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Grain size and shape have significant effects on grain weight and is also an important breeding target due to market and industry requirements. In this study, the genetic loci associated with six grain morphology traits including thousand grain weight, grain length (GL), grain width, grain length to width ratio (GLWR), grain area (GA) and grain perimeter (GP) in a population of 300 Chinese wheat germplasm were investigated through genome wide association mapping. A total of 189 SNPs were significantly related to the six grain traits based on the BLUP data from three environments. Of these SNPs, 24 SNPs for GA, GL, GLWR, and GP were continuously detected under all three experiments. These SNPs were distributed on chromosome 2A, 2B, 2D, 4D, 5A, and 6D, respectively. Some SNPs such as Affx-88368872 on 2A, Affx-109472887 on 6D, and Affx-108968182, Affx-111286671, Affx-110418048, Affx-109838962, Affx-88484232 on 5A were associated with multiple traits. In addition, a chromosome region tagged by 11 SNPs (585,117,524–595,658,182 bp) on chromosome 2D had significant effects on GLWR. Several genes inside the regions on chromosome 5A and 2D may play crucial roles in determining seed size and weight. We will characterize the function of these genes in grain development in our future experiments. This study provides novel insights into the genetic basis of grain morphology traits in wheat.

Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with the increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3LDat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field, and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[18F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3LDat/wt mice compared to Dnmt3a2/3Lwt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3LDat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[18F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3LDat/wt mice and controls. [18F]FDG PET showed that hypothalamic metabolic activity was tightly linked to motor behavior in Dnmt3a2/3LDat/wt mice. Furthermore, dopamine biosynthesis and motor-related metabolic activity were correlated in the hypothalamus. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, modulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis.

Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3LDat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[18F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3LDat/wt mice compared to Dnmt3a2/3Lwt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3LDat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[18F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3LDat/wt and controls. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, regulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis. Keywords: DNA methylation, transgenic mouse model, behavioral paradigms, positron emission tomography, dopaminergic system.

Activation of innate immunity by membrane-localized receptors is conserved across eukaryotes. Plant genomes contain hundreds of such receptor-like genes and those encoding proteins with an extracellular leucine-rich repeat (LRR) domain represent the largest family. Here, we develop a high-throughput approach to study LRR receptor-like genes on a genome-wide scale. In total, 257 tobacco rattle virus-based constructs are generated to target 386 of the 403 identified LRR receptor-like genes in Nicotiana benthamiana for silencing. Using this toolkit, we identify the LRR receptor-like protein Response to XEG1 (RXEG1) that specifically recognizes the glycoside hydrolase 12 protein XEG1. RXEG1 associates with XEG1 via the LRR domain in the apoplast and forms a complex with the LRR receptor-like kinases BAK1 and SOBIR1 to transduce the XEG1-induced defense signal. Thus, this genome-wide silencing assay is demonstrated to be an efficient toolkit to pinpoint new immune receptors, which will contribute to developing durable disease resistance.

Comparative proteomics is widely used to detect protein changes, especially differential abundance proteins (DAPs) that are involved in plant responses to development, disease, or environment. Once DAPs are identified, it is essential to validate any change in their abundance, and their role in the biological process under study. In addition to common confirmation by quantitative RT-PCR, immunoblot, and multiple reaction monitoring analysis, it has been proposed that enzyme activity assay (EAA) can be complementary to the standard proteomics results, especially regarding the elucidation of protein (enzyme) function and the mechanism of enzyme-associated biochemical or metabolic pathways. The enzymes discussed here are the DAPs identified in comparative plant proteomics. Despite the small number of enzymes in a proteome, they often make up a substantial proportion of the DAPs identified in comparative studies. Currently, only a few studies have performed EAA to complement the interpretation of proteomic data, especially activity-based protein profiling. This viewpoint aims to arouse the attention of proteomic researchers on the promising role of EAA in plant proteomics and highlights the need for high-throughput assays of enzyme activities in comparative plant proteomics.

