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1.  Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors 
BMC Biology  2015;13:55.
Background
Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades.
Results
We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage.
Conclusions
These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0163-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0163-z
PMCID: PMC4521377  PMID: 26231587
2.  Kinetic discrimination of self/non-self RNA by the ATPase activity of RIG-I and MDA5 
BMC Biology  2015;13:54.
Background
The cytoplasmic RIG-like receptors are responsible for the early detection of viruses and other intracellular microbes by activating the innate immune response mediated by type I interferons (IFNs). RIG-I and MDA5 detect virus-specific RNA motifs with short 5′-tri/diphosphorylated, blunt-end double-stranded RNA (dsRNA) and >0.5–2 kb long dsRNA as canonical agonists, respectively. However, in vitro, they can bind to many RNA species, while in cells there is an activation threshold. As SF2 helicase/ATPase family members, ATP hydrolysis is dependent on co-operative RNA and ATP binding. Whereas simultaneous ATP and cognate RNA binding is sufficient to activate RIG-I by releasing autoinhibition of the signaling domains, the physiological role of the ATPase activity of RIG-I and MDA5 remains controversial.
Results
A cross-analysis of a rationally designed panel of RNA binding and ATPase mutants and truncated receptors, using type I IFN promoter activation as readout, allows us to refine our understanding of the structure-function relationships of RIG-I and MDA5. RNA activation of RIG-I depends on multiple critical RNA binding sites in its helicase domain as confirmed by functional evidence using novel mutations. We found that RIG-I or MDA5 mutants with low ATP hydrolysis activity exhibit constitutive activity but this was fully reverted when associated with mutations preventing RNA binding to the helicase domain. We propose that the turnover kinetics of the ATPase domain enables the discrimination of self/non-self RNA by both RIG-I and MDA5. Non-cognate, possibly self, RNA binding would lead to fast ATP turnover and RNA disassociation and thus insufficient time for the caspase activation and recruitment domains (CARDs) to promote downstream signaling, whereas tighter cognate RNA binding provides a longer time window for downstream events to be engaged.
Conclusions
The exquisite fine-tuning of RIG-I and MDA5 RNA-dependent ATPase activity coupled to CARD release allows a robust IFN response from a minor subset of non-self RNAs within a sea of cellular self RNAs. This avoids the eventuality of deleterious autoimmunity effects as have been recently described to arise from natural gain-of-function alleles of RIG-I and MDA5.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0166-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0166-9
PMCID: PMC4517655  PMID: 26215161
RIG-I; MDA5; dsRNA; ATPase; self from non-self
3.  Poised chromatin and bivalent domains facilitate the mitosis-to-meiosis transition in the male germline 
BMC Biology  2015;13:53.
Background
The male germline transcriptome changes dramatically during the mitosis-to-meiosis transition to activate late spermatogenesis genes and to transiently suppress genes commonly expressed in somatic lineages and spermatogenesis progenitor cells, termed somatic/progenitor genes.
Results
These changes reflect epigenetic regulation. Induction of late spermatogenesis genes during spermatogenesis is facilitated by poised chromatin established in the stem cell phases of spermatogonia, whereas silencing of somatic/progenitor genes during meiosis and postmeiosis is associated with formation of bivalent domains which also allows the recovery of the somatic/progenitor program after fertilization. Importantly, during spermatogenesis mechanisms of epigenetic regulation on sex chromosomes are different from autosomes: X-linked somatic/progenitor genes are suppressed by meiotic sex chromosome inactivation without deposition of H3K27me3.
Conclusions
Our results suggest that bivalent H3K27me3 and H3K4me2/3 domains are not limited to developmental promoters (which maintain bivalent domains that are silent throughout the reproductive cycle), but also underlie reversible silencing of somatic/progenitor genes during the mitosis-to-meiosis transition in late spermatogenesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0159-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0159-8
PMCID: PMC4508805  PMID: 26198001
Germ cells; Epigenome; Sex chromosomes; Meiosis; Spermatogenesis
4.  Quantification of gait parameters in freely walking rodents 
BMC Biology  2015;13:50.
Background
Qualitative and quantitative measurements of motor performance are essential for characterizing perturbations of motor systems. Although several methods exist for analyzing specific motor tasks, few behavioral assays are readily available to researchers that provide a complete set of kinematic parameters in rodents.
