165 (55%) had significantly greater signal intensities for female cRNA and 136 (45%) for male cRNA. Of these, 113 with increased signals in female or male H. contortus had homologues in Caenorhabditis elegans, predicted to function in metabolism, information storage and processing, cellular processes and signalling, and embryonic and/or larval development. Of the rESTs with no known homologues in C. elegans, 24 (∼40%) had homologues in other nematodes, four had homologues in various other organisms and 30 (52%) had no homology to any sequence in current gene databases. A genetic interaction network was predicted for the C. elegans orthologues of the gender-enriched H. contortus genes, and a focused analysis of a subset revealed a tight network of molecules involved in amino acid, carbohydrate or lipid transport and metabolism, energy production and conversion, translation, ribosomal structure and biogenesis and, importantly, those associated with meiosis and/or mitosis in the germline during oogenesis or spermatogenesis. This study provides a foundation for the molecular, biochemical and functional exploration of selected molecules with differential transcription profiles in H. contortus, for further microarray analyses of transcription in different developmental stages of H. contortus, and for an extended functional analysis once the full genome sequence of this nematode is known.