The genetics of aging is typically concerned with lifespan determination that is associated with alterations in expression levels or mutations of particular genes. Previous reports in C. elegans have shown that the bmk-1 gene has important functions in chromosome segregation, and this has been confirmed with its mammalian homolog, KIF11. However, this gene has never been implicated in aging or lifespan regulation. Here we show that the bmk-1 gene is an important lifespan regulator in worms. We show that reducing bmk-1 expression using RNAi shortens worm lifespan by 32%, while over-expression of bmk-1 extends worm lifespan by 25%, and enhances heat-shock stress resistance. Moreover, bmk-1 over-expression increases the level of hsp-16 and decreases ced-3 in C. elegans. Genetic epistasis analysis reveals that hsp-16 is essential for the lifespan extension by bmk-1. These findings suggest that bmk-1 may act through enhanced hsp-16 function to protect cells from stress and inhibit the apoptosis pathway, thereby conferring worm longevity. Though it remains unclear whether this is a distinct function from chromosomal segregation, bmk-1 is a potential new target for extension of lifespan and enhancement of healthspan.

With ever-increasing elder population, the high incidence of age-related diseases such as neurodegenerative disorders has turned out to be a huge public concern. Especially the elders and their families dreadfully suffer from the learning, behavioral and cognitive impairments. The lack of effective therapies for such a horrible symptom makes a great demanding for biological mechanism study for cognitive aging. Epigenetics is an emerging field that broadens the dimensions of mammalian genome blueprint. It is, unlike genetics, not only inheritable but also reversible. Recent studies suggest that DNA methylation, one of major epigenetic mechanisms, plays a pivotal role in the pathogenesis of age-related neurodegenerations and cognitive defects. In this review, the evolving knowledge of age-related cognitive functions and the potential DNA methylation mechanism of cognitive aging are discussed. That indicates the impairment of DNA methylation may be a crucial but reversible mechanism of behavioral and cognitive related neurodegeneration. The methods to examine the dynamics of DNA methylation patterns at tissue and single cell level and at the representative scale as well as the whole genome single base resolution are also briefly discussed. Importantly, the challenges of DNA methylation mechanism of cognitive aging research are brought up, and the possible solutions to tackle these difficulties are put forward.

Age-related cognitive deficit is a growing concern for the contemporary society; however, the understanding of mechanisms underlying mammalian cognition at the molecular, cellular, and organismal levels is still largely elusive. Accumulated evidence suggests that epigenetics, an emerging field encompassing mechanisms regulating gene expression without changing sequences of genomic DNAs, is of increasing relevance to age-related neurodegenerative symptoms. The purpose of this chapter is to review and discuss the cutting-edge knowledge about major achievements regarding epigenetic regulation by DNA methylation and histone modifications, and their significant impact on brain aging as well as cognitive impairment.

Ageing, a leading cause of the decline/deficits in human learning, memory, and cognitive abilities, is a major risk factor for age-associated neurodegenerative disorders such as Alzheimer’s disease. Emerging evidence suggests that epigenetics, an inheritable but reversible biochemical process, plays a crucial role in the pathogenesis of age-related neurological disorders. DNA methylation, the best-known epigenetic mark, has attracted most attention in this regard. DNA methyltransferases (DNMTs) are key enzymes in mediating the DNA methylation process, by which a methyl group is transferred, faithfully or anew, to genomic DNA sequences. Biologically, DNMTs are important for gene imprinting. Accumulating evidence suggests that DNMTs not only play critical roles, including gene imprinting and transcription regulation, in early development stages of the central nervous system (CNS), but also are indispensable in adult learning, memory, and cognition. Therefore, the impact of DNMTs and DNA methylation on age-associated cognitive functions and neurodegenerative diseases has emerged as a pivotal topic in the field. In this review, the effects of each DNMT on CNS development and healthy and pathological ageing are discussed.

Glucagon-like peptide-1 (GLP-1), released from gut endocrine L cells in response to glucose, regulates appetite, insulin secretion, and gut motility. How glucose given orally, but not systemically, induces GLP-1 secretion is unknown. We show that human duodenal L cells express sweet taste receptors, the taste G protein gustducin, and several other taste transduction elements. Mouse intestinal L cells also express alpha-gustducin. Ingestion of glucose by alpha-gustducin null mice revealed deficiencies in secretion of GLP-1 and the regulation of plasma insulin and glucose. Isolated small bowel and intestinal villi from alpha-gustducin null mice showed markedly defective GLP-1 secretion in response to glucose. The human L cell line NCI-H716 expresses alpha-gustducin, taste receptors, and several other taste signaling elements. GLP-1 release from NCI-H716 cells was promoted by sugars and the noncaloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for alpha-gustducin. We conclude that L cells of the gut "taste" glucose through the same mechanisms used by taste cells of the tongue. Modulating GLP-1 secretion in gut "taste cells" may provide an important treatment for obesity, diabetes and abnormal gut motility.

