PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (1260851)

Clipboard (0)
None

Related Articles

1.  Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling 
Over 700 direct transcriptional targets of the dFOXO transcription factor are identified in the adult fruit fly. dFOXO-bound genes are conserved between worm and fly, but dFOXO is not the sole mediator of the transcriptional response to changes in insulin signalling in the fly.
More than 700 direct transcriptional targets of dFOXO were determined in the adult Drosophila female, in the wild-type or an insulin-signalling mutant.dFOXO has an important role in the wild-type fly and is important for transcription of numerous signalling components including TOR and Sos.While dFOXO is an important effector of the insulin signalling pathway, it is required for only a portion of the transcriptional changes that occur in response to alterations in the pathway in the fly, and in many cases indirectly.There is strong evolutionary conservation of dFOXO-bound genes between the worm and the fly, specifically enriched in regulatory genes.
Forkhead Box-O (FoxO) transcription factors are crucial players in numerous cellular and organismal processes including metabolism, stress protection, cellular differentiation, cell-cycle arrest, apoptosis and lifespan. FoxOs are regulated by a number of signalling pathways, including negative regulation by insulin/insulin-like growth factor signalling (IIS) (Partridge and Bruning, 2008; Salih and Brunet, 2008). The fruit fly Drosophila melanogaster has a single FoxO orthologue—dFOXO. dFOXO is capable of extending fly lifespan, as well as being required for lifespan extension in response to downregulation of IIS (Giannakou et al, 2004; Hwangbo et al, 2004; Slack et al, 2011). To further our understanding of dFOXO biology, we uncover over 700 direct dFOXO targets in the adult female fly, both in the wild-type fly and in a mutant with reduced IIS activity.
dFOXO is directly required for transcription of several components of IIS and interacting pathways, such as the gene encoding the target of rapamycin (TOR) kinase, in the wild-type fly. Indeed, the removal of dFOXO results in reduced signal through these pathways. The genomic locations occupied by dFOXO in adults are different from those observed by others in larvae or cultured cells (Puig et al, 2003; Teleman et al, 2008), indicating that dFOXO binding is influenced by developmental stage and/or cell type. These locations remain unchanged upon activation of dFOXO by stresses or reduced IIS in the adult, but the binding of dFOXO to the same sites is increased. Genetically induced reduction in IIS results in activation/repression of a greater number of direct dFOXO targets than observed in the wild-type fly.
To determine the relationship between dFOXO and IIS in the adult fly, we identify the part of the IIS transcriptional response that is controlled by dFOXO, both directly and indirectly. We observe that aspects of the transcriptional response to changes in IIS can take place in the absence of dFOXO, indicating that other transcriptional regulators must be involved. This is different to the situation in the worm Caenorhabditis elegans where the worm FoxO, encoded by the daf-16 gene, is required for all the effects of a reduction in IIS (Kenyon et al, 1993; Gems et al, 1998; Murphy et al, 2003). On the other hand, the existence of dFOXO-independent effects is in accordance with genetic experiments in the fly where lifespan extension and xenobiotic resistance caused by a reduction in IIS are dependent on dFOXO, while lowered fecundity and body size, delayed development and resistance to paraquat are not (Slack et al, 2011). Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the dFOXO-independent effects in the fly.
Despite the different topology of the transcriptomic response to IIS changes in the two organisms, there is genome-wide evolutionary conservation of dFOXO targets between the fly and the worm (Figure 9), enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.
FoxO transcription factors, inhibited by insulin/insulin-like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, we uncover over 700 direct dFOXO targets in adult female Drosophila. dFOXO is directly required for transcription of several IIS components and interacting pathways, such as TOR, in the wild-type fly. The genomic locations occupied by dFOXO in adults are different from those observed in larvae or cultured cells. These locations remain unchanged upon activation by stresses or reduced IIS, but the binding is increased and additional targets activated upon genetic reduction in IIS. We identify the part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects and show that parts of the transcriptional response to IIS reduction do not require dfoxo. Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the indirect and dfoxo-independent effects. We demonstrate genome-wide evolutionary conservation of dFOXO targets between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.
doi:10.1038/msb.2011.36
PMCID: PMC3159968  PMID: 21694719
dFOXO; Drosophila; insulin/insulin-like growth factor signalling; transcription
2.  Schistosomiasis Mansoni: Novel Chemotherapy Using a Cysteine Protease Inhibitor 
PLoS Medicine  2007;4(1):e14.
Background
Schistosomiasis is a chronic, debilitating parasitic disease infecting more than 200 million people and is second only to malaria in terms of public health importance. Due to the lack of a vaccine, patient therapy is heavily reliant on chemotherapy with praziquantel as the World Health Organization–recommended drug, but concerns over drug resistance encourage the search for new drug leads.
Methods and Findings
The efficacy of the vinyl sulfone cysteine protease inhibitor K11777 was tested in the murine model of schistosomiasis mansoni. Disease parameters measured were worm and egg burdens, and organ pathology including hepato- and splenomegaly, presence of parasite egg–induced granulomas in the liver, and levels of circulating alanine aminotransferase activity as a marker of hepatocellular function. K11777 (25 mg/kg twice daily [BID]), administered intraperitoneally at the time of parasite migration through the skin and lungs (days 1–14 postinfection [p.i.]), resulted in parasitologic cure (elimination of parasite eggs) in five of seven cases and a resolution of other disease parameters. K11777 (50 mg/kg BID), administered at the commencement of egg-laying by mature parasites (days 30–37 p.i.), reduced worm and egg burdens, and ameliorated organ pathology. Using protease class-specific substrates and active-site labeling, one molecular target of K11777 was identified as the gut-associated cathepsin B1 cysteine protease, although other cysteine protease targets are not excluded. In rodents, dogs, and primates, K11777 is nonmutagenic with satisfactory safety and pharmacokinetic profiles.
Conclusions
The significant reduction in parasite burden and pathology by this vinyl sulfone cysteine protease inhibitor validates schistosome cysteine proteases as drug targets and offers the potential of a new direction for chemotherapy of human schistosomiasis.
A significant reduction in parasite burden and pathology by a vinyl sulfone cysteine protease inhibitor suggests a new direction for chemotherapy of human schistosomiasis.
Editors' Summary
Background.
Schistosomiasis, a disease caused by a type of parasitic flatworm that lives in the blood, infects around 200 million people worldwide. The disease is a serious problem in sub-Saharan Africa, South America, China, and southeast Asia. Although this disease can kill, it is better known as a lifelong chronic infection with debilitating symptoms mainly due to an immune reaction raised against parasite eggs trapped in the liver, spleen, and gut. The worm's life cycle is complicated and involves a free-swimming form that emerges from certain types of snails that live in lakes and ponds. This can penetrate the skin of people in contact with the water. After a period spent in the skin and around the lungs, the parasites move to veins around the gut, and develop into adult worms that mate and lay eggs. These eggs eventually return to the water through the person's feces or urine. A particular group of proteins called cysteine proteases are thought to be very important in the biology of these worms, especially in their function as digestive enzymes in the parasite's gut. These proteases could represent an exciting opportunity for development of new drugs to treat schistosomiasis. The researchers are looking at whether it is possible to block the activity of cysteine proteases and, as a result, kill the worms or prevent them from developing and thriving.
Why Was This Study Done?
At the moment there is only one drug, praziquantel, in common use for treatment of schistosomiasis; it is cheap and effective. However many organizations are worried about relying on a single drug to treat a serious disease which affects so many people worldwide. The research group here has been looking at molecules that block cysteine protease activity, to see if any of these could be good drug candidates for schistosomiasis. One molecule they have been looking at goes by the name of K11777, which is under evaluation as a drug candidate for another parasitic infection (Chagas' disease). Here, the researchers wanted to find out whether K11777 had any activity against schistosome worms.
What Did the Researchers Do and Find?
In this study, the researchers deliberately infected laboratory mice with the schistosome parasite. These mice were then either injected with K11777 solution twice daily, or with equivalent volumes of water as a comparison. The researchers examined the effects of injecting K11777 either “early” in infection (using a 14 day course, starting 1 day after infection with the parasite) or “late” in the worms' development (using an 8 day treatment course starting 30 days after infection). The outcomes used as measures of success of treatment with K11777 included the number of worms recovered from mice after euthanasia, the number of worm eggs counted in the liver; the extent of the damage to the liver; and finally, the researchers also looked at activity levels of cysteine proteases in the worms themselves, in particular, those proteases associated with the parasite gut.
The results of the early-treatment experiment showed a substantial decrease in worm numbers and egg production. In five of the seven mice treated, eggs were eliminated entirely. Also, there was little measurable liver damage. For the late-treatment experiment, decreased burdens of worms and eggs in the livers of K11777 treated mice were also found, and there was less damage to the livers. Those worms surviving treatment and removed from mice also had much less activity of gut cysteine proteases suggesting that K11777 exerts its effects by targeting worm cysteine proteases.
What Do These Findings Mean?
These experiments show that K11777 is a potent antischistosomal agent in mice. It might therefore be a good ‘candidate' molecule for developing future treatments for human schistosomiasis. However, before that stage can be reached, it would be important to carry out clinical trials to test whether K11777 is both safe and effective in schistosomiasis patients. Full details as to which worm cysteine protease(s) is the critical target of K11777 would also need to be worked out, and more information would be needed as to whether the dosing plan used in this study (twice-daily injections for a week to 14 days) can be decreased.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040014.
World Health Organization pages about schistosomiasis including links to details on further research into the disease
Information from the US Centers for Disease Control for patients and health professionals about schistosomiasis
Wikipedia pages on schistosomiasis (Wikipedia is an internet encyclopedia anyone can edit)
PLoS Neglected Tropical Diseases is a new journal from the Public Library of Science that is devoted to publishing research on the world's most neglected tropical diseases, including schistosomiasis
doi:10.1371/journal.pmed.0040014
PMCID: PMC1764436  PMID: 17214506
3.  Genomic Treasure Troves: Complete Genome Sequencing of Herbarium and Insect Museum Specimens 
PLoS ONE  2013;8(7):e69189.
Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22–82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4–97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2–71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.
doi:10.1371/journal.pone.0069189
PMCID: PMC3726723  PMID: 23922691
4.  The rarity of gene shuffling in conserved genes 
Genome Biology  2005;6(6):R50.
The incidence of gene shuffling is estimated in conserved genes in 10 organisms from the three domains of life. Successful gene shuffling is found to be very rare among such conserved genes. This suggests that gene shuffling may not be a major force in reshaping the core genomes of eukaryotes.
Background
Among three sources of evolutionary innovation in gene function - point mutations, gene duplications, and gene shuffling (recombination between dissimilar genes) - gene shuffling is the most potent one. However, surprisingly little is known about its incidence on a genome-wide scale.
Results
We have studied shuffling in genes that are conserved between distantly related species. Specifically, we estimated the incidence of gene shuffling in ten organisms from the three domains of life: eukaryotes, eubacteria, and archaea, considering only genes showing significant sequence similarity in pairwise genome comparisons. We found that successful gene shuffling is very rare among such conserved genes. For example, we could detect only 48 successful gene-shuffling events in the genome of the fruit fly Drosophila melanogaster which have occurred since its common ancestor with the worm Caenorhabditis elegans more than half a billion years ago.
