The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes. FoxD3 is an important stem cell factor expressed in many types of embryonic cells including neural crest cells. In the embryo, neural crest cells are a type of stem cell that forms diverse derivatives, including nerve cells, pigment cells, and facial structures. To better understand neural crest development and differentiation, we have explored how FoxD3 expression is regulated in these cells. By examining non-coding DNA, we have identified distinct genomic regions that mediate expression of green fluorescent protein (GFP) in a pattern that recapitulates FoxD3 expression. Interestingly, we find two genomic “on–off” switches or enhancers, called NC1 and NC2, that drive GFP expression in a pattern that recapitulates FoxD3 expression at different times and places during neural crest development. We find that Pax and Msx proteins turn on both NC1 and NC2 enhancers by directly binding to them. In addition, cranial expression driven by NC1 requires a protein called Ets1, whereas trunk expression of NC2 requires a different protein called Zic1. The results show that FoxD3 in differentially regulated in distinct neural crest cell populations in a manner that is specifically encoded in the genome. These enhancers provide valuable tools for understanding neural crest development in birds and mammals. The neural crest (NC) is a transient population of cells that migrates throughout the embryo and forms many different cell types, including neurons and glia of the peripheral and enteric nervous systems, bone and cartilage of the craniofacial skeleton and melanocytes An important challenge is to establish direct connections within the neural crest GRN. For example, the neural plate border marker, Pax7, is essential for expression of a number of different neural crest specifier genes Xenopus and zebrafish Xenopus ectopic expression at the 8-cell stage increases the expression of neural crest markers, while expression of dominant-negative FoxD3 reduces expression of genes like Snail2, Twist and Ets1 Of the neural crest specifier genes, FoxD3 is one of the first markers of premigratory neural crest in many vertebrate species including mouse, chick, cis-regulatory regions of the critical neural crest gene, FoxD3. Taking advantage of the chick's compact genome and ability to assay putative regulatory regions by electroporation, we have identified two enhancers, NC1 and NC2, that mediate reporter expression in spatially and temporally distinct manners in the chick embryo, and in combination closely recapitulate the endogenous expression of FoxD3. Detailed regulatory analysis shows that initial expression of FoxD3 in both cranial and trunk neural crest requires direct input from neural plate border genes, Pax7 and Msx1/2. These factors function in combination with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene Zic1 to activate the NC2 enhancer. These results not only reveal region-specific enhancer activity in the neural crest, but also expand the neural crest GRN and inform upon direct interactions therein. Conserved between mouse and chick, these enhancers further provide excellent tools for assaying gene regulation and manipulation of neural crest gene expression in amniotes.Despite its important role both in stem and neural crest cells, no regulatory element(s) controlling the onset of FoxD3 expression are known. To define linkages and assess direct regulatory interactions in the neural crest gene regulatory network with particular interest in possible targets of Pax7, we set out to dissect the FoxD3 expression domain includes the neural folds of the forebrain and midbrain. Subsequently, at HH8+, FoxD3 expression expands posteriorly to the hindbrain . FoxD3 is expressed in most premigratory and migratory vagal and trunk neural crest, but is down-regulated in melanoblasts  NC1 and mNC2 constructs were electroporated into chick embryos at gastrula stages. The results show that the patterns of eGFP expression driven by mNC1 and mNC2 were identical to those observed with chick NC1 and NC2 , suggestTo examine the dynamic nature and combined activity of the two enhancers in the migrating cranial neural crest, we co-electroporated NC1 (green) and NC2 (blue) enhancers in combination with a previously identified cranial neural crest Sox10E element (red) Time-lapse movies revealed differential temporal and spatial activity of NC1 and NC2 enhancers. While NC1 activity was present in the premigratory neural crest, the expression it drove in the dorsal neural tube appeared transient in most cells and preceded that driven by the Sox10E enhancer  and NC2 (red) enhancers and observed neural crest formation and migration by time lapse microscopy. Analysis of the movies suggested that there was little overlap between cells showing activity of NC1 and those with NC2 , whereasXenopus, but showed no sequence conservation with zebrafish. Primers were designed to amplify fragments of NC1, which were tested for activity at stages HH9–10, corresponding to the time it drove strongest expression. Using this approach, NC1 was reduced to 553 bp (NC1.1) without loss of activity , which reflects transcript levels more accurately than At trunk levels, knock-down of Msx1/2, Pax7 and Zic1 resulted in a significant reduction of endogenous FoxD3 expression , whereasin vivo association of Pax7, Msx1 and Ets1 transcription factors with the NC1 enhancer, we performed quantitative chromatin immunoprecipitation (ChIP) experiments. Cross-linked chromatin isolated from the midbrain dorsal neural tube of HH8–9 embryos was immunoprecipitated using Pax7, Msx1 and Ets1 antibodies and ChIP-enriched DNA was used in site-specific qPCR, with primers designed to amplify fragments within the NC1 region. For all three factors, we found significant enrichment of the NC1 region amplicon, expressed as a percent of the total input chromatin, compared to IgG controls , as well as in different subpopulations of the cranial neural crest. The enhancer NC1 is active in premigratory and some migratory cranial neural crest rostral to R3, while enhancer NC2 activity initiates in a single continuous wave caudal to rhombomere 4, including vagal and trunk regions, but also later in a subpopulation of migrating cranial neural crest. In our analysis of the conserved regions within the FoxD3 locus, only these two regions were able to mediate reporter expression in patterns reflecting the distribution of neural crest. The proximity of the NC1 and NC2 enhancers to the FoxD3 coding region, the recapitulation of endogenous FoxD3 expression by the combined activity of the enhancers, and the effect of manipulating upstream regulators on both enhancers and endogenous FoxD3 expression, strongly suggest that NC1 and NC2 act as enhancers regulating endogenous expression of FoxD3 in the neural crest.de novo in actively migrating cranial neural crest cells, where its activity is preceded by that of Sox10E2. NC2 is active in only a few delaminating/emigrating cranial neural crest cells but in a majority of the migrating neural crest population. Interestingly, there is little overlap of NC1 and NC2 activity in the cranial neural crest, whereas both overlap with Sox10E2, which appears to be active in all of the migrating cranial crest population.Comparison of the activity of these two enhancers with the cranial Sox10 enhancer Sox10E2 The minimal overlap in activity of NC1 versus NC2 in cranial neural crest populations raises the interesting possibility that there may be a regulatory switch of enhancers from NC1 to NC2 at the endogenous promoter of FoxD3 when the cells reside within the dorsal neural tube and/or are emigrating. Such competition at the promoter could occur if only a single enhancer can be functional at any given time on the FoxD3 promoter. If this is the case, the very few double labeled cells expressing NC1- and NC2-driven reporter expression may represent perdurance of eGFP protein rather than the actual levels of enhancer activity. The finding that NC1 and NC2 enhancers are active in generally separate cranial neural crest populations further suggests that the cranial neural crest represents a heterogeneous population, even as the cells are delaminating from the neural tube, and that this heterogeneity may be encrypted at the regulatory level.It is intriguing to speculate that the differential activity of NC1 and NC2 in distinct subpopulations may reflect differential cell fate and commitment status of future neural crest derivatives. Consistent with the possibility that NC1 and NC2 activity may reflect commitment to different lineages, NC2 is later active in neural crest-derived dorsal root and trigeminal ganglia, whereas NC1 is active transiently in the branchial arches, but not in peripheral ganglia.The activity of NC2 in the vagal and trunk neural crest recapitulated expression of endogenous FoxD3 in premigratory and migratory neural crest cells. In addition, FoxD3 is retained by a subset of neural crest derivatives histone deacetylase 1 (hdac1) mutant, a severe loss of mitfa positive melanophores can be rescued by partial reduction of