Telomeres are specialized structures at the ends of chromosomes that consist of tandem repeats of the DNA sequence TTAGGG and several proteins that protect the DNA and regulate the plasticity of the telomeres. The telomere-associated protein TRF2 (telomeric repeat binding factor 2) is critical for the control of telomere structure and function; TRF2 dysfunction results in the exposure of the telomere ends and activation of ATM (ataxia telangiectasin mutated)-mediated DNA damage response. Recent findings suggest that telomere attrition can cause senescence or apoptosis of mitotic cells, but the function of telomeres in differentiated neurons is unknown. Here, we examined the impact of telomere dysfunction via TRF2 inhibition in neurons (primary embryonic hippocampal neurons) and mitotic neural cells (astrocytes and neuroblastoma cells). We demonstrate that telomere dysfunction induced by adenovirus-mediated expression of dominant-negative TRF2 (DN-TRF2) triggers a DNA damage response involving the formation of nuclear foci containing phosphorylated histone H2AX and activated ATM in each cell type. In mitotic neural cells DN-TRF2 induced activation of both p53 and p21 and senescence (as indicated by an up-regulation of β-galactosidase). In contrast, in neurons DN-TRF2 increased p21, but neither p53 nor β-galactosidase was induced. In addition, TRF2 inhibition enhanced the morphological, molecular and biophysical differentiation of hippocampal neurons. These findings demonstrate divergent molecular and physiological responses to telomere dysfunction in mitotic neural cells and neurons, indicate a role for TRF2 in regulating neuronal differentiation, and suggest a potential therapeutic application of inhibition of TRF2 function in the treatment of neural tumors.

The causes of neuronal dysfunction and degeneration in Alzheimer's disease (AD) are not fully understood, but increased production of neurotoxic forms of amyloid beta-peptide-42 (Abeta42) seems of major importance. Large extracellular deposits of aggregated Abeta42 (plaques) is a diagnostic feature of AD, but Abeta42 may be particularly cytotoxic when it accumulates inside neurons. The factors that may promote the intracellular accumulation of Abeta42 in AD are unknown, but recent findings suggest that individuals with elevated homocysteine levels are at increased risk for AD. We show that homocysteic acid (HA), an oxidized metabolite of homocysteine, induces intraneuronal accumulation of a Abeta42 that is associated with cytotoxicity. The neurotoxicity of HA can be attenuated by an inhibitor of gamma-secretase, the enzyme activity that generates Abeta42, suggesting a key role for intracellular Abeta42 accumulation in the neurotoxic action of HA. Concentrations of HA in cerebrospinal fluid (CSF) were similar in AD and control subjects. CSF homocysteine levels were elevated significantly in AD patients, however, and homocysteine exacerbated HA-induced neurotoxicity, suggesting a role for HA in the pathogenic action of elevated homocysteine levels in AD. These findings suggest that the intracellular accumulation of Abeta42 plays a role in the neurotoxic action of HA, and suggest a potential therapeutic benefit of agents that modify the production and neurotoxic actions of HA and homocysteine.

Schistosoma japonicum causes schistosomiasis in humans and livestock in the Asia-Pacific region. Knowledge of the genome of this parasite should improve understanding of schistosome-host interactions, biomedical aspects of schistosomiasis and invertebrate evolution. We assigned 43,707 expressed sequence tags (ESTs) derived from adult S. japonicum and their eggs to 13,131 gene clusters. Of these, 35% shared no similarity with known genes and 75% had not been reported previously in schistosomes. Notably, S. japonicum encoded mammalian-like receptors for insulin, progesterone, cytokines and neuropeptides, suggesting that host hormones, or endogenous parasite homologs, could orchestrate schistosome development and maturation and that schistosomes modulate anti-parasite immune responses through inhibitors, molecular mimicry and other evasion strategies.