Conclusion
The incidence of gene shuffling is roughly an order of magnitude smaller than the incidence of single-gene duplication in eukaryotes, but it can approach or even exceed the gene-duplication rate in prokaryotes. If true in general, this pattern suggests that gene shuffling may not be a major force in reshaping the core genomes of eukaryotes. Our results also cast doubt on the notion that introns facilitate gene shuffling, both because prokaryotes show an appreciable incidence of gene shuffling despite their lack of introns and because we find no statistical association between exon-intron boundaries and recombined domains in the two multicellular genomes we studied.
doi:10.1186/gb-2005-6-6-r50
PMCID: PMC1175970  PMID: 15960802
5.  TGF-β Signaling Controls Embryo Development in the Parasitic Flatworm Schistosoma mansoni 
PLoS Pathogens  2007;3(4):e52.
Over 200 million people have, and another 600 million are at risk of contracting, schistosomiasis, one of the major neglected tropical diseases. Transmission of this infection, which is caused by helminth parasites of the genus Schistosoma, depends upon the release of parasite eggs from the human host. However, approximately 50% of eggs produced by schistosomes fail to reach the external environment, but instead become trapped in host tissues where pathological changes caused by the immune responses to secreted egg antigens precipitate disease. Despite the central importance of egg production in transmission and disease, relatively little is understood of the molecular processes underlying the development of this key life stage in schistosomes. Here, we describe a novel parasite-encoded TGF-β superfamily member, Schistosoma mansoni Inhibin/Activin (SmInAct), which is key to this process. In situ hybridization localizes SmInAct expression to the reproductive tissues of the adult female, and real-time RT-PCR analyses indicate that SmInAct is abundantly expressed in ovipositing females and the eggs they produce. Based on real-time RT-PCR analyses, SmInAct transcription continues, albeit at a reduced level, both in adult worms isolated from single-sex infections, where reproduction is absent, and in parasites from IL-7R−/− mice, in which viable egg production is severely compromised. Nevertheless, Western analyses demonstrate that SmInAct protein is undetectable in parasites from single-sex infections and from infections of IL-7R−/− mice, suggesting that SmInAct expression is tightly linked to the reproductive potential of the worms. A crucial role for SmInAct in successful embryogenesis is indicated by the finding that RNA interference–mediated knockdown of SmInAct expression in eggs aborts their development. Our results demonstrate that TGF-β signaling plays a major role in the embryogenesis of a metazoan parasite, and have implications for the development of new strategies for the treatment and prevention of an important and neglected human disease.
Author Summary
Schistosomes are parasitic worms that infect hundreds of millions of people in developing countries. They cause disease by virtue of the fact that the eggs that they produce, which are intended for release from the host in order to allow transmission of infection, can become trapped in target organs such as the liver, where they induce damaging inflammation. Egg production by female schistosomes is critically dependent on the presence of male parasites, without which females never fully develop, and (counterintuitively) on the contribution of signals from the host's immune system. Very little is understood about the molecular basis of these interactions. Here, we describe a newly discovered schistosome gene, which is expressed in the reproductive tract of the female parasite and in parasite eggs. The protein encoded by this gene is made only when females are paired with males in an immunologically competent setting. Using recently developed tools that allow gene function to be inhibited in schistosomes, we show that the product of this gene plays a crucial role in egg development. Examining how the expression of this gene is controlled has the potential to provide insight into the molecular nature of the interactions between male and female parasites and their hosts. Moreover, the pivotal role of this gene in the egg makes it a potential target for blocking transmission and disease development.
doi:10.1371/journal.ppat.0030052
PMCID: PMC1847691  PMID: 17411340
6.  The Role of Abcb5 Alleles in Susceptibility to Haloperidol-Induced Toxicity in Mice and Humans 
PLoS Medicine  2015;12(2):e1001782.
Background
We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study.
Methods and Findings
A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered.
Conclusions
ABCB5 alleles alter susceptibility to HIT in mouse and humans. This discovery leads to a new model that (at least in part) explains inter-individual differences in susceptibility to a drug-induced CNS toxicity.
Gary Peltz and colleagues examine the role of ABCB5 alleles in haloperidol-induced toxicity in a murine genetic model and humans treated with haloperidol.
Editors' Summary
Background
The brain is the control center of the human body. This complex organ controls thoughts, memory, speech, and movement, it is the seat of intelligence, and it regulates the function of many organs. The brain comprises many different parts, all of which work together but all of which have their own special functions. For example, the forebrain is involved in intellectual activities such as thinking whereas the hindbrain controls the body’s vital functions and movements. Messages are passed between the various regions of the brain and to other parts of the body by specialized cells called neurons, which release and receive signal molecules known as neurotransmitters. Like all the organs in the body, blood vessels supply the brain with the oxygen, water, and nutrients it needs to function. Importantly, however, the brain is protected from infectious agents and other potentially dangerous substances circulating in the blood by the “blood-brain barrier,” a highly selective permeability barrier that is formed by the cells lining the fine blood vessels (capillaries) within the brain.
Why Was This Study Done?
Although drugs have been developed to treat various brain disorders, more active and less toxic drugs are needed to improve the treatment of many if not most of these conditions. Unfortunately, relatively little is known about how the blood-brain barrier regulates the entry of drugs into the brain or about the genetic factors that affect the brain’s susceptibility to drug-induced toxicities. It is not known, for example, why about half of patients given haloperidol—a drug used to treat psychotic disorders (conditions that affect how people think, feel, or behave)—develop tremors and other symptoms caused by alterations in the brain region that controls voluntary movements. Here, to improve our understanding of how drugs enter the brain and impact its function, the researchers investigate the genetic factors that affect haloperidol-induced toxicity by genetically analyzing several inbred mouse strains (every individual in an inbred mouse strain is genetically identical) with different susceptibilities to haloperidol-induced toxicity and by undertaking a human genetic association study (a study that looks for non-chance associations between specific traits and genetic variants).
What Did the Researchers Do and Find?
The researchers used a database of genetic variants called single nucleotide polymorphisms (SNPs) and a computational genetic mapping approach to show first that variations within the gene encoding Abcb5 affected susceptibility to haloperidol-induced toxicity (indicated by changes in the length of time taken by mice to move their paws when placed on an inclined wire-mesh screen) among inbred mouse strains. Abcb5 is an ATP-binding cassette transporter, a type of protein that moves molecules across cell membranes. The researchers next showed that Abcb5 is expressed in brain capillaries, which is the location of the blood-brain barrier. Abcb5 was also expressed in cerebellar Purkinje cells, which help to control motor (intentional) movements. They also measured the measured the effect of haloperidol and the haloperidol concentration in brain tissue sections in mice that were genetically engineered to make no Abcb5 (Abcb5 knockout mice). Finally, the researchers investigated whether specific alleles (alternative versions) of ABCB5 are associated with haloperidol-induced toxicity in people. Among a group of 85 patients treated with haloperidol for a psychotic illness, one specific ABCB5 allele was associated with haloperidol-induced toxicity during the first few days of treatment.
What Do These Findings Mean?
These findings indicate that Abcb5 is a component of the blood-brain barrier in mice and suggest that genetic variants in the gene encoding this protein underlie, at least in part, the differences in susceptibility to haloperidol-induced toxicity seen among inbred mice strains. Moreover, the human genetic association study indicates that a specific ABCB5 allele also affects the susceptibility of people to haloperidol-induced toxicity. The researchers note that other ABCB5 alleles or other genetic factors that affect haloperidol-induced toxicity in people might emerge if larger groups of patients were studied. However, based on their findings, the researchers propose a new model for the genetic mechanisms that underlie inter-individual and cell type-specific differences in susceptibility to haloperidol-induced brain toxicity. If confirmed in future studies, this model might facilitate the development of more effective and less toxic drugs to treat a range of brain disorders.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001782.
The US National Institute of Neurological Disorders and Stroke provides information about a wide range of brain diseases (in English and Spanish); its fact sheet “Brain Basics: Know Your Brain” is a simple introduction to the human brain; its “Blueprint Neurotherapeutics Network” was established to develop new drugs for disorders affecting the brain and other parts of the nervous system
MedlinePlus provides links to additional resources about brain diseases and their treatment (in English and Spanish)
Wikipedia provides information about haloperidol, about ATP-binding cassette transporters and about genetic association (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1001782
PMCID: PMC4315575  PMID: 25647612
7.  A Deep Sequencing Approach to Comparatively Analyze the Transcriptome of Lifecycle Stages of the Filarial Worm, Brugia malayi 
Background
Developing intervention strategies for the control of parasitic nematodes continues to be a significant challenge. Genomic and post-genomic approaches play an increasingly important role for providing fundamental molecular information about these parasites, thus enhancing basic as well as translational research. Here we report a comprehensive genome-wide survey of the developmental transcriptome of the human filarial parasite Brugia malayi.
Methodology/Principal Findings
Using deep sequencing, we profiled the transcriptome of eggs and embryos, immature (≤3 days of age) and mature microfilariae (MF), third- and fourth-stage larvae (L3 and L4), and adult male and female worms. Comparative analysis across these stages provided a detailed overview of the molecular repertoires that define and differentiate distinct lifecycle stages of the parasite. Genome-wide assessment of the overall transcriptional variability indicated that the cuticle collagen family and those implicated in molting exhibit noticeably dynamic stage-dependent patterns. Of particular interest was the identification of genes displaying sex-biased or germline-enriched profiles due to their potential involvement in reproductive processes. The study also revealed discrete transcriptional changes during larval development, namely those accompanying the maturation of MF and the L3 to L4 transition that are vital in establishing successful infection in mosquito vectors and vertebrate hosts, respectively.
Conclusions/Significance
Characterization of the transcriptional program of the parasite's lifecycle is an important step toward understanding the developmental processes required for the infectious cycle. We find that the transcriptional program has a number of stage-specific pathways activated during worm development. In addition to advancing our understanding of transcriptome dynamics, these data will aid in the study of genome structure and organization by facilitating the identification of novel transcribed elements and splice variants.
Author Summary
Lymphatic filariasis, also known as elephantiasis, is a tropical disease affecting over 120 million people worldwide. More than 40 million people live with painful, disfiguring symptoms that can cause severe debilitation and social stigma. The disease is caused by infection with thread-like filarial nematodes (roundworms) that have a complex parasitic lifecycle involving both human and mosquito hosts. In the study, the authors profiled the transcriptome (the set of genes transcribed into messenger RNA rather than all of those in the genome) of the human filarial worm Brugia malayi in different lifecyle stages using deep sequencing technology. The analysis revealed major transitions in RNA expression from eggs through larval stages to adults. Using statistical approaches, the authors identified groups of genes with distinct life stage dependent transcriptional patterns, with particular emphasis on genes displaying sex-biased or germline-enriched patterns and those displaying significant changes during larval development. This study presents a first comprehensive analysis of the lifecycle transcriptome of B. malayi, providing fundamental molecular information that should help researchers better understand parasite biology and could provide clues for the development of more effective interventions.
doi:10.1371/journal.pntd.0001409
PMCID: PMC3236722  PMID: 22180794
8.  The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific 
PLoS Biology  2007;5(3):e77.