Results
Here we present MouseWalker, an integrated hardware and software system that provides a comprehensive and quantitative description of kinematic features in freely walking rodents. Footprints are visualized with high spatial and temporal resolution by a non-invasive optical touch sensor coupled to high-speed imaging. A freely available and open-source software package tracks footprints and body features to generate a comprehensive description of many locomotion features, including static parameters such as footprint position and stance patterns and dynamic parameters, such as step and swing cycle duration, and inter-leg coordination. Using this method, we describe walking by wild-type mice including several previously undescribed parameters. For example, we demonstrate that footprint touchdown occurs instantaneously by the entire paw with no obvious rostral–caudal or lateral–medial bias.
Conclusions
The readily available MouseWalker system and the large set of readouts it generates greatly increases the currently available toolkit for the analysis of wild type and aberrant locomotion in rodents.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0154-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0154-0
PMCID: PMC4511453  PMID: 26197889
5.  Macrolides rapidly inhibit red blood cell invasion by the human malaria parasite, Plasmodium falciparum 
BMC Biology  2015;13:52.
Background
Malaria invasion of red blood cells involves multiple parasite-specific targets that are easily accessible to inhibitory compounds, making it an attractive target for antimalarial development. However, no current antimalarial agents act against host cell invasion.
Results
Here, we demonstrate that the clinically used macrolide antibiotic azithromycin, which is known to kill human malaria asexual blood-stage parasites by blocking protein synthesis in their apicoplast, is also a rapid inhibitor of red blood cell invasion in human (Plasmodium falciparum) and rodent (P. berghei) malarias. Multiple lines of evidence demonstrate that the action of azithromycin in inhibiting parasite invasion of red blood cells is independent of its inhibition of protein synthesis in the parasite apicoplast, opening up a new strategy to develop a single drug with multiple parasite targets. We identified derivatives of azithromycin and erythromycin that are better invasion inhibitors than parent compounds, offering promise for development of this novel antimalarial strategy.
Conclusions
Safe and effective macrolide antibiotics with dual modalities could be developed to combat malaria and reduce the parasite’s options for resistance.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0162-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0162-0
PMCID: PMC4506589  PMID: 26187647
Plasmodium; Merozoite; Invasion; Macrolide; Dual modality
6.  Pest control and resistance management through release of insects carrying a male-selecting transgene 
BMC Biology  2015;13:49.
Background
Development and evaluation of new insect pest management tools is critical for overcoming over-reliance upon, and growing resistance to, synthetic, biological and plant-expressed insecticides. For transgenic crops expressing insecticidal proteins from the bacterium Bacillus thuringiensis (‘Bt crops’) emergence of resistance is slowed by maintaining a proportion of the crop as non-Bt varieties, which produce pest insects unselected for resistance. While this strategy has been largely successful, multiple cases of Bt resistance have now been reported.
One new approach to pest management is the use of genetically engineered insects to suppress populations of their own species. Models suggest that released insects carrying male-selecting (MS) transgenes would be effective agents of direct, species-specific pest management by preventing survival of female progeny, and simultaneously provide an alternative insecticide resistance management strategy by introgression of susceptibility alleles into target populations. We developed a MS strain of the diamondback moth, Plutella xylostella, a serious global pest of crucifers. MS-strain larvae are reared as normal with dietary tetracycline, but, when reared without tetracycline or on host plants, only males will survive to adulthood. We used this strain in glasshouse-cages to study the effect of MS male P. xylostella releases on target pest population size and spread of Bt resistance in these populations.
Results
Introductions of MS-engineered P. xylostella males into wild-type populations led to rapid pest population decline, and then elimination. In separate experiments on broccoli plants, relatively low-level releases of MS males in combination with broccoli expressing Cry1Ac (Bt broccoli) suppressed population growth and delayed the spread of Bt resistance. Higher rates of MS male releases in the absence of Bt broccoli were also able to suppress P. xylostella populations, whereas either low-level MS male releases or Bt broccoli alone did not.
Conclusions
These results support theoretical modeling, indicating that MS-engineered insects can provide a powerful pest population suppressing effect, and could effectively augment current Bt resistance management strategies. We conclude that, subject to field confirmation, MS insects offer an effective and versatile control option against P. xylostella and potentially other pests, and may reduce reliance on and protect insecticide-based approaches, including Bt crops.
doi:10.1186/s12915-015-0161-1
PMCID: PMC4504119  PMID: 26179401
Bacillus thuringiensis; Diamondback moth; Insect; Insecticide resistance management; Pest; Plutella xylostella; Sterile insect technique; Transgenic
7.  Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor 
BMC Biology  2015;13:48.
