The impact of parasite dispersal on antagonistic host-parasite coevolution

Coevolving populations of hosts and parasites are often subdivided into a set of patches connected by dispersal. Higher relative rates of parasite compared with host dispersal are expected to lead to parasite local adaptation. However, we know of no studies that have considered the implications of higher relative rates of parasite dispersal for other aspects of the coevolutionary process, such as the rate of coevolution and extent of evolutionary escalation of resistance and infectivity traits. We investigated the effect of phage dispersal on coevolution in experimental metapopulations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage SBW25Phi2. Both the rate of coevolution and the breadth of evolved infectivity and resistance ranges peaked at intermediate rates of parasite dispersal. These results suggest that parasite dispersal can enhance the evolutionary potential of parasites through provision of novel genetic variation, but that high rates of parasite dispersal can impede the evolution of parasites by homogenizing genetic variation between patches, thereby constraining coevolution.     

HOW SPECIFICITY AND EPIDEMIOLOGY DRIVE THE COEVOLUTION OF STATIC TRAIT DIVERSITY IN HOSTS AND PARASITES

There is typically considerable variation in the level of infectivity of parasites and the degree of resistance of hosts within populations. This trait variation is critical not only to the evolutionary dynamics but also to the epidemiology, and potentially the control of infectious disease. However, we lack an understanding of the processes that generate and maintain this trait diversity. We examine theoretically how epidemiological feedbacks and the characteristics of the interaction between host types and parasites strains determine the coevolution of host–parasite diversity. The interactions include continuous characterizations of the key phenotypic features of classic gene‐for‐gene and matching allele models. We show that when there are costs to resistance in the hosts and infectivity in the parasite, epidemiological feedbacks may generate diversity but this is limited to dimorphism, often of extreme types, in a broad range of realistic infection scenarios. For trait polymorphism, there needs to be both specificity of infection between host types and parasite strains as well as incompatibility between particular strains and types. We emphasize that although the high specificity is well known to promote temporal “Red Queen” diversity, it is costs and combinations of hosts and parasites that cannot infect that will promote static trait diversity.     

Novel gene signatures for prognosis prediction in ovarian cancer

Ovarian cancer (OV) is one of the leading causes of cancer deaths in women worldwide. Late diagnosis and heterogeneous treatment result to poor survival outcomes for patients with OV. Therefore, we aimed to develop novel biomarkers for prognosis prediction from the potential molecular mechanism of tumorigenesis. Eight eligible data sets related to OV in GEO database were integrated to identify differential expression genes (DEGs) between tumour tissues and normal. Enrichment analyses discovered DEGs were most significantly enriched in G2/M checkpoint signalling pathway. Subsequently, we constructed a multi‐gene signature based on the LASSO Cox regression model in the TCGA database and time‐dependent ROC curves showed good predictive accuracy for 1‐, 3‐ and 5‐year overall survival. Utility in various types of OV was validated through subgroup survival analysis. Risk scores formulated by the multi‐gene signature stratified patients into high‐risk and low‐risk, and the former inclined worse overall survival than the latter. By incorporating this signature with age and pathological tumour stage, a visual predictive nomogram was established, which was useful for clinicians to predict survival outcome of patients. Furthermore, SNRPD1 and EFNA5 were selected from the multi‐gene signature as simplified prognostic indicators. Higher EFNA5 expression or lower SNRPD1 indicated poorer outcome. The correlation between signature gene expression and clinical characteristics was observed through WGCNA. Drug‐gene interaction was used to identify 16 potentially targeted drugs for OV treatment. In conclusion, we established novel gene signatures as independent prognostic factors to stratify the risk of OV patients and facilitate the implementation of personalized therapies.     