The world's oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed “fragment recruitment,” addressed questions of genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes and gene families. A second method, termed “extreme assembly,” made possible the assembly and reconstruction of large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3) hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into genetically isolated populations that have overlapping but independent distributions, implying distinct environmental preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show how they may be grouped into several community types. Specific functional adaptations can be identified both within individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or absence of the phosphate-binding gene PstS.
Author Summary
Marine microbes remain elusive and mysterious, even though they are the most abundant life form in the ocean, form the base of the marine food web, and drive energy and nutrient cycling. We know so little about the vast majority of microbes because only a small percentage can be cultivated and studied in the lab. Here we report on the Global Ocean Sampling expedition, an environmental metagenomics project that aims to shed light on the role of marine microbes by sequencing their DNA without first needing to isolate individual organisms. A total of 41 different samples were taken from a wide variety of aquatic habitats collected over 8,000 km. The resulting 7.7 million sequencing reads provide an unprecedented look at the incredible diversity and heterogeneity in naturally occurring microbial populations. We have developed new bioinformatic methods to reconstitute large portions of both cultured and uncultured microbial genomes. Organism diversity is analyzed in relation to sampling locations and environmental pressures. Taken together, these data and analyses serve as a foundation for greatly expanding our understanding of individual microbial lineages and their evolution, the nature of marine microbial communities, and how they are impacted by and impact our world.
TheSorcerer II GOS expedition, data sampling, and analysis is described. The immense diversity in the sequence data required novel comparative genomic assembly methods, which uncovered genomic differences that marker-based methods could not.
doi:10.1371/journal.pbio.0050077
PMCID: PMC1821060  PMID: 17355176
9.  Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development 
PLoS Medicine  2013;10(11):e1001551.
TB filled in by Laureen
Please see later in the article for the Editors' Summary
Background
Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development.
Methods and Findings
Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression.
Conclusions
HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Cancer, which is responsible for 13% of global deaths, can develop anywhere in the body, but all cancers are characterized by uncontrolled cell growth and reduced cellular differentiation (the process by which unspecialized cells such as “stem” cells become specialized during development, tissue repair, and normal cell turnover). Genetic alterations—changes in the sequence of nucleotides (DNA's building blocks) in specific genes—are required for this cellular transformation and subsequent cancer development (carcinogenesis). However, recent evidence suggests that epigenetic modifications—reversible, heritable changes in gene function that occur in the absence of nucleotide sequence changes—may also be involved in carcinogenesis. For example, the addition of methyl groups to a set of genes called stem cell polycomb group target genes (PCGTs; polycomb genes control the expression of their target genes by modifying their DNA or associated proteins) is one of the earliest molecular changes in human cancer development, and increasing evidence suggests that hypermethylation of PCGTs is an epigenetic hallmark of cancer.
Why Was This Study Done?
The methylation of PCGTs, which is triggered by age and by environmental factors that are associated with cancer development, reduces cellular differentiation and leads to the accumulation of undifferentiated cells that are susceptible to cancer development. It is unclear, however, whether epigenetic modifications have a causal role in carcinogenesis. Here, the researchers investigate the involvement of epigenetic factors in the development of endometrial (womb) cancer. The risk of endometrial cancer (which affects nearly 50,000 women annually in the United States) is largely determined by environmental and lifestyle factors. Specifically, the risk of this cancer is increased in women in whom estrogen (a hormone that drives cell proliferation in the endometrium) is functionally dominant over progesterone (a hormone that inhibits endometrial proliferation and causes cell differentiation); obese women and women who have taken estrogen-only hormone replacement therapies fall into this category. Thus, endometrial cancer is an ideal model in which to study whether epigenetic mechanisms underlie carcinogenesis.
What Did the Researchers Do and Find?
The researchers collected data on genome-wide DNA methylation at cytosine- and guanine-rich sites in endometrial cancers and normal endometrium and integrated this information with the human interactome and transcriptome (all the physical interactions between proteins and all the genes expressed, respectively, in a cell) using an algorithm called Functional Epigenetic Modules (FEM). This analysis identified HAND2 as the hub of the most highly ranked differential methylation hotspot in endometrial cancer. HAND2 is a progesterone-regulated stem cell PCGT. It encodes a transcription factor that is expressed in the endometrial stroma (the connective tissue that lies below the epithelial cells in which most endometrial cancers develop) and that suppresses the production of the growth factors that mediate the growth-inducing effects of estrogen on the endometrial epithelium. The researchers hypothesized, therefore, that epigenetic deregulation of HAND2 could be a key step in endometrial cancer development. In support of this hypothesis, the researchers report that HAND2 methylation was increased in premalignant endometrial lesions (cancer-prone, abnormal-looking tissue) compared to normal endometrium, and was associated with suppression of HAND2 expression. Moreover, a high level of endometrial HAND2 methylation in premalignant lesions predicted a poor response to progesterone treatment (which stops the growth of some endometrial cancers), and analysis of HAND2 methylation in endometrial secretions collected from women with postmenopausal bleeding (a symptom of endometrial cancer) accurately identified individuals with early stage endometrial cancer. Finally, mice in which the Hand2 gene was specifically deleted in the endometrium developed precancerous endometrial lesions with age.
What Do These Findings Mean?
These and other findings identify HAND2 methylation as a common, key molecular alteration in endometrial cancer. These findings need to be confirmed in more women, and studies are needed to determine the immediate molecular and cellular consequences of HAND2 silencing in endometrial stromal cells. Nevertheless, these results suggest that HAND2 methylation could potentially be used as a biomarker for the early detection of endometrial cancer and for predicting treatment response. More generally, these findings support the idea that methylation of HAND2 (and, by extension, the methylation of other PCGTs) is not a passive epigenetic feature of cancer but is functionally involved in cancer development, and provide a framework for identifying other genes that are epigenetically regulated and functionally important in carcinogenesis.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001551
The US National Cancer Institute provides information on all aspects of cancer and has detailed information about endometrial cancer for patients and professionals (in English and Spanish)
The not-for-profit organization American Cancer Society provides information on cancer and how it develops and specific information on endometrial cancer (in several languages)
The UK National Health Service Choices website includes an introduction to cancer, a page on endometrial cancer, and a personal story about endometrial cancer
The not-for-profit organization Cancer Research UK provides general information about cancer and specific information about endometrial cancer
Wikipedia has a page on cancer epigenetics (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Eve Appeal charity that supported this research provides useful information on gynecological cancers
doi:10.1371/journal.pmed.1001551
PMCID: PMC3825654  PMID: 24265601
10.  Anti-Fungal Innate Immunity in C. elegans Is Enhanced by Evolutionary Diversification of Antimicrobial Peptides 
PLoS Pathogens  2008;4(7):e1000105.
Encounters with pathogens provoke changes in gene transcription that are an integral part of host innate immune responses. In recent years, studies with invertebrate model organisms have given insights into the origin, function, and evolution of innate immunity. Here, we use genome-wide transcriptome analysis to characterize the consequence of natural fungal infection in Caenorhabditis elegans. We identify several families of genes encoding putative antimicrobial peptides (AMPs) and proteins that are transcriptionally up-regulated upon infection. Many are located in small genomic clusters. We focus on the nlp-29 cluster of six AMP genes and show that it enhances pathogen resistance in vivo. The same cluster has a different structure in two other Caenorhabditis species. A phylogenetic analysis indicates that the evolutionary diversification of this cluster, especially in cases of intra-genomic gene duplications, is driven by natural selection. We further show that upon osmotic stress, two genes of the nlp-29 cluster are strongly induced. In contrast to fungus-induced nlp expression, this response is independent of the p38 MAP kinase cascade. At the same time, both involve the epidermal GATA factor ELT-3. Our results suggest that selective pressure from pathogens influences intra-genomic diversification of AMPs and reveal an unexpected complexity in AMP regulation as part of the invertebrate innate immune response.
Author Summary
We are interested in how exactly the nematode Caenorhabditi elegans, widely used in biological research, defends itself against fungal infection. Like most animals, this worm responds to infection by switching on defense genes. We used DNA chips to measure the levels of all the worm's 20,000 genes and discovered new inducible defense genes. Many of them encode small proteins or peptides that can probably kill microbes. By looking in other nematode species, we saw that these antimicrobial peptide genes are evolving rapidly. This means that they could be important for the worms' survival in their natural environment. We also looked at how some of these genes are regulated and uncovered a sophisticated control mechanism involving a series of proteins called kinases that relay signals within cells. The genes we looked at are active in the worm's skin. Some of the antimicrobial peptide genes that we looked at are also switched on in the skin by high salt, but in this case, the regulation doesn't involve the same cascade of kinases. The responses to both infection and high salt do, however, require the same transcription factor (the protein that actually switches genes on), in this case called a GATA factor.
doi:10.1371/journal.ppat.1000105
PMCID: PMC2453101  PMID: 18636113
11.  Modification of Gene Duplicability during the Evolution of Protein Interaction Network 
PLoS Computational Biology  2011;7(4):e1002029.
Duplications of genes encoding highly connected and essential proteins are selected against in several species but not in human, where duplicated genes encode highly connected proteins. To understand when and how gene duplicability changed in evolution, we compare gene and network properties in four species (Escherichia coli, yeast, fly, and human) that are representative of the increase in evolutionary complexity, defined as progressive growth in the number of genes, cells, and cell types. We find that the origin and conservation of a gene significantly correlates with the properties of the encoded protein in the protein-protein interaction network. All four species preserve a core of singleton and central hubs that originated early in evolution, are highly conserved, and accomplish basic biological functions. Another group of hubs appeared in metazoans and duplicated in vertebrates, mostly through vertebrate-specific whole genome duplication. Such recent and duplicated hubs are frequently targets of microRNAs and show tissue-selective expression, suggesting that these are alternative mechanisms to control their dosage. Our study shows how networks modified during evolution and contributes to explaining the occurrence of somatic genetic diseases, such as cancer, in terms of network perturbations.
Author Summary
Gene copy number is often tightly controlled because it directly affects the gene dosage. In several species, including yeast, worm, and fly, genes that have a single gene copy (singleton genes) encode proteins with several connections in the protein interaction network (hubs) as well as essential proteins. Surprisingly, in mouse and human essential proteins and hubs are encoded by genes with more than one copy in the genome (duplicated genes). Here we show that these two distinct groups of hubs were acquired at different times during the evolution of protein interaction network and contribute in different ways to the cell life. Singleton hubs are ancestral genes that are conserved from prokaryotes to vertebrates and accomplish basic functions that deal with the cell survival. Duplicated hubs were acquired mostly within metazoans and duplicated through vertebrate-specific whole genome duplication. These genes are involved in processes that are crucial for the organization of multicellularity. Although duplicated, also recent hubs are subject to gene dosage control through microRNAs and tissue-selective expression. The clarification of how the protein interaction network evolves enables us to understand the adaptation to the progressive increase in complexity and to better characterize the genes involved in diseases such as cancer.
doi:10.1371/journal.pcbi.1002029
PMCID: PMC3072358  PMID: 21490719
12.  Atypical Haemolytic Uraemic Syndrome Associated with a Hybrid Complement Gene 
PLoS Medicine  2006;3(10):e431.
