Ehrlichia, phylogenetically distant from E. ruminantium, was found in 2010 infecting cattle in Canada. In 2012 and 2013, we reported the in vitro propagation, molecular and ultrastructural characterization of Ehrlichia sp. UFMG-EV (E. mineirensis), a new species of Ehrlichia isolated from the haemolymph of Brazilian Rhipicephalus (Boophilus) microplus ticks. A new organism, named Ehrlichia sp. UFMT-BV, closely related to Ehrlichia sp. UFMG-EV, was recently described in Brazil and after experimental infection it was shown to be pathogenic for cattle. This new emerging clade of cattle Ehrlichia pathogens is closely related to E. canis. The major immunogenic Tandem Repeat Protein  is extensively used to characterize the genetic diversity of E. canis. Homologs of TRP36 were found in both Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV.A new species of Ehrlichia clade using TRP36 sequences. Our working hypothesis is that Ehrlichia sp. UFMG-EV and related microorganisms evolved from a highly variable E. canis clade. In support of our hypothesis we found that Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV TRP36 evolved from a highly divergent and variable clade within E. canis and this clade evolved under episodic diversifying selection with a high proportion of sites under positive selection.Herein, we characterized the evolution of this new Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV evolved from a variable clade within E. canis.Our results suggest that The online version of this article (doi:10.1186/s13071-014-0584-5) contains supplementary material, which is available to authorized users. Anaplasma, Ehrlichia, Neorickettsia and Wolbachia. From these genera, Ehrlichia and Anaplasma are important pathogens affecting animals and humans. Ehrlichia are obligate intracellular gram-negative, tick-borne bacteria that grow within membrane-bound vacuoles in human and animal leukocytes causing ehrlichiosis. With a worldwide distribution ehrlichioses are considered emerging diseases that can cause serious illness in a variety of hosts, including humans, livestock and pets. Three news species of cattle-related Ehrlichia spp have been recently reported: (i) a new species that naturally infect cattle from British Columbia, Canada [Ehrlichia sp. UFMG-EV  Ehensis in ,3) that emolymph -4, and  of homology , but also other variants – AQVSADSGA (Ehrlichia sp. UFMT-BV), EASVVPEA (New Brazilian variant of E. canis) and VPAASGDAQ (Ehrlichia sp. UFMG-EV)  residue in the second position making it improbable that the asparagine (N) will be glycosylated  showed that many sites experienced positive or negative selection Table . The higE. canis), codon 116 was selected in branch 2 . This data therefore suggests that putative N-glycosylation associated with this sequon might be important in the host shift (see below) observed in Ehrlichia sp. UFMT-BV and Ehrlichia sp. UFMG-EV.Codon 77 evolved under diversifying (positive) and codon 116 evolved under negative selection. These two codons code for amino acids involved in the formation of sequons among TRP36 homologs  [Ehrlichia sp. UFMG-EV may have diverged a long time ago from E. canis.Our evidence supports the idea of differential evolutionary pressures on the glycoprotein TRP36 along different strains of ew hosts . In agreE. canis . Taking E. canis sensu stricto and has become ecologically independent from the parental species. In agreement with the new hosts association of this group of microorganisms, it was found that Ehrlichia sp. UFMG-EV was able to propagate in bovine aorta BA886 cell line, while E. canis did not [in vitro observation supports the above conclusions regarding the new host specificity of this novel group of cattle related agents. At the ultrastructural level, Ehrlichia sp. UFMG-EV shares ultrastructural features with other members of the genus Ehrlichia . We found cells, however, with unusual structures (invagination of the cellular membrane) for which we yet do not have an explanation [E. mineirensis (Ehrlichia sp. UFMG-EV) might be an important contribution to these studies.Altogether, these results suggest that this new group of organisms evolved from  did not . This inlanation . Further


Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate.Neutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of L. braziliensis promastigotes in mice induced neutrophil accumulation in vivo, peaking at 24 h. In vitro, exposure of thyoglycollate-elicited inflammatory or bone marrow neutrophils to L. braziliensis modulated the expression of surface molecules such as CD18 and CD62L, and induced the oxidative burst. Using mCherry-expressing L. braziliensis, we determined that such effects were mainly observed in infected and not in bystander cells. Neutrophil activation following contact with L. braziliensis was also confirmed by the release of TNF-α and neutrophil elastase. Lastly, neutrophils infected with L. braziliensis but not with L. major displayed markers of early apoptosis.Inoculation of serum-opsonized L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection.We show that  Leishmania is the parasite responsible for the disease leishmaniasis, present in all continents. Leishmania parasites are spread through infected sand-flies and, during transmission into the vertebrate host, neutrophils are among the first cells to arrive at the infection site. Since neutrophils are key players at the frontline of defense against invading organisms, we investigated their response to Leishmania braziliensis. Importantly, L. braziliensis causes both Cutaneous and Mucocutaneous Leishmaniasis, two clinical manifestations characterized by their chronic development and by the presence of skin lesions with tissue destruction. Upon inoculation of mice with L. braziliensis, neutrophils rapidly arrive at the site of infection. We then observed that culture of mouse neutrophils with L. braziliensis induced the expression of adhesion molecules, production of Reactive Oxygen Species and secretion of elastase and TNF-α, two important inflammatory mediators. Also, infection with L. braziliensis induced neutrophil apoptosis, a cell death mechanism key for regulating inflammation. Our results show that neutrophils respond to presence of the L. braziliensis parasites by becoming activated and undergoing apoptosis. We suggest that this outcome modifies the local environment at the site of parasite inoculation and thus contributes with parasite killing in the infected host. Neutrophils are essential components of the early inflammatory response, acting as the first line of defense against invading pathogens (rev. in ). NeutroLeishmania infection, neutrophils play distinct roles depending on the combination of mouse strain and parasite species. For L. donovani and L. infantum, neutrophils contributed to parasite killing . Analyses were conducted using Prism (GraphPad software) and a p-value of <0.05 was considered significant.L. braziliensis [L. braziliensis infection. Mice were inoculated in the ear dermis with L. braziliensis parasites and recruited cells were selected based on size and granularity; within this population, we defined Ly6G+ neutrophils . These results