Application of gene silencing in plants

Recent studies of gene silencing in plants have revealed two RNA-mediated epigenetic processes, RNA-directed RNA degradation and RNA-directed DNA methylation. These natural processes have provided new avenues for developing high-efficiency, high-throughput technology for gene suppression in plants.

Acquired differential regulation of caspase-8 in cisplatin-resistant non-small-cell lung cancer

Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism.

Expression, regulation and putative nutrient-sensing function of taste GPCRs in the heart

G protein-coupled receptors (GPCRs) are critical for cardiovascular physiology. Cardiac cells express >100 nonchemosensory GPCRs, indicating that important physiological and potential therapeutic targets remain to be discovered. Moreover, there is a growing appreciation that members of the large, distinct taste and odorant GPCR families have specific functions in tissues beyond the oronasal cavity, including in the brain, gastrointestinal tract and respiratory system. To date, these chemosensory GPCRs have not been systematically studied in the heart. We performed RT-qPCR taste receptor screens in rodent and human heart tissues that revealed discrete subsets of type 2 taste receptors (TAS2/Tas2) as well as Tas1r1 and Tas1r3 (comprising the umami receptor) are expressed. These taste GPCRs are present in cultured cardiac myocytes and fibroblasts, and are enriched in myocytes, which we corroborated using in situ hybridization. Tas1r1 gene-targeted mice (Tas1r1Cre/Rosa26tdRFP) strikingly recapitulated these data. In vivo taste receptor expression levels were developmentally regulated in the postnatal period. Intriguingly, several Tas2rs were upregulated in cultured rat myocytes and in mouse heart in vivo following starvation. The discovery of taste GPCRs in the heart opens an exciting new field of cardiac research. We predict that these taste receptors may function as nutrient sensors in the heart.

mTOR expression in colorectal adenoma--reply

"...The mTOR protein expression in colorectal adenomas has not been widely reported in the literature. Our recent study demonstrated no significant difference in mTOR protein expression in adenomas compared to carcinomas of the large intestine [1]. However, mTOR mRNA showed lower expression in colorectal adenomas compared to colorectal adenocarcinomas..."

Clinical impacts of mammalian target of rapamycin expression in human colorectal cancers

This study investigated the clinicopathologic roles of mammalian target of rapamycin (mTOR) expression and its relationship to carcinogenesis and tumor progression in a colorectal adenoma-adenocarcinoma model. Two colon cancer cell lines with different pathologic stages (SW480 and SW48) and 1 normal colonic epithelial cell line (FHC) were used, in addition to 119 colorectal adenocarcinomas and 32 adenomas. mTOR expression profiles at messenger RNA (mRNA) and protein levels were investigated in the cells and tissues using real-time quantification polymerase chain reaction and immunohistochemistry. The findings were correlated with the clinicopathologic features of the tumors. The colon cell line from stage III cancer (SW48) showed higher expression of mTOR mRNA than that from stage II cancer (SW480). At the tissue level, mTOR showed higher mRNA and protein expression in colorectal carcinoma than in adenoma. The mRNA and protein expression was correlated with each other in approximately one-third of the carcinomas and adenomas. High levels of mTOR mRNA expression were noted more in carcinoma or adenoma arising from the distal portion of the large intestine (P = .025 and .019, respectively). Within the colorectal cancer population, a high level of expression of mTOR mRNA was related to the presence of lymph node metastases (P = .031), advanced pathologic stage (P = .05), and presence of persistent disease or tumor recurrence (P = .035). To conclude, the study has indicated that mTOR is likely to be involved in the development and progression of colorectal cancer and is linked to cancer initiation, invasiveness, and progression.

Altered JS-2 expression in colorectal cancers and its clinical pathological relevance