Background
Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery.
Results
Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation.
Conclusions
With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a “proteinous drill” for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0160-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0160-2
PMCID: PMC4495774  PMID: 26152191
Bacillus thuringiensis; Bombyx mori; Cry toxin; Drosophila melanogaster; Genetic ablation; Necrosis
8.  Mechanotransduction: use the force(s) 
BMC Biology  2015;13:47.
Mechanotransduction - how cells sense physical forces and translate them into biochemical and biological responses - is a vibrant and rapidly-progressing field, and is important for a broad range of biological phenomena. This forum explores the role of mechanotransduction in a variety of cellular activities and highlights intriguing questions that deserve further attention.
doi:10.1186/s12915-015-0150-4
PMCID: PMC4491211  PMID: 26141078
9.  Models in biology: lessons from modeling regulation of the eukaryotic cell cycle 
BMC Biology  2015;13:46.
In this essay we illustrate some general principles of mathematical modeling in biology by our experiences in studying the molecular regulatory network underlying eukaryotic cell division. We discuss how and why the models moved from simple, parsimonious cartoons to more complex, detailed mechanisms with many kinetic parameters. We describe how the mature models made surprising and informative predictions about the control system that were later confirmed experimentally. Along the way, we comment on the ‘parameter estimation problem’ and conclude with an appeal for a greater role for mathematical models in molecular cell biology.
doi:10.1186/s12915-015-0158-9
PMCID: PMC4486427  PMID: 26129844
Mathematical models; Mitosis-promoting factor; Cell cycle checkpoints; Regulated kinases; Regulated phosphatases; Regulated proteolysis
10.  Q&A: How do plants sense and respond to UV-B radiation? 
BMC Biology  2015;13:45.
Plants are able to sense UV-B through the UV-B photoreceptor UVR8. UV-B photon absorption by a UVR8 homodimer leads to UVR8 monomerization and interaction with the downstream signaling factor COP1. This then initiates changes in gene expression, which lead to several metabolic and morphological alterations. A major response is the activation of mechanisms associated with UV-B acclimation and UV-B tolerance, including biosynthesis of sunscreen metabolites, antioxidants and DNA repair enzymes. To balance the response, UVR8 is inactivated by regulated re-dimerization. Apart from their importance for plants, UVR8 and its interacting protein COP1 have already proved useful for the optogenetic toolkit used to engineer synthetic light-dependent responses.
doi:10.1186/s12915-015-0156-y
PMCID: PMC4484705  PMID: 26123292
11.  Sexual conflict maintains variation at an insecticide resistance locus 
BMC Biology  2015;13:34.
Background
The maintenance of genetic variation through sexually antagonistic selection is controversial, partly because specific sexually-antagonistic alleles have not been identified. The Drosophila DDT resistance allele (DDT-R) is an exception. This allele increases female fitness, but simultaneously decreases male fitness, and it has been suggested that this sexual antagonism could explain why polymorphism was maintained at the locus prior to DDT use. We tested this possibility using a genetic model and then used evolving fly populations to test model predictions.
Results
Theory predicted that sexual antagonism is able to maintain genetic variation at this locus, hence explaining why DDT-R did not fix prior to DDT use despite increasing female fitness, and experimentally evolving fly populations verified theoretical predictions.
Conclusions
This demonstrates that sexually antagonistic selection can maintain genetic variation and explains the DDT-R frequencies observed in nature.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0143-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0143-3
PMCID: PMC4484701  PMID: 26032845
12.  The role of juvenile hormone and insulin/TOR signaling in the growth of Manduca sexta 
BMC Biology  2015;13:44.
Background
In many insect species, fitness trade-offs exist between maximizing body size and developmental speed. Understanding how various species evolve different life history strategies requires knowledge of the physiological mechanisms underlying the regulation of body size and developmental timing. Here the roles of juvenile hormone (JH) and insulin/target of rapamycin (TOR) signaling in the regulation of the final body size were examined in the tobacco hornworm, Manduca sexta.
Results
Feeding rapamycin to wild-type larvae decreased the growth rate but did not alter the peak size of the larvae. In contrast, feeding rapamycin to the JH-deficient black mutant larvae caused the larvae to significantly increase the peak size relative to the DMSO-fed control animals by lengthening the terminal growth period. Furthermore, the critical weight was unaltered by feeding rapamycin, indicating that in Manduca, the critical weight is not influenced by insulin/TOR signaling. In addition, post-critical weight starved black mutant Manduca given rapamycin underwent metamorphosis sooner than those that were fed, mimicking the “bail-out mechanism”.