FoxD3; suggesting hdac1 is required to repress FoxD3 in melanophores NC2 not only was active in neuronal derivatives, but also directed activity in neural crest cells migrating along the dorsolateral pathway, which are melanocyte precursors that migrate 24 h after the ventrolateral population migrate to the ganglia. Cells on the dorsolateral population do not normally express FoxD3 Xenopus, lamprey and mouse, and provides further support for a conserved gene regulatory network in the neural crest. Pax7 and Pax3 are closely related paralogs which have overlapping expression and function Xenopus, Pax3 is expressed in premigratory neural crest along the neural axis, and Pax7 is restricted to cranial levels (and very weak in Xenopus) Xenopus, mouse and lamprey suggests that Pax3 and/or Pax7 is required for FoxD3 expression and neural crest specification Xenopus, Pax3 is necessary for expression of FoxD3 The current results establish for the first time a direct regulatory connection between the neural plate border genes, Pax7 and Msx1/2, and FoxD3, suggesting it is an immediate downstream target. This confirms and validates previous indirect evidence in XenopusMsx1 has been proposed to lie upstream of Pax3, FoxD3 and Snail2 during neural crest induction in in situ hybridization suggests that Ets1 and FoxD3 are expressed concomitantly in the cranial neural crest. The difference in results between these two studies likely rests in the stages at which the knock-down reagents were effective, with the present results uncovering an earlier role for Ets1.Our data also show that Ets1 is necessary for initial FoxD3 expression since electroporation of Ets1 morpholinos during gastrulation (at HH5) depletes FoxD3 expression at HH9. In contrast, a dominant-negative Ets1 inhibited cranial neural crest migration but did not result in decreased FoxD3 expression XenopusRecent work on the Sox10E2 enhancer showed that Sox10 expression in the cranial neural crest is regulated by Ets1, Sox9 and cMyb Xenopus neural crest Snail2 expression domains Although there is little published information regarding the molecular players are involved in the establishment of the more caudal neural crest populations, the present results implicate the neural plate border specifier, Zic1, as a critical factor in the control of FoxD3 expression at vagal and trunk levels. Zic1 has been shown to be required for FoxD3 expression in The present study expands the number of known direct regulatory interactions in the cranial neural crest gene regulatory network, confirming a direct regulation of FoxD3 by Pax3/7 and Msx1/2, and revealing a previously unknown regulation of FoxD3 by Ets1. We have also identified Zic1 as a key player in setting up the FoxD3 expression domain in the trunk neural crest. Several other genes, like Hairy2, Sox10 and Sox5, have been suggested to regulate FoxD3 expression It is well known that the developmental potential of neural crest cells varies along different levels of the neural axis. Quail/chick chimeras have elegantly demonstrated that both the pathways of migration and derivatives differ depending upon the axial level from which neural crest cells emigrate Our data show that the inputs to FoxD3 in the vagal/trunk region are distinct from those functioning at cranial levels, suggesting a model for region-specific expression of FoxD3 . Whereashttp://ecrbrowser.dcode.org) and comparative analysis tracks of UCSC Genome Browser (http://genome.ucsc.edu/). We analyzed a 160 kb genomic region encompassing the FoxD3 locus up to the first upstream (Atg4C) and downstream (Alg6) of neighboring genes. Regions containing elements found to be highly conserved across most vertebrates including human, mouse and Xenopus were amplified using Expand High Fidelity Plus system  with CH261-166E22 and CH261-100C15  BAC clones as templates and directionally cloned into the ptkeGFP or ptkCherry vectors The genomic region of chicken FoxD3 was compared to other vertebrates using the ECR Browser (Chicken embryos were electroporated at HH4 using previously described techniques in situ hybridization for FoxD3 were performed using previously described procedures in situ hybridization for eGFP was modified using the guidelines in in situ hybridization was performed according to Whole mount and section Some embryos expressing RFP and eGFP were processed and cryosectioned at 14 mm. Select sections were labeled using the HNK-1 antibody (diluted 1/50), secondarily detected using goat anti-mouse IgM Alexa 350 . For whole mount immunostaining we used the protocol described by tggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacgg, for 30 bp substitutions acaagcagaagaacggcatcaaggtgaact and for 20 bp substitutions tggagtacaactacaacagc. Fragments were amplified using primers detailed in http://rvista.dcode.org/) and Jaspar database (http://jaspar.genereg.net/cgi-bin/jaspar_db.pl) were used to predict and analyze binding motifs within highly conserved regions. Individual sites were mutated by substituting 6–8 adjacent critical base pairs with GFP coding sequence, using fusion PCR and sub-cloning into ptkeGFP. Primers used are listed in Regions of NC1 and NC2 were replaced with eGFP coding sequence using fusion PCR protocol. For 100 bp substitutions, the region of eGFP used was ChIP was performed using chromatin prepared from dorsal neural tube regions of HH8–10 (4–10 somite) chicken embryos using Ets1 , Pax7 (ab34360. Abcam) and Msx1 antibodies (Sigma M0944) with normal rabbit IgGs  as previously described Figure S1In situ hybridization for FoxD3 showing expression in a dorsal root ganglion (DRG) adjacent to the neural tube (NT) in the trunk. No FoxD3 expression is present underneath the ectoderm where melanocytes are localized. (B) Transverse section through the trunk region similar to that shown in (A). eGFP (green) driven by enhancer NC2 is observed in the DRG as well as underneath the ectoderm (arrows) in presumptive melanocytes on the dorsolateral pathway. (C) Whole mount view of NC2 driven eGFP activity at HH19. Expression can be seen in the dorsal root ganglia (arrow) and melanoblasts (arrowheads). (D) In situ hybridization for eGFP shows expression in DRGs (arrow) and melanoblasts (arrowheads). (E) Transverse section of (D) confirms expression of eGFP in migrating melanoblasts (arrows). (F) NC2 activity can be seen in neural crest cells in the gut at HH27.Expression of NC2 in dorsal root ganglia, melanocytes and enteric nervous system. (A) (TIF)Click here for additional data file.Figure S2Putative transcription factor binding sites in the NC1 core region were subsequently mutated to examine effects on activity. (A) Core region of the enhancer with several binding sites highlighted. Mutation of sites in blue had no effect on the activity of the enhancer; sites in red abolished activity of enhancer when mutated. (TIF)Click here for additional data file.Figure S3Sox9 and HNK-1 expression in neural crest persists after knock-down of Pax7, Msx1/2 and Ets1 morpholinos. (A–C) Embryos in which NC1 enhancer-driven Cherry was depleted via knockdown of Pax7, Msx1/2 and Ets1 see  were ana(TIF)Click here for additional data file.Table S1Mutational analysis of the NC2 enhancer reveals importance of Zic binding sites. Mutation M11, which impairs a Zic binding site, causes complete loss of trunk NC2 activity. Mutations M11, M15, M18 and M20 suppress cranial NC2 activity but only result in a slight reduction of enhancer expression in the trunk.(DOCX)Click here for additional data file.Table S2Primers used for NC1, NC2 deletions and substitutions. Text in capitals indicates enhancer sequence, and text in small letters indicates replacement GFP sequence. To make the mutated constructs, mutated primers were paired with flanking primers NC1.1 or NC2.9, amplified and joined in a fusion PCR reaction using the flanking primers NC1.1 or NC2.9.(DOCX)Click here for additional data file.Table S3Primers used for binding site mutations of NC1. Text in capitals indicates mutated sequence. To make the mutated constructs, primers were paired with flanking primers NC1.1, amplified and joined in a fusion PCR reaction using the flanking NC1.1 primers.(DOCX)Click here for additional data file.Video S1Dynamic regulation of FoxD3 and Sox10 enhancers in the cranial neural crest. Time-lapse movie shows differential temporal and spatial activity of NC1 (green), NC2 (blue) and the Sox10E2 (red) enhancers in a chick cranial slice preparation. NC1 drives expression of the reporter in the premigratory neural crest, preceding Sox10E enhancer activity. NC2 activity was observed in few cells within the neural tube, and few delaminating neural crest cells in which Sox10E2 is also active.(M4V)Click here for additional data file.Video S2Time lapse movie of migrating cranial neural crest cells electroporated with NC1:eGFP and NC2:Cherry. Time-lapse movie shows little overlap between cells with activity of NC1 and NC2. NC1 is active transiently in early neural crest cells, while NC2 seems to be primarily responsible for FoxD3 expression in migratory neural crest at cranial levels.(M4V)Click here for additional data file.


Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a “proof-of-concept” of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci.Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode  Haemonchus contortus is a parasitic nematode of sheep that has a high propensity to develop resistance and is the most widely used model system in which to study anthelmintic drug resistance. Ivermectin is an extremely important drug for parasite control in both humans and animals. Here, we report a novel approach using genetic crossing to transfer a region of the H. contortus genome containing ivermectin resistance genes from resistant strains into a susceptible strain. During our backcrossing approach, we have identified a genetic marker showing evidence of genetic linkage to ivermectin resistance. The susceptible strain we have used is currently having its complete genome sequenced making the information and strains generated here extremely valuable for the identification of ivermectin resistance genes. This work represents an important proof of concept for using genetic approaches to identify genomic regions containing drug resistant genes in parasitic nematodes.The use of drugs (anthelmintics) to control nematode parasites (roundworms) is common in both humans and animals. This has led to the widespread development of drug resistance in livestock parasites and serious concerns regarding its emergence in human parasites.  Parasitic nematode worms are important human and animal pathogens. Human parasites infect well over 1 billion people worldwide and livestock parasites cause major economic production loss to grazing ruminants. Control is dependent on the use of a limited number of anthelmintic drugs and intensive use of these has already led to widespread resistance in livestock parasites Haemonchus contortus is a parasitic nematode of sheep which has a high propensity to develop anthelmintic. It is also one of the most amenable parasitic nematodes to experimental manipulation which, together with recent progress in sequencing its genome makes it a potentially powerful model system to study drug resistance in the strongylid nematode group Unfortunately, parasitic nematodes of humans make extremely difficult experimental subjects and so there is a need to develop model systems to study potential mechanisms of anthelmintic resistance. The genetic basis of anthelmintic resistance is still relatively poorly understood. To date, most research has focussed on the investigation of possible associations between the resistance phenotype and polymorphisms in candidate genes. This approach has been successful in identifying polymorphisms in the isotype-1 β-tubulin gene as important determinants of benzimidazole resistance in vitro). However, a major limitation of such approaches is that selection in the real world is very different to that applied in the laboratory H. contortus making genetic mapping in this organism a feasible objective in the future The artificial selection of resistance by serial passage and underdosing of susceptible laboratory strains has been undertaken by a number of groups in the past in vitroIn this paper we report the introgression of ivermectin resistance-conferring loci from two different ivermectin resistant strains, into the genetic background of the susceptible genome reference strain MHco3(ISE) All experimental procedures described in this manuscript were examined and approved by the Moredun Research Institute Experiments and Ethics Committee and were conducted under approved British Home Office licenses in accordance with the Animals (Scientific Procedures) Act of 1986. The Home Office licence number is PPL 60/03899 and experimental IDs for these studies were E06/58, E06/75 and E09/36.H. contortus sequencing project at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/H_contortus/). In spite of its inbreeding history it retains high levels of genetic polymorphism H. contortus, were originally isolated from South Africa and Australia respectively. Subsequently they have been experimentally passaged through sheep for a number of years at the Moredun Research Institute, and these versions of the strains are designated as MHco4(WRS) and MHco10(CAVR) respectively throughout the manuscript. These strains were chosen for this work for several reasons: Firstly, their origins were from the field and have subsequently been well characterized in the laboratory The MHco3(ISE) strain 1 female worms derived from each generation of backcross were crossed again with male MHco3(ISE) worms (1 progeny of the first genetic crosses between MHco3(ISE) and the two ivermectin resistant strains MHco4(WRS) and MHco10(CAVR) were designated as MHco3/4 and MHco3/10 respectively. The nomenclature for subsequent backcrosses was MHco3/4.BCn and MHco3/10.BCn, denoting the Moredun Research Institute (M), H. contortus (Hco), the unique numbers allocated to both parental strains (3/4 or 3/10), and the backcross generation  . The passage of these larvae through three more rounds of ivermectin selection and crossing against the ivermectin susceptible isolate (MHco3(ISE)) produced a final fourth backcross generation (MHco3/4.BC4 or MHco3/10.BC4)  susceptible strain with female worms from the resistant strains in the initial cross. Subsequently FE) worms . The F1 3 and 4) . Progeny/10.BC4) .4 male worms from one strain and 50 late L4/adult female worms from the other strain directly into the abomasum of a recipient sheep. In order to produce L4 for transplantation male worm-free donor lambs were orally dosed with between 5,000–10,000 L3 of either MHco3(ISE), or the ivermectin resistant strain to be crossed against. H. contortus donors with the ivermectin resistant strains were treated with 0.1 mg/kg of ivermectin  on day 10 or 11 post infection to select for ivermectin resistant progeny prior to transplantation. Donor sheep were euthanased on day 14 post infection and worms were harvested from their abomasa  H. contortus and 50–100 female late L4/immature adult H. contortus were surgically transferred into the abomasa of male worm free recipient lambs, within 2 hours of recovery from the donor sheep.Crosses between two strains were performed by surgically transplanting approximately 50 late L4/immature adult MHco3(ISE) H. contortus and 50 female late L4/immature adult MHco4(WRS), or MHco10(CAVR) H. contortus (or subsequent backcross generations) were introduced into the abomasum in approx 5 ml RPMI, using a 5 mm diameter blunt ended, glass pipette. The purse-string suture was then closed and the surgical incision repaired allowing the completion of surgical transfers within about 2 hours from the recovery of the nematodes from the donor sheep. Sheep were routinely injected with 1 mg/kg meloxicam  for post-surgical analgesia and 7 mg/kg amoxicillin/1.75 mg/kg clavulanic acid  and closely monitored on completion of the surgery. No adverse effects were noted during the course of this study. Eggs were identified in the faeces approx 3 days post surgery and collected daily and coprocultured to produce L3.Recipient sheep were anaesthetised to allow a 10 cm vertical incision to be made through the skin, underlying fascia, muscle and peritoneum, over the right flank, midway between the last rib and pelvis and about 10 cm above the midline. The abomasum was located and partially exteriorised, to enable a 1 cm diameter sub-serosal purse-string suture to be placed. A stab incision was then made in the centre of the purse-string suture, through which 50 male late L4, and MHco3/10.BC4 alongside the three original parental strains used in this study, namely MHco3(ISE), MHco4(WRS) and MHco10(CAVR). Seventy-five parasite naïve lambs were divided equally between the five different strains. For each strain, the 15 lambs were further allocated into three treatment groups of five animals: no treatment, ivermectin (0.2 mg/kg BW) or ivermectin (0.1 mg/kg BW). Lambs were initially allocated randomly to strain and treatment groupings that were subsequently balanced, where needed on the basis of sex and the weight of the animal just prior to the experiment, to ensure that groups were as similar as possible. The lambs were infected with 5,000 H. contortus L3 on day 0. Faecal worm egg counts (FEC) were conducted H. contortus burdens in abomasal saline washings and digests H. contortus recovered from 2% (MHco4 and MHco10) and 10%  sub-samples of the abomasal washings and digests were counted and sexed (only adults were seen), the higher sub-sample volume was examined in the backcross strains due to the smaller numbers of worms present. The percentage efficacies of each anthelmintic treatment were calculated using the equation 100 (1−T/C), where T and C are the arithmetic mean total H. contortus burdens of the treated and control groups respectively A controlled efficacy test (CET) using ivermectin was undertaken on the two separate fourth generation backcross strains, MHco3/4.BCAll microsatellite genotyping, on