Comparative evolutionary genetics of spontaneous mutations affecting fitness in rhabditid nematodes

Deleterious mutations are of fundamental importance to all aspects of organismal biology. Evolutionary geneticists have expended tremendous effort to estimate the genome-wide rate of mutation and the effects of new mutations on fitness, but the degree to which genomic mutational properties vary within and between taxa is largely unknown, particularly in multicellular organisms. Beginning with two highly inbred strains from each of three species in the nematode family Rhabditidae (Caenorhabditis briggsae, Caenorhabditis elegans, and Oscheius myriophila), we allowed mutations to accumulate in the relative absence of natural selection for 200 generations. We document significant variation in the rate of decay of fitness because of new mutations between strains and between species. Estimates of the per-generation mutational decay of fitness were very consistent within strains between assays 100 generations apart. Rate of mutational decay in fitness was positively associated with genomic mutation rate and negatively associated with average mutational effect. These results provide unambiguous experimental evidence for substantial variation in genome-wide properties of mutation both within and between species and reinforce conclusions from previous experiments that the cumulative effects on fitness of new mutations can differ markedly among related taxa.     

Discordance between changes in the gut microbiota and pathogenicity in a mouse model of spontaneous colitis

Under conventional conditions, mice deficient in core 1-derived O-glycans (TM-IEC C1galt1^−/−), which have a defective mucus layer, experienced spontaneous inflammation of the colon. Analysis of fecal bacterial populations by pyrosequencing of 16S rRNA gene showed that disease in conventional TM-IEC C1galt1^−/− was associated with shifts in the microbiota manifested by increases in Lactobacillus and Clostridium species, and decreases in unclassified Ruminococcaceae and Lachnospiraceae. Under germ-free (GF) conditions, TM-IEC C1galt1^−/− presented decreased goblet cells, but did not develop inflammation. Monoassociation of GF TM-IEC C1galt1^−/− revealed that bacterial species differ significantly in their ability to induce inflammatory changes. Bacteroides thetaiotaomicron caused inflammation, while Lactobacillus johnsonii (enriched during colitis) did not. These observations demonstrate that not all microbiota shifts that correlate with disease contribute to pathogenesis.     

Evolution of host range in Coleosporium ipomoeae,a plant pathogen with multiple hosts

Plants and their pathogens coevolve locally. Previous investigations of one host–one pathogen systems have demonstrated that natural selection favors pathogen genotypes that are virulent on a broad range of host genotypes. In the present study, we examine a system consisting of one pathogen species that infects three host species in the morning glory genus Ipomoea. We show that many pathogen genotypes can infect two or three of the host species when tested on plants from nonlocal communities. By contrast, pathogen genotypes are highly host-specific, infecting only one host species, when tested on host species from the local community. This pattern indicates that within-community evolution narrows the host breadth of pathogen genotypes. Possible evolutionary mechanisms include direct selection for narrow host breadth due to costs of virulence and evolution of ipomoea resistance in the host species.Much of plant-pathogen coevolution is mediated by “gene-for-gene” (GFG) interactions. These interactions involve R genes in plants and corresponding virulence/avirulence genes in the pathogen (1). At a given pair of corresponding loci, a host may carry either a resistant (Res) or a susceptible (Sus) allele, or both, with Res typically being dominant. The pathogen may carry either a virulent (Vir) allele or an avirulent (Avr) allele. Infection results, unless at one pair of corresponding loci, the plant R locus has a Res allele and the pathogen has the Avr allele. Models of the evolution of GFG systems generally predict that generalist pathogens (those able to infect multiple host-resistance genotypes) will be favored by natural selection over highly specialized genotypes that can infect only one resistance genotype (2–6). Experimental analyses of pathogen host breadth in natural plant–pathogen systems are consistent with these expectations in that pathogen isolates are generally able to infect multiple host-resistance genotypes, especially in host populations with high levels of resistance (7–10).With very few exceptions (11, 12), the evolution of pathogen host range has been examined, both theoretically and empirically, for a single pathogen species interacting with a single host species. Many pathogens, however, are capable of infecting multiple host species. Predictions of evolutionary models based on a single evolving host species cannot be clearly extrapolated to this situation. Moreover, there are reasons to believe that, with multiple host species, selection for generalism may not be as prevalent. Maintaining infectivity on multiple hosts requires continued success in the coevolutionary arms race with more than one independently evolving host genome. The conditions under which this maintained infectivity can occur are likely more restrictive than with only one host, although this possibility has not been examined theoretically. In addition, selection to maintain infectivity on a particular host is likely weaker when the pathogen population can successfully reproduce on another host (see ref. 13 for an analogous argument with respect to partial resistance). Finally, costs associated with the ability to infect multiple host species (e.g., ref. 14) are likely greater than costs associated with the ability to infect multiple genotypes within the same host. All of these factors would tend to weaken selection for a broad host range and thus promote the evolution of specialist pathogen genotypes within populations.One approach to determining whether there is an evolutionary tendency for host breadth to be narrowed within populations is to compare pathogen host breadth in its local native community with host breadth on hosts from outside its native community (e.g., refs. 9 and 13). The latter constitutes an estimate of host breadth on host species with which the pathogen has presumably not recently coevolved and is also an estimate of host breadth for a pathogen strain that has recently immigrated into a new community. If evolutionary processes within local communities act to promote specialization, host breadth should be lower on hosts from the native community. In this report, we demonstrate that this pattern is exhibited for a host–pathogen system consisting of one pathogen and three host species.     