Conclusions
Our study demonstrates that JH masks the effects of insulin/TOR signaling in the determination of the final body size and that the critical weights in Drosophila and Manduca rely on distinct mechanisms that reflect different life history strategies. Our study also suggests that TOR signaling lengthens the terminal growth period in Manduca as it does in Drosophila, and that JH levels determine the relative contributions of nutrient- and body size-sensing pathways to metamorphic timing.
doi:10.1186/s12915-015-0155-z
PMCID: PMC4499214  PMID: 26108483
Developmental timing; Critical weight; Life history trade-offs; Manduca sexta; Drosophila melanogaster; Metamorphosis; Insulin/TOR signaling; Juvenile hormone; Prothoracic gland; Molt timer
13.  Shell neurons of the master circadian clock coordinate the phase of tissue clocks throughout the brain and body 
BMC Biology  2015;13:43.
Background
Daily rhythms in mammals are programmed by a master clock in the suprachiasmatic nucleus (SCN). The SCN contains two main compartments (shell and core), but the role of each region in system-level coordination remains ill defined. Herein, we use a functional assay to investigate how downstream tissues interpret region-specific outputs by using in vivo exposure to long day photoperiods to temporally dissociate the SCN. We then analyze resulting changes in the rhythms of clocks located throughout the brain and body to examine whether they maintain phase synchrony with the SCN shell or core.
Results
Nearly all of the 17 tissues examined in the brain and body maintain phase synchrony with the SCN shell, but not the SCN core, which indicates that downstream oscillators are set by cues controlled specifically by the SCN shell. Interestingly, we also found that SCN dissociation diminished the amplitude of rhythms in core clock gene and protein expression in brain tissues by 50–75 %, which suggests that light-driven changes in the functional organization of the SCN markedly influence the strength of rhythms in downstream tissues.
Conclusions
Overall, our results reveal that body clocks receive time-of-day cues specifically from the SCN shell, which may be an adaptive design principle that serves to maintain system-level phase relationships in a changing environment. Further, we demonstrate that lighting conditions alter the amplitude of the molecular clock in downstream tissues, which uncovers a new form of plasticity that may contribute to seasonal changes in physiology and behavior.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0157-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0157-x
PMCID: PMC4489020  PMID: 26099272
Circadian; Coupling; Peripheral and central oscillators; Photoperiod; Suprachiasmatic nucleus
14.  Sequencing strategies and characterization of 721 vervet monkey genomes for future genetic analyses of medically relevant traits 
BMC Biology  2015;13:41.
Background
We report here the first genome-wide high-resolution polymorphism resource for non-human primate (NHP) association and linkage studies, constructed for the Caribbean-origin vervet monkey, or African green monkey (Chlorocebus aethiops sabaeus), one of the most widely used NHPs in biomedical research. We generated this resource by whole genome sequencing (WGS) of monkeys from the Vervet Research Colony (VRC), an NIH-supported research resource for which extensive phenotypic data are available.
Results
We identified genome-wide single nucleotide polymorphisms (SNPs) by WGS of 721 members of an extended pedigree from the VRC. From high-depth WGS data we identified more than 4 million polymorphic unequivocal segregating sites; by pruning these SNPs based on heterozygosity, quality control filters, and the degree of linkage disequilibrium (LD) between SNPs, we constructed genome-wide panels suitable for genetic association (about 500,000 SNPs) and linkage analysis (about 150,000 SNPs). To further enhance the utility of these resources for linkage analysis, we used a further pruned subset of the linkage panel to generate multipoint identity by descent matrices.
Conclusions
The genetic and phenotypic resources now available for the VRC and other Caribbean-origin vervets enable their use for genetic investigation of traits relevant to human diseases.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0152-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0152-2
PMCID: PMC4494155  PMID: 26092298
Vervet; Non-human primate; Whole genome sequencing; SNP; Linkage; Association
15.  Lhx8 regulates primordial follicle activation and postnatal folliculogenesis 
BMC Biology  2015;13:39.