Generation of a Mig-6 conditional null allele

Mitogen-inducible gene 6 (Mig-6) is a stress-induced gene that serves as a negative regulator of epidermal growth factor (EGF) signaling and acts as a tumor suppressor. Ablation of Mig-6 results in a significant percentage of embryo lethality as well as abnormalities in multiple tissues. To understand the physiological roles of Mig-6, a conditional null allele, Mig-6(f/f) was generated by introducing LoxP sites that flank exons 2 and 4. The Mig-6(f/f) allele was validated by generating recombined Mig-6(-/-) mice using the Zp3-Cre system. The conditional null allele was confirmed by assaying for Mig-6 gene expression in liver, lung, uterus, and skin. The recombined Mig-6(-/-) mice developed pathological changes, such as degenerative joint diseases and skin hyperplasia similar to the previously reported Mig-6 germline null allele. In addition, these mice also had enlarged uteri with endometrial hyperplasia. In summary, this Mig-6(f/f) mouse is a useful tool for the functional study of the Mig-6 gene in a tissue-specific fashion.     

Unmodified oligodeoxynucleotides require single-strandedness to induce targeted repair of a chromosomal EGFP gene

BACKGROUND: A number of genetic defects in humans are due to point mutations in a single, often tightly regulated gene. Genetic treatment of such defects is preferably done by correcting only the altered base pair at the endogenous locus rather than by a gene replacement strategy involving viral vectors. Promisingly high repair rates have been achieved in some systems with the non-viral approach of transfecting chimeric RNA/DNA oligonucleotides (chimeraplasts). However, since this technique does not yet perform robustly, several parameters thought to be important in oligonucleotide-mediated gene repair were examined. METHODS: A series of transgenic HEK-293 cell clones has been established harboring high or low copy numbers of a point-mutated 'enhanced green fluorescent protein' (EGFP) gene as the target. At the level of single living cells, repair efficiencies were measured by fluorescence-activated cell sorting (FACS) regarding topology (single-stranded, double-stranded), exonuclease protection (four phosphorothioate linkages at both ends), polarity (sense, antisense), and length (13mer, 19mer, 35mer, 69mer) of the oligonucleotide. RESULTS: When targeting chromosomal loci, up to 0.2% corrected cells were obtained with single-stranded unmodified oligodeoxynucleotides, whereas a chimeraplast, its DNA analogue, and double-stranded DNA fragments were practically non-functional. Correction efficiencies correlated with target gene copy numbers. Modifying exonuclease resistance, polarity or length of single-stranded oligodeoxynucleotides did not enhance repair efficacy above the sub-percentage range. CONCLUSIONS: Successful chromosomal reporter gene repair in HEK-293 cells required an oligodeoxynucleotide to be single-stranded. In concert with the gene copy number correlation, functional interaction between the repair molecule and the target site seems to be one bottleneck in targeted gene repair.     

The effects of a stimulating intron on the expression of heterologous genes in Arabidopsis thaliana

Introns are often added to transgenes to increase expression, although the mechanism through which introns stimulate gene expression in plants and other eukaryotes remains mysterious. While introns vary in their effect on expression, it is unknown whether different genes respond similarly to the same stimulatory intron. Furthermore, the degree to which gene regulation is preserved when expression is increased by an intron has not been thoroughly investigated. To test the effects of the same intron on the expression of a range of genes, GUS translational fusions were constructed using the promoters of eight Arabidopsis genes whose expression was reported to be constitutive (GAE1, CNGC2 and ROP10), tissue specific (ADL1A, YAB3 and AtAMT2) or regulated by light (ULI3 and MSBP1). For each gene, a fusion containing the first intron from the UBQ10 gene was compared to fusions containing the gene's endogenous first intron (if the gene has one) or no intron. In every case, the UBQ10 intron increased expression relative to the intronless control, although the magnitude of the change and the level of expression varied. The UBQ10 intron also changed the expression patterns of the CNGC2 and YAB3 fusions to include strong activity in roots, indicating that tissue specificity was disrupted by this intron. In contrast, the regulation of the ULI3 and MSBP1 genes by light was preserved when their expression was stimulated by the intron. These findings have important implications for biotechnology applications in which a high level of transgene expression in only certain tissues is desired.     