Background
Sequence analysis of the regulators of complement activation (RCA) cluster of genes at chromosome position 1q32 shows evidence of several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for factor H (CFH) and the genes for the five factor H-related proteins (CFHL1–5; aliases CFHR1–5). CFH mutations have been described in association with atypical haemolytic uraemic syndrome (aHUS). The majority of the mutations are missense changes that cluster in the C-terminal region and impair the ability of factor H to regulate surface-bound C3b. Some have arisen as a result of gene conversion between CFH and CFHL1. In this study we tested the hypothesis that nonallelic homologous recombination between low-copy repeats in the RCA cluster could result in the formation of a hybrid CFH/CFHL1 gene that predisposes to the development of aHUS.
Methods and Findings
In a family with many cases of aHUS that segregate with the RCA cluster we used cDNA analysis, gene sequencing, and Southern blotting to show that affected individuals carry a heterozygous CFH/CFHL1 hybrid gene in which exons 1–21 are derived from CFH and exons 22/23 from CFHL1. This hybrid encodes a protein product identical to a functionally significant CFH mutant (c.3572C>T, S1191L and c.3590T>C, V1197A) that has been previously described in association with aHUS.
Conclusions
CFH mutation screening is recommended in all aHUS patients prior to renal transplantation because of the high risk of disease recurrence post-transplant in those known to have a CFH mutation. Because of our finding it will be necessary to implement additional screening strategies that will detect a hybrid CFH/CFHL1 gene.
Tim Goodship and colleagues have identified a heterozygousCFH/CFHL1 hybrid gene which encodes a protein product identical to one previously described in association with atypical hemolytic uremic syndrome.
Editors' Summary
Background.
Atypical hemolytic uremic (aHUS) syndrome is a rare, chronic disease that can run in families. People with the condition are prone to developing kidney failure and high blood pressure, and are likely to have a shorter life span than healthy people. Previous work done by a group of researchers in Newcastle-on-Tyne, UK looked at the genetic underpinnings of aHUS in three families suffering from the condition. They found a region of the genome that was linked with the disease in all three families. That region was known to contain a gene for a protein called “factor H,” as well as a number of other genes for proteins that are involved in the same pathway as factor H in controlling an ancient defence system called complement. This system helps antibodies to kill invaders by marking any cell that is not protected by proteins such as factor H. Our own cells would be under constant threat without protective proteins such as factor H. Later studies found simple genetic mutations in people with aHUS, in the genes coding for factor H. However, other work suggested that in some families with aHUS, simple genetic mutations might not be the cause; instead more complicated rearrangements of the genome might occur which would then result in an abnormal factor H that incorporated part of the gene for another protective protein called factor H related protein 1.
Why Was This Study Done?
The researchers knew that it was important to understand the exact genetic mutations linked with aHUS in different families. This was because the exact type of mutation would help them predict whether a kidney transplant is likely to be successful in treating an individual with aHUS who has developed kidney failure. In people with mutations affecting proteins produced by the kidney, a kidney transplant would be likely to work; but in people with mutations affecting factor H, which is produced by the liver, the disease would probably recur after a kidney transplant.
What Did the Researchers Do and Find?
In this study, the researchers went back to one of the three families with aHUS they had previously studied. The researchers had shown before that in this family, the disease was linked with the genome region containing factor H, but no precise mutation in that region had been found. This time, the researchers screened the genome of the family members and looked in particular for a specific rearrangement of the genome that they suspected might be involved. They found that the genomes in this family had been shuffled in the factor H region, resulting in an abnormal version of factor H being produced.
What Do These Findings Mean?
The mutation these researchers identified is likely to result in development of aHUS that does not get better after a kidney transplant, because the abnormal factor H would still be produced in the liver after a transplant had been done. Therefore, the researchers suggest that patients with aHUS be checked for this particular mutation before it is decided whether to go ahead with a transplant.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030431.
US National Institutes of Health Office of Rare Diseases information about atypical hemolytic uremic syndrome
The Online Mendelian Inheritance in Man (OMIM) contains an entry on hemolytic uremic syndrome. OMIM is a database of human genes and genetic disorders developed by the US National Center for Biotechnology Information
The US National Kidney and Urologic Diseases has a page about hemolytic uremic syndrome
The Wikipedia has a page about HUS (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030431
PMCID: PMC1626556  PMID: 17076561
13.  Genes That Act Downstream of Sensory Neurons to Influence Longevity, Dauer Formation, and Pathogen Responses in Caenorhabditis elegans 
PLoS Genetics  2012;8(12):e1003133.
The sensory systems of multicellular organisms are designed to provide information about the environment and thus elicit appropriate changes in physiology and behavior. In the nematode Caenorhabditis elegans, sensory neurons affect the decision to arrest during development in a diapause state, the dauer larva, and modulate the lifespan of the animals in adulthood. However, the mechanisms underlying these effects are incompletely understood. Using whole-genome microarray analysis, we identified transcripts whose levels are altered by mutations in the intraflagellar transport protein daf-10, which result in impaired development and function of many sensory neurons in C. elegans. In agreement with existing genetic data, the expression of genes regulated by the transcription factor DAF-16/FOXO was affected by daf-10 mutations. In addition, we found altered expression of transcriptional targets of the DAF-12/nuclear hormone receptor in the daf-10 mutants and showed that this pathway influences specifically the dauer formation phenotype of these animals. Unexpectedly, pathogen-responsive genes were repressed in daf-10 mutant animals, and these sensory mutants exhibited altered susceptibility to and behavioral avoidance of bacterial pathogens. Moreover, we found that a solute transporter gene mct-1/2, which was induced by daf-10 mutations, was necessary and sufficient for longevity. Thus, sensory input seems to influence an extensive transcriptional network that modulates basic biological processes in C. elegans. This situation is reminiscent of the complex regulation of physiology by the mammalian hypothalamus, which also receives innervations from sensory systems, most notably the visual and olfactory systems.
Author Summary
The senses provide animals with information about their environment, which affects not only their behavior but also their internal state and physiological outputs. How this information is processed is still unclear. In this study, we used mutant C. elegans roundworms that had defective sensory neurons to investigate how changes in sensation alter the expression of genes and regulate physiology, specifically the worms' choice to hibernate during growth and their longevity as fully-grown adults. We showed that defects in sensory neurons change the pattern of gene expression and regulate these outputs through known hormonal pathways, including insulin/IGF-1 and steroid pathways. We also identified a new regulator of longevity, MCT-1, that is predicted to transport small metabolites and hormones in the body. Unexpectedly, we found that sensory impairment altered yet another physiological output, the response to infectious agents. It prevented the worms from avoiding infectious bacteria and reduced the expression of potentially protective factors, but also increased the worms' resistance to infection, suggesting a complex network of responses to environmental stimuli. Understanding how sensory information is relayed in this relatively simple organism may inform our understanding of sensory processing in higher organisms like mammals.
doi:10.1371/journal.pgen.1003133
PMCID: PMC3527274  PMID: 23284299
14.  A question of size: the eukaryotic proteome and the problems in defining it 
Nucleic Acids Research  2002;30(5):1083-1090.
We discuss the problems in defining the extent of the proteomes for completely sequenced eukaryotic organisms (i.e. the total number of protein-coding sequences), focusing on yeast, worm, fly and human. (i) Six years after completion of its genome sequence, the true size of the yeast proteome is still not defined. New small genes are still being discovered, and a large number of existing annotations are being called into question, with these questionable ORFs (qORFs) comprising up to one-fifth of the ‘current’ proteome. We discuss these in the context of an ideal genome-annotation strategy that considers the proteome as a rigorously defined subset of all possible coding sequences (‘the orfome’). (ii) Despite the greater apparent complexity of the fly (more cells, more complex physiology, longer lifespan), the nematode worm appears to have more genes. To explain this, we compare the annotated proteomes of worm and fly, relating to both genome-annotation and genome evolution issues. (iii) The unexpectedly small size of the gene complement estimated for the complete human genome provoked much public debate about the nature of biological complexity. However, in the first instance, for the human genome, the relationship between gene number and proteome size is far from simple. We survey the current estimates for the numbers of human genes and, from this, we estimate a range for the size of the human proteome. The determination of this is substantially hampered by the unknown extent of the cohort of pseudogenes (‘dead’ genes), in combination with the prevalence of alternative splicing. (Further information relating to yeast is available at http://genecensus.org/yeast/orfome)
PMCID: PMC101239  PMID: 11861898
15.  Epistatic Interaction Maps Relative to Multiple Metabolic Phenotypes 
PLoS Genetics  2011;7(2):e1001294.
An epistatic interaction between two genes occurs when the phenotypic impact of one gene depends on another gene, often exposing a functional association between them. Due to experimental scalability and to evolutionary significance, abundant work has been focused on studying how epistasis affects cellular growth rate, most notably in yeast. However, epistasis likely influences many different phenotypes, affecting our capacity to understand cellular functions, biochemical networks adaptation, and genetic diseases. Despite its broad significance, the extent and nature of epistasis relative to different phenotypes remain fundamentally unexplored. Here we use genome-scale metabolic network modeling to investigate the extent and properties of epistatic interactions relative to multiple phenotypes. Specifically, using an experimentally refined stoichiometric model for Saccharomyces cerevisiae, we computed a three-dimensional matrix of epistatic interactions between any two enzyme gene deletions, with respect to all metabolic flux phenotypes. We found that the total number of epistatic interactions between enzymes increases rapidly as phenotypes are added, plateauing at approximately 80 phenotypes, to an overall connectivity that is roughly 8-fold larger than the one observed relative to growth alone. Looking at interactions across all phenotypes, we found that gene pairs interact incoherently relative to different phenotypes, i.e. antagonistically relative to some phenotypes and synergistically relative to others. Specific deletion-deletion-phenotype triplets can be explained metabolically, suggesting a highly informative role of multi-phenotype epistasis in mapping cellular functions. Finally, we found that genes involved in many interactions across multiple phenotypes are more highly expressed, evolve slower, and tend to be associated with diseases, indicating that the importance of genes is hidden in their total phenotypic impact. Our predictions indicate a pervasiveness of nonlinear effects in how genetic perturbations affect multiple metabolic phenotypes. The approaches and results reported could influence future efforts in understanding metabolic diseases and the role of biochemical regulation in the cell.
