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Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis., Competing Interests: The authors have declared that no competing interests exist.

Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a "chaperone" to promote 3' to 5' filament growth via highly dynamic engagement with 5' filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators., Competing Interests: Declaration of interests S.J.B. is also scientific co-founder and VP Science Strategy at Artios Pharma Ltd., Babraham Research Campus, Cambridge, UK. The other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cysticercosis caused by the metacestode larval stage of Taenia hydatigena formerly referred to as Cysticercus tenuicollis is a disease of veterinary importance that constitutes a significant threat to livestock production worldwide, especially in endemic regions due to condemnation of visceral organs and mortality rate of infected young animals. While the genetic diversity among parasites is found to be potentially useful in many areas of research including molecular diagnostics, epidemiology and control, that of T. hydatigena across the globe remains poorly understood. In this study, analysis of the mitochondrial DNA (mtDNA) of adult worms and larval stages of T. hydatigena isolated from dogs, sheep and a wild boar in China showed that the population structure consists of two major haplogroups with very high nucleotide substitutions involving synonymous and non-synonymous changes. Compared with other cestodes such as Echinococcus spp., the genetic variation observed between the haplogroups is sufficient for the assignment of major haplotype or genotype division as both groups showed a total of 166 point-mutation differences between the 12 mitochondrial protein-coding gene sequences. Preliminary analysis of a nuclear protein-coding gene (pepck) did not reveal any peculiar changes between both groups which suggests that these variants may only differ in their mitochondrial makeup.

DNA double-strand breaks (DSBs) are one of the most dangerous assaults on the genome, and yet their natural and programmed production are inherent to life. When DSBs arise close together they are particularly deleterious, and their repair may require an altered form of the DNA damage response. Our understanding of how clustered DSBs are repaired in the germline is unknown. Using laser microirradiation, we examine early events in the repair of clustered DSBs in germ cells within Caenorhabditis elegans. We use precise temporal resolution to show how the recruitment of MRE-11 to complex damage is regulated, and that clustered DNA damage can recruit proteins from various repair pathways. Abrogation of non-homologous end joining or COM-1 attenuates the recruitment of MRE-11 through distinct mechanisms. The synaptonemal complex plays both positive and negative regulatory roles in these mutant contexts. These findings indicate that MRE-11 is regulated by modifying its accessibility to chromosomes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

DNA damage causes cancer, impairs development and accelerates aging. Transcription-blocking lesions and transcription-coupled repair defects lead to developmental failure and premature aging in humans. Following DNA repair, homeostatic processes need to be reestablished to ensure development and maintain tissue functionality. Here, we report that, in Caenorhabditis elegans, removal of the WRAD complex of the MLL/COMPASS H3K4 methyltransferase exacerbates developmental growth retardation and accelerates aging, while depletion of the H3K4 demethylases SPR-5 and AMX-1 promotes developmental growth and extends lifespan amid ultraviolet-induced damage. We demonstrate that DNA-damage-induced H3K4me2 is associated with the activation of genes regulating RNA transport, splicing, ribosome biogenesis and protein homeostasis and regulates the recovery of protein biosynthesis that ensures survival following genotoxic stress. Our study uncovers a role for H3K4me2 in coordinating the recovery of protein biosynthesis and homeostasis required for developmental growth and longevity after DNA damage.

Like mammalian cells, helminth parasites are equipped with an array of enzymatic anti-oxidant system which has an adaptive strategy to cope up with several conditions of stress that arise from host immune response or drug treatment. Earlier, we had reported that three species of Senna, viz. S. alata, S. alexandrina and S. occidentalis leaf extracts caused severe morphological and biochemical alterations in the zoonotic parasite Hymenolepis diminuta. To understand whether the leaf extracts of the three species of Senna have any effect on the enzymatic anti-oxidant system in H.diminuta or not, the present study was investigated on the mechanism of action of these leaf extracts on the anti-oxidant system of the parasite. The viability of the parasite was assessed by MTT reduction assay, chromatin condensation through Hoechst staining of tissue and DNA fragmentation assay, and the oxidative enzymes of the parasite were estimated biochemically. Activity of superoxide dismutase, catalase, glutathione S- transferase and glutathione peroxidase were found to be increased in all the treated parasites from that of the control, with S. alata showed the highest increased amongst the three plant species in all the enzymes, at 331.0 %, 215.4 %, 85.4 % and 65.5 % respectively. Upliftment of apoptotic protein CED-3, CED-4 and EGL-1 and down regulation of anti-apototic protein CED-9 was visualised in all treated paraites. The redox imbalance triggered by these leaf extracts resulted in the activation of apoptotic pathway that led to death of the parasite. Our results demonstrated that the leaf extracts of the three Senna plant species could open new insight for an affordable natural anthelmintic with high efficacy and less toxicity., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Changes in parasite communities might result in new host-parasite dynamics and may threaten local fish populations. This phenomenon has been suggested for acanthocephalan parasites in the river Rhine and Danube where the species Pomphorhynchus tereticollis is becoming replaced by the Ponto-Caspian P. laevis. Developing knowledge on morphologic, genetic and behavioural differences between such species is important to follow such changes. However, disagreements on the current phylogeny of these two acanthocephalan species are producing conflicts that is affecting their correct identification. This study is offering a clearer morphological and genetic distinction between these two species. As P. tereticollis is found in rhithral tributaries of the Rhine, it was questioned whether the local salmonid populations were hosts for this species and whether P. laevis was expanding into the Rhine watershed as well. In order to test for this, brown trout, Salmo trutta, and grayling, Thymallus thymallus from South-Western Germany watersheds have been samples and screened for the occurrence of acanthocephalan parasites. For the first time, both species were confirmed to be hosts for P. tereticollis in continental Europe. P. tereticollis was found to be common, whereas P. leavis was found only at a single location in the Danube. This pattern suggest either that the expansion of P. laevis through salmonid hosts into rhithral rivers has not yet occurred, or that not yet ascertained biotic or abiotic features of rhithral rivers hinder P. laevis to spread into these areas., Competing Interests: The authors have declared that no competing interests exist.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a causative agent of pine wilt disease (PWD). To date, although several molecular diagnostic methods have been developed, rapid on-site diagnostic tools for detecting PWN in pinewood are limited. In this study, a point of care diagnostic (POCD) method for detecting PWN in pinewood using recombinase polymerase amplification (RPA) assay was developed. This method comprises quick gDNA extraction buffer (DAP buffer) for the direct extraction of gDNA of PWN from pinewood and a battery-mounted portable optical isothermal device (POID) for the detection of PWD in the field. The RPA assay can distinguish between the PWN and its conspecies which exist in pinewood and can complete diagnostic procedures within 25 min in the field. Moreover, the RPA assay can detect PWN in old wood samples in both natural and stored conditions. The POCD-RPA assay to detect PWN will be useful for epidemiological investigations in the field as well as for quarantine processes in the wood trade., Competing Interests: 2NCBio provided support in the form of salaries for SP. 2NCBio provided the portable optical isothermal device in development. The funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The specific roles of the authors are articulated in the ‘author contributions’ section.