both bulk and single worm DNA lysates, was performed using the same PCR amplification methods and parameters previously described 3 worms from each generation of the backcrossing procedure , the F1 progeny of the initial genetic crosses (MHco3/4 and MHco3/10) and L3 from the three parental strains MHco3(ISE), MHco4(WRS) and MHco10(CAVR).Bulk worm DNA lysates were made as previously described 3 or adult) worm DNA lysates were prepared from seven strains for more detailed genetic analysis: the three parental strains MHco3(ISE), MHco4(WRS) and MHco10(CAVR); the two backcross strains, MHco3/4.BC4 and MHco3/10.BC4 and the two populations of survivors (0.1 mg/kg ivermectin) of both the backcross strains .In addition to the bulk worm DNA preparations, 30–40 individual .The genotyping of parasite populations/strains by amplifying a microsatellite from “bulk” DNA lysates made from a population of worms has been previously described ST values were calculated using Arlequin version 3.11 For the single worm genotype data, Pairwise FH. contortus arithmetic mean burdens of treated and control groups  and MHco4(WRS) strains respectively  and MHco4(WRS) strains were 100, 89, 69, 41 and 0% at 0.1 mg/kg IVM and 100, 92, 93, 37 and 36% at 0.2 mg/kg IVM respectively when compared to untreated controls , MHco3/10.BC1 strains and each backcross generation  but were lost at the second backcross generation (MHco3/10.BC2). These were relatively rare alleles in MHco3(ISE) strain - allele 215 bp present at 3.6% and allele 217 bp present at 11.1% - and therefore their loss could be due to purely stochastic reasons. In contrast, almost all alleles specific to the two ivermectin-resistant strains MHco4(WRS) or MHc010(CAVR), disappear during the backcross procedure and are absent in the MHco3/4BC4 and MHco3/10BC4 strains. There are only two exceptions to this. First, the HcX256 allele 243 bp, which is retained in MHco3/10BC4. However, it was only detected at a frequency of 4.6% in MHco3/10BC4 compared with its original frequency of 38% in the MHco10(CAVR) parental strain and so although not completely eliminated, this allele has undergone a dramatic reduction in frequency during the backcrossing procedure (Supplementary 4 and MHco3/10BC4 strains have a similar genetic background to the MHco3(ISE) parental strain based on bulk genotyping with a panel of 18 microsatellite loci as would be predicted from the backcrossing scheme.The genotyping of parasite populations/strains by amplifying a microsatellite from “bulk” DNA lysates made from a population of worms has been previously described 4 and MHco3/10.BC4) were analysed in more detail by genotyping 30–40 individual worms with 9 of the most discriminatory microsatellite markers  and MHco4(WRS) had a high level of genetic differentiation  parental strain (8 loci FST = 0.0299 and 9 loci FST = 0.0265 for MHco3/4.BC4 and 8 loci FST = 0.0045 and 9 loci FST 0.0040 for MHco3/10.BC4). In contrast they show a high level of genetic differentiation from the ivermectin resistant parental isolates (8 loci FST = 0.1930 and 9 loci FST = 0.1767 between MHco4(WRS) and MHco3/4.BC4 (ST of 0.3821 and 9 loci FST of 0.3657 between MHco10(CAVR) and MHco3/10.BC4 (4 and MHco3/10.BC4 populations are distinct from the MHco4(WRS) and MHco10(CAVR) strains respectively and cluster with the MHco3(ISE) strain (th generation backcross strains is similar to that of the MHco3(ISE) parental strain.Both the BCo3/4.BC4  and 8 lo3/10.BC4 ). Indeed) strain . This de4 and MHco3/10.BC4 backcross populations are ivermectin resistant based on the CET, they are significantly less resistant than the original MHco4(WRS) and MHco10(CAVR) parental strains. This is not unexpected given the nature of the backcrossing scheme (see discussion below) and means the backcross strains consist of a mixed population of worms of differing ivermectin resistant phenotypes. In order to genetically characterize those worms that were phenotypically resistant to ivermectin at a dose which is 100% effective for the MHco3(ISE) isolate, we infected two sheep each with MHco3/4.BC4 and MHco3/10.BC4, treated with 0.1 mg/kg and harvested and prepared DNA from adult worms that survived this drug treatment. These worms surviving ivermectin treatment were then individually genotyped with the 9 microsatellite markers and FST and PCA analysis was performed initially with 8 loci (loci Hcms8a20 excluded) and subsequently with loci Hcms8a20 included in the analysis (9 loci) (ST = 0.0379) and genetically divergent from the ivermectin resistant parental strain (8 loci FST of 0.2195 for MHco4(WRS))  parental strain (8 loci FST = 0.0181) than to the MHco10(CAVR) parental strain (8 loci FST = 0.3927) (Although the MHco3/4.BC(9 loci) . On the o4(WRS)) . Similar 0.3927) .ST = 0.2137 vs 8 loci FST = 0.2195 between the MHco3/4.BC4 ivermectin survivors and MHco4(WRS) strain and 9 loci FST = 0.3647 vs 8 loci FST = 0.3927 between MHco3/10.BC4 ivermectin survivors and MHco10(CAVR) (ST = 0.0487 vs 8 loci FST = 0.0379 between the MHco3/4.BC4 ivermectin survivors and MHco3(ISE) and 9 loci FST = 0.1129 vs 8 loci FST = 0.0181 between the MHco3/10.BC4 ivermectin survivors and MHco3(ISE)  ). ConverST analysis, examination of the allele frequency data derived from the single worm genotyping revealed that for eight of the nine markers, the allele frequency histograms of the ivermectin treatment survivors of strains MHco3/4.BC4 and MHco3/10.BC4 were very similar to the MHco3(ISE) susceptible parental strain and divergent from the resistant parental strains MHc04(WRS) and MHco10(CAVR) respectively  susceptible strain. This latter strain is susceptible to the main classes of anthelmintics and is currently being used as the reference strain for the H. contortus genome sequencing project (http://www.sanger.ac.uk/resources/downloads/helminths/haemonchus-contortus.html). The introgression of resistance genes into this strain was achieved by repeated backcrossing of the MHco4(WRS) and MHco10(CAVR) strains against the MHco3(ISE) strain with the application of ivermectin selection at each backcross. A therapeutic dose of 0.1 mg/kg ivermectin was chosen as an appropriate discriminatory dose for selection because it is 100% effective against the parental MHco3(ISE) strain . This was confirmed by our controlled efficacy test; not a single worm of the MHco3(ISE) strain could be found surviving treatment at this dose rate in any of the five treated sheep  parental strain and highly differentiated from the MHco4(WRS) and MHco10(CAVR) resistant parental strains  and MHco4(WRS) parental strains and hence harbouring resistance conferring loci. Such analyses could include genome-wide polymorphism analysis, RNAseq analysis (to examine expression profiles and coding-region polymorphisms) or targeted analysis of candidate genes. It is important to note that it is likely that the introgressed regions of the MHco4(WRS) and MHco10(CAVR) are relatively large since just four generations of backcrossing have been performed and recombination will have had limited opportunity to break down genetic linkage. Nevertheless analysis of these strains should provide the locations of major ivermectin resistance loci in the H. contortus genome. Further backcrossing, together with improving genomic resources for this parasite, will provide the opportunity to iteratively interrogate these strains to identify the genomic location of resistance loci more accurately.The important point is that backcross strains contain a proportion of individuals that are phenotypically resistant to ivermectin (unlike the MHco3(ISE) parental susceptible strain). The observation that some worms in the backcross strains survive treatment at this dose demonstrates that the resistance-conferring alleles have been successfully introgressed from the parental ivermectin resistant isolates MHco4(WRS) and MHco10(CAVR). Importantly, when individual worms from the MHco3/4.BC strains . This deth generation backcross progeny for both back crosses. This was marker Hcms8a20. Furthermore, when the FST and PCA analysis were performed using the nine most discriminatory markers, each single marker was iteratively excluded to check for distortions of the data due to any effects of single markers. The only marker whose exclusion had any discordant effect on the data was Hcms8a20 (Data not shown). The exclusion of the loci Hcms8a20 from the FST and PCA analysis revealed that MHco3/4.BC4 and MHco3/10.BC4 ivermectin resistant worms (survivors), the susceptible MHco3(ISE) strain and their respective backcross strains were all genetically indistinguishable. The inclusion of the loci Hcms8a20 into the same analysis increased the level of genetic differentiation between these aforementioned strains of worms to the point of statistical significance. Examination of the individual allele frequencies for this marker confirms that the allelic profile was more similar to the resistant parental strains than the MHco3(ISE) parental strain . However, these are present at much higher frequencies in the populations of backcross worms that survive 0.1 mg/ml ivermectin treatment . It is impossible to predict the precise changes in allele frequency one would expect at a single locus during the backcrossing procedure, or following drug selection, when several loci may have differing additive contributions to the overall resistance phenotype. However, the fact that the same locus, Hcms8a20, shows evidence of retention of alleles specific for the parental resistant isolates in both fourth generation backcross strains, together with the dramatic increase in frequency of these in the phenotypically ivermectin resistant worms (relative to the unselected backcross populations) provides strong evidence that this locus is linked to a resistance conferring polymorphism. The fact that different alleles appear to be selected from the two different parental resistant strains is not necessarily surprising. These two strains – MHco4(WRS) and MHco10(CAVR) - are genetically divergent and originally derived from disparate geographical regions. Consequently, it is entirely possible that a resistance-conferring polymorphism would be genetically linked to different haplotypes of adjacent markers. As the H. contortus genome project progresses it will be interesting to “walk out” from the Hcms8a20 marker to examine additional linked markers to define the size of the region showing evidence of linkage disequilibrium. Furthermore, we hypothesize that additional loci contribute to the ivermectin resistant phenotype of the MHco4(WRS) and MHco10(CAVR) parental strains. We anticipate that these may be identified as we iteratively interrogate the backcross strains with larger marker panels as they become available form the H. contortus genome sequencing project. Similarly, the backcross strains now represent a powerful genetic resource with which to determine if the various candidate genes identified from other studies contribute to the ivermectin resistance phenotype of the MHco4(WRS) or the MHco10(CAVR) strains.Of the 18 microsatellite markers that were used to monitor the backcrossing procedure, based on the presence and absence of strain-specific alleles, there was only one in which alleles specific to the parental resistance strains were retained in the 4l strain . Indeed,H. contortus. This is a novel approach that provides a powerful adjunct to both candidate gene and whole genome analysis aimed at identifying anthelmintic drug resistance loci. The continued advancement of such genetic approaches, alongside genomic resources for H. contortus, should allow this organisms to be used in an increasingly powerful manner to study the genetic basis of anthelmintic resistance in strongylid nematode parasites.In summary, we describe the introgression of resistance-conferring loci from two independent ivermectin resistant strains into a susceptible reference strain of Figure S1Total worm burden of lambs. Total worm burden of lambs following therapeutic dose (0.1 mg/kg and 0.2 mg/kg) of ivermectin against the three parental isolates and the backcross isolates. Mean worm burden per treatment group indicated by trend lines.(EPS)Click here for additional data file.Figure S2Allele frequency histograms of loci HcmsX256 for H. contortus parental and backcross strains. Allele frequencies resulting from individual worm genotyping of populations of 30 single worms confirm the presence of the MHco10(CAVR)-specific allele, 243 bp at the very low level of 4.6% in the MHco3/10.BC4 strain.(EPS)Click here for additional data file.Figure S3Allele frequency histograms of eight loci for H. contortus parental and backcross strains. Allele frequencies of the microsatellite loci, Hcms27 (A), Hcms36 (B), Hcms40 (C), Hc53265 (D), Hcmc22c03 (E), Hc22193 (F), Hcms3086A (G) and Hc44104 (H) for parental strains , the two 4th generation backcross strains  and 4th backcross generation survivors of ivermectin treatment (0.1 mg/kg ivermectin) .(EPS)Click here for additional data file.Table S1Summary information for the five new H. contortus microsatellite markers. Sequence of repeat and primers used to amplify microsatellite loci.(DOC)Click here for additional data file.Table S2Treatment efficacies based on worm burden. Arithmetic mean (±SEM) and range of H. contortus counts, sex differentiation of worm burdens and percentage efficacies.(DOC)Click here for additional data file.Table S3Treatment efficacies based on faecal egg count reduction. Arithmetic mean (±SEM) and range of faecal egg count and percentage efficacy seven days post-treatment.(DOC)Click here for additional data file.Table S4Genetic profiles of backcrossed strains derived from MHco4(WRS) monitored by bulk worm microsatellite “finger-printing”. Alleles present in the bulk worm preparations of the parental (MHco3(ISE) and MHco4(WRS)), F1 and backcross strains.(XLS)Click here for additional data file.Table S5Genetic profiles of backcrossed strains derived from MHco10(CAVR) monitored by bulk worm microsatellite “finger-printing”. Alleles present in the bulk worm preparations of the parental (MHco3(ISE) and MHco10(CAVR)), F1 and backcross strains.(XLS)Click here for additional data file.


Autosomal dominant FMF due to deletion of residue 694 was first described in 3 British families in 2000. It has subsequently been reported in other patients of Northern European ancestry but the phenotype has not been fully characterised.To describe the presenting symptoms, complications and treatment responses of familial Mediterranean fever due to deletion M694.We sought patients with a genetic finding of deletion M694 from our database and reviewed their case records.A total of 19 patients (11 M:8 F) had been found to carry the Del M694 variant. Clinical details were available on 16 patients who had been assessed at our centre. 13 were of white British ancestry, the other 3 were of Irish ancestry. 2 patients gave no relevant family history, 1 was adopted and unaware of any family details, 10 patients (from 5 kindreds) gave a history of similar symptoms in at least 1 relative and the final patient reported that his mother died of renal failure of unknown cause raising the possibility of AA amyloidosis although without suggestive symptoms. 13 patients had symptoms: median age at onset 18 years (range 6-48), median age at diagnosis 48.1 years, median attack duration 2.5 days with a median of 1 attack per month, all described fever with peritonitic abdominal pain, pleuritic symptoms occurred in 4 cases, and erysipelas like erythema rash in 2. 3 patients had an appendectomy and 2 cholecystectomy prior to diagnosis. 5 of 7 symptomatic women reported attacks with menstruation and 3 had partial remissions with oral contraception. 3 patients had presented with AA amyloidosis of unrecognised aetiology of whom only 1 was truly asymptomatic. 2 children detected as part of family screening also denied any symptoms. 11 patients are on colchicine with good clinical and inflammatory responses, median age at starting treatment was 50 years, 2 felt that oral contraceptives provided adequate symptom relief, 1 declined treatment.Familial Mediterranean fever associated with deletion M694 is an autosomal dominant condition in Northern European Caucasians with variable penetrance. Disease onset appears slightly later but symptoms and colchicine responsiveness are very similar to classical autosomal recessive FMF. The high rate of AA amyloidosis (19%) may reflect the very delayed diagnosis and late initiation of colchicine treatment.None declared


CLN3, by Xbp1. Xbp1 is induced as glucose is depleted and it is among the most abundant transcripts in quiescent cells. Xbp1 binds and represses CLN3 transcription and in the absence of Xbp1, or with extra copies of CLN3, cells undergo ectopic divisions and produce very small cells. The Rad53-mediated replication stress checkpoint reinforces the arrest and becomes essential when Cln3 is overproduced. The XBP1 transcript also undergoes metabolic oscillations under glucose limitation and we identified many additional transcripts that oscillate out of phase with XBP1 and have Xbp1 binding sites in their promoters. Further global analysis revealed that Xbp1 represses 15% of all yeast genes as they enter the quiescent state and over 500 of these transcripts contain Xbp1 binding sites in their promoters. Xbp1-repressed transcripts are highly enriched for genes involved in the regulation of cell growth, cell division and metabolism. Failure to repress some or all of these targets leads xbp1 cells to enter a permanent arrest or senescence with a shortened lifespan.Pure populations of quiescent yeast can be obtained from stationary phase cultures that have ceased proliferation after exhausting glucose and other carbon sources from their environment. They are uniformly arrested in the G1 phase of the cell cycle, and display very high thermo-tolerance and longevity. We find that G1 arrest is initiated before all