show that neutrophil recruitment peaks one day after L. braziliensis inoculation.Previously, we reported that neutrophils are present throughout the course of lesion development in BALB/c mice inoculated with iliensis . Herein,trophils . Six houh saline . Twenty-L. braziliensis inoculation  and bystander neutrophils (Ly6G+mCherry-), the latter defined as neutrophils that remained uninfected in spite of exposure to L. braziliensis  neutrophils was approximately 36% (-). The percentage of CD18+ cells among infected (mCherry+) neutrophils was higher (p<0.01) in comparison to bystanders (mCherry-) (+ cells was very low (+ cells was high (+ cells was lower (p<0.05) among infected (mCherry+) neutrophils compared to bystanders (mCherry-)   whereas very low . In non-was high  and diffCherry-) . Incubat among infected (mCherry+) neutrophils compared to bystanders (mCherry-) (+ cells was highest in non-exposed neutrophils (-) compared to infected (mCherry+) neutrophils. These data indicate that neutrophils infected with L. braziliensis upregulate CD18 and downregulate CD62L, regardless of their activation state.To expand on these findings, we performed experiments with bone marrow neutrophils, which, comparatively have an enhanced capacity to become primed . Bone-mately 48%  whereas trophils , the perCherry-) . Also reCherry-) , the pertrophils , and sigL. braziliensis displayed a significant increase in superoxide production  (L. braziliensis (+ cells (Neutrophils produce Reactive Oxygen Species (ROS), which form a central component of the defense mechanism against foreign pathogens during infection. Inflammatory neutrophils co-cultured with oduction , which wCherry-) . Additioiliensis . Co-cultiliensis . Similar increase in the percentage of early apoptotic (Annexin V+/PI-) neutrophils (+/PI+) neutrophils was similar  and late apoptotic/necrotic (Annexin V+/PI+) neutrophils, compared to neutrophils cultured with L. braziliensis  and PS exposure can be detected by Annexin V staining and quantified by flow cytometry. Inflammatory neutrophils were co-cultured for 18 h with trophils , compare similar . On the iliensis . Analysiarasites . Moreovelization . In co-c vacuole , reinforpoptosis , differeL. braziliensis induced apoptosis in inflammatory neutrophils, we then examined whether this would also occur with bone marrow neutrophils. Indeed, upon co-culture with L. braziliensis, a significant increase in the percentage of late apoptotic/necrotic (Annexin V+/DAPI+) neutrophils  was mostly detected in infected neutrophils (mCherry+) when compared with bystanders (mCherry-) (+ neutrophils was ~28% (Following the observation that trophils  was obseCherry-) . At thiswas ~28% .L. braziliensis inoculation into the ear dermis of BALB/c mice leads to the development of a cutaneous ulcer, which heals spontaneously after ten weeks of infection [L. braziliensis and neutrophils decreased lesion size whereas depletion of neutrophils and monocytes had an opposing effect, significantly increasing parasite load and lesion size [Leishmania parasites, the purpose of the current study was to investigate how neutrophils respond to L. braziliensis exposure, evaluating neutrophil activation and downstream events such as apoptosis.Numerous studies have demonstrated that neutrophils play a crucial role in immunity against bacterial, fungal  and intrnfection . Additioion size . Given tion size  and, thuLeishmania promastigotes are inoculated. Following this event, there is rapid accumulation of neutrophils [L. braziliensis inoculation by syringe and showed maximal accumulation at 24h. Of interest, syringe inoculation of L. amazonensis, also induced maximal neutrophil accumulation at 24 h [Leishmania species.Sand flies probe the human host to obtain blood and, in this process, lacerate capillaries forming a blood pool into which trophils  and it htrophils ,34. Here at 24 h , indicatL. braziliensis inoculation, we then performed a series of in vitro experiments to investigate how neutrophils respond to this type of stimulation and, in addition, we compared the responses of inflammatory and bone marrow neutrophils. Initially, we evaluated the expression of adhesion molecules. β2 integrins are leukocyte-specific integrins required for neutrophil adhesion and transmigration across the activated endothelium [2 integrin present in LFA1 (CD11aCD18), Mac-1/ CR3 (CD11bCD18) and p150/94/CR4 (CD11cCD18). In the presence of L. braziliensis we detected an increase in the percentage of neutrophils (inflammatory and bone marrow) expressing CD18 and this increase was associated with infected neutrophils (mCherry+), indicating that L. braziliensis were readily internalized. Indeed, Mac-1/CR3 (CD11bCD18) plays a major role in the phagocytosis of complement-opsonized L. major promastigotes by both macrophages [Following our observation that neutrophils are recruited in response to othelium  and CD18rophages –38 and hrophages .L. braziliensis, we detected a lower percentage of inflammatory CD62L+/mCherry+ neutrophils, compared to bystanders (mCherry-). Similar results were obtained with bone marrow neutrophils, indicating that L. braziliensis phagocytosis induced more CD62L shedding, marking neutrophil activation [+/mCherry- neutrophils was lower compared to bone marrow (CD62L+/mCherry-) neutrophils, possibly reflecting their already primed nature and their extravasation to the peritoneum following thyoglycollate stimulation. Such difference may also be related to the priming potential of bone marrow neutrophils vs. inflammatory, as shown by fMLP stimulation and induction of ROS [L. amazonensis LPG activates human neutrophils in levels similar to those observed with promastigotes [L-selectin (CD62L) participates in neutrophil tethering and rolling  but it itivation . With ren of ROS . Also, wstigotes .L. braziliensis, both inflammatory and bone marrow neutrophils displayed a significant increase in the production of superoxide, a hallmark of neutrophil activation, and ROS detection was significantly higher in infected (mCherry+) neutrophils. Similar results were obtained in experiments with other Leishmania spp. [L. braziliensis, we can suggest that ROS produced by infected cells contributed with TNF-α secretion and CD18 (a Mac1/CR3 component) expression. Furthermore, elastase production was also elevated in neutrophils cultured with L. braziliensis and, importantly, elastase was associated with the killing of intracellular Leishmania in macrophages cultured with neutrophils [In the presence of nia spp. ,44–46. Nnia spp. ,48 and Tnia spp. . Since Ttrophils , a procetrophils . IL-10 ptrophils ,51.L. braziliensis induced neutrophil apoptosis, findings that were confirmed by transmission electron microscopy analysis. Indeed, infected neutrophils displayed condensed chromatin and degraded intracellular parasites. Similar results were obtained with bone marrow neutrophils: Annexin+/DAPI+ staining was significantly higher in infected cells (mCherry+) compared to bystanders (mCherry-) and the percentage of infected neutrophils (mCherry+) was lower compared to bystanders (mCherry-). We can suggest that phagocytosis of L. braziliensis results in apoptosis and, in