Genomic footprints of speciation in Atlantic eels (Anguilla anguilla and A. rostrata)

The importance of speciation‐with‐geneflow scenarios is increasingly appreciated. However, the specific processes and the resulting genomic footprints of selection are subject to much discussion. We studied the genomics of speciation between the two panmictic, sympatrically spawning sister species; European (Anguilla anguilla) and American eel (A. rostrata). Divergence is assumed to have initiated more than 3 Ma, and although low gene flow still occurs, strong postzygotic barriers are present. Restriction‐site‐associated DNA (RAD) sequencing identified 328 300 SNPs for subsequent analysis. However, despite the presence of 3757 strongly differentiated SNPs (F_ST > 0.8), sliding window analyses of F_ST showed no larger genomic regions (i.e. hundreds of thousands to millions of bases) of elevated differentiation. Overall F_ST was 0.041, and linkage disequilibrium was virtually absent for SNPs separated by more than 1000 bp. We suggest this to reflect a case of genomic hitchhiking, where multiple regions are under directional selection between the species. However, low but biologically significant gene flow and high effective population sizes leading to very low genetic drift preclude accumulation of strong background differentiation. Genes containing candidate SNPs for positive selection showed significant enrichment for gene ontology (GO) terms relating to developmental processes and phosphorylation, which seems consistent with assumptions that differences in larval phase duration and migratory distances underlie speciation. Most SNPs under putative selection were found outside coding regions, lending support to emerging views that noncoding regions may be more functionally important than previously assumed. In total, the results demonstrate the necessity of interpreting genomic footprints of selection in the context of demographic parameters and life‐history features of the studied species.     

Virus‐induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation

We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so‐called virus‐induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5′ end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3′ end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3′ end, and an overall increase in CG methylation in the 5′ end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.     

Comparison of cDNA and genomic forms of tyrosine hydroxylase gene therapy of the brain with Trojan horse liposomes

BACKGROUND: The present study examines whether chromosomal derived forms of therapeutic genes can be delivered to brain following intravenous administration. The brain expression of a rat tyrosine hydroxylase (TH) cDNA is compared to the brain expression of a plasmid DNA encoding the 18 kb rat TH gene. METHODS: TH gene expression is measured in cell culture and in vivo in brain in experimental Parkinson's disease (PD). A total of four eukaryotic expression plasmids encoding rat TH were engineered wherein the size of the TH expression cassette ranged from 1.5 kb, in the case of the cDNA form of the gene, to 17.5 kb, in the case of the largest size genomic construct. The TH expression plasmids were delivered to either cultured cells or to rat brain in vivo with Trojan horse liposomes (THLs), which target the non-viral plasmid DNA to cells via cell membrane receptors. RESULTS: The pattern of TH gene expression in cell culture and in vivo was similar: the cDNA form of the TH gene was fast-acting with short duration of action, and the genomic form of the TH gene was slow-acting with longer duration of action. The most sustained replacement of striatal TH enzyme activity in experimental PD was produced by combination gene therapy where both the cDNA and the genomic forms of the TH gene were administered simultaneously. CONCLUSIONS: Eukaryotic expression plasmids encoding genomic forms of therapeutic genes, as large as 18 kb, can be successfully incorporated in THLs and delivered to brain following intravenous administration.     

Association study of catechol-O-methyltransferase gene polymorphisms with schizophrenia and psychopathological symptoms in Han Chinese

Although dysfunction of catechol‐O‐methyltransferase (COMT)‐mediated dopamine transmission is implicated in the etiology of schizophrenia, the human COMT gene has not been associated consistently with schizophrenia. The purpose of this study was to investigate whether the COMT gene is associated with the development of schizophrenia and whether the polymorphisms of this gene influence the psychopathological symptoms in patients with schizophrenia. Fourteen polymorphisms of the COMT gene were analyzed in a case–control study of 876 Han Chinese individuals (434 patients and 442 controls). All participants were screened using a Chinese version of the modified Schedule for Affective Disorders and Schizophrenia‐Lifetime Version (SADS‐L) and all patients met the criteria for schizophrenia. Furthermore, pretreatment of psychopathology was assessed using the Positive and Negative Syndrome Scale (PANSS) in a subset of 224 hospitalized schizophrenia patients, who were drug‐naÏve or drug‐free, to examine the association between clinical symptomatology and COMT polymorphisms. No significant differences in allele or genotype frequencies were observed between schizophrenia patients and controls, for all variants investigated. Haplotype analysis showed that three haplotype blocks of the COMT gene were not associated with the development of schizophrenia. Moreover, these COMT polymorphisms did not influence the PANSS scores of schizophrenia patients. This study suggests that the COMT gene may not contribute to the risk of schizophrenia and to the psychopathological symptoms of schizophrenia among Han Chinese.     