the glucose has been scavenged from the media. Maintaining G1 arrest requires transcriptional repression of the G1 cyclin,  Complex organisms depend on populations of non-dividing quiescent cells for their controlled growth, development and tissue renewal. These quiescent cells are maintained in a resting state, and divide only when stimulated to do so. Unscheduled exit or failure to enter this quiescent state results in uncontrolled proliferation and cancer. Yeast cells also enter a stable, protected and reversible quiescent state. As with higher cells, they exit the cell cycle from G1, reduce growth, conserve and recycle cellular contents. These similarities, and the fact that the mechanisms that start and stop the cell cycle are fundamentally conserved lead us to think that understanding how yeast enter, maintain and reverse quiescence could give important leads into the same processes in complex organisms. We show that yeast cells maintain G1 arrest by expressing a transcription factor that represses conserved activators (cyclins) and hundreds of other genes that are important for cell division and cell growth. Failure to repress some or all of these targets leads to extra cell divisions, prevents reversible arrest and shortens life span. Many Xbp1 targets are conserved cell cycle regulators and may also be actively repressed in the quiescent cells of more complex organisms. Budding yeast that are grown in rich glucose-containing media and are allowed to naturally exhaust their carbon source undergo a series of changes that enable a significant fraction of the cells, primarily daughter cells, to enter a protective quiescent (Q) state CLN3 is transcribed at the M/G1 border CLN1 and CLN2 cyclins and other genes that trigger budding and DNA replication An important characteristic of all quiescent cells is that they arrest their cell cycle in G1. This requires the G1 to S transition to be stably halted by a mechanism that can be readily reversed when conditions permit. In cycling cells, progression through G1 into the next S phase involves two consecutive waves of G1 cyclin (Cln) expression. UBI4 promoter, prevents G1 arrest and causes loss of viability CLN3 genes Cln3/Cdk activity is rate limiting for the G1 to S transition during exponential growth. Excess Cln3 results in shorter G1 phases and smaller cells, while loss of Cln3 function prolongs G1 and results in larger cells CLN3 is a critical target of repression for G1 arrest and for the transition to quiescence. Rad53 checkpoint activity reinforces this arrest in wild type cells and becomes essential when Cln3 is overproduced. Xbp1 is also important for maintaining G1 arrest. Xbp1 is a repressor of CLN3 transcription XBP1 transcript is induced and it is among the most abundant transcripts in Q cells. Xbp1 binds and represses hundreds of genes, including CLN3 during the post-DS phase of growth. In the absence of Xbp1, cells undergo extra post-DS cell divisions and produce very small cells. These phenotypes are Cln3-dependent. xbp1 mutant Q cells are also defective in the maintenance of and recovery from the Q state. xbp1 Q cells maintain viability, but lose the ability to re-enter the cell cycle. Using Next Generation Sequencing In this work, we demonstrate that G1 arrest is initiated before the diauxic shift (DS), which is when all the glucose has been scavenged from the media. 600) of about 24, but the cell number only doubles once after the DS, which occurs between the 12 and 14 hour time points. We have monitored the DNA content of these cells to determine what fraction of cells are in G1, S and G2/M over this time course. Interestingly, the 12 to 14 hour interval shows the sharpest increase in the percentage of cells in G1. This indicates that the signal to slow proliferation is occurring at or before the DS and cells respond by extending or arresting in G1. Yeast cells spend most of their time in a non-dividing state triggered by nutrient depletion from their environment. Under the conditions we employ see , yeast uCLN3 gene (5XCLN3). This strategy maintains all the regulatory features of the wild type CLN3 gene, while it increases the Cln3 expression level. We first verified that the 5XCLN3 construct produces about five-fold higher levels of CLN3 mRNA than wild type as cells grow from log to stationary phase  By day five, 43% of these cells contained ROS and 31% showed DNA fragmentation, as detected by TUNEL staining  and DNA fragmentation has been associated with DNA damage and replication stress in yeast and metazoan cells staining . ROS wasday five . This in5XCLN3 with rad9, which is a DNA damage-specific checkpoint protein 5XCLN3 showed no toxicity in the absence of Rad9  and continues for 48 hours. It is also present at very high levels in Q cells purified from a seven day old culture. In fact, XBP1 ranks within the top 100 most abundant transcripts in Q cells. CLN3 mRNA levels over this same time course in wild type and xbp1 cells. The initial pre-DS drop in CLN3 mRNA still occurs, but we see a two to three-fold de-repression of CLN3 from 14 to 48 hours in the absence of Xbp1. It then drops to a very low level in Q cells, and that drop is also Xbp1-independent. This pattern suggests that there may be three distinct mechanisms for establishing and maintaining CLN3 repression and that Xbp1 plays a role in maintaining CLN3 repression during post-diauxic growth.Repression of 21 alone . This suCLN3 levels still occurs in xbp1 cells indicates that the initial signaling to slow proliferation is intact. xbp1 cells in G1 is very similar to wild type for the first 18 hours. Direct comparison of the FACS profiles of wild type  also display metabolic oscillations, and peak out of phase with Xbp1. Using microarray and motif search tools PIS1, DOG2, and CDC10 are bound in vivo by Xbp1 after the DS, just like CLN3 , which we also verified to be in vivo binding sites for Xbp1 by chromatin immunoprecipitation  are not significantly affected by the absence of Xbp1 during log phase (8 hours). A few transcripts begin to rise in the xbp1 cells at the DS (14 hours), and this trend continues throughout the time course and in purified Q cells. Very few direct targets are down-regulated. This is consistent with our previous findings that Xbp1 functions as a repressor To show that the repression of these transcripts is Xbp1-mediated and to identify other targets, we used our Next-Generation RNA sequencing se. CLN3 , and allXbp1 expression is induced at 14 hours and remains high across this time course , but botCLN3 and CLN1) that drive the G1 to S transition SWI6NDD1MSS11 and MGA1IME1To look more closely at all Xbp1-mediated repression, we identified transcripts that are derepressed three-fold or greater at each time point . SignifiCLB4, CLB2PCL9After 24 hours of growth, 65 known genes are derepressed in the absence of Xbp1. At this time point, cell wall proteins are highly enriched. These include most of the daughter-specific genes By 48 hours, nearly 10% of all genes (515) are derepressed in the absence of Xbp1. At this time point almost half of the known targeted genes are involved in metabolism and the other large class is involved in cell wall biogenesis. 45 cell cycle genes and 25 transcription regulators are also derepressed at this time point. Only one-third of these derepressed genes are also derepressed in Q cells. Xbp1 affects a more diverse group of genes in purified Q cells. Metabolic genes are the largest class. In addition, 42 genes involved in transmembrane transport, including five glucose transporters are repressed by Xbp1 in Q cells.We also analyzed direct and indirect targets separately. What is striking is that direct and indirect targets are largely in the same pathways. At 18 and 24 hours, mitosis, cell cycle, cell division and cytokinesis are significantly enriched classes in both direct and indirect targets  and nitrogen metabolism . In Q cells, they are highly enriched for ribosomal proteins  and genes involved in monosaccharide catabolism . The ribosome biogenesis and ribosomal protein transcripts are tightly and coordinately regulated in response to nutrient conditions xbp1 cells at 48 hours versus purified Q cells suggests that these are fundamentally different states.The dot plots of In rich glucose-containing medium, yeast cells cease growth and division after about 48 hours due to carbon limitation. The resulting culture is a heterogeneous population of live and dead cells. Most of the daughter cells enter a quiescent state and can be purified due to their increased density CLN3 gene, interferes with Q cell formation, and cells lacking Cln3 produce more Q cells. Cells transitioning to quiescence with excess Cln3 accumulate in G1 more slowly than wild type cells, but they eventually arrest and remain viable due to the activation of the checkpoint kinase Rad53.We are investigating the events that differentiate Q cells from nonQ cells and promote their longevity 5XCLN3 cells, so we conclude that