The functional genomic studies of resveratrol in respect to its anti-cancer effects

Resveratrol has anti-cancer effects in vitro, and hypothetical chemopreventive effects in vivo. Effects are pleiotropic, mediated by changes in expression of many genes and epigenetic reprogramming. Thus, they are well suited for functional genomic studies. We carried out systematic review of such studies (reflecting also on technological progress). Differentially expressed genes commonly linked to resveratrol treatment were linked to cell cycle, proliferation, and apoptosis. However, it is unclear if these are primary and specific targets of resveratrol. We conclude by discussing areas where additional functional genomic studies are desirable, including experiments that better model in vivo effects of dietary intake.     

Functional genomics in the post-genome era

As the biomedical research community enters the post-genome era, studying gene expression patterns and phenotypes in model organisms will be an important part of analyzing the role of genes in human health and disease. New technologies involving DNA chips will improve the ability to evaluate the differential expression of a large number of genes simultaneously. Also, new approaches for generating mutations in mice will significantly decrease the cost and increase the rate of generating mutant lines that model human disease.     

Defining the identity of mouse embryonic dermal fibroblasts

Embryonic dermal fibroblasts in the skin have the exceptional ability to initiate hair follicle morphogenesis and contribute to scarless wound healing. Activation of the Wnt signaling pathway is critical for dermal fibroblast fate selection and hair follicle induction. In humans, mutations in Wnt pathway components and target genes lead to congenital focal dermal hypoplasias with diminished hair. The gene expression signature of embryonic dermal fibroblasts during differentiation and its dependence on Wnt signaling is unknown. Here we applied Shannon entropy analysis to identify the gene expression signature of mouse embryonic dermal fibroblasts. We used available human DNase‐seq and histone modification ChiP‐seq data on various cell‐types to demonstrate that genes in the fibroblast cell identity signature can be epigenetically repressed in other cell‐types. We found a subset of the signature genes whose expression is dependent on Wnt/β‐catenin activity in vivo. With our approach, we have defined and validated a statistically derived gene expression signature that may mediate dermal fibroblast identity and function in development and disease. genesis 54:415–430, 2016. © 2016 Wiley Periodicals, Inc.     

High-throughput screening of soluble recombinant proteins

The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure-function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well-expressed soluble fusion protein, total cell lysates of bacteria culture ( approximately 1.5 mL) were subjected to high-speed centrifugation in a 96-tube format and analyzed by multiwell denaturing SDS-PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis.     

Dynamical analysis of gene networks requires both mRNA and protein expression information

One of the important goals of biology is to understand the relationship between DNA sequence information and nonlinear cellular responses. This relationship is central to the ability to effectively engineer cellular phenotypes, pathways, and characteristics. Expression arrays for monitoring total gene expression based on mRNA can provide quantitative insight into which gene or genes are on or off; but this information is insufficient to fully predict dynamic biological phenomena. Using nonlinear stability analysis we show that a combination of gene expression information at the message level and at the protein level is required to describe even simple models of gene networks. To help illustrate the need for such information we consider a mechanistic model for circadian rhythmicity which shows agreement with experimental observations when protein and mRNA information are included and we propose a framework for acquiring and analyzing experimental and mathematically derived information about gene networks.     

Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO

Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green fluorescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar KM values, but SUMO protease had a 25-fold higher kcat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.     

A less laborious approach to the high-throughput production of recombinant proteins in Escherichia coli using 2-liter plastic bottles

Contemporary approaches to biology often call for the high-throughput production of large amounts of numerous proteins for structural or functional studies. Even with the highly efficient protein expression systems developed in Escherichia coli, production of these proteins is laborious and time-consuming. We have simplified established protocols by the use of disposable culture vessels: common 2-liter polyethylene terephthalate beverage bottles. The bottles are inexpensive, fit conveniently in commonly available flask holders, and, because they are notched, provide sufficient aeration to support the growth of high-density cultures. The use of antibiotics and freshly prepared media alleviates the need for sterilization of media and significantly reduces the labor involved. Uninoculated controls exhibited no growth during the time required for protein expression in experimental cultures. The yield, solubility, activity, and pattern of crystallization of proteins expressed in bottles were comparable to those obtained under conventional culture conditions. After use, the bottles are discarded, reducing the risk of cross-contamination of subsequent cultures. The approach appears to be suitable for high-throughput production of proteins for structural or functional studies.     