Microrchidia (MORC) ATPases are critical for gene silencing and chromatin compaction in multiple eukaryotic systems, but the mechanisms by which MORC proteins act are poorly understood. Here, we apply a series of biochemical, single-molecule, and cell-based imaging approaches to better understand the function of the Caenorhabditis elegans MORC-1 protein. We find that MORC-1 binds to DNA in a length-dependent but sequence non-specific manner and compacts DNA by forming DNA loops. MORC-1 molecules diffuse along DNA but become static as they grow into foci that are topologically entrapped on DNA. Consistent with the observed MORC-1 multimeric assemblies, MORC-1 forms nuclear puncta in cells and can also form phase-separated droplets in vitro. We also demonstrate that MORC-1 compacts nucleosome templates. These results suggest that MORCs affect genome structure and gene silencing by forming multimeric assemblages to topologically entrap and progressively loop and compact chromatin., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Trichinella spiralis infection in skeletal muscle culminates with nurse cell formation. The participation of excretory-secretory products of the muscle larvae has been implicated in this process through different studies performed in infected muscle and the muscle cell line C2C12. In this work, we developed primary myoblast cultures to analyse the changes induced by excretory-secretory products of the muscle larvae in muscle cells. Microarray analyses revealed expression changes in muscle cell differentiation, proliferation, cytoskeleton organisation, cell motion, transcription, cell cycle, apoptosis and signalling pathways such as MAPK, Jak-STAT, Wnt and PI3K-Akt. Some of these changes were further evaluated by other methodologies such as quantitative real-time PCR (qRT-PCR) and western blot, confirming that excretory-secretory products of the muscle larvae treated primary mouse myoblasts undergo increased proliferation, decreased expression of MHC and up-regulation of α-actin. In addition, changes in relevant muscle transcription factors (Pax7, Myf5 and Mef2c) were observed. Taken together, these results provide new information about how T. spiralis could alter the normal process of skeletal muscle repair after ML invasion to accomplish nurse cell formation., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa. TpLAP contains two characteristic motifs of the M17LAP family in the C-terminal sequence: the metal binding site 265-[VGKG]-271 and the catalytic domain motif 351-[NTDAEGRL]-357. The soluble GST-TpLAP protein was expressed in Escherichia coli Transetta (DE3) and four specific anti-TpLAP monoclonal antibodies (mAbs) were prepared. In enzymatic assays, the optimal activity was observed at pH 9.5 at 45 °C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/ml. The enzymatic activity was significantly enhanced by Mn 2+ and completely inhibited by 20 nM bestatin and 0.15 mM EDTA. The native TpLAP was detected specifically in ES components of adult T. pisiformis by western blotting using anti-TpLAP mAb as a probe. Quantitative real-time PCR revealed that the TpLAP gene was expressed at a high level in adult worm tissues, especially in the gravid proglottids (50.71-fold). Immunolocalization analysis showed that TpLAP was located primarily in the subtegumental parenchyma zone and the uterine wall of adult worms. Our results indicate that TpLAP is a new member of the M17LAP family and can be considered as a stage-differentially expressed protein. These findings might provide new insights into the study of the mechanisms of growth, development and survival of T. pisiformis in the final host and have potential value as an attractive target for drug therapy or vaccine intervention., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: High-throughput sequencing has made it theoretically possible to obtain high-quality de novo assembled genome sequences but in practice DNA extracts are often contaminated with sequences from other organisms. Currently, there are few existing methods for rigorously decontaminating eukaryotic assemblies. Those that do exist filter sequences based on nucleotide similarity to contaminants and risk eliminating sequences from the target organism., Results: We introduce a novel application of an established machine learning method, a decision tree, that can rigorously classify sequences. The major strength of the decision tree is that it can take any measured feature as input and does not require a priori identification of significant descriptors. We use the decision tree to classify de novo assembled sequences and compare the method to published protocols., Conclusions: A decision tree performs better than existing methods when classifying sequences in eukaryotic de novo assemblies. It is efficient, readily implemented, and accurately identifies target and contaminant sequences. Importantly, a decision tree can be used to classify sequences according to measured descriptors and has potentially many uses in distilling biological datasets.

Infliction of DNA damage initiates a complex cellular reaction - the DNA damage response - that involves both signaling and DNA repair networks with many redundancies and parallel pathways. Here, we reveal the three strategies that the simple multicellular eukaryote, C. elegans, uses to deal with DNA damage induced by light. Separately inactivating repair or replicative bypass of photo-lesions results in cellular hypersensitivity towards UV-light, but impeding repair of replication associated DNA breaks does not. Yet, we observe an unprecedented synergistic relationship when these pathways are inactivated in combination. C. elegans mutants that lack nucleotide excision repair (NER), translesion synthesis (TLS) and alternative end joining (altEJ) grow undisturbed in the dark, but become sterile when grown in light. Even exposure to very low levels of normal daylight impedes animal growth. We show that NER and TLS operate to suppress the formation of lethal DNA breaks that require polymerase theta-mediated end joining (TMEJ) for their repair. Our data testifies to the enormous genotoxicity of light and to the demand of multiple layers of protection against an environmental threat that is so common., (Copyright © 2017 Elsevier B.V. All rights reserved.)