Components or downstream targets of many signaling pathways such as Insulin/IGF-1 and TOR, as well as genes involved in cellular metabolism and bioenergetics can extend worm lifespan 20% or more. The C. elegans gene pch-2 and its homologs, including TRIP13 in humans, have been studied for their functions in cell mitosis and meiosis, but have never been implicated in lifespan regulation. Here we show that over-expression of TRIP13 in human fibroblasts confers resistance to environmental stressors such as UV radiation and oxidative stress. Furthermore, pch-2 overexpression in C. elegans extends worm lifespan, and enhances worm survival in response to various stressors. Conversely, reducing pch-2 expression with RNAi shortens worm lifespan. Additional genetic epistasis analysis indicates that the molecular mechanism of pch-2 in worm longevity is tied to functions of the sirtuin family, implying that pch-2 is another chromatin regulator for worm longevity. These findings suggest a novel function of the pch-2 gene involved in lifespan determination.

Understanding molecular mechanisms of skin ageing is critical for developing effective anti-ageing strategies. Recently, it has been suggested that epigenetics maybe be involved in tissue ageing and age-related diseases; however, the evidence regarding skin ageing has been very limited. We ran a pilot study in mouse skin to test whether DNA methyltransferases (Dnmts), DNA demethylases such as ten-eleven translocation enzymes (Tets) and DNA methylation of gene promoters change with age by quantitative RT-PCR and methylated DNA immunoprecipitation (MeDIP)-chip. We discovered that the expression of Dnmt3a, Dnmt3b and Tet2 declines significantly with skin ageing. The genome-wide DNA methylation analysis indicates that both hypermethylation and hypomethylation in promoters of genes are taken place. Functional category of those genes suggests that inhibition of cell proliferation and activation of immune response are important adaptations likely induced by skin ageing. These findings shed new light on epigenetic regulation of skin ageing.

The ten-eleven translocation family of proteins (Tet1/2/3, Tets) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can be further oxidized and repaired by thymine DNA glycosylase (TDG), to influence gene transcription in embryonic and adult tissues. However the mechanisms of how Tets and TDG levels are regulated are unknown. We show that miR-29 can directly regulate Tet1-3 and TDG mRNA levels through binding to their 3′UTRs. miR-29 mimic decreases global 5hmC levels, a hallmark of Tet activity. Moreover, the mRNA levels for Tet3 and TDG are inversely correlated with the levels of miR-29 in aged mouse aorta implying that aging may affect methylation patterns via miRNA. In summary, our data show that Tets and TDG are direct targets of miR-29 and unravel a novel regulatory role for this miRNA in epigenetic DNA demethylation pathways.

Because the olfactory system plays a major role in food consumption, and because 'food addiction' and associated morbidities have reached epidemic proportions, we tested the hypothesis that dietary energy restriction can modify adverse effects of cocaine on behavior and olfactory cellular and molecular plasticity. Mice maintained on an alternate day fasting (ADF) diet exhibited increased baseline locomotion and increased cocaine-sensitized locomotion during cocaine conditioning, despite no change in cocaine conditioned place preference, compared with mice fed ad libitum. Levels of dopamine and its metabolites in the olfactory bulb (OB) were suppressed in mice on the ADF diet compared with mice on the control diet, independent of acute or chronic cocaine treatment. The expression of several enzymes involved in dopamine metabolism including tyrosine hydroxylase, monoamine oxidases A and B, and catechol-O-methyltransferase were significantly reduced in OBs of mice on the ADF diet. Both acute and chronic administration of cocaine suppressed the production of new OB cells, and this effect of cocaine was attenuated in mice on the ADF diet. Cocaine administration to mice on the control diet resulted in up-regulation of OB genes involved in mitochondrial energy metabolism, synaptic plasticity, cellular stress responses, and calcium- and cAMP-mediated signaling, whereas multiple olfactory receptor genes were down-regulated by cocaine treatment. ADF abolished many of the effects of cocaine on OB gene expression. Our findings reveal that dietary energy intake modifies the neural substrates underlying some of the behavioral and physiological responses to repeated cocaine treatment, and also suggest novel roles for the olfactory system in addiction. The data further suggest that modification of dietary energy intake could provide a novel potential approach to addiction treatments.