Background
The early stages of ovarian follicle formation—beginning with the breakdown of germ cell cysts and continuing with the formation of primordial follicles and transition to primary and secondary follicles—are critical in determining reproductive life span and fertility. Previously, we discovered that global knockouts of germ cell-specific transcriptional co-regulators Sohlh1, Sohlh2, Lhx8, and Nobox, cause rapid oocyte loss and ovarian failure. Also factors such as Nobox and Sohlh1 are associated with human premature ovarian failure. In this study, we developed a conditional knockout of Lhx8 to study oocyte-specific pathways in postnatal folliculogenesis.
Results
The conditional deficiency of Lhx8 in the oocytes of primordial follicles leads to massive primordial oocyte activation, in part, by indirectly interacting with the PI3K-AKT pathway, as shown by synergistic effects on FOXO3 nucleocytoplasmic translocation and rpS6 activation. However, LHX8 does not directly regulate members of the PI3K-AKT pathway; instead, we show that LHX8 represses Lin28a expression, a known regulator of mammalian metabolism and of the AKT/mTOR pathway. LHX8 can bind to the Lin28a promoter, and the depletion of Lin28a in Lhx8-deficient oocytes partially suppresses primordial oocyte activation. Moreover, unlike the PI3K-AKT pathway, LHX8 is critical beyond primordial follicle activation, and blocks the primary to secondary follicle transition.
Conclusions
Our results indicate that the LHX8-LIN28A pathway is essential in the earliest stages of primordial follicle activation, and LHX8 is an important oocyte-specific transcription factor in the ovary for regulating postnatal folliculogenesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0151-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0151-3
PMCID: PMC4487509  PMID: 26076587
Lhx8; Primordial follicle; Oocyte; Lin28a; Ovarian reserve
16.  Layering genetic circuits to build a single cell, bacterial half adder 
BMC Biology  2015;13:40.
Background
Gene regulation in biological systems is impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit. Here, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of compiling a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits.
Results
Using a synthetic biology approach that is supported by computational modelling and rigorous characterisation, AND, OR and NOT biological logic gates were layered in both parallel and serial arrangements to generate a repertoire of Boolean operations that include NIMPLY, XOR, half adder and half subtractor logics in a single cell. Subsequent evaluation of these near-digital biological systems revealed critical design pitfalls that triggered genetic context-dependent effects, including 5′ UTR interferences and uncontrolled switch-on behaviour of the supercoiled σ54 promoter. In particular, the presence of seven consecutive hairpins immediately downstream of the promoter transcription start site severely impeded gene expression.
Conclusions
As synthetic biology moves forward with greater focus on scaling the complexity of engineered genetic circuits, studies which thoroughly evaluate failure modes and engineering solutions will serve as important references for future design and development of synthetic biological systems. This work describes a representative case study for the debugging of genetic context-dependent effects through principles elucidated herein, thereby providing a rational design framework to integrate multiple genetic circuits in a single prokaryotic cell.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0146-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0146-0
PMCID: PMC4490610  PMID: 26078033
17.  DNA transposons have colonized the genome of the giant virus Pandoravirus salinus 
BMC Biology  2015;13:38.
Background
Transposable elements are mobile DNA sequences that are widely distributed in prokaryotic and eukaryotic genomes, where they represent a major force in genome evolution. However, transposable elements have rarely been documented in viruses, and their contribution to viral genome evolution remains largely unexplored. Pandoraviruses are recently described DNA viruses with genome sizes that exceed those of some prokaryotes, rivaling parasitic eukaryotes. These large genomes appear to include substantial noncoding intergenic spaces, which provide potential locations for transposable element insertions. However, no mobile genetic elements have yet been reported in pandoravirus genomes.
Results
Here, we report a family of miniature inverted-repeat transposable elements (MITEs) in the Pandoravirus salinus genome, representing the first description of a virus populated with a canonical transposable element family that proliferated by transposition within the viral genome. The MITE family, which we name Submariner, includes 30 copies with all the hallmarks of MITEs: short length, terminal inverted repeats, TA target site duplication, and no coding capacity. Submariner elements show signs of transposition and are undetectable in the genome of Pandoravirus dulcis, the closest known relative Pandoravirus salinus. We identified a DNA transposon related to Submariner in the genome of Acanthamoeba castellanii, a species thought to host pandoraviruses, which contains remnants of coding sequence for a Tc1/mariner transposase. These observations suggest that the Submariner MITEs of P. salinus belong to the widespread Tc1/mariner superfamily and may have been mobilized by an amoebozoan host. Ten of the 30 MITEs in the P. salinus genome are located within coding regions of predicted genes, while others are close to genes, suggesting that these transposons may have contributed to viral genetic novelty.