parallel, parasite destruction, hence the lower percentage of infected cells. In addition, ROS [L. braziliensis.Cell death and the subsequent clearance of apoptotic neutrophils is crucial for maintaining homeostasis and, at the same time, necessary for resolution of inflammation. At inflammatory sites, neutrophils can undergo spontaneous apoptosis  or apoption, ROS  and TNF-ion, ROS  also triL. amazonensis [L. major, on the other hand, delays neutrophil apoptosis [+/PI+) staining was low in neutrophils cultured with L. major in contrast to neutrophils cultured alone and to neutrophils cultured with L. braziliensis, both of which were positive for Annexin/PI . In . In L. aite load , also suL. brazilensis inoculation and upon contact with promastigotes, in vitro, neutrophils become activated producing superoxide, TNF-α and elastase. Later, we observed neutrophil apoptosis, particularly of infected cells. However, once amastigotes become predominant, a different scenario may ensue since this stage is more resistant to these same effector mechanisms, as recently described for L. amazonensis [L. braziliensis develop cutaneous ulcers, despite the presence of neutrophils [L. braziliensis-infected macrophages, as previously shown in vitro [L. braziliensis. However this remains to be investigated. Thus, the strong impact of L. braziliensis on neutrophils phenotype and function reported here in vitro are likely to occur at the onset of infection with the parasite, suggesting that these cells are playing a crucial role following infection.We showed that neutrophils are recruited to the site of zonensis , impactitrophils . Howeverin vitro . Neutropin vitro ,61,62 anS1 FigInflammatory neutrophils were purified using MACS and the 1A8 (Ly6G) mAb. In this sample cells were first gated for size and granularity (SSC x FSC). The gated cells were further analyzed for expression of CD11b/1A8 or 1A8/Ly6C.(TIF)Click here for additional data file.S2 FigBone marrow neutrophils were purified using MACS and the 1A8 (Ly6G) mAb. In this sample cells were first gated for size and granularity (SSC x FSC). The gated cells were further analyzed for expression of CD11b/1A8 or Gr-1/Ly6C.(TIF)Click here for additional data file.S3 FigL. braziliensis or with Zymozan (SIGMA (100ug/ml). (A) Representative dot plots showing the gating strategy used to identify neutrophils (Ly6G) following exposure to L. braziliensis. Cells were stained with anti-CD18 (B) and were analyzed by FACS. Orange histograms: isotype control. Gray histogram: neutrophils cultured in medium only. Red histograms: neutrophils cultured with Zymozan. Black histograms, neutrophils exposed to L. braziliensis. Bar graphs represent the percentage of neutrophils positive for CD18. Data shown (mean ± SEM) are pooled from two independent repeats. *p<0.05 (One Way ANOVA).Inflammatory neutrophils were co-cultured with (TIF)Click here for additional data file.


Caenorhabditis elegans and, to a lesser extent, the mammalian gastrointestinal parasite Ascaris suum are, historically, the primary sources of knowledge regarding nematode neurobiology. Despite differences in size and habitat, C. elegans and A. suum share a surprisingly similar neuroanatomy. Here, we examined species across several clades in the phylum Nematoda and show that there is a surprising degree of neuroanatomical variation both within and among nematode clades when compared to C. elegans and Ascaris. We found variation in the numbers of neurons in the ventral nerve cord and dye-filling pattern of sensory neurons. For example, we found that Pristionchus pacificus, a bacterial feeding species used for comparative developmental research had 20% fewer ventral cord neurons compared to C. elegans. Steinernema carpocapsae, an insect-parasitic nematode capable of jumping behavior, had 40% more ventral cord neurons than C. elegans. Interestingly, the non-jumping congeneric nematode, S. glaseri showed an identical number of ventral cord neurons as S. carpocapsae. There was also variability in the timing of neurodevelopment of the ventral cord with two of five species that hatch as second-stage juveniles showing delayed neurodevelopment. We also found unexpected variation in the dye-filling of sensory neurons among examined species. Again, sensory neuron dye-filling pattern did not strictly correlate with phylogeny. Our results demonstrate that variation in nematode neuroanatomy is more prevalent than previously assumed and recommend this diverse phylum for future “evo-devo-neuro” studies.Nematodes are considered excellent models for understanding fundamental aspects of neuron function. However, nematodes are less frequently used as models for examining the evolution of nervous systems. While the habitats and behaviors of nematodes are diverse, the neuroanatomy of nematodes is often considered highly conserved. A small number of nematode species greatly influences our understanding of nematode neurobiology. The free-living species  Caenorhabditis elegans consists of only 302 neurons in the adult hermaphrodite and remains the only nervous system to be completely reconstructed  species has been examined using both light and transmission electron microscopy (TEM).For the past 200 years, nematodes received significant attention from neurobiologists due to their relatively simple anatomy   . Signifiigure 1) . Howeveronserved .C. elegans (Clade 9) and the gastrointestinal parasitic nematode Ascaris suum (Clade 8). The VNC consists of a series of motor neurons that innervate body-wall muscles and regulate movement  of movement . While A muscles . In A. sle cells .C. elegans, there is one pair of amphid sensilla each containing 12 sensory neurons. The anterior nervous system of C. elegans also contains six inner labial, six outer labial and four cephalic sensilla each with an invariant number of neurons  and fluorescence microscopy to examine the neuroanatomy of the VNC and sensory neurons in nematodes from clades 9 to 12 . We founMeloidogyne hapla was isolated by the senior author from infected tomato plants and identified using morphological characters. Pratylenchus penetrans was isolated by Dr. Terry Niblack (formerly University of Illinois). Both M. hapla and P. penetrans were cultured on monoxenic excised corn and tomato root cultures, respectively  were received from Dr. Albrecht Koppenhöfer at Rutgers University and reared on living greater wax moth larvae Galleria mellonella , C. elegans (strain N2), and P. pacificus (strain PS312) were received from the Caenorhabditis Genetics Center and cultured on NGM agar with Escherichia coli OP50 using standard methods  of H. glycines were extracted from soybean roots using sugar centrifugation and washed three times with distilled water . In cases where a cell could not be unambiguously identified as a neuron an independent count was made by a researcher blind to the species. If the cell identity was still in doubt, it was excluded from the total neuron count.Nematodes were fixed in 4% formaldehyde at 4°C overnight in microcentrifuge tubes. Following formaldehyde fixation, nematodes were washed three times with Phosphate buffered saline with Triton X-100  and incubated in methanol for at