Numb is an evolutionarily conserved protein that controls the differentiation of neuronal progenitor cells by unknown mechanisms. Here we report that the neural cells expressing Numb isoforms with short phosphotyrosine-binding (SPTB) domain undergo extensive neurite outgrowth, an effect that can be blocked by voltage-gated Ca 2+ channel (VGCC) inhibitor or by Ca 2+ chelator. In contrast, tyrosine kinase inhibitor, genistein, and selective receptor tyrosine kinase (TrkA) inhibitor, K252α did not affect SPTB Numb-mediated neurite outgrowth. MAP kinase inhibitor, PD98059 partially reduced SPTB Numb-mediated neurite outgrowth. Cells expressing SPTB Numbs exhibit increased whole-cell Ca 2+ current densities (ICa) which can be prevented by preincubation of either nifedipine or PD98095. Cells expressing LPTB Numbs expressed little ICa (density) and were not able to grow neurites. Our results indicate that Ca 2+ influx through VGCC may be required for SPTB Numb-mediated neurite outgrowth, suggesting that Numb promotes neuronal differentiation by a mechanism involving PTB domain-specific regulation of Ca 2+ influx and MAP kinase activation.

Leptin plays a pivotal role in the regulation of energy homeostasis and metabolism, primarily by acting on neurons in the hypothalamus that control food intake. However, leptin receptors are more widely expressed in the brain suggesting additional, as yet unknown, functions of leptin. Here we show that both embryonic and adult hippocampal neurons express leptin receptors coupled to activation of STAT3 and phosphatidylinositol 3-kinase-Akt signaling pathways. Leptin protects hippocampal neurons against cell death induced by neurotrophic factor withdrawal and excitotoxic and oxidative insults. The neuroprotective effect of leptin is antagonized by the JAK2-STAT3 inhibitor AG-490, STAT3 decoy DNA, and phosphatidylinositol 3-kinase/Akt inhibitors but not by an inhibitor of MAPK. Leptin induces the production of manganese superoxide dismutase and the anti-apoptotic protein Bcl-xL, and stabilizes mitochondrial membrane potential and lessens mitochondrial oxidative stress. Leptin receptor-deficient mice (db/db mice) are more vulnerable to seizure-induced hippocampal damage, and intraventricular administration of leptin protects neurons against seizures. By enhancing mitochondrial resistance to apoptosis and excitotoxicity, our findings suggest that leptin signaling serves a neurotrophic function in the developing and adult hippocampus.

We present the AGEMAP (Atlas of Gene Expression in Mouse Aging Project) gene expression database, which is a resource that catalogs changes in gene expression as a function of age in mice. The AGEMAP database includes expression changes for 8,932 genes in 16 tissues as a function of age. We found great heterogeneity in the amount of transcriptional changes with age in different tissues. Some tissues displayed large transcriptional differences in old mice, suggesting that these tissues may contribute strongly to organismal decline. Other tissues showed few or no changes in expression with age, indicating strong levels of homeostasis throughout life. Based on the pattern of age-related transcriptional changes, we found that tissues could be classified into one of three aging processes: (1) a pattern common to neural tissues, (2) a pattern for vascular tissues, and (3) a pattern for steroid-responsive tissues. We observed that different tissues age in a coordinated fashion in individual mice, such that certain mice exhibit rapid aging, whereas others exhibit slow aging for multiple tissues. Finally, we compared the transcriptional profiles for aging in mice to those from humans, flies, and worms. We found that genes involved in the electron transport chain show common age regulation in all four species, indicating that these genes may be exceptionally good markers of aging. However, we saw no overall correlation of age regulation between mice and humans, suggesting that aging processes in mice and humans may be fundamentally different., Author Summary This work studies the aging process in mice using DNA microarrays to identify genes that change expression during aging. The entire set of age-regulated genes constitutes a transcriptional profile that can be used to measure ages of different individuals. Furthermore, the aging expression profile highlights genetic and metabolic pathways that change with age, providing key insights about possible molecular changes that may contribute to cell senescence and physiological decline. This aging study is massive in scope, involving gene expression measurements from 16 different tissues at four different ages. Expression data for genes of interest can be queried over the web by researchers interested in aging. Different tissues were found to have strikingly different levels of age-related change, and could be divided into three groups based on their patterns of aging: a neural group, a vascular group, and a steroid-responsive group. The electron transport chain pathway stands out because it is the only genetic pathway that shows a similar pattern of age-related change in mice, humans, worms, and flies. However, there is little overall similarity between changes in gene expression during aging of humans and mice, consistent with evolutionary theories suggesting that aging lies outside the force of natural selection.