Conclusions
Our discovery highlights the remarkable ability of DNA transposons to colonize and shape genomes from all domains of life, as well as giant viruses. Our findings continue to blur the division between viral and cellular genomes, adhering to the emerging view that the content, dynamics, and evolution of the genomes of giant viruses do not substantially differ from those of cellular organisms.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0145-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0145-1
PMCID: PMC4495683  PMID: 26067596
Genome evolution; Miniature inverted-repeat transposable element (MITE); Virus
18.  The initiation of nocturnal dormancy in Synechococcus as an active process 
BMC Biology  2015;13:36.
Background
Most organisms, especially photoautotrophs, alter their behaviours in response to day–night alternations adaptively because of their great reliance on light. Upon light-to-dark transition, dramatic and universal decreases in transcription level of the majority of the genes in the genome of the unicellular cyanobacterium, Synechococcus elongatus PCC 7942 are observed. Because Synechococcus is an obligate photoautotroph, it has been generally assumed that repression of the transcription in the dark (dark repression) would be caused by a nocturnal decrease in photosynthetic activities through the reduced availability of energy (e.g. adenosine triphosphate (ATP)) needed for mRNA synthesis.
Results
However, against this general assumption, we obtained evidence that the rapid and dynamic dark repression is an active process. Although the addition of photosynthesis inhibitors to cells exposed to light mimicked transcription profiles in the dark, it did not significantly affect the cellular level of ATP. By contrast, when ATP levels were decreased by the inhibition of both photosynthesis and respiration, the transcriptional repression was attenuated through inhibition of RNA degradation. This observation indicates that Synechococcus actively downregulates genome-wide transcription in the dark. Even though the level of total mRNA dramatically decreased in the dark, Synechococcus cells were still viable, and they do not need de novo transcription for their survival in the dark for at least 48 hours.
Conclusions
Dark repression appears to enable cells to enter into nocturnal dormancy as a feed-forward process, which would be advantageous for their survival under periodic nocturnal conditions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0144-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0144-2
PMCID: PMC4494158  PMID: 26058805
Cyanobacteria; Light/Dark; Transcription; Feed-forward regulation
19.  Nuclear myosin 1 contributes to a chromatin landscape compatible with RNA polymerase II transcription activation 
BMC Biology  2015;13:35.
Background
Nuclear myosin 1c (NM1) is emerging as a regulator of transcription and chromatin organization.
Results
Using chromatin immunoprecipitation and deep sequencing (ChIP-Seq) in combination with molecular analyses, we investigated the global association of NM1 with the mammalian genome. Analysis of the ChIP-Seq data demonstrates that NM1 binds across the entire mammalian genome with occupancy peaks correlating with distributions of RNA Polymerase II (Pol II) and active epigenetic marks at class II gene promoters. In mouse embryonic fibroblasts subjected to RNAi mediated NM1 gene silencing, we show that NM1 synergizes with polymerase-associated actin to maintain active Pol II at the promoter. NM1 also co-localizes with the nucleosome remodeler SNF2h at class II promoters where they assemble together with WSTF as part of the B-WICH complex. A high resolution micrococcal nuclease (MNase) assay and quantitative real time PCR shows that this mechanism is required for local chromatin remodeling. Following B-WICH assembly, NM1 mediates physical recruitment of the histone acetyl transferase PCAF and the histone methyl transferase Set1/Ash2 to maintain and preserve H3K9acetylation and H3K4trimethylation for active transcription.
Conclusions
We propose a novel genome-wide mechanism where myosin synergizes with Pol II-associated actin to link the polymerase machinery with permissive chromatin for transcription activation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0147-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0147-z
PMCID: PMC4486089  PMID: 26044184
RNA polymerase II transcription; NM1; Epigenetics; Genome-wide analysis
20.  Q&A: insulin secretion and type 2 diabetes: why do β-cells fail? 
BMC Biology  2015;13:33.
doi:10.1186/s12915-015-0140-6
PMCID: PMC4435650  PMID: 25982967
21.  A dynamic evolutionary and functional landscape of plant phased small interfering RNAs 
BMC Biology  2015;13:32.
Background
Secondary, phased small interfering RNAs (phasiRNAs) derived from protein-coding or noncoding loci (PHAS) are emerging as a new type of regulators of gene expression in plants. However, the evolution and function of these novel siRNAs in plant species remain largely unexplored.