least 4 h. Nematodes were then washed three times with PBST and incubated in 0.2–0.5 μg/ ml of 4′, 6-diamidino-2-phenylindole  overnight in dark at room temperature. Nematodes were store at 4°C prior to examination. We were unable to distinguish the sex of e nuclei . Counts M. hapla. Nematodes were recovered from tomato root cultures and transferred to 0.2X Finney-Ruvkun buffer with 5% methanol and 2% formaldehyde . In C. elegans, the VNC contains a series of 57 motorneurons lying between the RVG and the PAG  is used as a satellite nematode species for evolutionary studies  suggesting that the number of neurons does not strictly correlate with phylogeny. Furthermore, this data indicates that evolved behaviors such as predation do not necessarily require an increase in the number of motorneurons. To test if other, more distantly related bacterial feeding nematodes, show even greater divergence in ventral cord neuron number, we examined the Clade 11 bacterial feeding nematode Acrobeles sp. Interestingly, Acrobeles sp. showed a nearly identical number of VNC neurons as C. elegans .The free-living nematode,  studies , 2006. Sematodes . We founHeterorhabditis sp. (Clade 9) and Steinernema sp. (Clade 10) infect a wide range of insect hosts. Though phylogenetically distinct, the two genera have similar lifestyles. Heterorhabditis sp. is more closely related to C. elegans than to Steinernema sp. (Clade 10). We found that two species of Heterorhabditis had 59 ventral cord neurons, similar to C. elegans . However, the ventral cord of S. carpocapsae contained 76 neurons ; approximately 40% more than C. elegans and the largest number of VNC neurons among all examined species. S. carpocapsae is capable of an unusual jumping behavior wherein it stands on its tail, curls to form a loop and quickly extends to jump into the air . This data suggests either that jumping behavior does not specifically require additional motor neurons or that an ancestor to S. glaseri could jump and the additional neurons in S. glaseri are remnants of this ancestor. Steinernema sp. are male-female species. During the IJ stage sexes are easily distinguishable based on the shape of the gonad. However, we did not observe major differences in the number of VNC nuclei between sexes (data not shown).The entomopathogenic nematode genera  the air . We hypoAphelenchus avenae and the plant-parasitic nematodes, Heterodera glycines and Meloidogyne hapla each had approximately 65 VNC neurons . However, the plant-parasitic nematode P. penetrans ventral cord contained fewer neurons than any other Clade 12 species . Pratylenchus is considered basal to Meloidogyne  with 15 VNC neurons (P. pacificus (Clade 9), A. avenae (Clade 12) and P. penetrans (Clade 12). Both P. pacificus and A. avenae showed delayed development in the VNC. P. pacificus hatched as a J2 with 20 VNC neurons while the adult hermaphrodite has 45 VNC neurons (Table 1). Similarly, A. avenae hatched as a J2 with 26 VNC neurons, while the adult female has 66 (Table 1). Interestingly, the delayed development of the VNC was not conserved among all species that hatch as J2s. We found no apparent difference in the number of VNC neurons between newly hatched J2 and adult P. penetrans (Clade 12) (data not shown). These data suggest an independent evolution of neuronal heterochrony in nematodes. neurons . During  neurons . This poA. avenae and P. pacificus may be due to a shift in development from the C. elegans-like J1 VNC development to a J2 VNC development. Alternatively, the delayed development may be due to a progressive increase in VNC neuron number from J1 to the adult stage. To test these options, we collected time-series data on the development of the VNC in A. avenae using DIC microscopy. Aphelenchus avenae is an easily cultured fungal feeding nematode, closely related to, and a possible transitional model for, plant-parasitic nematodes. We found that eggs developed from single-cell embryos to hatched J2s in approximately 48 h. We observed 26 neurons in the VNC of newly hatched J2s using DIC microscopy. Cell division occurs after hatching and during feeding. Immediately prior to the J3 molt (25–35 h after hatching), we observed the most intensive increase of neurons in the VNC. By 48 h after hatching, A. avenae had a fully developed VNC with 66 neurons . Thus, the delayed development of the VNC in A. avenae is a shift from a C. elegans-like J1 developmental sequence to a J2 post-hatching developmental sequence.The delayed development seen in C. elegans research. Specific ciliated sensory neurons in the head and tail of C. elegans will fluoresce following exposure to the lipophilic compound DiI  and Steinernema carpocapsae (Clade 10). These entomopathogenic nematodes infect their hosts as IJs, a non-feeding developmentally arrested stage analogous to the C. elegans dauer stage. Following infection, the nematodes will resume development and feed until resources are depleted. The nematodes then reenter the IJ stage and disperse to find a new host. Interestingly, we did not observe any dye-filling in IJs of either species. Therefore, we isolated non-infective stages of these species using both in vitro and in vivo methods. We observed dye-filling in six putative IL2 orthologs in non-IJs and adults of S. carpocapsae . Two pairs of putative phasmid neurons also dye-filled in the tail of males of S. carpocapsae . Surprisingly, dye-filling of amphid neurons was only periodically observed in both of these insect-parasitic species. Based on these results, we examined the dauer stage of C. elegans. As previously shown, five of the six amphid neurons routinely dye-filled in C. elegans dauers   . Howevern by TEM . TogetheH. bacteriophora (Clade 9) and S. carpocapsae (Clade 10) showed different dye-filling patterns than C. elegans. However, the bacterial-feeding nematode Acrobeles sp. (Clade 11) showed an identical dye-filling pattern to C. elegans . The fungal-feeding nematode A. avenae (Clade 12) also displayed a similar dye-filling pattern as C. elegans. Specifically, six putative IL2 orthologs and twelve putative amphid orthologs dye-filled in A. avenae . In A. avenae, an additional four neurons were stained anterior to amphid neurons that we identified as cephalic neurons. Previous TEM data demonstrated that A. avenae females have two cephalic neurons (CEP1 and CEP2) in each sensillum  in C. elegans are exposed to the external environment, they do not dye-fill . Based on axon morphology and nuclear position we identified these neurons as likely ADL orthologs. We observed only occasional and weak dye-filling in P. penetrans phasmid neurons (data not shown). Two plant-parasitic nematodes, H. glycines and M. hapla, did not show dye-filling in any neuron suggesting a possible modification of these neurons.Unlike the fungal feeding Clade 12 nematode Nematodes have made substantial contributions to our understanding of the molecular basis of evolution. These studies have generally focused on non-neuronal structures such as vulva development and male-tail morphology . Fewer sC. elegans and A. suum consist of a series of motor neurons that regulate movement through coordinated excitation and inhibition