The innate immune system senses the invasion of pathogenic microorganisms and tissue injury through Toll-like receptors (TLR), a mechanism thought to be limited to immune cells. We now report that neurons express several TLRs, and that the levels of TLR2 and -4 are increased in neurons in response to IFN-γ stimulation and energy deprivation. Neurons from both TLR2 knockout and -4 mutant mice were protected against energy deprivation-induced cell death, which was associated with decreased activation of a proapoptotic signaling cascade involving jun N-terminal kinase and the transcription factor AP-1. TLR2 and -4 expression was increased in cerebral cortical neurons in response to ischemia/reperfusion injury, and the amount of brain damage and neurological deficits caused by a stroke were significantly less in mice deficient in TLR2 or -4 compared with WT control mice. Our findings establish a proapoptotic signaling pathway for TLR2 and -4 in neurons that may render them vulnerable to ischemic death.

We have examined the gene structure and regulatory regions of octamer-binding transcription factor 3/4 (Oct 3/4), sex determining region Y box 2 (Sox2), signal transducer and activator of transcription 3 (Stat3), embryonal stem cell-specific gene 1 (ESG), Nanog homeobox (Nanog), and several other genes highly expressed in embryonic stem (ES) cells across different species. Our analysis showed that ES cell-expressed Ras (ERAS) was orthologous to a human pseudogene Harvey Ras (HRASP) and that the promoter and other regulatory sequences were highly divergent. No ortholog of (ES) cell-derived homeobox containing gene (Ehox) could be identified in human, and the closest paralogs PEPP gene subfamily 1 (PEPP1), PEPP2, and extraembryonic, spermatogenesis, homeobox 1 (Esx1) were not expressed by ES cells and shared little homology. The Sox2 promoter was the most conserved across species and the Oct3/4 promoter region showed significant homology particularly in the distal enhancer active in ES cells. Analysis suggested common and divergent pathways of regulation. Conserved Oct3/4 and Sox2 co-binding domains were identified in most ES expressed genes, highlighting the importance of this transcriptional pathway. Conserved fibroblast growth factor response element sites were identified in regulatory regions, suggesting a potential parallel pathway for regulation by FGFs. A central role of Stat3 activation in self-renewal and in a regulatory feedback loop was suggested by the identification of the conserved binding sites in most pathways. Although most pathways were evolutionarily conserved, promoters and genomic structure of the leukemia inhibitory factor (LIF) pathway components were divergent, likely explaining the differential requirement of LIF for human and rodent cells. Our analysis further suggested that the Nanog regulatory pathway was relatively independent of the LIF/Oct pathway and may interact with the Nodal/transforming growth factor-beta pathway. These results provide a framework for examining the current reported differences between rodent and human ES cells and define targets for future perturbation studies.

Human hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. In this work, we report on a comprehensive characterization of gene expression profiles of hepatitis B virus-positive HCC through the generation of a large set of 5′-read expressed sequence tag (EST) clusters (11,065 in total) from HCC and noncancerous liver samples, which then were applied to a cDNA microarray system containing 12,393 genes/ESTs and to comparison with a public database. The commercial cDNA microarray, which contains 1,176 known genes related to oncogenesis, was used also for profiling gene expression. Integrated data from the above approaches identified 2,253 genes/ESTs as candidates with differential expression. A number of genes related to oncogenesis and hepatic function/differentiation were selected for further semiquantitative reverse transcriptase–PCR analysis in 29 paired HCC/noncancerous liver samples. Many genes involved in cell cycle regulation such as cyclins, cyclin-dependent kinases, and cell cycle negative regulators were deregulated in most patients with HCC. Aberrant expression of the Wnt-β-catenin pathway and enzymes for DNA replication also could contribute to the pathogenesis of HCC. The alteration of transcription levels was noted in a large number of genes implicated in metabolism, whereas a profile change of others might represent a status of dedifferentiation of the malignant hepatocytes, both considered as potential markers of diagnostic value. Notably, the altered transcriptome profiles in HCC could be correlated to a number of chromosome regions with amplification or loss of heterozygosity, providing one of the underlying causes of the transcription anomaly of HCC.