replicative stress, not DNA damage, triggers the checkpoint during the transition to quiescence. We detect delayed G1 arrest and increased ROS accumulation as nutrients become limiting, even in wild type cells carrying rad53-21. This suggests that replication stress occurs and this checkpoint pathway plays a role in restricting cell cycle progression during the wild type transition to quiescence. With excess Cln3, checkpoint function becomes essential and cells lacking it fail to arrest in G1 and undergo apoptosis. Related effects have been observed with excess cyclin E, and other activated oncogenes in higher cells Rad53 is an effector of the DNA damage and replication stress checkpoints CLN3 repression is mechanistically different before and after the DS, and that only its post-DS repression is Xbp1-dependent. The initial drop in CLN3 levels and the halt to S phase that we observe at the DS are Xbp1-independent. Only after the DS, the CLN3 promoter is bound and repressed by Xbp1. Cells lacking Xbp1 resume DNA replication and continue to divide after the DS, and this results in a significant population of very small cells. These phenotypes are Cln3-dependent. These data are consistent with Xbp1 playing a role in maintaining repression of CLN3 and G1 arrest as cells transition from growth to quiescence. However, unlike 5XCLN3, xbp1 mutants are not dependent on the Rad53 replication stress checkpoint for viability. We suspect that either the timing or the extent of derepression of CLN3 by xbp1 could explain the Rad53-independence of these cells. A third possibility is that Rad53 acts in the same pathway and upstream of Xbp1 to restrict cell cycle progression. Rad53 has been shown to increase the level of Xbp1 in response to DNA damage Our data indicate that in vivo binding sites for Xbp1. Binding is only detected after the DS, which explains why these targets were not identified in previous studies. None of the Xbp1 targets identified by genome-wide location analysis xbp1 deletion Xbp1 mutant Q cells remain viable, but they are profoundly delayed in cell cycle re-entry upon re-feeding. They are also short-lived as Q cells, entering an irreversible, senescent state more rapidly than wild type. We have not identified the genes responsible for these phenotypes because our data show that Xbp1 plays a global and continuous repressive role in cells as they transition from a dividing to a non-dividing quiescent state. We have identified 520 targets of Xbp1-mediated repression that contain Xbp1 binding sites in their promoters. All seven that we tested are direct XBP1 mRNA oscillates dramatically in cells that are undergoing yeast metabolic and cell cycle (YMC) oscillations XBP1. YMC oscillations are achieved by growing the cells to maximum density, starving them for glucose, then restoring a limited amount of glucose, which is immediately imported and cannot be detected in the media XBP1. Cell division stops and storage carbohydrates accumulate. This is the quiescence-like phase of the YMC XBP1 is turned off, and it's targets peak. Then, DNA replication takes place and cells divide. The striking parallels between the events associated with the transitions in and out of quiescence, and those associated with the YMC suggest that YMC oscillations may be the result of switching on and off the signal to arrest in G1 and enter quiescence. It also seems likely that the oscillation of XBP1 expression is responsible for the subsequent YMC oscillations of its many targets. One such verified target, CLN3, and many other cell cycle regulated transcripts have been shown to have different peak time or multiple peaks in the synchronized cell cycles induced by the YMC protocol CLN3 and 800 other transcripts is also oscillating during the YMC time course may explain some of those altered peak times.Xbp1 negatively regulates the mRNA levels of 15% of yeast genes during post-diauxic growth. When Xbp1 was ectopically expressed during logarithmic growth, only a small number of Xbp1 targets were identified xbp1 early in the transition to quiescence are largely cell cycle and growth regulators. At 18 hours, even the gene products associated with the cell periphery are largely sensors and regulators, rather then structural proteins. This is true of both direct and indirect targets. One possible explanation is that xbp1 mutant cells continue to divide during this interval. About 20% of Xbp1 targets are cell cycle regulated at the transcript level (data not shown). These transcripts are not made when cells stop dividing, so anything that promotes ectopic cell division would increase the transcription of these genes. However, only one-third of the 600 most cell cycle regulated transcripts It is striking that transcripts derepressed by xbp1 mutant. These were prominent only in the last two time points and they do not show any enrichment for Xbp1 binding sites. This supports the view that Xbp1 functions primarily, if not solely, as a repressor. We expect that the reduced levels of these transcripts are an indirect effect of the many perturbations that arise in xbp1 cells where 15% of genes are expressed at a time when they should be off.We also looked for transcripts that were under-represented in the XBP1 mRNA is among the top 1% highest level transcripts in Q cells. Xbp1 has also been shown to be translationally up-regulated in response to both glucose and amino acid starvation xbp1 mutant Q cells demonstrate the importance of this repression. Xbp1 shares homology within its DNA binding domain with four other S. cerevisiae transcription factors that specify cell fate. Swi4 and Mbp1 associate with Swi6 and serve as activators of mitotic growth Candida family members, Efg1, is critical for biofilm formation, which renders these pathogens drug-resistant PMT1, 2, and 4ECM33SMI1 and FKS1CLN3 were generated by integrating additional copies of CLN3 at four different marker loci using the integrating vectors, pRS303-306 rad53-21 mutant  was crossed into the W303 background above to generate BY6741 and subsequently crossed with the 5XCLN3 strain to generate BY6698. CLN3, XBP1 and RAD9 were deleted with KanMX as described The yeast strains were all derived from W303. The auxotrophic markers were corrected in all strains. The strains carrying five copies of 600) of 0.02 and allowed to grow at 30°C, shaking at 200 RPM. The diauxic shift was defined as the point at which no glucose was detected in the media, which was determined with glucose detection strips . Quiescent (Q) cells were purified from YEPD cultures that were seven days old using a 25 ml percoll density gradient 600 units loaded that sediment to the bottom nine ml of the gradient. Cell size and cell count was measured on a Z2 Beckman Coulter Counter. All time course data was collected in duplicate or triplicate, averaged and error bars are shown.Reproducible growth curves were obtained by patching cells from fresh plates onto YEP plus 2% glycerol and growing them overnight to eliminate petites. This patch was used to inoculate 5 ml YEPD, then a further 1/50 dilution was made and grown overnight. This culture was used to inoculate 25 ml YEPD in a 250 ml flask to an optical density (ODCell viability was monitored using the FungaLight Yeast Viability Kit (Molecular Probes) according to the manufacturer's protocol and the percentage of live cells was plotted over time. Reproductive capacity was assayed as the ability to resume cell division and produce colonies. Serial dilutions were plated on YEPD plates in duplicate and the percentage of colony forming units (CFU) was plotted, using the CFU from the freshly harvested Q cell sample as 100%. The FungaLight and CFU viability data are averages from at least two independent experiments.7 cells were collected and mixed with Calcofluor white M2R  at a final concentration of 100 µg/ml. Cells were incubated at room temperature for 15 min in the dark then were washed twice with H2O. The stained cells were examined with a Nikon Eclipse E600 microscope with a Nikon Plan Apochromat 60XA/1.40 oil immersion objective and a UV-2E/C DAPI filter (excitation at 330–380 nm). Photomicrographs of cells were taken on a Photometrics Cascade 512B camera and analyzed with MetaMorph version 6.3r2 software . TUNEL Assay: Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, spun down at 5000 rpm for 5 min and washed once with 0.1 M potassium phosphate 1.2 M sorbitol buffer pH 7.5. Stationary phase cells were first resuspended in 100–200 µl fresh pretreatment buffer , and then pelleted in a microfuge at 2000 rpm for 3 min at room temperature. These cells were resuspended in 1 M sorbitol and pelleted as before. Cell walls were digested with 50 µg/ml Zymolyase 100T in 1 M Sorbitol buffer (pH 5.8) for 10–45 min at 30°C. Cells were pelleted at 2000 rpm for 3 min, gently washed and resuspended in 15 µl potassium phosphate/sorbitol buffer, transferred to 0.1% polylysine-coated wells of an eight well microscope slide and allowed to settle for 20 min at room temperature. The slide was washed twice with phosphate-buffered saline (PBS). Each well was incubated with 40 µl fresh permeabilization solution (0.1% Triton X-100 in a 0.1% sodium citrate solution) for 2 min on ice, then rinsed with PBS buffer. 