of body wall muscles. Amazingly, these phylogenetically and ecologically separate species show a nearly identical number and pattern of cholinergic and GABAergic VNC neurons  than A. suum (Clade 8), P. pacificus (Clade 9) had 20% fewer VNC neurons than either species. Similarly, P. penetrans had 20% fewer VNC neurons than all other examined Clade 12 species  and Acrobeles sp. (Clade 11) show a nearly identical dye-filling pattern, the entomopathogenic nematode Heterorhabditis bacteriophora (Clade 9) showed striking differences. This variability in dye-filling is consistent with previous results showing only a single pair of amphid neurons dye-fill in the Clade 10 mammalian-parasitic nematode Parastrongyloides trichosuri (Heterodera glycines and Meloidogyne sp. clearly indicate that the amphids are exposed to the environment (Heterodera glycines (Clade 12), which showed no dye-filling with DiI, was previously shown to undergo amphid dye-filling with fluorescein isothiocyanate . Following migration, the P cells divide to form an anterior neuroblast cell (Pn.a) and a posterior cell (Pn.p) which either forms a hypodermal nucleus or a vulval precursor cell. Certain ancestors of the C. elegans Pn.a cell undergo programmed cell death eventually leading to the defined 57 VNC neurons. Previous lineage analysis in P. pacificus demonstrated that the non-vulva forming Pn.P cells undergo apoptosis prior to hatch  and P. pacificus (Clade 9) is suggestive of neuronal heterochrony. As P. penetrans (Clade 12), which also hatches as a J2, shows no post-hatch VNC development we propose that the heterochronic developmental events in A. avenae and P. pacificus arose independently. In C. elegans, several mutants have been isolated that result in altered timing of development events . However, our methods do not allow for the identification of individual neuron types. TEM could be used to study the neuron types in order to classify repeated segments on select species. In addition, immunohistochemistry may be utilized to distinguish the neurotransmitter identity of individual cells. Using immunohistochemistry, C. elegans and P. pacificus (Our data suggest that the neuroanatomy of nematodes is not as highly conserved as previously described. The evolution of nematode nervous systems has been relatively neglected, in part, due to previous assumptions of high anatomical conservation. It will be valuable to use higher resolution imaging techniques and antibody staining to define the neuronal subtypes found in these species. For example, ectivity . In our acificus , we propZH and NS conceived project and designed experiments. ZH, SB, and NS performed experiments and analyzed results. ZH, SB, and NS wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pectobacterium species are economically important bacteria that cause soft rotting of potato tubers in the field and in storage. Here, we report the draft genome sequence of the type strain for P. carotovorum subsp. carotovorum, ICMP 5702 (ATCC 15713). The genome sequence of ICMP 5702 will provide an important reference for future phylogenomic and taxonomic studies of the phytopathogenic Enterobacteriaceae. P. carotovorum are ubiquitous in nature and infect a broad range of crops including Brussels sprout, carrot, celery, cucumber, banana, tomato, capsicum, and potato . The quality of the sequence reads was checked, and low-quality reads (score of <Q30) were trimmed using FastQC . A vo v2.04 , resultihttp://www.ncbi.nlm.nih.gov/genome/annotation_prok/). Annotation revealed the presence of 72 tRNAs and 4,109 coding sequences, a number encoding pathogenicity- or virulence-related factors such as plant cell wall degrading enzymes, flagella-based motility, cell membrane structures such as lipopolysaccharides and secretion systems. The identification of these factors provides insight into the mechanisms used by P. carotovorum subsp. carotovorum to invade potato tubers. As the type strain for this species/subspecies, the draft genome of ICMP 5702 also provides an important resource for future phylogenomic and taxonomic studies of phytopathogenic enterobacterial pathogens.The draft genome sequence of ICMP 5702 was annotated using the PGAAP annotation pipeline (P. carotovorum subsp. carotovorum ICMP 5702 has been deposited in the GenBank database under the accession no. AODT00000000. The version described in this paper is the first version, AODT01000000.The draft genome sequence of


Clostridium difficile, multistep algorithms are recommended. This study aimed to compare the current, representative diagnostic algorithms for detecting toxigenic C. difficile, using VIDAS C. difficile toxin A&B (toxin ELFA), VIDAS C. difficile GDH , and Xpert C. difficile . In 271 consecutive stool samples, toxigenic culture, toxin ELFA, GDH ELFA, and Xpert C. difficile were performed. We simulated two algorithms: screening by GDH ELFA and confirmation by Xpert C. difficile (GDH + Xpert) and combined algorithm of GDH ELFA, toxin ELFA, and Xpert C. difficile (GDH + Toxin + Xpert). The performance of each assay and algorithm was assessed. The agreement of Xpert C. difficile and two algorithms (GDH + Xpert and GDH+ Toxin + Xpert) with toxigenic culture were strong . The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of algorithms (GDH + Xpert and GDH + Toxin + Xpert) were 96.7%, 95.8%, 85.0%, 98.1%, and 94.5%, 95.8%, 82.3%, 98.5%, respectively. There were no significant differences between Xpert C. difficile and two algorithms in sensitivity, specificity, PPV and NPV. The performances of both algorithms for detecting toxigenic C. difficile were comparable to that of Xpert C. difficile. Either algorithm would be useful in clinical laboratories and can be optimized in the diagnostic workflow of C. difficile depending on costs, test volume, and clinical needs.Since every single test has some limitations for detecting toxigenic  Clostridium difficile is a primary pathogen causing antibiotic-associated colitis and responsible for 15–25% of cases of nosocomial antibiotic-associated diarrhea [C. difficile infection (CDI) range from mild diarrhea to pseudomembranous colitis and cannot be distinguished with those from other causes, making laboratory confirmation essential [ssential .C. difficile are the toxigenic culture and cell cytotoxicity assay [C. difficile had been toxin detection using enzyme immunoassay (EIA). It is fast and inexpensive; however, its low sensitivity is a limitation for its use [The two reference methods for detecting toxigenic ty assay , 5. Howety assay . In prac its use , 8.C. difficile cell wall and GDH testing detects both non-toxigenic and toxigenic C. difficile. It shows high sensitivity and negative predictive value (NPV), but it should be paired with a test detecting toxin [C. difficile [Glutamate dehydrogenase (GDH) is a common antigen of the ng toxin , 9. Receng toxin . Howeverng toxin , 4, 9–13ifficile , 13–15.C. difficile, using VIDAS C. difficile toxin A&B, VIDAS C. difficile GDH , and Xpert C. difficile .This study aimed to compare the current, representative diagnostic algorithms for detecting toxigenic in vitro study was approved by the Institutional Review Board of the Konkuk