Results
We systematically analyzed PHAS loci in 23 plant species covering major phylogenetic groups spanning alga, moss, gymnosperm, basal angiosperm, monocot, and dicot. We identified over 3,300 PHAS loci, among which ~1,600 were protein-coding genes. Most of these PHAS loci were novel and clade- or species-specific and showed distinct expression patterns in association with particular development stages, viral infection, or abiotic stresses. Unexpectedly, numerous PHAS loci produced phasiRNAs from introns or exon–intron junction regions. Our comprehensive analysis suggests that phasiRNAs predominantly regulate protein-coding genes from which they are derived and genes from the same families of the phasiRNA-deriving genes, in contrast to the dominant trans-regulatory mode of miRNAs. The stochastic occurrence of many PHAS loci in the plant kingdom suggests their young evolutionary origins.
Conclusions
Our study discovered an unprecedented diversity of protein-coding genes that produce phasiRNAs in a wide variety of plants, and set a kingdom-wide foundation for investigating the novel roles of phasiRNAs in shaping phenotype diversities of plants.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0142-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0142-4
PMCID: PMC4457045  PMID: 25980406
Evolution; Function; Plant phasiRNAs; Protein-coding genes
22.  A chromatin code for alternative splicing involving a putative association between CTCF and HP1α proteins 
BMC Biology  2015;13:31.
Background
Alternative splicing is primarily controlled by the activity of splicing factors and by the elongation of the RNA polymerase II (RNAPII). Recent experiments have suggested a new complex network of splicing regulation involving chromatin, transcription and multiple protein factors. In particular, the CCCTC-binding factor (CTCF), the Argonaute protein AGO1, and members of the heterochromatin protein 1 (HP1) family have been implicated in the regulation of splicing associated with chromatin and the elongation of RNAPII. These results raise the question of whether these proteins may associate at the chromatin level to modulate alternative splicing.
Results
Using chromatin immunoprecipitation sequencing (ChIP-Seq) data for CTCF, AGO1, HP1α, H3K27me3, H3K9me2, H3K36me3, RNAPII, total H3 and 5metC and alternative splicing arrays from two cell lines, we have analyzed the combinatorial code of their binding to chromatin in relation to the alternative splicing patterns between two cell lines, MCF7 and MCF10. Using Machine Learning techniques, we identified the changes in chromatin signals that are most significantly associated with splicing regulation between these two cell lines. Moreover, we have built a map of the chromatin signals on the pre-mRNA, that is, a chromatin-based RNA-map, which can explain 606 (68.55%) of the regulated events between MCF7 and MCF10. This chromatin code involves the presence of HP1α, CTCF, AGO1, RNAPII and histone marks around regulated exons and can differentiate patterns of skipping and inclusion. Additionally, we found a significant association of HP1α and CTCF activities around the regulated exons and a putative DNA binding site for HP1α.
Conclusions
Our results show that a considerable number of alternative splicing events could have a chromatin-dependent regulation involving the association of HP1α and CTCF near regulated exons. Additionally, we find further evidence for the involvement of HP1α and AGO1 in chromatin-related splicing regulation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0141-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0141-5
PMCID: PMC4446157  PMID: 25934638
Chromatin; Splicing; Histones; Splicing code
23.  Expression of leukemia inhibitory factor in Müller glia cells is regulated by a redox-dependent mRNA stability mechanism 
BMC Biology  2015;13:30.
Background
Photoreceptor degeneration is a main hallmark of many blinding diseases making protection of photoreceptors crucial to prevent vision loss. Thus, regulation of endogenous neuroprotective factors may be key for cell survival and attenuation of disease progression. Important neuroprotective factors in the retina include H2O2 generated by injured photoreceptors, and leukemia inhibitory factor (LIF) expressed in Müller glia cells in response to photoreceptor damage.
Results
We present evidence that H2O2 connects to the LIF response by inducing stabilization of Lif transcripts in Müller cells. This process was independent of active gene transcription and p38 MAPK, but relied on AU-rich elements (AREs), which we identified within the highly conserved Lif 3′UTR. Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs. Among those, interleukin enhancer binding factor 3 (ILF3) was confirmed to participate in the redox-dependent Lif mRNA stabilization. Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells.
Conclusions
Our results suggest that H2O2-induced redox signaling increases Lif transcript levels through ILF3 mediated mRNA stabilization. Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0137-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0137-1
PMCID: PMC4462110  PMID: 25907681
LIF; Redox signaling; mRNA stability; Retina; Müller glial cells; ILF3; KHSRP; p38 MAPK; Neuroprotection
24.  The study of Priapulus caudatus reveals conserved molecular patterning underlying different gut morphogenesis in the Ecdysozoa 
BMC Biology  2015;13:29.