River blindness, caused by infection with the filarial nematode Onchocerca volvulus, is a neglected tropical disease affecting millions of people. There is a clear need for diagnostic tools capable of identifying infected patients, but that can also be used for monitoring disease progression and treatment efficacy. Plasma-derived parasitic microRNAs have been suggested as potential candidates for such diagnostic tools. We have investigated whether these parasitic microRNAs are present in sufficient quantity in plasma of Onchocerca-infected patients to be used as a diagnostic biomarker for detection of O. volvulus infection or treatment monitoring. Plasma samples were collected from different sources (23 nodule-positive individuals and 20 microfilaridermic individuals), microRNAs (miRNAs) were extracted using Qiagen miRNeasy kit, and a set of 17 parasitic miRNAs was evaluated on these miRNA extracts using miRCURY Locked Nucleic Acid (LNA) Universal RT microRNA PCR system. Of the 17 miRNAs evaluated, only 7 miRNAs were found to show detectable signal in a number of samples: bma-miR-236-1, bma-miR-71, ov-miR71-22nt, ov-miR-71-23nt, ov-miR-100d, ov-bantam-a, and ov-miR-87-3p. Subsequent melting curve analysis, however, indicated that the signals observed for ov-miR-71 variants and ov-miR-87-3p are non-specific. The other miRNAs only showed positive signal in one or few samples with Cq values just below the cutoff. Our data indicate that parasitic miRNAs are not present in circulation at a sufficiently high level to be used as biomarker for O. volvulus infection or treatment monitoring using LNA-based RT-qPCR analysis.

14-3-3 proteins have been found to be an excreted/secreted antigen and assumed to be released into the host-parasite interface and described in several unicellular and multicellular parasites. However, little is known about the immunomodulatory effects of H. controtus 14-3-3 protein on host cell. In present study, 14-3-3 isoform 2 gene, designated as Hcftt-2, was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from the adult H. contortus cDNA and cloned into expression plasmid pET32a (+) and expression of the recombinant protein (rHcftt-2) was induced by IPTG. Binding activity of rHcftt-2 to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay (IFA) and modulatory effects on cytokine production, cell proliferation, cell migration and nitric oxide (NO) production were observed by co-incubation of rHcftt-2 with goat PBMCs. Sequence analysis showed that it had significant homology with the known 14-3-3 protein isoform 2. Results of IFA revealed that, the rHcftt-2 was bound to the cell surface. We found that, the productions of IL10, IL-17, IFN-γ and cell migration of PBMCs were increased after the cells were incubated with rHCftt-2. However, the productions of IL-4, NO and cell proliferation of the PBMCs were significantly decreased in dose depended manner. Our results showed that the Hcftt-2 played important suppressive regulatory effects on the goat PBMCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Histone H3 lysine 9 (H3K9) methylation is a conserved modification that generally represses transcription. In Caenorhabditis elegans it is enriched on silent tissue-specific genes and repetitive elements. In met-2 set-25 double mutants, which lack all H3K9 methylation (H3K9me), embryos differentiate normally, although mutant adults are sterile owing to extensive DNA-damage-driven apoptosis in the germ line. Transposons and simple repeats are derepressed in both germline and somatic tissues. This unprogrammed transcription correlates with increased rates of repeat-specific insertions and deletions, copy number variation, R loops and enhanced sensitivity to replication stress. We propose that H3K9me2 or H3K9me3 stabilizes and protects repeat-rich genomes by suppressing transcription-induced replication stress.

Haemonchus contortus is the leading parasitic nematode species used to study anthelmintic drug resistance. A variety of candidate loci have been implicated as being associated with ivermectin resistance in this parasite but definitive evidence of their importance is still lacking. We have previously performed two independent serial backcross experiments to introgress ivermectin resistance loci from two H. contortus ivermectin-resistant strains - MHco4(WRS) and MHco10(CAVR) - into the genetic background of the ivermectin-susceptible genome reference strain MHco3(ISE). We have interrogated a number of candidate ivermectin resistance loci in the resulting backcross populations and assessed the evidence for their genetic linkage to an ivermectin resistance locus. These include the microsatellite marker Hcms8a20 and six candidate genes Hco-glc-5, Hco-avr-14, Hco-lgc-37 (previously designated Hco-hg-1), Hco-pgp-9 (previously designated Hco-pgp-1), Hco-pgp-2 and Hco-dyf-7. We have sampled the haplotype diversity of amplicon markers within, or adjacent to, each of these loci in the parental strains and fourth generation backcross populations to assess the evidence for haplotype introgression from the resistant parental strain into the genomic background of the susceptible parental strain in each backcross. The microsatellite Hcms8a20 locus showed strong evidence of such introgression in both independent backcrosses, suggesting it is linked to an important ivermectin resistance mutation in both the MHco4(WRS) and MHco10(CAVR) strains. In contrast, Hco-glc-5, Hco-avr-14, Hco-pgp-9 and Hco-dyf-7 showed no evidence of introgression in either backcross. Hco-lgc-37 and Hco-pgp-2 showed only weak evidence of introgression in the MHco3/4 backcross but not in the MHco3/10 backcross. Overall, these results suggest that microsatellite marker Hcms8a20, but not the other candidate genes tested, is linked to a major ivermectin resistance locus in the MHco4(WRS) and MHco10(CAVR) strains. This work also emphasises the need for genome-wide approaches to identify mutations responsible for the ivermectin resistance in this parasite., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.