University Medical Center, Seoul, Korea . From January 2015 to April 2015, we collected 271 consecutive, remnant diarrheal stool samples submitted to the clinical microbiology laboratory from patients admitted to our hospital.They included 258 adults and 13 children . Since we used remnant samples after routine tests and the data were analyzed anonymously, informed consent was exempted. As a routine practice of C. difficile testing in our hospital, we performed toxigenic culture and VIDAS C. difficile toxin A&B (bioMérieux) simultaneously. The VIDAS C. difficile GDH (bioMérieux) and Xpert C. difficile (Cepheid) were additionally performed in the same samples. Duplicated samples from the same patients and from patients on treatment for CDI were excluded. The samples were tested within 2 hours of collection; otherwise, they were kept at 2–8°C for up to 2 days.This C. difficile were initially investigated by Gram staining and finally identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) using VITEK MS system (bioMérieux). Isolates of C. difficile were examined for toxin production using in-house PCR as described previously [The alcohol-shocked stool samples were inoculated onto on a chromogenic agar plate , and the plates were incubated at 37°C under anaerobic conditions  for 48 hrs. The isolates of typical morphology with gray-to-black colonies with irregular or smooth borders suspicious for eviously , 17.C. difficile toxin A&B and VIDAS C. difficile GDH kit (bioMérieux), respectively, according to the manufacturer's instructions. The assay principle combines a two-step enzyme immunoassay sandwich method with a final fluorescent detection . An aliquot of liquid stool was added in the specific diluent, and supernatant after centrifugation was tested. The VIDAS C. difficile toxin A&B is completed within 75 minutes, and results are reported as negative, equivocal, or positive with cut-offs of 0.13 and 0.37 test value (TV). There were 10 equivocal results in our study and they were considered negative for the performance calculations in this study. The VIDAS C. difficile GDH is completed within 50 minutes, and results are reported as negative or positive with a cut-off of 0.10 of TV.Stool samples were tested for toxin and GDH using VIDAS C. difficile system(Cepheid) is an automated, real-time multiplex PCR assay using disposable cartridge. This assay detects toxin B (tcdB), binary toxin (cdt), and a point mutation associated with PCR ribotype 027. The result of the toxin B target means the presence of toxigenic C. difficile. The results of other targets indicate additional information regarding ribotype 027. The invalid result is reported when sample processing control is failed, indicating that the sample is not properly processed or PCR is inhibited. Using a maximum valid cycle threshold setting of 37 for tcdB, the limit of detection point estimate for toxigenic C. difficile is 1,657 CFU/swab , according to the manufacturer’s insert.Xpert C. difficile toxin A&B (toxin ELFA), VIDAS C. difficile GDH (GDH ELFA), and Xpert C. difficile ; GDH-based 2-step algorithm, in which GDH ELFA is initially performed and followed by toxin gene detection using Xpert C. difficile (GDH + Xpert)[C. difficile, in which the initial screening is performed with GDH ELFA and toxin ELFA simultaneously and followed by Xpert C. difficile in discordant samples (GDH + Toxin + Xpert)[We selected the following diagnostic algorithms using VIDAS  + Xpert) and comb + Xpert). The ave + Xpert), 19 and The sensitivity, specificity, positive predictive values (PPV),and NPV with 95% CI of each assay and algorithm were determined based on the results of toxigenic culture as a gold standard. McNemar’s test was used for the statistical differences of sensitivity and specificity between assays and algorithms and chi-square test for the statistical differences of PPV and NPV between them. Agreement between assays were determined using Cohen’s Kappa  [. The agreement of toxin ELFA and GDH ELFA with toxigenic culture were weak and moderate . The agreement of the Xpert C. difficile and the two algorithms (GDH + Xpert and GDH + Toxin + Xpert) with toxigenic culture were strong .The result of each assay and algorithm was compared with that of toxigenic culture , while GDH ELFA showed significantly lower specificity (P < 0.0001) and PPV  than toxin GDH, Xpert C. difficile, GDH + Xpert, and GDH + Toxin + Xpert (Table 2). There were no significant differences between the Xpert C. difficile, GDH + Xpert, and GDH + Toxin + Xpert in sensitivity, specificity, PPV, and NPV. Their sensitivity and specificity ranged from 95% to 97%. Their PPV ranged from 82% to 85%, and their NPV were all above 98%.Based on toxigenic culture, toxin ELFA showed significantly lower sensitivity and NPV than the other assays and algorithms  by GDH + Xpert and 78.6% (213/271) by GDH + Toxin + Xpert (Table 3). Based on our distribution of samples, estimated average costs and assay times are also shown in Table 3. Cost per sample included reagent costs only and other costs like labor or quality control were not included. The assay time is calculated when GDH and toxin assays are performed concurrently in separate racks. The assay time per 30 samples was simulated based on capacity of our laboratory which has 1 GeneXpert with 4 cartilages spaces and 1 VIDAS with 5 racks (30 samples load).C difficile toxin EIA, GDH EIA, and NAAT, and they have different targets. The toxin EIA directly detects free toxin in feces, GDH test detects a common antigen produced by C difficile, and NAAT detects toxin genes but not free toxin [C. difficile using updated, automated, and widely used assays in clinical samples. Compared with toxigenic culture, two diagnostic algorithms, GDH + Xpert and GDH + Toxin + Xpert, showed strong agreements that are comparable to that of the Xpert C. difficile (Table 1).The best laboratory method for diagnosis of CDI remains controversial. The widely used assays have been ee toxin , 21. Altee toxin . Accordiee toxin , 13–15. C. difficile positive. When we considered equivocal results as positive category, toxin ELFA showed higher sensitivity and lower specificity  but they were not significant.In this study, toxin ELFA showed significantly lower sensitivity 49.1%) and NPV (88.3%) with comparable specificity and PPV to the other assays and algorithms. This finding is generally in line with most previous reports. Toxin EIA including ELFA, however, have shown widely varying sensitivity (32–99%) and specificity (65–100%), depending types of assays and used reference methods % and NPV, the algC. difficile, suggesting that it is suitable for initial screening [Most previous studies on GDH tests used ELISA kits . Only a creening –24.C. difficile and both algorithms (GDH + Xpert and GDH + Toxin + Xpert). The performance of the Xpert C. difficile was comparable to those of recent reports  [C. difficile and each algorithm. This finding demonstrates that we can choose either one in terms of performance (Table 2).Our study demonstrated the high sensitivities and specificities of the Xpert 0–98.8%) , 25, 26.Table 3). The cost of Xpert C. difficile was about $50 per sample. Based