Author Summary
An epistatic interaction between two genes occurs when the phenotypic impact of one gene is dependent on the other. While different phenotypes have been used to uncover epistasis in different contexts, little is known about how cell-scale genetic interaction networks vary across multiple phenotypes. Here we use a genome-scale mathematical model of yeast metabolism to compute a three-dimensional matrix of interactions between any two gene deletions with respect to all metabolic flux phenotypes. We find that this multi-phenotype epistasis map contains many more interactions than found relative to any single phenotype. The unique contribution of examining multiple phenotypes is further demonstrated by the fact that individual interactions may be synergistic relative to some phenotypes and antagonistic relative to others. This observation indicates that different phenotypes are indeed capturing different aspects of the functional relationships between genes. Furthermore, the observation that genes involved in many epistatic interactions across all metabolic flux phenotypes are found to be highly expressed and under strong selective pressure seems to indicate that these interactions are important to the cell and are not just the unavoidable consequence of the connectivity of biological networks. Multi-phenotype epistasis maps may help elucidate the functional organization of biological systems and the role of epistasis in the manifestation of complex genetic diseases.
doi:10.1371/journal.pgen.1001294
PMCID: PMC3037399  PMID: 21347328
16.  Molecular characterization of the evolution of phagosomes 
First large-scale comparative proteomics/phosphoproteomics study characterizing some of the key steps that contributed to the remodeling of phagosomes that occurred during evolution. Comparison of profiling analyses of isolated phagosomes from three distant organisms (Dictyostelium, Drosophila, and mouse) revealed a protein core that defines a potential ‘ancient' phagosome and a set of 50 proteins that emerged while adaptive immunity was already well established.Gene duplication events of mouse phagosome paralogs occurred mostly in Bilateria and Euteleostomi, coinciding with the emergence of innate and adaptive immunity, and thus, provided the functional innovations needed for the establishment of these two crucial evolutionary steps of the immune system.Phosphoproteomics of isolated phagosomes from the same three distant species indicate that the phagosome phosphoproteome has been extensively modified during evolution. Still, some phosphosites have been maintained for >1.2 billion years, and thus, highlight their particular significance in the regulation of key phagosomal functions.
Phagocytosis is the process by which multiple cell types internalize large particulate material from the external milieu. The functional properties of phagosomes are acquired through a complex maturation process, referred to as phagolysosome biogenesis. This pathway involves a series of rapid interactions with organelles of the endocytic apparatus, enabling the gradual transformation of newly formed phagosomes into phagolysosomes in which proteolytic degradation occurs. The degradative environment encountered in the phagosome lumen has enabled the use of phagocytosis as a predation mechanism for feeding (phagotrophy) in amoeba, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in jawed vertebrates (fish), initiate a sustained immune response.
High-throughput proteomics profiling of isolated phagosomes has been tremendously helpful for the molecular comprehension of this organelle. This approach is achieved by feeding low buoyancy latex beads to phagocytic cells, enabling the subsequent isolation of latex bead-containing phagosomes, away from all the other cell organelles, by a single-isopicnic centrifugation in sucrose gradient. In order to characterize some of the key steps that contributed to the remodeling of phagosomes during evolution, we isolated this organelle from three distant organisms: the amoeba Dictyostelium discoideum, the fruit fly Drosophila melanogaster, and mouse (Mus musculus) that use phagocytosis for different purposes, and performed detailed proteomics and phosphoproteomics analyses with unparallel protein coverage for this organelle (two- to four-fold enhancements in identified proteins).
In order to establish the origin of the mouse phagosome proteome, we performed comparative analyses among 39 taxa including plants/algea, unicellular organisms, fungi, and more complex animal multicellular organisms. These genomic comparisons indicated that a large proportion of the mouse phagosome proteome is of ancient origin (73.1% of the proteome is conserved in eukaryotic organisms) (Figure 2A). This stresses the fact that phagocytosis is a very ancient process, as shown by its possible involvement in the emergence of eukaryotic cells (eukaryogenesis). Indeed, we identified close to 300 phagosome mouse proteins also present on Drosophila and Dictyostelium phagosomes, defining a potential ‘ancient' core of proteins from which the immune functions of phagosomes likely evolved. Around 16.7% of the mouse phagosome proteins appeared in organisms that use phagocytosis for innate immunity (Bilateria to Chordata), whereas 10.2% appeared in Euteleostomi or Tetrapoda where phagosomes have an important function in linking the killing of microorganisms with the development of a specific sustained immune response following antigen recognition. The phagosome is made of molecules taken from a variety of sources within the cell, including the cytoplasm, the cytoskeleton and membrane organelles. Despite the evolution and diversification of these various cellular systems, the mammalian phagosome proteome is made preferentially of ancient proteins (Figure 2B). Comparison of functional annotation during evolution highlighted the emergence of specific phagosomal functions at various steps during evolution (Figure 2C). Some of these proteins and their point of origin during evolution are highlighted in Figure 2D. Strikingly, we identified in Tetrapods a set of 50 proteins that arose while adaptive immunity was already well established in teleosts (fish), indicating that the phagocytic system is still evolving.
Our study highlights the fact that the functional properties of phagosomes emerged by the remodeling of ancient molecules, the addition of novel components, and the duplication of existing proteins (paralogs) leading to the formation of molecular machines of mixed origin. Gene duplication is a process that contributed continuously to the complexification of the mouse proteome during evolution. In sharp contrast, paralog analysis indicated that the phagosome proteome was mainly reorganized through two periods of gene duplication, in Bilateria and Euteleostomi, coinciding with the emergence of adaptive immunity (in jawed fish), and innate immunity (at the split between Metazoa and Bilateria). These results strongly suggest that selective constraints may have favored the maintenance of phagosome paralogs to ensure the establishment of novel functions associated with this organelle at these two crucial evolutionary steps of the immune system.
The emergence of genes associated to the MHC locus in mammals that appeared originally in the genome of jawed fishes, contributed to the development of complex molecular mechanisms linking innate (our immune system that defends the host from infection in a non-specific manner) and adaptive immunity (the part of the immune system triggered specifically after antigen recognition). Several of the genes of this locus encode proteins known to have important functions in antigen presentation, such as subunits of the immunoproteasome (LMP2 and LMP7), MHC class I and class II molecules, as well as tapasin and the transporter associated with antigen processing (TAP1 and TAP2), involved in the transport and loading of peptides on MHC class I molecules (Figure 6). In addition to their ability to present peptides on MHC class II molecules, phagosomes of vertebrates have been shown to be competent for the presentation of exogenous peptides on MHC class I molecules, a process referred to as cross-presentation. From a functional point of view, the involvement of phagosomes in antigen cross-presentation is the outcome of the successful integration of a wide range of multimolecular components that emerged throughout evolution (Figure 6). The trimming of exogenous proteins into small peptides that can be loaded on MHC class I molecules is inherited from the phagotrophic properties of unicellular organisms, where internalized bacteria are degraded into basic molecules and used as a source of nutrients. Ancient processes have therefore been co-opted (the use of an existing biological structure or feature for a new function) for new functionalities. A summarizing model of the various steps that enabled phagosome antigen presentation is presented in Figure 6. This model highlights the fact that although antigen presentation is unique to evolutionary recent phagosomes (starting in jawed fishes about 450 million years ago), it uses and integrates molecular machines composed of proteins that emerged throughout evolution.
In summary, we present here the first large-scale comparative proteomics/phosphoproteomics study characterizing some of the key evolutionary steps that contributed to the remodeling of phagosomes during evolution. Functional properties of this organelle emerged by the remodeling of ancient molecules, the addition of novel components, the extensive adaption of protein phosphorylation sites and the duplication of existing proteins leading to the formation of molecular machines of mixed origin.
Amoeba use phagocytosis to internalize bacteria as a source of nutrients, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in vertebrates, initiate a sustained immune response. By using a large-scale approach to identify and compare the proteome and phosphoproteome of phagosomes isolated from distant organisms, and by comparative analysis over 39 taxa, we identified an ‘ancient' core of phagosomal proteins around which the immune functions of this organelle have likely organized. Our data indicate that a larger proportion of the phagosome proteome, compared with the whole cell proteome, has been acquired through gene duplication at a period coinciding with the emergence of innate and adaptive immunity. Our study also characterizes in detail the acquisition of novel proteins and the significant remodeling of the phagosome phosphoproteome that contributed to modify the core constituents of this organelle in evolution. Our work thus provides the first thorough analysis of the changes that enabled the transformation of the phagosome from a phagotrophic compartment into an organelle fully competent for antigen presentation.
doi:10.1038/msb.2010.80
PMCID: PMC2990642  PMID: 20959821
evolution; immunity; phosphoproteomics; phylogeny; proteomics
17.  A Gene Family Derived from Transposable Elements during Early Angiosperm Evolution Has Reproductive Fitness Benefits in Arabidopsis thaliana 
PLoS Genetics  2012;8(9):e1002931.
The benefits of ever-growing numbers of sequenced eukaryotic genomes will not be fully realized until we learn to decipher vast stretches of noncoding DNA, largely composed of transposable elements. Transposable elements persist through self-replication, but some genes once encoded by transposable elements have, through a process called molecular domestication, evolved new functions that increase fitness. Although they have conferred numerous adaptations, the number of such domesticated transposable element genes remains unknown, so their evolutionary and functional impact cannot be fully assessed. Systematic searches that exploit genomic signatures of natural selection have been employed to identify potential domesticated genes, but their predictions have yet to be experimentally verified. To this end, we investigated a family of domesticated genes called MUSTANG (MUG), identified in a previous bioinformatic search of plant genomes. We show that MUG genes are functional. Mutants of Arabidopsis thaliana MUG genes yield phenotypes with severely reduced plant fitness through decreased plant size, delayed flowering, abnormal development of floral organs, and markedly reduced fertility. MUG genes are present in all flowering plants, but not in any non-flowering plant lineages, such as gymnosperms, suggesting that the molecular domestication of MUG may have been an integral part of early angiosperm evolution. This study shows that systematic searches can be successful at identifying functional genetic elements in noncoding regions and demonstrates how to combine systematic searches with reverse genetics in a fruitful way to decipher eukaryotic genomes.
Author Summary
The genomes of complex organisms are mostly made up not of ordinary genes but of transposable elements. Transposable elements have been called “selfish DNA” because they normally persist by copying themselves, not by helping the organism to survive or reproduce. Yet transposable elements can help organisms to evolve; for instance, transposable element genes sometimes acquire new functions that do benefit the organism. Because they are difficult to distinguish from transposable elements, little is known about these “domesticated genes.” Although studies have attempted to identify them computationally, the predictions have not been verified experimentally. Here, we examine some of the first domesticated genes to be predicted computationally, the MUSTANG family of plant genes. We show that the predictions were correct: MUSTANGs are, like ordinary genes, functional. MUSTANG mutations result in serious defects in how plants grow, flower, and reproduce. Since they are present only in flowering plants, MUSTANG probably originated when flowers first evolved, perhaps taking on a key role. This study is important both because it shows that MUSTANG is critical to plant fitness and because, in the future, a similar approach can be used to find additional domesticated genes and to better understand how transposable elements contribute to evolution.
doi:10.1371/journal.pgen.1002931
PMCID: PMC3435246  PMID: 22969437
18.  Formation of Regulatory Modules by Local Sequence Duplication 
PLoS Computational Biology  2011;7(10):e1002167.