JS-2 is a novel gene located at 5p15.2 and originally detected in primary oesophageal cancer. There is no study on the role of JS-2 in colorectal cancer. The aim of this study is to determine the gene copy number and expression of JS-2 in a large cohort of patients with colorectal tumours and correlate these to the clinicopathological features of the cancer patients. We evaluated the DNA copy number and mRNA expression of JS-2 in 176 colorectal tissues (116 adenocarcinomas, 30 adenomas and 30 non-neoplastic tissues) using real-time polymerase chain reaction. JS-2 expression was also evaluated in two colorectal cancer cell lines and a benign colorectal cell line. JS-2 amplification was noted in 35% of the colorectal adenocarcinomas. Significant differences in relative expression levels for JS-2 mRNA between different colorectal tissues were noted (p = 0.05). Distal colorectal adenocarcinoma had significantly higher copy number than proximal adenocarcinoma (p = 0.005). The relative expression level of JS-2 was different between colonic and rectal adenocarcinoma (p = 0.007). Mucinous adenocarcinoma showed higher JS-2 expression than non-mucinous adenocarcinoma (p = 0.02). Early T-stage cancers appear to have higher JS-2 copy number and lower expression of JS-2 mRNA than later stage cancers (p = 0.001 and 0.03 respectively). Colorectal cancer cell lines showed lower expression of JS-2 than the benign colorectal cell line. JS-2 copy number change and expression were shown for the first time to be altered in the carcinogenesis of colorectal cancer. In addition, genetic alteration of JS-2 was found to be related to location, pathological subtypes and staging of colorectal cancer.

Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation

The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver:brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

Oestrogen-modulated gene expression in the human endometrium

To identify key regulatory mechanisms in the growth and development of the human endometrium, microarray analysis was performed on uncultured human endometrium collected during menstruation (M) and the late-proliferative (LATE-P)-phase of the menstrual cycle, as well as after 24 h incubation in the presence of oestradiol (17beta-E2). We demonstrate the expression of novel gene transcripts in the human endometrium. i.e. mucin-9, novel oestrogen-responsive gene transcripts, i.e. gelsolin and flotillin-1, and genes known to be expressed in human endometrium but not yet shown to be oestrogen responsive, i.e. connexin-37 and TFF1/pS2. Genes reported to be expressed during the implantation window and implicated in progesterone action, i.e. secretoglobin family 2A, member 2 (mammaglobin) and homeobox-containing proteins, were up-regulated in uncultured LATE-P-phase endometrium compared to M-phase endometrium. Some gene transcripts are regulated directly by 17beta-E2 alone, others are influenced by the in vivo environment as well. These observations emphasise that the regulation of endometrium maturation by oestrogen entails more then just stimulation of cell proliferation.

Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human

To date, research into the biological processes and molecular mechanisms associated with endometrial receptivity and embryo implantation has been a focus of attention, whereas the complex events that occur in the human endometrium during the menstrual and proliferative phase under the influence of estrogen have received little attention. The objective of this review is to provide an update of our current understanding of the actions of estrogen on both human and rodent endometrium, with special emphasis on the regulation of uterine growth and cell proliferation, and the value of global gene expression analysis, in increasing understanding of these processes.

The MS risk allele of CD40 is associated with reduced cell-membrane bound expression in antigen presenting cells: Implications for gene function

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS. © 2015 Field et al.

Gene expression profiling reveals a downregulation in immune-associated genes in patients with AS

Objective: To identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) compared with healthy individuals. Methods: RNA was extracted from PBMCs collected from 18 patients with active disease and 18 gender-matched and age-matched controls. Expression profiles of these cells were determined using microarray. Candidate genes with differential expressions were confirmed in the same samples using quantitative reverse transcription-PCR (qRT-PCR). These genes were then validated in a different sample cohort of 35 patients with AS and 18 controls by qRT-PCR. Results: Microarray analysis identified 452 genes detected with 485 probes which were differentially expressed between patients with AS and controls. Underexpression of NR4A2, tumour necrosis factor AIP3 (TNFAIP3) and CD69 was confirmed. These genes were further validated in a different sample group in which the patients with AS had a wider range of disease activity. Predictive algorithms were also developed from the expression data using receiver-operating characteristic curves, which demonstrated that the three candidate genes have ∼80% power to predict AS according to their expression levels. Conclusions: The findings show differences in global gene expression patterns between patients with AS and controls, suggesting an immunosuppressive phenotype in the patients. Furthermore, downregulated expression of three immune-related genes was confirmed. These candidate genes were also shown to be strong predictive markers for AS.