Polynucleobacter necessarius,a model for genome reduction in both free-living and symbiotic bacteria

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.Symbiosis, defined as a close relationship between organisms belonging to different species (1), is a ubiquitous, diverse, and important mechanism in ecology and evolution (e.g., refs. 2–4). In extreme cases, through the establishment of symbiotic relationships, quite unrelated lineages can functionally combine their genomes and generate advantageous emergent features or initiate parasite/host arms races. Ciliates, common unicellular protists of the phylum Ciliophora, are extraordinary receptacles for prokaryotic ecto- and endosymbionts (5, 6) that provide varied examples of biodiversity and ecological roles (6). Nevertheless, most of these symbionts are understudied, partially owing to the scarcity of available molecular data and the absence of sequenced genomes. Yet, thanks to their various biologies and the ease of sampling and cultivating their protist hosts, they are excellent potential models for symbioses between bacteria and heterotrophic eukaryotes. Until recently this field was dominated by studies on endosymbionts of invertebrates, especially insects (e.g., ref. 7), although unicellular systems like amoebas (e.g., refs. 8 and 9) have been shown to be suitable models.Polynucleobacter necessarius was first described as a cytoplasmic endosymbiont of the ciliate Euplotes aediculatus (10, 11). Further surveys detected its presence in a monophyletic group of fresh and brackish water Euplotes species (12, 13). All of the investigated strains of these species die soon after being cured of the endosymbiont (10, 12, 13). In the few cases in which P. necessarius is not present, a different and rarer bacterium apparently supplies the same function (12, 14). No attempt to grow symbiotic P. necessarius outside their hosts has yet been successful (15), strongly suggesting that the relationship is obligate for both partners, in contrast to most other known prokaryote/ciliate symbioses (6).Thus, the findings of many environmental 16S rRNA gene sequences similar to that of the symbiotic P. necessarius (16) but belonging to free-living freshwater bacteria came as a surprise. These free-living strains, which have been isolated and cultivated (17), are ubiquitous and abundant in the plankton of lentic environments (17, 18). They are smaller and do not show the most prominent morphological feature of the symbiotic form: the presence of multiple nucleoids, each containing one copy of the genome (10, 11). It is clear that free-living and endosymbiotic P. necessarius are not different life stages of the same organism (15). Nevertheless, these strikingly different bacteria, occupying separate ecological niches, exhibit >99% 16S rRNA gene sequence identity, and phylogenetic analyses fail to separate them into two distinct groups (15). Rather, several lines of evidence point to multiple, recent origins of symbiotic strains from the free-living bacterial pool (14, 15).Thus, the Euplotes–Polynucleobacter symbiosis provides a promising system for the study of changes promoting or caused by the shift to an intracellular lifestyle. The remarkably small (2.16 Mbp) genome of the free-living strain QLW-P1DMWA-1 has been sequenced and studied, especially for features that would explain the success of this lineage in freshwater systems worldwide (19, 20). Phylogenies based on the 16S rRNA gene (13, 14) and multiple-gene analyses (19, 21, 22) consistently cluster Polynucleobacter with bacteria of the family Burkholderiaceae (Betaproteobacteria), either in a basal position or as the sister group of Ralstonia and Cupriavidus.Here we provide the complete genomic sequence of a symbiotic P. necessarius harbored in the cytoplasm of E. aediculatus and present a comparative analysis of the two sequenced Polynucleobacter genomes, addressing the possible biological basis of the Euplotes–Polynucleobacter symbiosis. We also provide insights into the evolution of the unique two-step genome reduction in this bacterial species: the first step involving streamlining in a free-living ancestor and the second a more recent period of genome erosion confined to the symbiotic lineage.     