on the distribution of our samples, the average cost per sample in the two algorithms was estimated to be $25.7 and $30.7, respectively; it was lower than that of the Xpert C. difficile. However, rapid detection of CDI by Xpert C. difficile may offset laboratory costs by reducing hospitalization and C. difficile transmission from the view of total medical costs [Regarding the costs, 68.6% of samples by GDH + Xpert and 78.6% by GDH + Toxin + Xpert had the cost per sample of $10 and $20, respectively, and the other samples submitted to the second step had higher cost per sample of $60 and $70, respectively  would be extended in Xpert C. difficile when number of space for cartilages in GeneXpert system is limited, 4 cartilages in our system (Table 3). The system with more cartilages loading is needed for rapid reporting. The VIDAS system for GDH ELFA and toxin ELFA is also simple and easy to perform, offering routine batch or random access testing, whereas conventional ELISA system is difficult to perform as random access testing. Each laboratory can determine its own policy and can optimize the diagnostic workflow depending on its situations.The actual cost and turnaround time can vary across laboratories, according to the workflow, the number of requested tests, and many other factors. The Xpert C. difficile isolates and direct toxin, respectively [C. difficile excretes with diarrhea not due to CDI [In this study, we evaluated the performances of assays and algorithms based on toxigenic culture as a reference standard, which is suggested by many experts and guidelines , 9. Anotectively , 5. A ree to CDI . It alsoe to CDI , 9, 11. C. difficile showed comparable performances to that of the Xpert C. difficile. This data would be helpful to choose the diagnostic algorithms for the detection of CDI in clinical laboratories. Each laboratory can optimize its diagnostic workflow in accordance with its situation encompassing the costs, test volumes, and clinical needs.In conclusion, both algorithms screening with GDH and/or toxin ELFA confirmed by the Xpert


Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and, to a fewer occurrences, human erysipeloid. It is ubiquitous in nature and commensal in diverse species of animals, wild or domestic, from mammals and birds to reptiles and fish. Mechanisms of its virulence and pathogenicity are poorly understood.E. rhusiopathiae strain SY1027 and comparative genome analysis between the three highly pathogenic strains , the genomic structure and putative functional elements, such as pathogenicity island (PAI)-like regions, potential virulence factors and horizontal transferring genes of the bacteria are identified. Strain SY1027 genome is 1,752,910 base pairs long, just 30 kilobases smaller than strain Fujisawa, with the same GC level of 36.36%. It contains 1,845 open reading frames (ORF) predicted by GLIMMER 3.02, of which 1,775 were annotated by PGAAP, 1,757 (~95.23%) were annotated by NCBI nr blast, 1,209 by COG database and 1,076 by KEGG database. 37 potential virulence factors were annotated in strain SY1027 by VFDB, while 19 (~51.35%) of them are common in the 2 strains, 7 of which are potentially related to antibiotic resistance and highly conserved (~98-100% match identity (ID)) amongst the three strains of E. rhusiopathiae and modestly homologous to other gastrointestinal tract-inhabiting Firmicutes (~40% match ID), e.g. Clostridium spp., Enterococcus spp. Genomic island- and pathogenicity island-like regions were also predicted, in which some showed association with tRNA and potential virulence factors.Making use of the complete genome sequencing of Erysipelothrix rhusiopathiae, the causative agent of animal erysipelas, was performed. Molecular identification of various genomic elements pave the way to the better understanding of mechanisms underlying metabolic capabilities, pathogenicity of swine erysipelas and prospective vaccine targets besides the widely used SpaA antigens.Complete genome sequencing of  Erysipelothrix rhusiopathiae  tract by intake of contaminated water or food and causes great economical loss in husbandry worldwide. Given its ubiquitous nature in the environment and reservoir of asymptomatic carriers amongst both domestic and wild animals, the prevention and control of swine erysipelas is often challenging. More than 23 serovars have so far been described , howE. rhusiopathiae strain Fujisawa has just recently been published .A whole genome shotgun library was generated with 500 ng of E. rhusiopathiae strain SY1027 complete genome. All predicted ORF sequences were translated into amino acid sequences by in-house Perl scripts. BLASTp  was downloaded from NCBI Genbank. Orthologous genes were identified by reciprocal BLAT  General function prediction only (~12.08%), [J] Translation, ribosomal structure and biogenesis (~11.41%), [G] Carbohydrate transport and metabolism (~9.02%), [L] Replication, recombination and repair (~8.52%), [S] Function unknown (~7.28%), [K] Transcription (~6.20%), [P] Inorganic ion transport and metabolism (~5.46%), [E] Amino acid transport and metabolism (~5.29%), [V] Defense mechanisms (~4.63%), and [M] Cell wall/membrane/envelope biogenesis (~4.30%). As expected, majority of the genes were involved in basic cellular functions, such as replication, transcription, translation and metabolism, however, up to ~19.36% of them only have predicted or unknown functions on COG database. COG class distribution of strain SY1027 was illustrated  and ATCC 19414  Table  and 6.E. rhusiopathiae remain unresolved. A modestly higher number of virulence genes were identified in strain SY1027 than in strain Fujisawa in VFDB, while the number in strain ATCC19414 might be overestimated due to its draft genome nature  were identified by BLASTp search against VFDB. Categorized by their respective COG classes, their accession numbers and descriptions are listed in Additional file Clostridium spp. and other bacteria [E. rhusiopathiae. In addition to the difference in GC content to the rest of the genome, tRNA were found to flank GI-01, PAI-L03 and PAI-L04, providing further support to these putative genomic islands -like islands were identified in strain SY1027 via PAI finder in PAIDB Figure . In compbacteria ,23. SuggE. rhusiopathiae were identified in the strain SY1027 genome, and they span across various categories, including surface proteins, antioxidant proteins, phospholipases, hemolysins, capsular polysaccharide biosynthesis and extracellular proteins/enzymes , leading to the annotation of 2 RspA-like proteins, each representing the N-terminally and C-terminally truncated proteins in strain SY1027 genome  cycle were found missing in strain SY1027, with the exception of fumarate hydratase, citrate CoA-transferase, dihydrolipoamidesuccinyltransferase and dihydrolipoamide dehydrogenase. Genome reduction was previously hypothesized in E. rhusiopathiae strain Fujisawa, given the partial loss of fatty acid biosynthesis and DNA repair system similar in the Mollicutes [KEGG pathway analysis was performed for Erysipelothrix rhusiopathiae strain SY1027 was sequenced and assembled. It is 1,752,910 base pairs long, just 30 kilobases smaller than strain Fujisawa, with the same GC level of 36.36%. It contains 1845 open reading frames (ORF) predicted by GLIMMER 3.02, of which 1757 (95.23%) are annotated by NCBI nr blast, 1209 by COG database and 1076 by KEGG database. 