Schistosomiasis is a chronically debilitating helminth infection with a significant socio-economic and public health impact. Accurate diagnostics play a pivotal role in achieving current schistosomiasis control and elimination goals. However, many of the current diagnostic procedures, which rely on detection of schistosome eggs, have major limitations including lack of accuracy and the inability to detect pre-patent infections. DNA-based detection methods provide a viable alternative to the current tests commonly used for schistosomiasis diagnosis. Here we describe the optimisation of a novel droplet digital PCR (ddPCR) duplex assay for the diagnosis of Schistosoma japonicum infection which provides improved detection sensitivity and specificity. The assay involves the amplification of two specific and abundant target gene sequences in S. japonicum; a retrotransposon (SjR2) and a portion of a mitochondrial gene (nad1). The assay detected target sequences in different sources of schistosome DNA isolated from adult worms, schistosomules and eggs, and exhibits a high level of specificity, thereby representing an ideal tool for the detection of low levels of parasite DNA in different clinical samples including parasite cell free DNA in the host circulation and other bodily fluids. Moreover, being quantitative, the assay can be used to determine parasite infection intensity and, could provide an important tool for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Human dirofilariasis is a zoonotic infection that continues to spread to previously unaffected areas of Europe. In the South Moravian Region of the Czech Republic (CR), imported as well as autochthonous canine infections were recorded in the last decade, and parasite DNA was detected in mosquitoes of Aedes vexans. In the present paper, human Dirofilaria infections are reported from the country for the first time., Case Presentation: The samples from five patients with suspected tissue helminthiases were investigated. In particular cases, nematodes were isolated from various tissues including skin of lower leg, soft tissues of finger, subcutaneous tissue of hypogastrium, lymph node and peritoneum. The diagnosis was based on light microscopic morphology and/or DNA analysis of the worms. In addition, ELISA examination of patients' sera for anti-filaria IgG antibodies was performed., Conclusions: In the CR, five cases of human dirofilariasis caused by Dirofilaria repens were recorded during 2010-2014 (species determination for three of them was confirmed besides morphological also by DNA analysis). At least, three of the cases were of autochthonous origin (the patients are Czech citizens residing in South Moravian Region who have never travelled abroad). The findings confirm the natural setting of D. repens in South Moravian Region of the CR. Dirofilariasis should be therefore considered as endemic in this area where it may represent a significant risk factor for public health.

The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 and SmHMGB3 share highly conserved HMG box DNA binding domains but have significantly different C-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteins to examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengths of the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNA transactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to the structural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patterns of SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interference approaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms, whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phosphorylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBs were greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminal acidic tails dictate the differences in the structure, expression and function of schistosome HMGB proteins., (Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

C. elegans has recently emerged as a valuable model to understand the link between nuclear organization and cell fate, by combining microscopy approaches, genome-wide mapping techniques with advanced genetics. Crucial to these analyses are techniques to determine the genome-wide interaction pattern of proteins with DNA. Chromatin immunoprecipitation has proven valuable but it requires considerable amounts of starting material. This is sometimes difficult to achieve, in particular for specific genotypes (balanced strains, different sexes, severe phenotypes…). As an alternative to ChIP, DNA adenine methyltransferase identification by sequencing (DamID-seq) was recently shown to be able to characterize binding sites in single mammalian cells. Additionally, DamID can be achieved for cell-type specific analysis by expressing Dam fusion proteins under tissue specific promoters in a controlled manner. In this report, we present a user-friendly pipeline to analyse DamID-seq data in C. elegans. Based upon this pipeline, we provide a comparative analysis of libraries generated with different starting material and discuss important library features. Moreover, we introduce an adaptation of an imaging based tool to visualize in vivo the cell-specific tridimensional binding pattern of any protein of interest., (© 2016 Wiley Periodicals, Inc.)

Mass treatment of lymphatic filariasis with Albendazole (ABZ), a therapeutic benzimidazole, is fraught with serious limitations such as possible drug resistance and poor macrofilaricidal activity. Therefore, we need to develop new ABZ-based formulations to improve its antifilarial effectiveness. CuO nanoparticles were used as an adjuvant with ABZ to form ABZ-CuO nanocomposite, which was characterized by UV-vis spectroscopy, FT-IR, AFM and SEM. Antifilarial activity of nanocomposite was evaluated using relative motility assay and dye exclusion test in dark and under UV light. ROS generation, antioxidant levels, lipid peroxidation and DNA fragmentation in nanocomposite treated parasites were estimated. Biophysical techniques were employed to ascertain the mode of binding of nanocomposite to parasitic DNA. Nanocomposite increases parasite mortality as compared to ABZ in dark, and its antifilarial effect was increased further under UV light. Elevated ROS production and decline of parasitic-GST and GSH levels were observed in nanocomposite treated worms in dark, and these effects were pronounced further under UV light. Nanocomposite leads to higher DNA fragmentation as compared to ABZ alone. Further, we found that nanocomposite binds parasitic DNA in an intercalative manner where it generates ROS to induce DNA damage. Thus, oxidative stress production due to ROS generation and consequent DNA fragmentation leads to apoptosis in worms. This is the first report supporting CuO nanoparticles as a potential adjuvant with ABZ against filariasis along with enhanced antifilarial activity of nanocomposite under UV light. These findings, thus, indicate that development of ABZ-loaded nanoparticle compounds may serve as promising leads for filariasis treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Molecular dynamics simulations of the Caenorhabditis elegans transcription factor SKN-1 bound to its cognate DNA site show that the protein-DNA interface undergoes significant dynamics on the microsecond timescale. A detailed analysis of the simulation shows that movements of two key arginine side chains between the major groove and the backbone of DNA generate distinct conformational sub-states that each recognize only part of the consensus binding sequence of SKN-1, while the experimentally observed binding specificity results from a time-averaged view of the dynamic recognition occurring within this complex., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The neuromuscular system of helminths controls a variety of essential biological processes and therefore represents a good source of novel drug targets. The neuroactive substance, acetylcholine controls movement of Schistosoma mansoni but the mode of action is poorly understood. Here, we present first evidence of a functional G protein-coupled acetylcholine receptor in S. mansoni, which we have named SmGAR. A bioinformatics analysis indicated that SmGAR belongs to a clade of invertebrate GAR-like receptors and is related to vertebrate muscarinic acetylcholine receptors. Functional expression studies in yeast showed that SmGAR is constitutively active but can be further activated by acetylcholine and, to a lesser extent, the cholinergic agonist, carbachol. Anti-cholinergic drugs, atropine and promethazine, were found to have inverse agonist activity towards SmGAR, causing a significant decrease in the receptor's basal activity. An RNAi phenotypic assay revealed that suppression of SmGAR activity in early-stage larval schistosomulae leads to a drastic reduction in larval motility. In sum, our results provide the first molecular evidence that cholinergic GAR-like receptors are present in schistosomes and are required for proper motor control in the larvae. The results further identify SmGAR as a possible candidate for antiparasitic drug targeting., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been limited, as DNA-binding motifs are known for only 71 of the estimated 763 sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray experiments on representatives of canonical TF families in C. elegans, obtaining motifs for 129 TFs. Additionally, we predict motifs for many TFs that have DNA-binding domains similar to those already characterized, increasing coverage of binding specificities to 292 C. elegans TFs (∼40%). These data highlight the diversification of binding motifs for the nuclear hormone receptor and C2H2 zinc finger families and reveal unexpected diversity of motifs for T-box and DM families. Motif enrichment in promoters of functionally related genes is consistent with known biology and also identifies putative regulatory roles for unstudied TFs.