Genome-wide association identifies OBFC1 as a locus involved in human leukocyte telomere biology

Telomeres are engaged in a host of cellular functions, and their length is regulated by multiple genes. Telomere shortening, in the course of somatic cell replication, ultimately leads to replicative senescence. In humans, rare mutations in genes that regulate telomere length have been identified in monogenic diseases such as dyskeratosis congenita and idiopathic pulmonary fibrosis, which are associated with shortened leukocyte telomere length (LTL) and increased risk for aplastic anemia. Shortened LTL is observed in a host of aging-related complex genetic diseases and is associated with diminished survival in the elderly. We report results of a genome-wide association study of LTL in a consortium of four observational studies (n = 3,417 participants with LTL and genome-wide genotyping). SNPs in the regions of the oligonucleotide/oligosaccharide-binding folds containing one gene (OBFC1; rs4387287; P = 3.9 × 10⁻⁹) and chemokine (C-X-C motif) receptor 4 gene (CXCR4; rs4452212; P = 2.9 × 10⁻⁸) were associated with LTL at a genome-wide significance level (P < 5 × 10⁻⁸). We attempted replication of the top SNPs at these loci through de novo genotyping of 1,893 additional individuals and in silico lookup in another observational study (n = 2,876), and we confirmed the association findings for OBFC1 but not CXCR4. In addition, we confirmed the telomerase RNA component (TERC) as a gene associated with LTL (P = 1.1 × 10⁻⁵). The identification of OBFC1 through genome-wide association as a locus for interindividual variation in LTL in the general population advances the understanding of telomere biology in humans and may provide insights into aging-related disorders linked to altered LTL dynamics.     

Vacuoles of Candida yeast as a specialized niche for Helicobacter pylori

Helicobacter pylori(H.pylori)are resistant to hostile gastric environments and antibiotic therapy,reflecting the possibility that they are protected by an ecological niche,such as inside the vacuoles of human epithelial and immune cells.Candida yeast may also provide such an alternative niche,as fluorescently labeled H.pylori were observed as fast-moving and viable bacterium-like bodies inside the vacuoles of gastric,oral,vaginal and foodborne Candida yeasts.In addition,H.pylori-specific genes and proteins were detected in samples extracted from these yeasts.The H.pylori present within these yeasts produce peroxiredoxin and thiol peroxidase,providing the ability to detoxify oxygen metabolites formed in immune cells.Furthermore,these bacteria produce urease and VacA,two virulence determinants of H.pylori that influence phago-lysosome fusion and bacterial survival in macrophages.Microscopic observations of H.pylori cells in new generations of yeasts along with amplification of H.pylori-specific genes from consecutive generations indicate that new yeasts can inherit the intracellular H.pylori as part of their vacuolar content.Accordingly,it is proposed that yeast vacuoles serve as a sophisticated niche that protects H.pylori against the environmental stresses and provides essential nutrients,including ergosterol,for its growth and multiplication.This intracellular establishment inside the yeast vacuole likely occurred long ago,leading to the adaptation of H.pylori to persist in phagocytic cells.The presence of these bacteria within yeasts,including foodborne yeasts,along with the vertical transmission of yeasts from mother to neonate,provide explanations for the persistence and propagation of H.pylori in the human population.This Topic Highlight reviews and discusses recent evidence regarding the evolutionary adaptation of H.pylori to thrive in host cell vacuoles.     

A genome-wide view of the spectrum of spontaneous mutations in yeast

The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.     