Turnover of regulatory sequence and function is an important part of molecular evolution. But what are the modes of sequence evolution leading to rapid formation and loss of regulatory sites? Here we show that a large fraction of neighboring transcription factor binding sites in the fly genome have formed from a common sequence origin by local duplications. This mode of evolution is found to produce regulatory information: duplications can seed new sites in the neighborhood of existing sites. Duplicate seeds evolve subsequently by point mutations, often towards binding a different factor than their ancestral neighbor sites. These results are based on a statistical analysis of 346 cis-regulatory modules in the Drosophila melanogaster genome, and a comparison set of intergenic regulatory sequence in Saccharomyces cerevisiae. In fly regulatory modules, pairs of binding sites show significantly enhanced sequence similarity up to distances of about 50 bp. We analyze these data in terms of an evolutionary model with two distinct modes of site formation: (i) evolution from independent sequence origin and (ii) divergent evolution following duplication of a common ancestor sequence. Our results suggest that pervasive formation of binding sites by local sequence duplications distinguishes the complex regulatory architecture of higher eukaryotes from the simpler architecture of unicellular organisms.
Author Summary
Since Jacob and Monod stressed the importance of gene regulation in evolution, our understanding of the mechanisms of regulation has substantially advanced. In higher eukaryotes, genes often have complex regulatory input, which is encoded in cis-regulatory sequence with multiple transcription factor binding sites. However, the modes of genome evolution generating regulatory complexity are much less understood. This study reports a surprising finding: in fly regulatory modules, the majority of transcription factor binding sites show evidence of a local sequence duplication in their evolutionary history, which relates their sequence information to that of neighboring binding sites. Our analysis suggests that local sequence duplications are a pervasive production mode of regulatory information. This mode appears to be specific to higher eukaryotes; we have not found evidence of frequent local duplications in the yeast genome. Our results affect genomic sequence analysis, in particular, computational identification of cis-regulatory elements and alignment of regulatory DNA. At the same time, they address fundamental questions on the evolution of regulation: How much of the regulatory “grammar” observed in higher eukaryotes is due to optimization of function, and how much reflects the underlying sequence evolution modes? What is the result and what is the substrate of natural selection?
doi:10.1371/journal.pcbi.1002167
PMCID: PMC3188502  PMID: 21998564
19.  Convergence of Mutation and Epigenetic Alterations Identifies Common Genes in Cancer That Predict for Poor Prognosis  
PLoS Medicine  2008;5(5):e114.
Background
The identification and characterization of tumor suppressor genes has enhanced our understanding of the biology of cancer and enabled the development of new diagnostic and therapeutic modalities. Whereas in past decades, a handful of tumor suppressors have been slowly identified using techniques such as linkage analysis, large-scale sequencing of the cancer genome has enabled the rapid identification of a large number of genes that are mutated in cancer. However, determining which of these many genes play key roles in cancer development has proven challenging. Specifically, recent sequencing of human breast and colon cancers has revealed a large number of somatic gene mutations, but virtually all are heterozygous, occur at low frequency, and are tumor-type specific. We hypothesize that key tumor suppressor genes in cancer may be subject to mutation or hypermethylation.
Methods and Findings
Here, we show that combined genetic and epigenetic analysis of these genes reveals many with a higher putative tumor suppressor status than would otherwise be appreciated. At least 36 of the 189 genes newly recognized to be mutated are targets of promoter CpG island hypermethylation, often in both colon and breast cancer cell lines. Analyses of primary tumors show that 18 of these genes are hypermethylated strictly in primary cancers and often with an incidence that is much higher than for the mutations and which is not restricted to a single tumor-type. In the identical breast cancer cell lines in which the mutations were identified, hypermethylation is usually, but not always, mutually exclusive from genetic changes for a given tumor, and there is a high incidence of concomitant loss of expression. Sixteen out of 18 (89%) of these genes map to loci deleted in human cancers. Lastly, and most importantly, the reduced expression of a subset of these genes strongly correlates with poor clinical outcome.
Conclusions
Using an unbiased genome-wide approach, our analysis has enabled the discovery of a number of clinically significant genes targeted by multiple modes of inactivation in breast and colon cancer. Importantly, we demonstrate that a subset of these genes predict strongly for poor clinical outcome. Our data define a set of genes that are targeted by both genetic and epigenetic events, predict for clinical prognosis, and are likely fundamentally important for cancer initiation or progression.
Stephen Baylin and colleagues show that a combined genetic and epigenetic analysis of breast and colon cancers identifies a number of clinically significant genes targeted by multiple modes of inactivation.
Editors' Summary
Background.
Cancer is one of the developed world's biggest killers—over half a million Americans die of cancer each year, for instance. As a result, there is great interest in understanding the genetic and environmental causes of cancer in order to improve cancer prevention, diagnosis, and treatment.
Cancer begins when cells begin to multiply out of control. DNA is the sequence of coded instructions—genes—for how to build and maintain the body. Certain “tumor suppressor” genes, for instance, help to prevent cancer by preventing tumors from developing, but changes that alter the DNA code sequence—mutations—can profoundly affect how a gene works. Modern techniques of genetic analysis have identified genes such as tumor suppressors that, when mutated, are linked to the development of certain cancers.
Why Was This Study Done?
However, in recent years, it has become increasingly apparent that mutations are neither necessary nor sufficient to explain every case of cancer. This has led researchers to look at so-called epigenetic factors, which also alter how a gene works without altering its DNA sequence. An example of this is “methylation,” which prevents a gene from being expressed—deactivates it—by a chemical tag. Methylation of genes is part of the normal functioning of DNA, but abnormal methylation has been linked with cancer, aging, and some rare birth abnormalities.
Previous analysis of DNA from breast and colon cancer cells had revealed 189 “candidate cancer genes”—mutated genes that were linked to the development of breast and colon cancer. However, it was not clear how those mutations gave rise to cancer, and individual mutations were present in only 5% to 15% of specific tumors. The authors of this study wanted to know whether epigenetic factors such as methylation contributed to causing the cancers.
What Did the Researchers Do and Find?
The researchers first identified 56 of the 189 candidate cancer genes as likely tumor suppressors and then determined that 36 of these genes were methylated and deactivated, often in both breast and colon (laboratory-grown) cancer cells. In nearly all cases, the methylated genes were not active but could be reactivated by being demethylated. They further showed that, in normal colon and breast tissue samples, 18 of the 36 genes were unmethylated and functioned normally, but in cells taken from breast and colon cancer tumors they were methylated.
In contrast to the genetic mutations, the 18 genes were frequently methylated across a range of tumor types, and eight genes were methylated in both the breast and colon cancers. The authors found by reviewing the genetics and epigenetics of those 18 genes in breast and colon cancer that they were either mutated, methylated, or both. A literature review showed that at least six of the 18 genes were known to have tumor suppressor properties, and the authors determined that 16 were located in parts of DNA known to be missing from cells taken from a range of cancer tumors.
Finally, the researchers analyzed data on cancer cases to show that methylation of these 18 genes was correlated with reduced function of these genes in tumors and with a greater likelihood that a cancer will be terminal or spread to other parts of the body.
What Do These Findings Mean?
The researchers considered only the 189 candidate cancer genes found in one previous study and not other genes identified elsewhere. They also did not consider the biological effects of the individual mutations found in those genes. Despite this, they have demonstrated that methylation of specific genes is likely to play a role in the development of breast and/or colon cancer cells either together with mutations or independently, most likely by turning off their tumor suppression function.
More broadly, however, the study adds to the evidence that future analysis of the role of genes in cancer should include epigenetic as well as genetic factors. In addition, the authors have also shown that a number of these genes may be useful for predicting clinical outcomes for a range of tumor types.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050114.
A December 2006 PLoS Medicine Perspective article reviews the value of examining methylation as a factor in common cancers and its use for early detection
The Web site of the American Cancer Society has a wealth of information and resources on a variety of cancers, including breast and colon cancer
Breastcancer.org is a nonprofit organization providing information about breast cancer on the Web, including research news
Cancer Research UK provides information on cancer research
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins publishes background information on the authors' research on methylation, setting out its potential for earlier diagnosis and better treatment of cancer
doi:10.1371/journal.pmed.0050114
PMCID: PMC2429944  PMID: 18507500
20.  Characterization of Phlebotomus papatasi Peritrophins, and the Role of PpPer1 in Leishmania major Survival in its Natural Vector 
The peritrophic matrix (PM) plays a key role in compartmentalization of the blood meal and as barrier to pathogens in many disease vectors. To establish an infection in sand flies, Leishmania must escape from the endoperitrophic space to prevent excretion with remnants of the blood meal digestion. In spite of the role played regarding Leishmania survival, little is known about sand fly PM molecular components and structural organization. We characterized three peritrophins (PpPer1, PpPer2, and PpPer3) from Phlebotomus papatasi. PpPer1 and PpPer2 display, respectively, four and one chitin-binding domains (CBDs). PpPer3 on the other hand has two CBDs, one mucin-like domain, and a putative domain with hallmarks of a CBD, but with changes in key amino acids. Temporal and spatial expression analyses show that PpPer1 is expressed specifically in the female midgut after blood feeding. PpPer2 and PpPer3 mRNAs were constitutively expressed in midgut and hindgut, with PpPer3 also being expressed in Malpighian tubules. PpPer2 was the only gene expressed in developmental stages. Interestingly, PpPer1 and PpPer3 expression are regulated by Le. major infection. Recombinant PpPer1, PpPer2 and PpPer3 were obtained and shown to display similar biochemical profiles as the native; we also show that PpPer1 and PpPer2 are able to bind chitin. Knockdown of PpPer1 led to a 44% reduction in protein, which in spite of producing an effect on the percentage of infected sand flies, resulted in a 39% increase of parasite load at 48 h. Our data suggest that PpPer1 is a component for the P. papatasi PM and likely involved in the PM role as barrier against Le. major infection.
Author Summary
For a successful development within the midgut of the sand fly vector, Leishmania must overcome several barriers imposed by the vector that include the digestive proteases secreted within the midgut following a blood meal by the insect, the need to escape from the endoperitrophic space, and attachment to the midgut epithelia to prevent excretion with the remnants of the blood meal. The sand fly peritrophic matrix (PM) constitutes an important barrier against the establishment of Leishmania within the sand fly and if trapped within the PM these parasites will be passed along with the remnants of the blood meal. Despite the role of sand fly PM on Leishmania development, characterization of its molecular components and assessment of their roles against Leishmania are lacking. Thereby, we performed the molecular characterization of three P. papatasi peritrophins named PpPer1, PpPer2, and PpPer3. Overall, we demonstrated that: (1) PpPer3 displays a putative CBD domain that might have undertaken neo-functionalization, (2) PpPer1 and PpPer3 genes display differential gene expression upon Le. major infection; and (3) PpPer1 seems to be an important component for the function of P. papatasi PM as a barrier against Le. major infection.
doi:10.1371/journal.pntd.0002132
PMCID: PMC3597473  PMID: 23516661
21.  The Preclinical Natural History of Serous Ovarian Cancer: Defining the Target for Early Detection 
PLoS Medicine  2009;6(7):e1000114.