Three hundred cDNAs containing putatively entire open reading frames (ORFs) for previously undefined genes were obtained from CD34+ hematopoietic stem/progenitor cells (HSPCs), based on EST cataloging, clone sequencing, in silico cloning, and rapid amplification of cDNA ends (RACE). The cDNA sizes ranged from 360 to 3496 bp and their ORFs coded for peptides of 58–752 amino acids. Public database search indicated that 225 cDNAs exhibited sequence similarities to genes identified across a variety of species. Homology analysis led to the recognition of 50 basic structural motifs/domains among these cDNAs. Genomic exon–intron organization could be established in 243 genes by integration of cDNA data with genome sequence information. Interestingly, a new gene named as HSPC070 on 3p was found to share a sequence of 105bp in 3′ UTR with RAF gene in reversed transcription orientation. Chromosomal localizations were obtained using electronic mapping for 192 genes and with radiation hybrid (RH) for 38 genes. Macroarray technique was applied to screen the gene expression patterns in five hematopoietic cell lines (NB4, HL60, U937, K562, and Jurkat) and a number of genes with differential expression were found. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the function of genes involved in hematopoietic development and differentiation.[The sequence data described in this paper have been submitted to the GenBank data library under the accession nos. listed in Table 1, pp 1548–1552.]

The transcriptional profiles of five regions of the central nervous system (CNS) of mice varying in age, gender and dietary intake were measured by microarray. The resulting data provide insights into the mechanisms of age-, diet- and gender-related CNS plasticity and vulnerability in mammals., Background The structural and functional complexity of the mammalian central nervous system (CNS) is organized and modified by complicated molecular signaling processes that are poorly understood. Results We measured transcripts of 16,896 genes in 5 CNS regions from cohorts of young, middle-aged and old male and female mice that had been maintained on either a control diet or a low energy diet known to retard aging. Each CNS region (cerebral cortex, hippocampus, striatum, cerebellum and spinal cord) possessed its own unique transcriptome fingerprint that was independent of age, gender and energy intake. Less than 10% of genes were significantly affected by age, diet or gender, with most of these changes occurring between middle and old age. The transcriptome of the spinal cord was the most responsive to age, diet and gender, while the striatal transcriptome was the least responsive. Gender and energy restriction had particularly robust influences on the hippocampal transcriptome of middle-aged mice. Prominent functional groups of age- and energy-sensitive genes were those encoding proteins involved in DNA damage responses (Werner and telomere-associated proteins), mitochondrial and proteasome functions, cell fate determination (Wnt and Notch signaling) and synaptic vesicle trafficking. Conclusion Mouse CNS transcriptomes responded to age, energy intake and gender in a regionally distinctive manner. The systematic transcriptome dataset also provides a window into mechanisms of age-, diet- and sex-related CNS plasticity and vulnerability.

BackgroundA unique and essential property of embryonic stem cells is the ability to self-renew and differentiate into multiple cell lineages. However, the possible differences in proliferation and differentiation capabilities among independently-derived human embryonic stem cells (hESCs) are not well known because of insufficient characterization. To address this question, a side-by-side comparison of 1) the ability to maintain an undifferentiated state and to self-renew under standard conditions; 2) the ability to spontaneously differentiate into three primary embryonic germ lineages in differentiating embryoid bodies; and 3) the responses to directed neural differentiation was made between three NIH registered hES cell lines I3 (TE03), I6 (TE06) and BG01V. Lines I3 and I6 possess normal XX and a normal XY karyotype while BG01V is a variant cell line with an abnormal karyotype derived from the karyotypically normal cell line BG01.ResultsUsing immunocytochemistry, flow cytometry, qRT-PCR and MPSS, we found that all three cell lines actively proliferated and expressed similar "stemness" markers including transcription factors POU5F1/Oct3/4 and NANOG, glycolipids SSEA4 and TRA-1-81, and alkaline phosphatase activity. All cell lines differentiated into three embryonic germ lineages in embryoid bodies and into neural cell lineages when cultured in neural differentiation medium. However, a profound variation in colony morphology, growth rate, BrdU incorporation, and relative abundance of gene expression in undifferentiated and differentiated states of the cell lines was observed. Undifferentiated I3 cells grew significantly slower but their differentiation potential was greater than I6 and BG01V. Under the same neural differentiation-promoting conditions, the ability of each cell line to differentiate into neural progenitors varied.ConclusionOur comparative analysis provides further evidence for similarities and differences between three hESC lines in self-renewal, and spontaneous and directed differentiation. These differences may be associated with inherited variation in the sex, stage, quality and genetic background of embryos used for hESC line derivation, and/or changes acquired during passaging in culture.