SEX-LINKAGE OF SEXUALLY ANTAGONISTIC GENES IS PREDICTED BY FEMALE, BUT NOT MALE, EFFECTS IN BIRDS

Evolutionary theory predicts that sexually antagonistic loci will be preferentially sex-linked, and this association can be empirically testes with data on sex-biased gene expression with the assumption that sex-biased gene expression represents the resolution of past sexual antagonism. However, incomplete dosage compensating mechanisms and meiotic sex chromosome inactivation have hampered efforts to connect expression data to theoretical predictions regarding the genomic distribution of sexually antagonistic loci in a variety of animals. Here we use data on the underlying regulatory mechanism that produce expression sex-bias to test the genomic distribution of sexually antagonistic genes in chicken. Using this approach, which is free from problems associated with the lack of dosage compensation in birds, we show that female-detriment genes are significantly overrepresented on the Z chromosome, and female-benefit genes underrepresented. By contrast, male-effect genes show no over- or underrepresentation on the Z chromosome. These data are consistent with a dominant mode of inheritance for sexually antagonistic genes, in which male-benefit coding mutations are more likely to be fixed on the Z due to stronger male-specific selective pressures. After fixation of male-benefit alleles, regulatory changes in females evolve to minimize antagonism by reducing female expression.     

Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice

The adult brain contains small populations of neural precursor cells (NPC) that can give rise to new neurons and glia, and may play important roles in learning and memory, and recovery from injury. Growth factors can influence the proliferation, differentiation and survival of NPC, and may mediate responses of NPC to injury and environmental stimuli such as enriched environments and physical activity. We now report that neurotrophin expression and neurogenesis can be modified by a change in diet. When adult mice are maintained on a dietary restriction (DR) feeding regimen, numbers of newly generated cells in the dentate gyrus of the hippocampus are increased, apparently as the result of increased cell survival. The new cells exhibit phenotypes of neurons and astrocytes. Levels of expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are increased by DR, while levels of expression of high-affinity receptors for these neurotrophins (trkB and trkC) are unchanged. In addition, DR increases the ratio of full-length trkB to truncated trkB in the hippocampus. The ability of a change in diet to stimulate neurotrophin expression and enhance neurogenesis has important implications for dietary modification of neuroplasticity and responses of the brain to injury and disease.     

A roadmap for high‐throughput sequencing studies of wild animal populations using noninvasive samples and hybridization capture

Large‐scale genomic studies of wild animal populations are often limited by access to high‐quality DNA. Although noninvasive samples, such as faeces, can be readily collected, DNA from the sample producers is usually present in low quantities, fragmented, and contaminated by microorganism and dietary DNAs. Hybridization capture can help to overcome these impediments by increasing the proportion of subject DNA prior to high‐throughput sequencing. Here we evaluate a key design variable for hybridization capture, the number of rounds of capture, by testing whether one or two rounds are most appropriate, given varying sample quality (as measured by the ratios of subject to total DNA). We used a set of 1,780 quality‐assessed wild chimpanzee (Pan troglodytes schweinfurthii) faecal samples and chose 110 samples of varying quality for exome capture and sequencing. We used multiple regression to assess the effects of the ratio of subject to total DNA (sample quality), rounds of capture and sequencing effort on the number of unique exome reads sequenced. We not only show that one round of capture is preferable when the proportion of subject DNA in a sample is above ~2%–3%, but also explore various types of bias introduced by capture, and develop a model that predicts the sequencing effort necessary for a desired data yield from samples of a given quality. Thus, our results provide a useful guide and pave a methodological way forward for researchers wishing to plan similar hybridization capture studies.     