Anaerobic Bacteria as a Cause of Mycotic Aneurysm of the Aorta: Microbiology and Antimicrobial Therapy

This review summarizes the microbiology, and antimicrobial management of mycotic aneurysm of the aorta (MAA) due to anaerobic bacteria. Anaerobic bacteria are an uncommon but important cause of MAA. Most cases of anaerobic MAA are caused anaerobic gram-negative bacilli (mostly B. fragilis group), Clostridium spp. (mostly Clostridium septicum, and Propionobacterium spp. (mostly P. acnes). Clostridial infection is frequently associated with gastrointestinal or hematologic malignancy. A review of all the reported cases is presented. Treatment of MAA involving anaerobic bacteria includes the use of antimicrobial effective against these organisms.     

A hyperactive quantitative trait locus allele of Arabidopsis BRX contributes to natural variation in root growth vigor

Quantitative trait loci analysis of natural Arabidopsis thaliana accessions is increasingly exploited for gene isolation. However, to date this has mostly revealed deleterious mutations. Among them, a loss-of-function allele identified the root growth regulator BREVIS RADIX (BRX). Here we present evidence that BRX and the paralogous BRX-LIKE (BRXL) genes are under selective constraint in monocotyledons as well as dicotyledons. Unexpectedly, however, whereas none of the Arabidopsis orthologs except AtBRXL1 could complement brx null mutants when expressed constitutively, nearly all monocotyledon BRXLs tested could. Thus, BRXL proteins seem to be more diversified in dicotyledons than in monocotyledons. This functional diversification was correlated with accelerated rates of sequence divergence in the N-terminal regions. Population genetic analyses of 30 haplotypes are suggestive of an adaptive role of AtBRX and AtBRXL1. In two accessions, Lc-0 and Lov-5, seven amino acids are deleted in the variable region between the highly conserved C-terminal, so-called BRX domains. Genotyping of 42 additional accessions also found this deletion in Kz-1, Pu2-7, and Ws-0. In segregating recombinant inbred lines, the Lc-0 allele (AtBRX^Lc-0) conferred significantly enhanced root growth. Moreover, when constitutively expressed in the same regulatory context, AtBRX^Lc-0 complemented brx mutants more efficiently than an allele without deletion. The same was observed for AtBRXL1, which compared with AtBRX carries a 13 amino acid deletion that encompasses the deletion found in AtBRX^Lc-0. Thus, the AtBRX^Lc-0 allele seems to contribute to natural variation in root growth vigor and provides a rare example of an experimentally confirmed, hyperactive allelic variant.     

Evolution of host specialization in gut microbes: the bee gut as a model

Bacterial symbionts of eukaryotes often give up generalist lifestyles to specialize to particular hosts. The eusocial honey bees and bumble bees harbor two such specialized gut symbionts, Snodgrassella alvi and Gilliamella apicola. Not only are these microorganisms specific to bees, but different strains of these bacteria tend to assort according to host species. By using in-vivo microbial transplant experiments, we show that the observed specificity is, at least in part, due to evolved physiological barriers that limit compatibility between a host and a potential gut colonizer. How and why such specialization occurs is largely unstudied for gut microbes, despite strong evidence that it is a general feature in many gut communities. Here, we discuss the potential factors that favor the evolution of host specialization, and the parallels that can be drawn with parasites and other symbiont systems. We also address the potential of the bee gut as a model for exploring gut community evolution.     

The first deletion mutation in the TSP1-6 repeat domain of ADAMTS13 in a family with inherited thrombotic thrombocytopenic purpura

The inherited deficiency of ADAMTS13 is usually associated with severe forms of thrombotic thrombocytopenic purpura. Among the mutations identified in the ADAMTS13 gene, none have been described on the TSP1-6 repeat domain. We investigated an Iranian family with a history of chronic recurrent thrombotic thrombocytopenic purpura, severe ADAMTS13 deficiency and a heterogeneous pattern of clinical symptoms among affected members. Genetic analysis revealed a homozygous deletion of nucleotides 2930–2935 (GTGCCC) in exon 23 of ADAMTS13, leading to the replacement of Cys977 by a Trp and the deletion of Ala978 and Arg979 in the TSP1-6 repeat domain. To explore the mechanism of ADAMTS13 deficiency, in vitro expression studies were performed. Western blotting, pulse-chase labeling and immunofluorescence studies demonstrated a secretion pathway defect of the mutant protein, with no intracellular accumulation. This finding is consistent with the severe ADAMTS13 deficiency but does not explain the heterogeneous clinical picture of the 3 siblings carrying the same mutation.     