Analysis of differential gene expression based on Bayesian estimation of variance

Gene expression is arguably the most important indicator of biological function. Thus identifying differentially expressed genes is one of the main aims of high throughout studies that use microarray and RNAseq platforms to study deregulated cellular pathways. There are many tools for analysing differentia gene expression from transciptomic datasets. The major challenge of this topic is to estimate gene expression variance due to the high amount of ‘background noise’ that is generated from biological equipment and the lack of biological replicates. Bayesian inference has been widely used in the bioinformatics field. In this work, we reveal that the prior knowledge employed in the Bayesian framework also helps to improve the accuracy of differential gene expression analysis when using a small number of replicates. We have developed a differential analysis tool that uses Bayesian estimation of the variance of gene expression for use with small numbers of biological replicates. Our method is more consistent when compared to the widely used cyber-t tool that successfully introduced the Bayesian framework to differential analysis. We also provide a user-friendly web based Graphic User Interface for biologists to use with microarray and RNAseq data. Bayesian inference can compensate for the instability of variance caused when using a small number of biological replicates by using pseudo replicates as prior knowledge. We also show that our new strategy to select pseudo replicates will improve the performance of the analysis. - See more at: http://www.eurekaselect.com/node/138761/article#sthash.VeK9xl5k.dpuf

Standardization and validation of an induced ovulation model system in buffalo cows: Characterization of gene expression changes in the periovulatory follicle

In bovines characterization of biochemical and molecular determinants of the dominant follicle before and during different time intervals after gonadotrophin surge requires precise identification of the dominant follicle from a follicular wave. The objectives of the present study were to standardize an experimental model in buffalo cows for accurately identifying the dominant follicle of the first wave of follicular growth and characterize changes in follicular fluid hormone concentrations as well as expression patterns of various genes associated with the process of ovulation. From the day of estrus (day 0), animals were subjected to blood sampling and ultrasonography for monitoring circulating progesterone levels and follicular growth. On day 7 of the cycle, animals were administered a PGF2α analogue (Tiaprost Trometamol, 750 μg i.m.) followed by an injection of hCG (2000 IU i.m.) 36 h later. Circulating progesterone levels progressively increased from day 1 of the cycle to 2.26 ± 0.17 ng/ml on day 7 of the cycle, but declined significantly after PGF2α injection. A progressive increase in the size of the dominant follicle was observed by ultrasonography. The follicular fluid estradiol and progesterone concentrations in the dominant follicle were 600 ± 16.7 and 38 ± 7.6 ng/ml, respectively, before hCG injection and the concentration of estradiol decreased to 125.8 ± 25.26 ng/ml, but concentration of progesterone increased to 195 ± 24.6 ng/ml, 24 h post-hCG injection. Inh-α and Cyp19A1 expressions in granulosa cells were maximal in the dominant follicle and declined in response to hCG treatment. Progesterone receptor, oxytocin and cycloxygenase-2 expressions in granulosa cells, regarded as markers of ovulation, were maximal at 24 h post-hCG. The expressions of genes belonging to the super family of proteases were also examined; Cathepsin L expression decreased, while ADAMTS 3 and 5 expressions increased 24 h post-hCG treatment. The results of the current study indicate that sequential treatments of PGF2α and hCG during early estrous cycle in the buffalo cow leads to follicular growth that culminates in ovulation. The model system reported in the present study would be valuable for examining temporo-spatial changes in the periovulatory follicle immediately before and after the onset of gonadotrophin surge.

Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background: Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton. Results: We have used a custom cDNA microarray chip, developed for the blue swimmer crab Portunus pelagicus, to generate expression profiles of genes involved in exoskeletal formation across the moult cycle. A total of 21 distinct moult-cycle related differentially expressed transcripts representing crustacean cuticular proteins were isolated. Of these, 13 contained copies of the cuticle_1 domain previously isolated from calcified regions of the crustacean exoskeleton, four transcripts contained a chitin_bind_4 domain (RR consensus sequence) associated with both the calcified and un-calcified cuticle of crustaceans, and four transcripts contained an unannotated domain (PfamB_109992) previously isolated from C. pagurus. Additionally, cryptocyanin, a hemolymph protein involved in cuticle synthesis and structural integrity, also displays differential expression related to the moult cycle. Moult stage-specific expression analysis of these transcripts revealed that differential gene expression occurs both among transcripts containing the same domain and among transcripts containing different domains. Conclusion: The large variety of genes associated with cuticle formation, and their differential expression across the crustacean moult cycle, point to the complexity of the processes associated with cuticle formation and hardening. This study provides a molecular entry path into the investigation of the gene networks associated with cuticle formation.