Gene expression patterns associated with caste and reproductive status in ants: worker‐specific genes are more derived than queen‐specific ones

Variation in gene expression leads to phenotypic diversity and plays a central role in caste differentiation of eusocial insect species. In social Hymenoptera, females with the same genetic background can develop into queens or workers, which are characterized by divergent morphologies, behaviours and lifespan. Moreover, many social insects exhibit behaviourally distinct worker castes, such as brood‐tenders and foragers. Researchers have just started to explore which genes are differentially expressed to achieve this remarkable phenotypic plasticity. Although the queen is normally the only reproductive individual in the nest, following her removal, young brood‐tending workers often develop ovaries and start to reproduce. Here, we make use of this ability in the ant Temnothorax longispinosus and compare gene expression patterns in the queens and three worker castes along a reproductive gradient. We found the largest expression differences between the queen and the worker castes (~2500 genes) and the smallest differences between infertile brood‐tenders and foragers (~300 genes). The expression profile of fertile workers is more worker‐like, but to a certain extent intermediate between the queen and the infertile worker castes. In contrast to the queen, a high number of differentially expressed genes in the worker castes are of unknown function, pointing to the derived status of hymenopteran workers within insects.     

Interspecific Chromosome-Wide Transcription Profiles Reveal the Existence of Mammalian-Specific and Species-Specific Chromosome Domains

A long-range exploration of expression levels through wide chromosome territories was carried out in three species (pig, cattle, and chicken) by aligning EST counts against the human genome. This strategy made it possible to produce expression profiles that were very similar between pig and cattle and that were significantly correlated with chicken levels of expression. In parallel with these alignments, we developed a statistical approach enabling us to screen genomic regions for both underexpression and overexpression at the chromosome level within a given species, as well as interspecifically. The observed correlations are indicative of the existence of interspecifically conserved domains of gene expression, not only for housekeeping genes (which are highly expressed), but also for regions where genes are significantly underexpressed. Furthermore, our strategy made it possible to point out regions that are differentially regulated between species. These expression data were crossed with available comparative mapping information for pigs and cattle, suggesting that coregulated regions are syntenic in various mammals.     

Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale

Structural genomics requires the application of a standardised process for overexpression of soluble proteins that allows high-throughput purification and analysis of protein products. We have developed a highly parallel approach to protein expression, including the simultaneous expression screening of a large number of cDNA clones in an appropriate vector system and the use of a protease-deficient host strain. A set of 221 human genes coding for proteins of various sizes with unknown structures was selected to evaluate the system. We transferred the cDNAs from an E. coli vector to the yeast expression vector by recombinational cloning, avoiding time-consuming recloning steps and the use of restriction enzymes in the cloning process. The subcloning yield was 95%, provided that a PCR fragment of the correct size could be obtained. Sixty percent of these proteins were expressed as soluble products at detectable levels and 48% were successfully purified under native conditions using the His₆ tag fusion.The advantages of the developed yeast-based expression system are the ease of manipulation and cultivation of S. cerevisiae in the same way as with prokaryotic hosts and the ability to introduce post-translational modifications of proteins if required, thus being an attractive system for heterologous expression of mammalian proteins. The expression clones selected in this screening process are passed on to the fermentation process in order to provide milligram amounts of proteins for structure analysis within the Berlin Protein Structure Factory. All data generated is stored in a relational database and is available on our website(http://www.proteinstrukturfabrik.de).     

Inhibition of p38 MAPK during cellular activation modulate gene expression of head kidney leukocytes isolated from Atlantic salmon (Salmo salar) fed soy bean oil or fish oil based diets