Schistosomiasis is a chronically debilitating disease caused by parasitic worms of the genus Schistosoma, and it is a global problem affecting over 240 million people. Little is known about the regulatory proteins and mechanisms that control schistosome host invasion, gene expression, and development. Schistosome larvae, cercariae, are transiently free-swimming organisms and infectious to man. Cercariae penetrate human host skin directly using proteases that degrade skin connective tissue. These proteases are secreted from anucleate acetabular glands that contain many proteins, including heat shock proteins. Heat shock transcription factors are strongly conserved activators that play crucial roles in the maintenance of cell homeostasis by transcriptionally regulating heat shock protein expression. In this study, we clone and characterize the schistosome Heat shock factor 1 gene (SmHSF1). We verify its ability to activate transcription using a modified yeast one-hybrid system, and we show that it can bind to the heat shock binding element (HSE) consensus DNA sequence. Our quantitative RT-PCR analysis shows that SmHSF1 is expressed throughout several life-cycle stages from sporocyst to adult worm. Interestingly, using immunohistochemistry, a polyclonal antibody raised against an Hsf1-peptide demonstrates a novel localization for this conserved, stress-modulating activator. Our analysis suggests that schistosome Heat shock factor 1 may be localized to the acetabular glands of infective cercariae.

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

Little is known about the genetic variability of the soil-transmitted nematode, Strongyloides stercoralis, in humans. We sequenced portions of the small subunit rDNA (SSU), including the hyper variable regions (HVR) I and IV from S. stercoralis larvae derived from individuals living in a rural setting in Cambodia. We identified three polymorphic positions, including a previously reported one within the HVR I. HVR IV was invariable. Six different SSU alleles existed in our sample. Although different genotypes of S. stercoralis were found in the same individuals, no heterozygous larvae were found. This indicates that there is no or very little interbreeding between the different genotypes. Further studies are needed to examine if this is because sexual reproduction, which is facultative, is rare in our study area's S. stercoralis population or because what is considered to be S. stercoralis today is actually a complex of closely related species or subspecies., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, we aimed to identify transcription factors necessary for lin-39 expression in the VPCs, and in particular sought factors that initiate lin-39 expression in the embryo., Results: We used the yeast one-hybrid (Y1H) method to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and we characterized several factors by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations in lin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site we identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs., Conclusions: These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust.

The global identification of transcription factor (TF) binding sites is a critical step in the elucidation of the functional elements of the genome. Several methods have been developed that map TF binding in human cells, yeast, and other model organisms. These methods make use of chromatin immunoprecipitation, or ChIP, and take advantage of the fact that formaldehyde fixation of living cells can be used to cross-link DNA sequences to the TFs that bind them in vivo. In ChIP, the cross-linked TF-DNA complexes are sheared by sonication, size fractionated, and incubated with antibody specific to the TF of interest to generate a library of TF-bound DNA sequences. ChIP-chip was the first technology developed to globally identify TF-bound DNA sequences and involves subsequent hybridization of the ChIP DNA to oligonucleotide microarrays. However, ChIP-chip proved to be costly, labor-intensive, and limited by the fixed number of probes available on the microarray chip. ChIP-Seq combines ChIP with massively parallel high-throughput sequencing (see Explanatory Chapter: Next Generation Sequencing) and has demonstrated vast improvement over ChIP-chip with respect to time and cost, signal-to-noise ratio, and resolution. In particular, multiplex sequencing can be used to achieve a higher throughput in ChIP-Seq analyses involving organisms with genomes of lower complexity than that of human (Lefrançois et al., 2009) and thereby reduce the cost and amount of time needed for each result. The multiplex ChIP-Seq method described in this section has been developed for Caenorhabditis elegans, but is easily adaptable for other organisms., (© 2014 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are a subtype of small regulatory RNAs that are involved in numerous biological processes through small RNA-induced silencing networks. In an attempt to explore the phylogeny of miRNAs across five platyhelminths, we integrated annotated miRNAs and their full genomes. We identified conserved miRNA clusters and, in particular, miR-71/2 was conserved from planarian to parasitic flatworms and was expanded in free-living Schmidtea mediterranea. Analysis of 22 miRNA loci provided compelling evidence that most known miRNAs are conserved across platyhelminths. Meanwhile, we also observed alterations of known protein-coding genes flanking miRNA(s), such as transcriptional direction conversion and locus relocation, in around ~ 41% of 22 known miRNA loci. Compared with Echinococcus multilocularis, the majority of these events occurred in evolution-distant Hymenolepis microstoma, Schistosoma japonicum or/and S. mediterranea. These results imply rearrangement events occurred near the known miRNA loci., (© 2013 FEBS.)