Analysis of Adaptive Evolution in Lyssavirus Genomes Reveals Pervasive Diversifying Selection during Species Diversification

Lyssavirus is a diverse genus of viruses that infect a variety of mammalian hosts, typically causing encephalitis. The evolution of this lineage, particularly the rabies virus, has been a focus of research because of the extensive occurrence of cross-species transmission, and the distinctive geographical patterns present throughout the diversification of these viruses. Although numerous studies have examined pattern-related questions concerning Lyssavirus evolution, analyses of the evolutionary processes acting on Lyssavirus diversification are scarce. To clarify the relevance of positive natural selection in Lyssavirus diversification, we conducted a comprehensive scan for episodic diversifying selection across all lineages and codon sites of the five coding regions in lyssavirus genomes. Although the genomes of these viruses are generally conserved, the glycoprotein (G), RNA-dependent RNA polymerase (L) and polymerase (P) genes were frequently targets of adaptive evolution during the diversification of the genus. Adaptive evolution is particularly manifest in the glycoprotein gene, which was inferred to have experienced the highest density of positively selected codon sites along branches. Substitutions in the L gene were found to be associated with the early diversification of phylogroups. A comparison between the number of positively selected sites inferred along the branches of RABV population branches and Lyssavirus intespecies branches suggested that the occurrence of positive selection was similar on the five coding regions of the genome in both groups.     

Mutations in topoisomerase I as a self-resistance mechanism coevolved with the production of the anticancer alkaloid camptothecin in plants

Plants produce a variety of toxic compounds, which are often used as anticancer drugs. The self-resistance mechanism to these toxic metabolites in the producing plants, however, remains unclear. The plant-derived anticancer alkaloid camptothecin (CPT) induces cell death by targeting DNA topoisomerase I (Top1), the enzyme that catalyzes changes in DNA topology. We found that CPT-producing plants, including Camptotheca acuminata, Ophiorrhiza pumila, and Ophiorrhiza liukiuensis, have Top1s with point mutations that confer resistance to CPT, suggesting the effect of an endogenous toxic metabolite on the evolution of the target cellular component. Three amino acid substitutions that contribute to CPT resistance were identified: Asn421Lys, Leu530Ile, and Asn722Ser (numbered according to human Top1). The substitution at position 722 is identical to that found in CPT-resistant human cancer cells. The other mutations have not been found to date in CPT-resistant human cancer cells; this predicts the possibility of occurrence of these mutations in CPT-resistant human cancer patients in the future. Furthermore, comparative analysis of Top1s of CPT-producing and nonproducing plants suggested that the former were partially primed for CPT resistance before CPT biosynthesis evolved. Our results demonstrate the molecular mechanism of self-resistance to endogenously produced toxic compounds and the possibility of adaptive coevolution between the CPT production system and its target Top1 in the producing plants.     

Baculovirus resistance in codling moth is virus isolate-dependent and the consequence of a mutation in viral gene pe38