Pat Brown and colleagues carry out a modeling study and define what properties a biomarker-based screening test would require in order to be clinically useful.
Background
Ovarian cancer kills approximately 15,000 women in the United States every year, and more than 140,000 women worldwide. Most deaths from ovarian cancer are caused by tumors of the serous histological type, which are rarely diagnosed before the cancer has spread. Rational design of a potentially life-saving early detection and intervention strategy requires understanding the lesions we must detect in order to prevent lethal progression. Little is known about the natural history of lethal serous ovarian cancers before they become clinically apparent. We can learn about this occult period by studying the unsuspected serous cancers that are discovered in a small fraction of apparently healthy women who undergo prophylactic bilateral salpingo-oophorectomy (PBSO).
Methods and Findings
We developed models for the growth, progression, and detection of occult serous cancers on the basis of a comprehensive analysis of published data on serous cancers discovered by PBSO in BRCA1 mutation carriers. Our analysis yielded several critical insights into the early natural history of serous ovarian cancer. First, these cancers spend on average more than 4 y as in situ, stage I, or stage II cancers and approximately 1 y as stage III or IV cancers before they become clinically apparent. Second, for most of the occult period, serous cancers are less than 1 cm in diameter, and not visible on gross examination of the ovaries and Fallopian tubes. Third, the median diameter of a serous ovarian cancer when it progresses to an advanced stage (stage III or IV) is about 3 cm. Fourth, to achieve 50% sensitivity in detecting tumors before they advance to stage III, an annual screen would need to detect tumors of 1.3 cm in diameter; 80% detection sensitivity would require detecting tumors less than 0.4 cm in diameter. Fifth, to achieve a 50% reduction in serous ovarian cancer mortality with an annual screen, a test would need to detect tumors of 0.5 cm in diameter.
Conclusions
Our analysis has formalized essential conditions for successful early detection of serous ovarian cancer. Although the window of opportunity for early detection of these cancers lasts for several years, developing a test sufficiently sensitive and specific to take advantage of that opportunity will be a challenge. We estimated that the tumors we would need to detect to achieve even 50% sensitivity are more than 200 times smaller than the clinically apparent serous cancers typically used to evaluate performance of candidate biomarkers; none of the biomarker assays reported to date comes close to the required level of performance. Overcoming the signal-to-noise problem inherent in detection of tiny tumors will likely require discovery of truly cancer-specific biomarkers or development of novel approaches beyond traditional blood protein biomarkers. While this study was limited to ovarian cancers of serous histological type and to those arising in BRCA1 mutation carriers specifically, we believe that the results are relevant to other hereditary serous cancers and to sporadic ovarian cancers. A similar approach could be applied to other cancers to aid in defining their early natural history and to guide rational design of an early detection strategy.
Please see later in the article for Editors' Summary
Editors' Summary
Background
Every year about 190,000 women develop ovarian cancer and more than 140,000 die from the disease. Ovarian cancer occurs when a cell on the surface of the ovaries (two small organs in the pelvis that produce eggs) or in the Fallopian tubes (which connect the ovaries to the womb) acquires genetic changes (mutations) that allow it to grow uncontrollably and to spread around the body (metastasize). For women whose cancer is diagnosed when it is confined to the site of origin—ovary or Fallopian tube—(stage I disease), the outlook is good; 70%–80% of these women survive for at least 5 y. However, very few ovarian cancers are diagnosed this early. Usually, by the time the cancer causes symptoms (often only vague abdominal pain and mild digestive disturbances), it has spread into the pelvis (stage II disease), into the space around the gut, stomach, and liver (stage III disease), or to distant organs (stage IV disease). Patients with advanced-stage ovarian cancer are treated with surgery and chemotherapy but, despite recent treatment improvements, only 15% of women diagnosed with stage IV disease survive for 5 y.
Why Was This Study Done?
Most deaths from ovarian cancer are caused by serous ovarian cancer, a tumor subtype that is rarely diagnosed before it has spread. Early detection of serous ovarian cancer would save the lives of many women but no one knows what these cancers look like before they spread or how long they grow before they become clinically apparent. Learning about this occult (hidden) period of ovarian cancer development by observing tumors from their birth to late-stage disease is not feasible. However, some aspects of the early natural history of ovarian cancer can be studied by using data collected from healthy women who have had their ovaries and Fallopian tubes removed (prophylactic bilateral salpingo-oophorectomy [PBSO]) because they have inherited a mutated version of the BRCA1 gene that increases their ovarian cancer risk. In a few of these women, unsuspected ovarian cancer is discovered during PBSO. In this study, the researchers identify and analyze the available reports on occult serous ovarian cancer found this way and then develop mathematical models describing the early natural history of ovarian cancer.
What Did the Researchers Do and Find?
The researchers first estimated the time period during which the detection of occult tumors might save lives using the data from these reports. Serous ovarian cancers, they estimated, spend more than 4 y as in situ (a very early stage of cancer development), stage I, or stage II cancers and about 1 y as stage III and IV cancers before they become clinically apparent. Next, the researchers used the data to develop mathematical models for the growth, progression, and diagnosis of serous ovarian cancer (the accuracy of which depends on the assumptions used to build the models and on the quality of the data fed into them). These models indicated that, for most of the occult period, serous cancers had a diameter of less than 1 cm (too small to be detected during surgery or by gross examination of the ovaries or Fallopian tubes) and that more than half of serous cancers had advanced to stage III/IV by the time they measured 3 cm across. Furthermore, to enable the detection of half of serous ovarian cancers before they reached stage III, an annual screening test would need to detect cancers with a diameter of 1.3 cm and to halve deaths from serous ovarian cancer, an annual screening test would need to detect 0.5-cm diameter tumors.
What Do These Findings Mean?
These findings suggest that the time period over which the early detection of serous ovarian cancer would save lives is surprisingly long. More soberingly, the authors find that a test that is sensitive and specific enough to take advantage of this “window of opportunity” would need to detect tumors hundreds of times smaller than clinically apparent serous cancers. So far no ovarian cancer-specific protein or other biomarker has been identified that could be used to develop a test that comes anywhere near this level of performance. Identification of truly ovarian cancer-specific biomarkers or novel strategies will be needed in order to take advantage of the window of opportunity. The stages prior to clinical presentation of other lethal cancers are still very poorly understood. Similar studies of the early natural history of these cancers could help guide the development of rational early detection strategies.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000114.
The US National Cancer Institute provides a brief description of what cancer is and how it develops and information on all aspects of ovarian cancer for patients and professionals. It also provides a fact sheet on BRCA1 mutations and cancer risk (in English and Spanish)
The UK charity Cancerbackup also provides information about all aspects of ovarian cancer
MedlinePlus provides a list of links to additional information about ovarian cancer (in English and Spanish)
The Canary Foundation is a nonprofit organization dedicated to development of effective strategies for early detection of cancers including ovarian cancer.
doi:10.1371/journal.pmed.1000114
PMCID: PMC2711307  PMID: 19636370
22.  Mutations in a Novel, Cryptic Exon of the Luteinizing Hormone/Chorionic Gonadotropin Receptor Gene Cause Male Pseudohermaphroditism 
PLoS Medicine  2008;5(4):e88.
Background
Male pseudohermaphroditism, or Leydig cell hypoplasia (LCH), is an autosomal recessive disorder in individuals with a 46,XY karyotype, characterized by a predominantly female phenotype, a blind-ending vagina, absence of breast development, primary amenorrhea, and the presence of testicular structures. It is caused by mutations in the luteinizing hormone/chorionic gonadotropin receptor gene (LHCGR), which impair either LH/CG binding or signal transduction. However, molecular analysis has revealed that the LHCGR is apparently normal in about 50% of patients with the full clinical phenotype of LCH. We therefore searched the LHCGR for novel genomic elements causative for LCH.
Methods and Findings
In the present study we have identified a novel, primate-specific bona fide exon (exon 6A) within the LHCGR gene. It displays composite characteristics of an internal/terminal exon and possesses stop codons triggering nonsense-mediated mRNA decay (NMD) in LHCGR. Transcripts including exon 6A are physiologically highly expressed in human testes and granulosa cells, and result in an intracellular, truncated LHCGR protein of 209 amino acids. We sequenced exon 6A in 16 patients with unexplained LCH and detected mutations in three patients. Functional studies revealed a dramatic increase in the expression of the mutated internal exon 6A transcripts, indicating aberrant NMD. These altered ratios of LHCGR transcripts result in the generation of predominantly nonfunctional LHCGR isoforms, thereby preventing proper expression and functioning.
Conclusions
The identification and characterization of this novel exon not only identifies a new regulatory element within the genomic organization of LHCGR, but also points toward a complex network of receptor regulation, including events at the transcriptional level. These findings add to the molecular diagnostic tools for LCH and extend our understanding of the endocrine regulation of sexual differentiation.
Joerg Gromoll and colleagues describe the identification and characterization of a novel exon that appears to be a new regulatory element within the luteinizing hormone/chorionic gonadotropin receptor gene of three individuals with Leydig cell hypoplasia.
Editors' Summary
Background.
A person's sex is determined by their complement of X and Y (sex) chromosomes. Someone who has two X chromosomes is genetically female and usually has ovaries and female external sex organs. Someone who has an X and a Y chromosome is genetically male and has testes and male external sex organs. Sometimes, though, the development of the reproductive organs proceeds abnormally, resulting in a person with an “intersex” condition whose chromosomes, gonads (ovaries or testes), and external sex organs do not correspond. Leydig cell hypoplasia (LCH; also called male pseudohermaphroditism or a disorder of sex development) is an XY female intersex condition. People with this inherited condition develop testes but also have a vagina (which is not connected to a womb), and they do not develop breasts or have periods. This mixture of sexual characteristics arises because the Leydig cells in the testes are underdeveloped. Leydig cells normally secrete testosterone, the hormone that promotes the development and maintenance of male sex characteristics. Before birth, chorionic gonadotropin (CG; a hormone made by the placenta) stimulates Leydig cell development and testosterone production; after birth, luteinizing hormone (LH), which is made by the pituitary gland, stimulates testosterone production. Both hormones bind to the LH/CG receptor, a protein on the surface of Leydig cells. In LCH, this receptor either does not bind CG and LH or fails to tell the Leydig cells to make testosterone.
Why Was This Study Done?
The gene that encodes the LH/CG receptor is called LHCGR. Several mutations (genetic changes) that inactivate the LC/CG receptor have been identified in people with LCH. However, the LHCGR gene is apparently normal in 50% of people with this intersex condition. In this study, the researchers examine the LHCGR gene in detail to try to find the underlying genetic defect in these individuals.
What Did the Researchers Do and Find?