A mosquito survey was conducted to identify which species of mosquitoes carry Dirofilaria immitis (Leidy) (Nematoda: Filarioidea), dog heartworm, in northeast Arkansas. Using polymerase chain reaction, mosquitoes were analyzed for D. immitis, Dirofilaria repens Railliet & Henry, and Acanthocheilonema dracunculoides Cobbold. Mosquitoes were collected from April to October 2009 using black light ultraviolet traps baited with dry ice. Sixteen mosquito species were identified. D. immitis was identified in nine mosquito species, which included Aedes vexans (Meigen), Anopheles quadrimaculatus Say, Anopheles punctipennis (Say), Culex pipiens quinquefasciatus Say, Culex erraticus (Dyer & Knab), Culiseta inornata (Williston), Psorophora columbiae (Dyer & Knab), Psorophora ferox (Humboldt), and Psorophora howardii Coquillett. No D. repens or A. dracunculoides DNA was amplified. Of the 1,212 mosquito pools tested, 7.3% were positive for D. immitis. Frequency of D. immitis infections from six collection sites ranged from 2.1 to 19.4%. Ae. vexans and An. quadrimaculatus were the two most abundant species, composing 58.7 and 23.7% of the total mosquitoes collected, with 9.6 and 6.9% of pools positive for D. immitis, respectively. To investigate localized vector infection rates of D. immitis, mosquitoes were collected from inside the kennel of a heartworm-positive dog. Of the 114 mosquitoes collected, 84 (73.7%) were positive for D. immitis. The frequency of D. immitis-infected mosquitoes collected near a heartworm-positive dog was considerably higher than in the original six collection sites, suggesting a single heartworm-positive dog potentially increases infection pressure on susceptible animals sharing mosquito exposure.

The polymerase chain reaction (PCR) assay has turned out to be one of the most potential tools for diagnosis of schistosomiasis. However, the source and metabolic dynamics of Schistosoma japonicum DNA in the blood of hosts is not clear. In this study, rabbit models with monosexual and mixed sexual cercariae infection were established to interpret the source of the parasite DNA in serum of the hosts. Following administration of praziquantel at 7 weeks postinfection, the metabolic mechanism of S. japonicum DNA in serum of the hosts was studied. The findings showed that, for the monosexual cercariae infection, the parasite DNA was detectable in serum of the host from day 3 to week 3 postinfection, while for the mixed sexual cercariae infection, the detection results were continually positive during the 7 weeks after infection. After treatment with praziquantel, detection of S. japonicum DNA in rabbit sera became positive at the second day posttreatment, and the positive period lasted 3 weeks in the monosexual cercariae infection group. However, with the mixed sexual cercariae infection group, the PCR results remained positive for 16 weeks after treatment. We conclude that the S. japonicum DNA in host serum primarily comes from the residual body of dead schistosomula and/or tegument shedding of worm growing in the first 4 weeks postinfection, while during the spawning stage of the female schistosome, the parasite DNA mainly comes from the disintegration of inactive eggs. The duration from treatment to total elimination of worm origin DNA in serum is not exceeding 3 weeks. However, the DNA release from inactive eggs can last for more than 16 weeks. Further studies are needed to address the sources and metabolic dynamics of S. japonicum DNA in human serum.

Chromatin diminution is the programmed elimination of specific DNA sequences during development. It occurs in diverse species, but the function(s) of diminution and the specificity of sequence loss remain largely unknown. Diminution in the nematode Ascaris suum occurs during early embryonic cleavages and leads to the loss of germline genome sequences and the formation of a distinct genome in somatic cells. We found that ∼43 Mb (∼13%) of genome sequence is eliminated in A. suum somatic cells, including ∼12.7 Mb of unique sequence. The eliminated sequences and location of the DNA breaks are the same in all somatic lineages from a single individual and between different individuals. At least 685 genes are eliminated. These genes are preferentially expressed in the germline and during early embryogenesis. We propose that diminution is a mechanism of germline gene regulation that specifically removes a large number of genes involved in gametogenesis and early embryogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Base excision repair (BER) is an evolutionarily conserved DNA repair pathway that is critical for repair of many of the most common types of DNA damage generated both by endogenous metabolic pathways and exposure to exogenous stressors such as pollutants. Caenorhabditis elegans is an increasingly important model organism for the study of DNA damage-related processes including DNA repair, genotoxicity, and apoptosis, but BER is not well understood in this organism, and has not previously been measured in vivo. We report robust BER in the nuclear genome and slightly slower damage removal from the mitochondrial genome; in both cases the removal rates are comparable to those observed in mammals. However we could detect no deficiency in BER in the nth-1 strain, which carries a deletion in the only glycosylase yet described in C. elegans that repairs oxidative DNA damage. We also failed to detect increased lethality or growth inhibition in nth-1 nematodes after exposure to oxidative or alkylating damage, suggesting the existence of at least one additional as-yet undetected glycosylase., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was developed for the molecular identification of 2 introduced bucephalid trematodes, Prosorhynchoides ozakii and Parabucephalopsis parasiluri . The method was applied for sporocysts and cercariae obtained from the golden mussel Limnoperna fortunei collected in the Uji River, Japan. The PCR-RFLP method showed that L. fortunei is the intermediate host of both trematode species. The present study thus recognizes the risk of L. fortunei , an invasive molluscan species, as a potential host for pathogenic trematodes.

The genetic diversity within and among parasite populations provides clues to their evolutionary history. Here, we sought to determine whether mitochondrial and microsatellite DNA variation could be used to evaluate the extent of differentiation, gene flow and historical reproductive isolation among the freeze resistant parasites Trichinella nativa and the Trichinella T6 genotype infecting wolverines (Gulo gulo) in Nunavut, Canada. To this end, we genotyped Trichinella isolates derived from the diaphragms of 39 wolverines from this locale to reference strains of T. nativa and the Trichinella T6 genotype. Results showed that among a subset of 13 isolates examined, individuals resembled T. nativa in their mitochondrial DNA, but resembled the Trichinella T6 genotype when assayed at expansion segment V and the internal transcribed spacer of the nuclear rDNA. To adjudicate among these conflicting diagnoses, we further characterized each isolate at several nuclear microsatellite loci and again compared these to data from reference strains. Statistical assignment established that the nuclear genomes of most Nunavut isolates corresponded to those of the Trichinella T6 genotype; however, two isolates corresponded to T. nativa, and one isolate exhibited equal similarity to both reference strains. Taken as a whole, the evidence suggests that these isolates derive from the T. nativa matrilineage, but that their nuclear genomes resemble individuals previously designated as Trichinella T6. Assuming distinct lineages, this argues for cross-hybridization among these genotypes. Although introgression has occurred, recognizable genetic distinctions persist. One possibility is that selection disfavors the survival of hybrid offspring in most instances. Alternatively, the recent disappearance of glacial barriers might have increased contact, and therefore introgression. Broader geographic sampling will be required to determine the extent to which hybridization occurs beyond this particular geographic focus., (Published by Elsevier B.V.)