The baculovirus Cydia pomonella granulovirus (CpGV) is widely applied as a biocontrol agent of codling moth. After field resistance of codling moth populations had been observed against the commercially used Mexican (M) isolate of CpGV, infection experiments of larvae of the resistant codling moth strain CpRR1 showed that several other naturally occurring CpGV isolates (I12, S, E2, and I07) from different geographic origins are still infectious to resistant CpRR1. Whole-genome sequencing and phylogenetic analyses of these geographic CpGV variants revealed that their genomes share only a single common difference from that of CpGV-M, which is a mutation coding for a repeat of 24 nucleotides within the gene pe38; this mutation results in an additional repeat of eight amino acids that appears to be inserted to PE38 of CpGV-M only. Deletion of pe38 from CpGV-M totally abolished virus infection in codling moth cells and larvae, demonstrating that it is an essential gene. When the CpGV-M deletion mutant was repaired with pe38 from isolate CpGV-S, which originated from the commercial product Virosoft and is infectious for the resistant codling moth strain CpRR1, the repaired CpGV-M mutant was found to be fully infectious for CpRR1. Repair using pe38 from CpGV-M restored infectivity for the virus in sensitive codling moth strains, but not in CpRR1. Therefore, we conclude that CpGV resistance of codling moth is directed to CpGV-M but not to other virus isolates. The viral gene pe38 is not only essential for the infectivity of CpGV but it is also the key factor in overcoming CpGV resistance in codling moth.The codling moth Cydia pomonella L. is a worldwide occurring insect pest that infests apples, pears, and walnuts. The larvae of codling moth bore into the fruit and cause severe economic damage if not controlled. A number of chemical and biological agents are available for the control of codling moth. One of the most efficient biological control agents (1–5) is the Cydia pomonella granulovirus (CpGV), which belongs to the dsDNA virus family Baculoviridae (genus Betabaculovirus). CpGV was first discovered in Mexico (Mexican isolate, CpGV-M) in 1963 (6); this isolate was later developed to commercial products now registered in 34 countries worldwide. The genome sequence of the in vivo cloned strain CpGV-M1 is ∼123 kbp and encodes for 143 ORFs (7). Based on SNPs in highly conserved genes, different geographic CpGV isolates were classified into four genome types, A–D (8). CpGV exhibits an extremely narrow host range that is restricted to C. pomonella and a very few closely related tortricids (Lepidoptera) (9).The development of resistance to baculovirus infection was thought to be unlikely before 2005 (10, 11), which is when the first cases of resistant codling moth populations with a 1,000- to 100,000-fold reduced susceptibility to commercial CpGV products containing the isolate CpGV-M were reported from organic apple plantations in Germany and France, where CpGV products had been intensively applied (12, 13). Since then, 38 apple plantations with CpGV resistance have been identified in Austria (2 orchards), Czech Republic (1), France (3), Germany (22), Italy (6), Switzerland (2), and the Netherlands (2) (14). For the codling moth strain CpR, which originated from a resistant field population in south Germany, as well as the genetically homogenous laboratory strain CpRR1, which derived from CpR, the mode of inheritance was revealed to be incompletely dominant, monogenic, and linked to the Z (sex) chromosome (15, 16). A similar mode of inheritance was also observed in resistant Czech and French codling moth populations arguing for a more or less universal mode of resistance in Europe (17, 18). These data led to the hypothesis that a genetic adaptation of codling moth to CpGV-M infection had occurred and was selected for by the intensive use of products containing CpGV-M (15).Insects manifest miscellaneous strategies to resist pathogens but lack an adaptive immune system. Insect defense to viral infections involves nonspecific factors such as physical barriers, enzymatic responses, and increasing ejection of infected midgut cells as larvae age, as well as specific factors involving cellular and humoral immunity (19). Physical barriers to infection include the perithropic membrane (PM), an ultrafilter for particles with size exclusion greater than 30 nm (20). Melanization of the cuticula mediated by phenoloxidase enzymes is involved in the encapsulation reaction of pathogens, such as bacteria, fungi, or virus-infected cells (21, 22). Developmental resistance, the decreasing susceptibility of larvae with increasing age, is rather common, and is mediated by infected midgut cell-sloughing (23–25). Behavior modifications also count among insect defense strategies (19). Feeding behaviors, e.g., can strongly affect the risk of insects for baculovirus infection as shown for Lymantria dispar L., which exhibit heritable cadaver-avoidance behaviors (26).An alternative way for insects to get rid of virus-infected cells is via programmed cell death—the apoptosis pathway (27). Baculoviruses, however, are able to block apoptosis with the help of different virus encoded antiapoptotic proteins. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV), these proteins—namely P35 and P49—are inhibitors of the insect caspases. These inhibitors of apoptosis (IAPs), thereby, ensure a permissive virus infection (28).For codling moth, however, resistance based on the PM, the midgut, or the immune system has been excluded for CpGV (16, 29). Lack of CpGV DNA replication and a systemic resistance in all five instars indicated an early block to virus replication in resistant codling moth individuals (29, 30). At the same time, it was observed that certain CpGV isolates, such as I12 or NPP-R1/R4, were able to infect larvae from resistant codling moth strains (30, 31). Therefore, these naturally occurring CpGV isolates from different geographic origins were considered resistance-breaking isolates. Some of these isolates, meanwhile, replace CpGV-M in commercial biocontrol agents in Europe and demonstrate the importance of identifying resistance-breaking CpGV variants (30–32) for managing CpGV-resistant codling moth populations; however, their functional difference to CpGV-M allowing them to overcome CpGV resistance remained unknown.In this study we compared different naturally occurring geographic CpGV isolates representing all known CpGV genome types (8) in laboratory assays for their infectivity to susceptible (CpS) and resistant (CpRR1) codling moth strains. We found that all tested isolates—except CpGV-M—were able to overcome resistance in CpRR1 larvae. Whole-genome sequencing of these isolates revealed a single common difference in all resistance-breaking isolates, which was located in ORF24 (pe38). Therefore, occlusion bodies of bacmid-based recombinant CpGV (in the following termed pseudoviruses) knockout and recovery mutants of pe38 were established and tested for their activity in susceptible CpS and resistant CpRR1 larvae. The recovery of infectivity of a CpGV-M–based pseudovirus harboring the pe38 of the resistance-breaking CpGV-S in CpRR1 larvae demonstrated the key function of pe38 in overcoming baculovirus resistance in codling moth. In their entirety, these investigations demonstrate the significance of using multiple isolates of CpGV concurrently in field applications to avoid resistance in codling moth populations.     