Rmg8 and Rmg7, wheat genes for resistance to the wheat blast fungus,recognize the same avirulence gene AVR‐Rmg8

Rmg8 and Rmg7 are genes for resistance to the wheat blast fungus (Pyricularia oryzae), located on chromosome 2B in hexaploid wheat and chromosome 2A in tetraploid wheat, respectively. AVR‐Rmg8, an avirulence gene corresponding to Rmg8, was isolated from a wheat blast isolate through a map‐based strategy. The cloned fragment encoded a small protein containing a putative signal peptide. AVR‐Rmg8 was recognized not only by Rmg8, but also by Rmg7, suggesting that these two resistance genes are equivalent to a single gene from the viewpoint of resistance breeding.     

Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection

BACKGROUND: The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection. METHODS: We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with (35)S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. RESULTS: When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of (35)S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. CONCLUSIONS: Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.     

No evidence for germ-line transmission following prenatal and early postnatal AAV-mediated gene delivery

BACKGROUND: Recombinant adeno-associated viruses have been used successfully in a number of pre-clinical and clinical gene therapy studies. Since there is a broad consensus that gene therapy must not lead to germ-line transmission, the potential of such vectors for inadvertent gene transfer into germ cells deserves special attention. This applies in particular to pre- or perinatal vector application which has been considered for diseases presenting with morbidity already at birth. METHODS: AAV serotype 2 derived vectors carrying a beta-galactosidase reporter gene or human clotting factor IX cDNA were injected intraperitoneally or via a yolk sac vein into mouse fetuses or administered intravascularly to newborn mice. Tissue samples of the treated animals including the gonads as well as sperm DNA, obtained by differential lysis of one testis of each male animal, and the offspring of all treated mice were investigated for the presence of vector DNA by nested PCR. In positive samples, the copy number of the vector was determined by quantitative real-time PCR. RESULTS: AAV vectors administered intraperitoneally or intravascularly to fetal or newborn mice reached the gonads of these animals and persisted there for time periods greater than one year. Intravascular injection of the vector resulted more frequently in gene transfer to the gonads than intraperitoneal injection. Vector copy numbers in the gonads ranged from 0.3 to 74 per 10(4) cell equivalents. However, neither in isolated sperm DNA from the treated animals nor in their offspring were vector sequences detectable. CONCLUSIONS: These data suggest the risk of inadvertent germ-line transmission following prenatal or early postnatal AAV type 2 mediated gene delivery to be very low.     

Maladaptation in wild populations of the generalist plant pathogen Pseudomonas syringae

Multihost pathogens occur widely on both natural and agriculturally managed hosts. Despite the importance of such generalists, evolutionary studies of host-pathogen interactions have largely focused on tightly coupled interactions between species pairs. We characterized resistance in a collection of Arabidopsis thaliana hosts, including 24 accessions collected from the Midwest USA and 24 from around the world, and patterns of virulence in a collection of Pseudomonas syringae strains, including 24 strains collected from wild Midwest populations of A. thaliana (residents) and 18 from an array of cultivated species (nonresidents). All of the nonresident strains and half of the resident strains elicited a resistance response on one or more A. thaliana accessions. The resident strains that failed to elicit any resistance response possessed an alternative type III secretion system (T3SS) that is unable to deliver effectors into plant host cells; as a result, these seemingly nonpathogenic strains are incapable of engaging in gene for gene interactions with A. thaliana. The remaining resident strains triggered greater resistance compared to nonresident strains, consistent with maladaptation of the resident bacterial population. We weigh the plausibility of two explanations: general maladaptation of pathogen strains and a more novel hypothesis whereby community level epidemiological dynamics result in adaptive dynamics favoring ephemeral hosts like A. thaliana.