During some estimations of the nuclear DNA content, based on determinations propidium iodide (PI) binding through fluorocytometry for Taenia crassiceps cysticerci, significant variation in the results were found. This initial observation led to a series of experiments designed to explain the variation. These changes could be induced by the diameter of the needles in the syringes used for the mouse to mouse transfer of the cysts. Nuclei from cysts transferred through 27-gauge needles showed 30% less PI staining than those transferred through 21 gauge needles, after 2 months infections. Reduction in PI capture induced by 27-gauge needle was reversible when the cysts were maintained in their mice hosts during 5 months. Moreover, variation in PI binding to cysticercal DNA was also found when comparing parasites grown in male versus female mice. The use of agents that homogenize the chromatin structure during PI staining, allowed demonstrating that variation were entirely due to differences in the chromatin relaxation/compaction. Additional experiments demonstrated that the higher compaction is accompanied by a reduced ability of cysts to grow in the peritoneal cavity of BALB/cAnN mice. Furthermore, proteomic analysis also showed that these changes in chromatin relaxation/compaction resulted in different levels and patterns of protein expression. Our results strongly suggest that chromatin is involved in several well characterized phenomena of the T. crassiceps murine model, and open new avenues for a detailed approach to understand such a complex host-parasite relationship., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Endonuclease G (EndoG) is a mitochondrial protein that traverses to the nucleus and participates in chromosomal DNA degradation during apoptosis in yeast, worms, flies, and mammals. However, it remains unclear how EndoG binds and digests DNA. Here we show that the Caenorhabditis elegans CPS-6, a homolog of EndoG, is a homodimeric Mg(2+)-dependent nuclease, binding preferentially to G-tract DNA in the optimum low salt buffer at pH 7. The crystal structure of CPS-6 was determined at 1.8 Å resolution, revealing a mixed αβ topology with the two ββα-metal finger nuclease motifs located distantly at the two sides of the dimeric enzyme. A structural model of the CPS-6-DNA complex suggested a positively charged DNA-binding groove near the Mg(2+)-bound active site. Mutations of four aromatic and basic residues: Phe(122), Arg(146), Arg(156), and Phe(166), in the protein-DNA interface significantly reduced the DNA binding and cleavage activity of CPS-6, confirming that these residues are critical for CPS-6-DNA interactions. In vivo transformation rescue experiments further showed that the reduced DNase activity of CPS-6 mutants was positively correlated with its diminished cell killing activity in C. elegans. Taken together, these biochemical, structural, mutagenesis, and in vivo data reveal a molecular basis of how CPS-6 binds and hydrolyzes DNA to promote cell death.

Selfish DNA poses a significant challenge to genome stability and organismal fitness in diverse eukaryotic lineages. Although selfish mitochondrial DNA (mtDNA) has known associations with cytoplasmic male sterility in numerous gynodioecious plant species and is manifested as petite mutants in experimental yeast lab populations, examples of selfish mtDNA in animals are less common. We analyzed the inheritance and evolution of mitochondrial DNA bearing large heteroplasmic deletions including nad5 gene sequences (nad5Δ mtDNA), in the nematode Caenorhabditis briggsae. The deletion is widespread in C. briggsae natural populations and is associated with deleterious organismal effects. We studied the inheritance patterns of nad5Δ mtDNA using eight sets of C. briggsae mutation-accumulation (MA) lines, each initiated from a different natural strain progenitor and bottlenecked as single hermaphrodites across generations. We observed a consistent and strong drive toward higher levels of deletion-bearing molecules in the heteroplasmic pool of mtDNA after ten generations of bottlenecking. Our results demonstrate a uniform transmission bias whereby nad5Δ mtDNA accumulates to higher levels relative to intact mtDNA in multiple genetically diverse natural strains of C. briggsae. We calculated an average 1% per-generation transmission bias for deletion-bearing mtDNA relative to intact genomes. Our study, coupled with known deleterious phenotypes associated with high deletion levels, shows that nad5Δ mtDNA are selfish genetic elements that have evolved in natural populations of C. briggsae, offering a powerful new system to study selfish mtDNA dynamics in metazoans.

DNA injected into the Caenorhabditis elegans germline forms extrachromosomal arrays that segregate during cell division [1, 2]. The mechanisms underlying array formation and segregation are not known. Here, we show that extrachromosomal arrays form de novo centromeres at high frequency, providing unique access to a process that occurs with extremely low frequency in other systems [3-8]. De novo centromerized arrays recruit centromeric chromatin and kinetochore proteins and autonomously segregate on the spindle. Live imaging following DNA injection revealed that arrays form after oocyte fertilization via homologous recombination and nonhomologous end-joining. Individual arrays gradually transition from passive inheritance to active segregation during the early embryonic divisions. The heterochromatin protein 1 (HP1) family proteins HPL-1 and HPL-2 are dispensable for de novo centromerization even though arrays become strongly enriched for the heterochromatin-associated H3K9me3 modification over time. Partial inhibition of HP1 family proteins accelerates the acquisition of segregation competence. In addition to reporting the first direct visualization of new centromere formation in living cells, these findings reveal that naked DNA rapidly builds de novo centromeres in C. elegans embryos in an HP1-independent manner and suggest that, rather than being a prerequisite, HP1-dependent heterochromatin antagonizes de novo centromerization., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Mitochondrial supercomplexes containing complexes I, III, and IV of the electron transport chain are now regarded as an established entity. Supercomplex I·III·IV has been theorized to improve respiratory chain function by allowing quinone channeling between complexes I and III. Here, we show that the role of the supercomplexes extends beyond channeling. Mutant analysis in Caenorhabditis elegans reveals that complex III affects supercomplex I·III·IV formation by acting as an assembly or stabilizing factor. Also, a complex III mtDNA mutation, ctb-1, inhibits complex I function by weakening the interaction of complex IV in supercomplex I·III·IV. Other complex III mutations inhibit complex I function either by decreasing the amount of complex I (isp-1), or decreasing the amount of complex I in its most active form, the I·III·IV supercomplex (isp-1;ctb-1). ctb-1 suppresses a nuclear encoded complex III defect, isp-1, without improving complex III function. Allosteric interactions involve all three complexes within the supercomplex and are necessary for maximal enzymatic activities.