A de novo germline MLH1 mutation in a Lynch syndrome patient with discordant immunohistochemical and molecular biology test results

We describe a patient with a Homo sapiens mutL homolog 1 (MLH1)-associated Lynch syndrome with previous diagnoses of two distinct primary cancers: a sigmoid colon cancer at the age of 39 years, and a right colon cancer at the age of 50 years. The mutation identified in his blood and buccal cells, c.1771delG, p.Asp591Ilefs*25, appears to be a de novo event, as it was not transmitted by either of his parents. This type of de novo event is rare in MLH1 as only three cases have been reported in the literature so far. Furthermore, the discordant results observed between replication error phenotyping and immunohistochemistry highlight the importance of the systematic use of both pre-screening tests in the molecular diagnosis of Lynch syndrome.     

Mutations of PHF6 are associated with mutations of NOTCH1, JAK1 and rearrangement of SET-NUP214 in T-cell acute lymphoblastic leukemia

Background

Mutations in the PHF6 gene were recently described in patients with T-cell acute lymphoblastic leukemia and in those with acute myeloid leukemia. The present study was designed to determine the prevalence of PHF6 gene alterations in T-cell acute lymphoblastic leukemia.

Design and Methods

We analyzed the incidence and prognostic value of PHF6 mutations in 96 Chinese patients with T-cell acute lymphoblastic leukemia. PHF6 deletions were screened by real-time quantitative polymerase chain reaction and array-based comparative genomic hybridization. Patients were also investigated for NOTCH1, FBXW7, WT1, and JAK1 mutations together with CALM-AF10, SET-NUP214, and SIL-TAL1 gene rearrangements.

Results

PHF6 mutations were identified in 11/59 (18.6%) adult and 2/37 (5.4%) pediatric cases of T-cell acute lymphoblastic leukemia, these incidences being significantly lower than those recently reported. Although PHF6 is X-linked and mutations have been reported to occur almost exclusively in male patients, we found no sex difference in the incidences of PHF6 mutations in Chinese patients with T-cell acute lymphoblastic leukemia. PHF6 deletions were detected in 2/79 (2.5%) patients analyzed. NOTCH1 mutations, FBXW7 mutations, WT1 mutations, JAK1 mutations, SIL-TAL1 fusions, SET-NUP214 fusions and CALM-AF10 fusions were present in 44/96 (45.8%), 9/96 (9.4%), 4/96 (4.1%), 3/49 (6.1%), 9/48 (18.8%), 3/48 (6.3%) and 0/48 (0%) of patients, respectively. The molecular genetic markers most frequently associated with PHF6 mutations were NOTCH1 mutations (P=0.003), SET-NUP214 rearrangements (P=0.002), and JAK1 mutations (P=0.005). No differences in disease-free survival and overall survival between T-cell acute lymphoblastic leukemia patients with and without PHF6 mutations were observed in a short-term follow-up.

Conclusions

Overall, these results indicate that, in T-cell acute lymphoblastic leukemia, PHF6 mutations are a recurrent genetic abnormality associated with mutations of NOTCH1, JAK1 and rearrangement of SET-NUP214.