Background
The digestive systems of animals can become highly specialized in response to their exploration and occupation of new ecological niches. Although studies on different animals have revealed commonalities in gut formation, the model systems Caenorhabditis elegans and Drosophila melanogaster, which belong to the invertebrate group Ecdysozoa, exhibit remarkable deviations in how their intestines develop. Their morphological and developmental idiosyncrasies have hindered reconstructions of ancestral gut characters for the Ecdysozoa, and limit comparisons with vertebrate models. In this respect, the phylogenetic position, and slow evolving morphological and molecular characters of marine priapulid worms advance them as a key group to decipher evolutionary events that occurred in the lineages leading to C. elegans and D. melanogaster.
Results
In the priapulid Priapulus caudatus, the gut consists of an ectodermal foregut and anus, and a mid region of at least partial endodermal origin. The inner gut develops into a 16-cell primordium devoid of visceral musculature, arranged in three mid tetrads and two posterior duplets. The mouth invaginates ventrally and shifts to a terminal anterior position as the ventral anterior ectoderm differentially proliferates. Contraction of the musculature occurs as the head region retracts into the trunk and resolves the definitive larval body plan. Despite obvious developmental differences with C. elegans and D. melanogaster, the expression in P. caudatus of the gut-related candidate genes NK2.1, foxQ2, FGF8/17/18, GATA456, HNF4, wnt1, and evx demonstrate three distinct evolutionarily conserved molecular profiles that correlate with morphologically identified sub-regions of the gut.
Conclusions
The comparative analysis of priapulid development suggests that a midgut formed by a single endodermal population of vegetal cells, a ventral mouth, and the blastoporal origin of the anus are ancestral features in the Ecdysozoa. Our molecular data on P. caudatus reveal a conserved ecdysozoan gut-patterning program and demonstrates that extreme morphological divergence has not been accompanied by major molecular innovations in transcriptional regulators during digestive system evolution in the Ecdysozoa. Our data help us understand the origins of the ecdysozoan body plan, including those of C. elegans and D. melanogaster, and this is critical for comparisons between these two prominent model systems and their vertebrate counterparts.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0139-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0139-z
PMCID: PMC4434581  PMID: 25895830
C. elegans; Drosophila; Ecdysozoa; Endoderm; Gut development; Hindgut; Mesoderm; Midgut; Mouth; Priapulid
25.  Dissection of jasmonate functions in tomato stamen development by transcriptome and metabolome analyses 
BMC Biology  2015;13:28.
Background
Jasmonates are well known plant signaling components required for stress responses and development. A prominent feature of jasmonate biosynthesis or signaling mutants is the loss of fertility. In contrast to the male sterile phenotype of Arabidopsis mutants, the tomato mutant jai1-1 exhibits female sterility with additional severe effects on stamen and pollen development. Its senescence phenotype suggests a function of jasmonates in regulation of processes known to be mediated by ethylene. To test the hypothesis that ethylene involved in tomato stamen development is regulated by jasmonates, a temporal profiling of hormone content, transcriptome and metabolome of tomato stamens was performed using wild type and jai1-1.
Results
Wild type stamens showed a transient increase of jasmonates that is absent in jai1-1. Comparative transcriptome analyses revealed a diminished expression of genes involved in pollen nutrition at early developmental stages of jai1-1 stamens, but an enhanced expression of ethylene-related genes at late developmental stages. This finding coincides with an early increase of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in jai1-1 and a premature pollen release from stamens, a phenotype similarly visible in an ethylene overproducing mutant. Application of jasmonates to flowers of transgenic plants affected in jasmonate biosynthesis diminished expression of ethylene-related genes, whereas the double mutant jai1-1 NeverRipe (ethylene insensitive) showed a complementation of jai1-1 phenotype in terms of dehiscence and pollen release.
Conclusions
Our data suggest an essential role of jasmonates in the temporal inhibition of ethylene production to prevent premature desiccation of stamens and to ensure proper timing in flower development.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0135-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-015-0135-3
PMCID: PMC4443647  PMID: 25895675
Array hybridization; Desiccation; Ethylene; Flower development; Jasmonic acid; Jasmonic acid-insensitive; Metabolite profiling; Pollen development

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