Head kidney leukocytes isolated from Atlantic salmon fed either a diet based on fish oil (FO) or soy bean oil (VO) were used in order to evaluate if different lipid sources could contribute to cellular activation of the salmon innate immune system. A specific inhibitor of p38 MAPK, SB202190, was used to investigate the effect of lipopolysaccharide (LPS) signalling in the head kidney leukocytes. The results show that LPS up regulate IL-1β, TNF-α, Cox2 expression in leukocytes isolated from fish fed either diet. The p38 MAPK inhibitor, SB202190, reduced the LPS induced expression of these genes in both dietary groups. In LPS stimulated leukocytes isolated from VO fed fish, SB202190 showed a clear dose dependent inhibitory effect on IL-1β, TNF-α and Cox2 expression. This effect was also observed for Cox2 in leukocytes isolated from FO fed fish. Furthermore, there was a stronger mean induction of Cox2 in LPS stimulated leucocytes isolated from the VO-group compared to LPS stimulated leukocytes isolated from the FO-group. In both dietary groups, LPS stimulation of salmon head kidney leukocytes increased the induction of CD83, a dendrite cell marker, while the inhibitor reduced CD83 expression in the VO fed fish only. The inhibitor also clearly reduced hsp27 expression in VO fed fish. Indicating a p38 MAPK feedback loop, LPS significantly inhibited the expression of p38MAPK itself in both diets, while SB202190 increased p38MAPK expression especially in the VO diet group. hsp70 expression was not affected by any treatment or feed composition. There were also differences in p38MAPK protein phosphorylation comparing treatment groups but no obvious difference comparing the two dietary groups. The results indicate that dietary fatty acids have the ability to modify signalling through p38 MAPK which may have consequences for the fish's ability to handle infections and stress. Signalling through p38MAPK is ligand dependent and affects gene and protein expression differently.     

植物功能基因组学研究进展

植物功能基因组学是从整体水平研究基因的功能及表达规律的科学。对植物功能基因组学的研究将助于我们对基因功能的理解和对植物性状的定性改造和利用。本文简要介绍了植物功能基因组学的概念、研究方法和最新研究进展。     

Can dietary intake of phase 2 protein inducers affect the rising epidemic of diseases such as type 2 diabetes?

I propose that those populations with a long history of agriculture, particularly where survival was dependent upon relatively few food types, were selected for resistance to diseases such as type 2 diabetes. The basis for this resistance is hypothesized to be a higher basal level of ability to scavenge strong oxidants than found in populations whose recent ancestors depended to a great extent on gathering for their subsistence. Specifically, I hypothesize that selective pressures on populations whose ancestors ate a very restricted diet resulted in the development of populations with high basal levels of phase 2 protein gene expression; a consequence of which is that their tissues have a greater ability to cope with oxidative stress. In contrast, populations with a recent ancestry of hunting and gathering are more dependent upon the consumption of dietary phase 2 protein inducers for adequate phase 2 gene expression necessary for maintaining their tissues' abilities to cope with oxidative stress. This hypothesis can be readily tested by examining basal and induced phase 2 protein gene expression in umbilical cord fibroblasts, obtained from infants of different genetic backgrounds, grown in cell culture.     

Effects of dietary lipid levels on mitochondrial gene expression in low and high‐feed efficient families of rainbow trout Oncorhynchus mykiss

A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on mitochondrial gene expression in mixed sex rainbow trout Oncorhynchus mykiss. Practical diets with a fixed crude protein content of 42%, formulated to contain 10% (42/10), 20% (42/20) and 30% (42/30) dietary lipid, were fed to triplicate groups of either low‐feed efficient (F129; mean ± s.d. = 105·67 ± 3·04 g initial average mass) or high‐feed efficient (F134; mean ± s.d. = 97·86 ± 4·02 g) families of fish, to apparent satiety, twice per day, for 108 days. At the end of the experiment, diets 42/20 and 42/30 led to similar fish condition factors, which were higher than that observed with diet 42/10 (P < 0·05). F134 fish fed diet 42/10 showed the highest hepato‐somatic index, while there was no significant difference among all the other treatments (P < 0·05). When the group of F134 fish fed diet 42/10 was used as the calibrator for gene expression analysis, the five genes selected for their involvement in lipid metabolism (complex I‐nd1, complex III‐cytb, complex IV‐cox1, complex IV‐cox2 and complex V‐atp6) were up‐regulated in the muscle and down‐regulated in both the liver and the intestine. There was a significant family × diet interaction regarding nd1, cox2 and atp6 in the liver; nd1, cytb, cox1, cox2 and atp6 in the intestine, and nd1, cytb, cox1, cox2 and atp6 in the muscle (P < 0·05). The overall results of this study constitute basic information for the understanding of molecular mechanisms of lipid metabolism at the mitochondrial level in fishes.