15 µl TUNEL reaction mixture  was added to each well, slides were covered and incubated for 60 min at 37°C, then rinsed twice with PBS. Cells were observed under the microscope with an FITC filter (excitation at 460–500 nm). 100–200 cells per sample were evaluated.Calcofluor staining of bud scars: Approximately 10For flow cytometry, cells were fixed in 70% ethanol for two hours or overnight, washed once with water, then resuspended in .5 ml 50 mM Tris-HCl (pH 8.0) containing 0.2 mg/ml RNAse A and incubated at 37°C for four hours. These cells were spun down, resuspended in .5 ml 50 mM Tris-HCl (pH 7.5) containing 2 mg/ml Proteinase K, and incubated at 50°C for one hour. They were then spun down again and resuspended in .5 ml 50 mM Tris-HCl (pH 7.5) and stored at 4°C. Before analysis, they were sonicated, pelleted, and resuspended in .5 ml I.0 µM Sytox Green (Invitrogen). Percent of cells in G1, S or G2/M phase of the cell cycle were quantified with FlowJo V9.6 cells were pelleted, gently washed with PBS, then resuspended in 1 mL PBS. 2.5 µL of a 10 mM carboxy-H2DCFDA (Invitrogen) stock solution was added and the cells were incubated for 30 minutes at 37°C. Cells were washed twice with PBS, resuspended in 1 mL 50 mM Tris-HCl pH 7.5. Cultures were then sonicated and 30,000 cells per sample were collected on a Fluorescence Activated Cell Sorter FACScan cytometer  and analyzed using Cell Quest software. FACS parameters were set at excitation and emission settings of 495 nm and 529 nm (filter FL-1), respectively. Average from two experiments is reported.For ROS assays, approximately 1×10600 of cells were collected every 10 minutes, washed with RNA buffer  and frozen for later RNA purification. The levels of CLN3 and ACT1 mRNA were monitored by an S1 nuclease protection assay as previously described CLN3 and ACT1 transcript levels were measured in each sample of wild type and 5XCLN3 cells. The ACT1, though not invariant, was not affected by excess CLN3 so it could be used to normalize the RNA levels between the two strains.To generate enough cells for RNA measurements during growth from log phase to stationary phase, 5 ODNext-Generation RNA sequencing was carried out with RNA prepared as above from log phase cells, purified Q cells, and cells grown in YEPD to log phase (8 hours) the DS (14 hours), then 18, 24 and 48 hours. mRNA expression levels following polyA selection were assayed using the HiSeq 2000 next generation sequencing system from Illumina Saccharomyces cerevisiae W303 reference genome using the Tophat application, a fast splice junction mapper for RNA-Seq reads The W303 reference genome in FASTA format and gene annotations in GFF were obtained from the Wellcome Trust Sanger Institute's SGRP group. Sequences from each read were mapped to the in vivo binding sites. PCR primers used to amplify potential targets as well as an unregulated DNA (IRV) are provided as Supplementary Cells carrying Xbp1 tagged with a Tandem Affinity Purification (TAP) tag Figure S1XBP1 (green), and three of its direct targets: PIS1 (blue), CLN3 (red) and DOG2 (purple), reproduced from the Periodic Transcript Server CLN3, DOG2 and PIS1 in the metabolic oscillation data set XBP1 transcript oscillates out of phase with its targets in cells synchronized with limiting glucose. (A) Metabolic oscillations of mRNA for (TIF)Click here for additional data file.Table S1xbp1. Open reading frame number and classification, gene name and gene description are provided. TRUE indicates genes with Xbp1 binding sites within 800 base pairs of translational start sites. FALSE indicates indirect targets.All transcripts derepressed three-fold or more by (XLSX)Click here for additional data file.Table S2Xbp1 repressed genes after different intervals of growth. (A) Targets containing Xbp1 binding sites or (B) that lack Xbp1 binding sites that are listed based on when they are derepressed, that is after 18, 24, 48 hours of growth or in purified Q cells. Genes are organized by gene ontology (GO) process. Corrected P value and the number of genes in each class are shown in parentheses.(DOC)Click here for additional data file.Table S3xbp1 and wild type cells are normalized and provided as a log base 2 value for xbp1/wild type. Rank is provided as an indicator of the likelihood that the genes are differentially expressed in the two strains at each time.Next Generation RNA sequence data for Xbp1 targets. Data for all transcripts derepressed three-fold or more at one or more time point are provided. Open reading frame number and classification, and gene name are provided. TRUE indicates genes with Xbp1 binding sites within 800 base pairs of translational start sites. FALSE indicates indirect targets. For each target gene at each time point, the data for (XLSX)Click here for additional data file.Table S4PCR primers used in chromatin immunoprecipitations.(DOCX)Click here for additional data file.


Human parvovirus 4 has been considered to be transmitted only parenterally. However, after novel genotype 3 of parvovirus 4 was found in 2 patients with no parenteral risks, we tested infants in Ghana. A viremia rate of 8.6% over 2 years indicates that this infection is common in children in Africa. Parvovirinae.In 2005, a novel human parvovirus, termed parvovirus 4 (PARV4), was identified in a plasma sample from a patient with symptoms resembling those of acute HIV infection  or HIV   and who did not live close to a river  of 279 samples were positive for PARV4 genotype 3 DNA. At the time of blood collection, no infant had signs of acute infection . Positive samples were found for infants in 7 of 9 studied villages, indicating widespread prevalence. To test whether socioeconomic factors might influence prevalence, we conducted a χ a river .Two age groups were randomly selected from the cohort: 1) 94 infants with a median age of 14.9 months  and 2) 185 infants with a median age of 24.0 months . Significantly more positive results were seen among infants in the older versus the younger age group . Viral loads in the whole study group ranged from 420 to 56,000 copies/mL of whole blood . Median viral loads did not differ significantly between the age groups .A 746-nt fragment of open reading frame (ORF) 1 and a 558-nt fragment of the ORF2 gene and the noncoding region between them were sequenced. Maximal nucleotide distances from previously known PARV4 sequences were 7%–8%. To obtain highly informative sequence datasets for phylogenetic analysis, we concatenated ORF1 and 2 sequences and excluded interfragment recombination by using SimPlot and GARD analysis with the HYPHY package . The novAlthough infection with human parvovirus PARV4 has been considered to be restricted to adults and transmitted parenterally, we found high prevalence of PARV4 genotype 3 in blood of infants in Africa. In agreement with findings for the 2 adults with PARV4 genotype 3 infection  are thus suspected. In conclusion, in Africa, novel PARV4 genotype 3 is prevalent among infants who are most likely not at risk for parenteral exposure.


The legend for Table 6 should read: "Table 6. Identified 43 biomarkers of the 99 biomarkers that correlated with height adjusted TKV."There were errors in Table 6 and Supplementary Table 2. Correct versions of these tables are available below:Table 6: Supplementary Table 2: Click here for additional data file.


In the present study, we examined the influence of a histamine H1 receptor antagonist, fexofenadine hydrochloride (FEX) on CC10 production in vitro and in vivo.Uteroglobin (CC10) is well known to be an immuno-suppressive protein secreted from airway epithelial cells after inflammatory stimulation and function in development of allergic disorders. Although histamine H6 cells/ml) were stimulated with 20 ng/ml TNF-α in the presence of various concentrations of FEX for 24 h. CC10 levels in culture supernatants were examined by ELISA. Patients with Japanese cedar pollinosis were orally treated with FEX twice a day at a single dose of 60 mg for two weeks during Japanese cedar pollen season (February 2011 to April 2011). CC10 levels in nasal secretions were also examined by ELISA.Nasal epithelial cells (5 x 10The addition of FEX into epithelial cell cultures caused dose-dependent increase in the ability of cells to produce CC10 in response to TNF-α stimulation, and the minimum concentration that caused significant increase was 200 ng/ml. Oral administration of FEX for two weeks also increased CC10 levels in nasal secretions from pollinosis patients along with attenuation of clinical symptoms.The ability of FEX to enhance CC10 production may account, at least in part, for the clinical efficacy of the agent on allergic disorders, including allergic rhinitis