The researchers used several molecular biology techniques to identify a new exon—exon 6A—within the human LHCGR gene. (Exons are DNA sequences that contain the information for making proteins; introns are DNA sequences that interrupt the coding sequence of a gene. Both introns and exons are transcribed into messenger RNA [mRNA] and the exons are then “spliced” together to make the mature mRNA, which is translated into protein.) The researchers identify several differently spliced LHCGR mRNA transcripts that contain exon 6A—a terminal exon 6A mRNA that contains exons 1–6 and exon 6A, and two internal exon 6A mRNAs that also contain exons 7–11. The researchers report that human testes express high levels of the terminal exon 6A transcript, which is translated into a short version of LHCGR protein that remains within the cell (full-length LHCGR moves to the cell surface). By contrast, testes contain low levels of the internal exon 6A mRNAs. This is because exon 6A contains two premature stop codons (DNA sequences that mark the end of a protein), which trigger “nonsense-mediated decay” (NMD), a cellular surveillance mechanism that regulates protein synthesis by degrading mRNAs that contain internal stop codons. When the researchers screened 16 people with LCH but without known mutations in the LHCGR gene, three had mutations in exon 6A. Laboratory experiments show that these mutations greatly increased the amounts of the internal exon 6A transcripts present in cells and interfered with the cells' normal response to chorionic gonadotropin.
What Do These Findings Mean?
These findings identify a new, functional exon in the LHCGR gene and show that mutations in this exon cause some cases of LCH. This is the first time that a human disease has been associated with mutations in an exon that is a target for NMD. In addition, these findings provide important insights into how the LHCGR is regulated. The researchers speculate that a complex network that involves the exon 6A-containing transcripts and NMD normally tightly regulates the production of functional LHCGR already at the transcriptional level. When mutations are present in exon 6A, they suggest, NMD is the predominant pathway for all the exon 6A-containing transcripts, thereby drastically decreasing the amount of functional LHCGR.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050088.
The MedlinePlus Encyclopedia has a page on intersex conditions (in English and Spanish)
Wikipedia has pages on intersexuality and on the LH/CG receptor (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Intersex Society of North America provides information and support for the parents of children with intersex conditions
The Androgen Insensitivity Syndrome Support Group also provides some general information about intersex conditions, including information about LCH and other XY female conditions (in several languages)
Sequence-Structure-Function-Analysis (SSFA), run by a group of researchers in Germany (Leibniz-Institut für Molekulare Pharmakologie; Humboldt-Universitätzu Berlin), is a database dealing the sequence, structure, and function of glycoprotein hormone receptors
Glycoprotein-hormone Receptors Information System (GRIS), from Université Libre de Bruxelles and Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, is a database giving structural information on the LHCGR
doi:10.1371/journal.pmed.0050088
PMCID: PMC2323302  PMID: 18433292
23.  Ecology and evolution of viruses infecting uncultivated SUP05 bacteria as revealed by single-cell- and meta-genomics 
eLife  2014;3:e03125.
Viruses modulate microbial communities and alter ecosystem functions. However, due to cultivation bottlenecks, specific virus–host interaction dynamics remain cryptic. In this study, we examined 127 single-cell amplified genomes (SAGs) from uncultivated SUP05 bacteria isolated from a model marine oxygen minimum zone (OMZ) to identify 69 viral contigs representing five new genera within dsDNA Caudovirales and ssDNA Microviridae. Infection frequencies suggest that ∼1/3 of SUP05 bacteria is viral-infected, with higher infection frequency where oxygen-deficiency was most severe. Observed Microviridae clonality suggests recovery of bloom-terminating viruses, while systematic co-infection between dsDNA and ssDNA viruses posits previously unrecognized cooperation modes. Analyses of 186 microbial and viral metagenomes revealed that SUP05 viruses persisted for years, but remained endemic to the OMZ. Finally, identification of virus-encoded dissimilatory sulfite reductase suggests SUP05 viruses reprogram their host's energy metabolism. Together, these results demonstrate closely coupled SUP05 virus–host co-evolutionary dynamics with the potential to modulate biogeochemical cycling in climate-critical and expanding OMZs.
DOI: http://dx.doi.org/10.7554/eLife.03125.001
eLife digest
Microorganisms help to drive a number of processes that recycle energy and nutrients, including elements such as carbon, nitrogen, and sulfur, around the Earth's ecosystems. Viruses that infect microbes can also affect these cycles by killing and breaking open microbial cells, or by reprogramming the cell's metabolism. However, as there are many different species of microbes and viruses —the vast majority of which cannot easily be grown in the laboratory— little is known about most virus–host interactions in natural ecosystems, especially in the oceans.
In the world's oceans, the concentration of oxygen dissolved in the water changes in different regions and at different depths. ‘Oxygen minimum zones’ occur globally throughout the oceans at depths of 200–1000 meters, and climate change is causing these zones to expand and intensify. Although a lack of oxygen is sometimes considered detrimental to living organisms, oxygen minimum zones appear to be rich with microbial life that is adapted to thrive under oxygen-starved conditions.
Sulfur-oxidizing bacteria are one of the most abundant groups of microbes in these oxygen minimum zones, and several of these bacteria are known to influence the recycling of chemical substances. Now, Roux et al. introduce a new method to identify viruses that infect the microbes in this environment, including those microbes that cannot be grown in the laboratory and which have previously remained largely unexplored.
The genomes of 127 individual bacterial cells —collected from an oxygen minimum zone in western Canada— were examined. Roux et al. estimate that about a third of the sulfur-oxidizing bacterial cells are infected by at least one virus, but often multiple viruses infected the same bacterium. Five new genera (groups of one or more species) of viruses were also discovered and found to infect these bacteria. Looking for these new viral sequences in the DNA of this oxygen minimum zone's microbial community revealed that these newly discovered viruses persist in this region over several years. It also revealed that these viruses appear to only be found within the oxygen minimum zone. Roux et al. uncovered that these viruses carry genes that could manipulate how an infected bacterium processes sulfur-containing compounds; this is similar to previous observations showing that other viruses also influence cellular process (such as photosynthesis) in infected bacteria. As such, these newly discovered viruses might also influence the recycling of chemical elements within oxygen minimum zones.
Together, Roux et al.'s findings provide an unprecedented look into a wild virus community using a method that can be generalized to uncover viruses in a data type that is quickly becoming more widespread: single cell genomes. This effort to understand virus–host interactions by looking in the genomes of individual cells now sets the stage for future efforts aimed to uncover the impact of viruses on bacteria in other environments across the globe.
DOI: http://dx.doi.org/10.7554/eLife.03125.002
doi:10.7554/eLife.03125
PMCID: PMC4164917  PMID: 25171894
SUP05; bacteriophages; viruses; single cell genomics; oxygen minimum zone; viral dark matter; other
24.  A Regulatory Code for Neuron-Specific Odor Receptor Expression 
PLoS Biology  2008;6(5):e125.
Olfactory receptor neurons (ORNs) must select—from a large repertoire—which odor receptors to express. In Drosophila, most ORNs express one of 60 Or genes, and most Or genes are expressed in a single ORN class in a process that produces a stereotyped receptor-to-neuron map. The construction of this map poses a problem of receptor gene regulation that is remarkable in its dimension and about which little is known. By using a phylogenetic approach and the genome sequences of 12 Drosophila species, we systematically identified regulatory elements that are evolutionarily conserved and specific for individual Or genes of the maxillary palp. Genetic analysis of these elements supports a model in which each receptor gene contains a zip code, consisting of elements that act positively to promote expression in a subset of ORN classes, and elements that restrict expression to a single ORN class. We identified a transcription factor, Scalloped, that mediates repression. Some elements are used in other chemosensory organs, and some are conserved upstream of axon-guidance genes. Surprisingly, the odor response spectra and organization of maxillary palp ORNs have been extremely well-conserved for tens of millions of years, even though the amino acid sequences of the receptors are not highly conserved. These results, taken together, define the logic by which individual ORNs in the maxillary palp select which odor receptors to express.
Author Summary
Odors are detected by olfactory receptor neurons (ORNs). Which odor an individual neuron detects is dictated by the odor receptors it expresses. Odor receptors are encoded by large families of genes, and an individual neuron must thus select the gene it expresses from among many possibilities. The mechanism underlying this choice is largely unknown. We have examined the problem of receptor gene choice in the fruit fly Drosophila, whose maxillary palp contains six functional classes of ORNs, each expressing different odor receptor genes. By comparing the DNA sequences flanking these genes in 12 different species of Drosophila, we have identified regulatory elements that are evolutionarily conserved and specific to each odor receptor. Genetic analysis of these elements showed that some act positively to dictate expression in a subset of ORNs, while others act negatively to restrict the expression of a receptor gene to a particular ORN class. We identified a transcription factor, Scalloped, that mediates repression. We were surprised to find that the odor response spectra of these neurons have been well-conserved for tens of millions of years, even though the amino acid sequences of their receptors have diverged considerably.
How does an olfactory receptor neuron select which odor receptor to express? A computational analysis of 12Drosophila genomes combined with mutational analysis identifies conservedcis elements and defines a regulatory code.
doi:10.1371/journal.pbio.0060125
PMCID: PMC2430909  PMID: 18846726
25.  Genetic Basis for Spontaneous Hybrid Genome Doubling during Allopolyploid Speciation of Common Wheat Shown by Natural Variation Analyses of the Paternal Species 
PLoS ONE  2013;8(8):e68310.
The complex process of allopolyploid speciation includes various mechanisms ranging from species crosses and hybrid genome doubling to genome alterations and the establishment of new allopolyploids as persisting natural entities. Currently, little is known about the genetic mechanisms that underlie hybrid genome doubling, despite the fact that natural allopolyploid formation is highly dependent on this phenomenon. We examined the genetic basis for the spontaneous genome doubling of triploid F1 hybrids between the direct ancestors of allohexaploid common wheat (Triticum aestivum L., AABBDD genome), namely Triticumturgidum L. (AABB genome) and Aegilopstauschii Coss. (DD genome). An Ae. tauschii intraspecific lineage that is closely related to the D genome of common wheat was identified by population-based analysis. Two representative accessions, one that produces a high-genome-doubling-frequency hybrid when crossed with a T. turgidum cultivar and the other that produces a low-genome-doubling-frequency hybrid with the same cultivar, were chosen from that lineage for further analyses. A series of investigations including fertility analysis, immunostaining, and quantitative trait locus (QTL) analysis showed that (1) production of functional unreduced gametes through nonreductional meiosis is an early step key to successful hybrid genome doubling, (2) first division restitution is one of the cytological mechanisms that cause meiotic nonreduction during the production of functional male unreduced gametes, and (3) six QTLs in the Ae. tauschii genome, most of which likely regulate nonreductional meiosis and its subsequent gamete production processes, are involved in hybrid genome doubling. Interlineage comparisons of Ae. tauschii’s ability to cause hybrid genome doubling suggested an evolutionary model for the natural variation pattern of the trait in which non-deleterious mutations in six QTLs may have important roles. The findings of this study demonstrated that the genetic mechanisms for hybrid genome doubling could be studied based on the intrinsic natural variation that exists in the parental species.
doi:10.1371/journal.pone.0068310
PMCID: PMC3738567  PMID: 23950867

Results 1-25 (1260851)