Aging is a complex phenotype responsive to a plethora of environmental inputs; yet only a limited number of transcriptional regulators are known to influence life span. How the downstream expression programs mediated by these factors (or others) are coordinated into common or distinct set of aging effectors is an addressable question in model organisms, such as C. elegans. Here, we establish the transcription factor ETS-4, an ortholog of vertebrate SPDEF, as a longevity determinant. Adult worms with ets-4 mutations had a significant extension of mean life span. Restoring ETS-4 activity in the intestine, but not neurons, of ets-4 mutant worms rescued life span to wild-type levels. Using RNAi, we demonstrated that ets-4 is required post-developmentally to regulate adult life span; thus uncoupling the role of ETS-4 in aging from potential functions in worm intestinal development. Seventy ETS-4-regulated genes, identified by gene expression profiling of two distinct ets-4 alleles and analyzed by bioinformatics, were enriched for known longevity effectors that function in lipid transport, lipid metabolism, and innate immunity. Putative target genes were enriched for ones that change expression during normal aging, the majority of which are controlled by the GATA factors. Also, some ETS-4-regulated genes function downstream of the FOXO factor, DAF-16 and the insulin/IGF-1 signaling pathway. However, epistasis and phenotypic analyses indicate that ets-4 functioned in parallel to the insulin/IGF-1 receptor, daf-2 and akt-1/2 kinases. Furthermore, ets-4 required daf-16 to modulate aging, suggesting overlap in function at the level of common targets that affect life span. In conclusion, ETS-4 is a new transcriptional regulator of aging, which shares transcriptional targets with GATA and FOXO factors, suggesting that overlapping pathways direct common sets of lifespan-related genes., Competing Interests: The authors have declared that no competing interests exist.

Diet can have profound effects on an organism's health. Metabolic studies offer an effective way to measure and understand the physiological effects of diet or disease. The metabolome is very sensitive to dietary, lifestyle and genetic changes. Caenorhabditis elegans, a soil nematode, is an attractive model organism for metabolic studies because of the ease with which genetic and environmental factors can be controlled. In this work, we report significant effects of diet, mutation and RNA interference on the C.elegans metabolome. Two strains of Escherichia coli, OP50 and HT115 are commonly employed as food sources for maintaining and culturing the nematode. We studied the metabolic and phenotypic effects of culturing wild-type and mutant worms on these two strains of E. coli. We report significant effects of diet on metabolic profile, on mitochondrial DNA copy number and on phenotype. The dietary effects we report are similar in magnitude to the effects of mutations or RNA interference-mediated gene suppression. This is the first critical evaluation of the physiological and metabolic effects on C.elegans of two commonly used culture conditions., (Copyright 2010 Elsevier Inc. All rights reserved.)

Our recent investigation in the protist Trichomonas vaginalis suggested a DNA sequence periodicity with a unit length of 120.9 nt, which represents a sequence signature for nucleosome positioning. We now extended our observation in higher eukaryotes and identified a similar periodicity of 175 nt in length in Caenorhabditis elegans. In the process of defining the sequence compositional characteristics, we found that the 10.5-nt periodicity, the sequence signature of DNA double helix, may not be sufficient for cross-nucleosome positioning but provides essential guiding rails to facilitate positioning. We further dissected nucleosome-protected sequences and identified a strong positive purine (AG) gradient from the 5'-end to the 3'-end, and also learnt that the nucleosome-enriched regions are GC-rich as compared to the nucleosome-free sequences as purine content is positively correlated with GC content. Sequence characterization allowed us to develop a hidden Markov model (HMM) algorithm for decoding nucleosome positioning computationally, and based on a set of training data from the fifth chromosome of C. elegans, our algorithm predicted 60%-70% of the well-positioned nucleosomes, which is 15%-20% higher than random positioning. We concluded that nucleosomes are not randomly positioned on DNA sequences and yet bind to different genome regions with variable stability, well-positioned nucleosomes leave sequence signatures on DNA, and statistical positioning of nucleosomes across genome can be decoded computationally based on these sequence signatures., (Copyright 2010 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.)

Chromosome fragmentation is a hallmark of apoptosis, conserved in diverse organisms. In mammals, caspases activate apoptotic chromosome fragmentation by cleaving and inactivating an apoptotic nuclease inhibitor. We report that inactivation of the Caenorhabditis elegans dcr-1 gene, which encodes the Dicer ribonuclease important for processing of small RNAs, compromises apoptosis and blocks apoptotic chromosome fragmentation. DCR-1 was cleaved by the CED-3 caspase to generate a C-terminal fragment with deoxyribonuclease activity, which produced 3' hydroxyl DNA breaks on chromosomes and promoted apoptosis. Thus, caspase-mediated activation of apoptotic DNA degradation is conserved. DCR-1 functions in fragmenting chromosomal DNA during apoptosis, in addition to processing of small RNAs, and undergoes a protease-mediated conversion from a ribonuclease to a deoxyribonuclease.

Meiotic crossovers (COs) are tightly regulated to ensure that COs on the same chromosome are distributed far apart (crossover interference, COI) and that at least one CO is formed per homolog pair (CO homeostasis). CO formation is controlled in part during meiotic double-strand break (DSB) creation in Caenorhabditis elegans, but a second level of control must also exist because meiotic DSBs outnumber COs. We show that the antirecombinase RTEL-1 is required to prevent excess meiotic COs, probably by promoting meiotic synthesis-dependent strand annealing. Two distinct classes of meiotic COs are increased in rtel-1 mutants, and COI and homeostasis are compromised. We propose that RTEL-1 implements the second level of CO control by promoting noncrossovers.