37 potential virulence factors are annotated in strain SY1027 by VFDB, while 19 (51.35%) of them are common in the 2 strains, 7 of which are potentially related to antibiotic resistance and highly conserved (~98-100% match ID) amongst the three strains of E. rhusiopathiae and only modestly homologous to other gastrointestinal tract-inhabiting Firmicutes (~40% match ID), e.g. Clostridium spp., Enterococcus spp. Molecular identification of these genomic elements and potential virulence factors offer insights into testing prospective vaccine targets besides the widely used SpaA antigens in swine erysipelas and development of more effective treatment or prevention in control of the disease.The complete genome of The authors declare that they have no competing interests.AHYK prepared the genomic DNA for next-generation sequencing (NGS), post-NGS sequence alignment, genome assembly and annotation and comparative genomic analyses, and the drafting of the manuscript. YL and PJ isolated the bacteria from field sample and participated in design of study and coordination. JJ helped in writing in-house perl script and subsequent bioinformatics analyses. FCL participated in the design of study and coordination. All authors read and approved the final manuscript.Local Chinese swine erysipelas outbreak reports. Three articles (titles shaded in yellow) on local swine erysipelas outbreaks published in Chinese scientific journals.Click here for fileNCBI nr database annotation for E. rhusiopathiae strain. The full list of orfs in E. rhusiopathiae strain SY1027 annotated against NCBI non-redundant database.Click here for fileCOG-annotated genes in E. rhusiopathiae strain SY1027 genome. Tabulated summary of orfs in E. rhusiopathiae strain SY1027 annotated against COG database under various COG classes.Click here for fileCOG functional annotation for E. rhusiopathiae strain SY1027 genome. The full list of orfs in E. rhusiopathiae strain SY1027 annotated against COG database.Click here for fileCOG class annotation distribution between E. rhusiopathiae strains SY1027 and Fujisawa genomes. A bar chart comparing the percentage of each single-letter COG class found between the 2 complete E. rhusiopathiae genomes.Click here for fileComparison of COG-annotated genes between E. rhusiopathiae strains SY1027 and Fujisawa. Tabulated comparison between COG-annotated genes under each single-letter COG class from the 2 complete E. rhusiopathiae genomes.Click here for fileVFDB-annotated genes between E. rhusiopathiae strain SY1027 genome. The list of VFDB-annotated genes in strain SY1027 genome, classified by COG classes. Genes shared between strains SY1027, Fujisawa and ATCC19414 were shaded in light gray while those shared between strains SY1027 and Fujisawa were highlighted in dark gray.Click here for fileOther potential virulence factors in E. rhusiopathiae strain SY1027 genome. A panel of virulence factors mentioned in the literature was searched in strain SY1027 genome and listed here. ^orf00231, orf00462 and orf00466 are pseudogenes with frame-shift or point mutation(s).Click here for fileARDB-annotated genes in E. rhusiopathiae strain SY1027 genome. List of ARDB-annotated genes and their putative antibiotic resistance.Click here for fileACLAME database-annotated genes in E. rhusiopathiae strain SY1027 genome. Full list of orf annotated via ACLAME database in strain SY1027 genome.Click here for fileKEGG database-annotated genes in E. rhusiopathiae strain SY1027 genome. Full list of KEGG database-annotated genes in strain SY1027 genome.Click here for file


Clinicians should be aware of these infections, especially in patients from disease-endemic regions. Talaromyces (formerly Penicillium) marneffei are rare in patients who do not have AIDS. We report disseminated T. marneffei infection in 4 hematology patients without AIDS who received targeted therapy with monoclonal antibodies against CD20 or kinase inhibitors during the past 2 years. Clinicians should be aware of this emerging complication, especially in patients from disease-endemic regions.Infections with the fungus  Medscape, LLC is pleased to provide online continuing medical education (CME) for this journal article, allowing clinicians the opportunity to earn CME credit. This activity has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education through the joint providership of Medscape, LLC and Emerging Infectious Diseases. Medscape, LLC is accredited by the ACCME to provide continuing medical education for physicians.TMAMA PRA Category 1 Credit(s). Physicians should claim only the credit commensurate with the extent of their participation in the activity.Medscape, LLC designates this Journal-based CME activity for a maximum of 1.0 http://www.medscape.org/journal/eid; (4) view/print certificate.All other clinicians completing this activity will be issued a certificate of participation. To participate in this journal CME activity: (1) review the learning objectives and author disclosures; (2) study the education content; (3) take the post-test with a 75% minimum passing score and complete the evaluation at Release date: June 15, 2015; Expiration date: June 15, 2016Upon completion of this activity, participants will be able to:T. marneffei infection, based on a case series reportDistinguish the clinical and epidemiologic characteristics of T. marneffei infection in non-AIDS patients with hematologic malignant neoplasms treated with targeted therapiesDiscuss the recent emergence of disseminated T. marneffei infection in non-AIDS patients with hematologic malignant neoplasms treated with targeted therapiesIdentify possible mechanisms of action underlying disseminated Thomas J. Gryczan, MS, Technical Writer/Editor, Emerging Infectious Diseases. Disclosure: Thomas J. Gryczan, MS, has disclosed no relevant financial relationships.Laurie Barclay, MD, freelance writer and reviewer, Medscape, LLC. Disclosure: Laurie Barclay, MD, has disclosed no relevant financial relationships.Disclosures: Harinder Gill, MBBS; Frank Y.F. Lam, MBBS, MRCP(UK), FHKAM; Nigel J. Trendell-Smith, MBBS, FRCPath; Siddharth Sridhar, MBBS, MRCP(UK); Herman Tse, MBBS; Susanna K.P. Lau, MD; and Kwok-Yung Yuen, MD, have disclosed no relevant financial relationships. Jasper F.W. Chan, MBBS, FRCPath, FRCP(Edin), has disclosed the following relevant financial relationships: received travel grants from Pfizer Co. HK Ltd. and Astellas Pharma HK Co. Ltd. Thomas S.Y. Chan, MBBS(Hons), MRCP(UK), FHKAM(Med), has disclosed the following relevant financial relationships: served as a speaker or a member of a speakers bureau for Pfizer. Ivan F.N. Hung, MD, has disclosed the following relevant financial relationships: served as an advisor or consultant for Pfizer , MSD; received conference sponsorships from AstraZeneca, Ferring. Patrick C.Y. Woo, MD, has disclosed the following relevant financial relationships: involved in Tigecycline Evaluation Surveillance Trial with Pfizer. Talaromyces (formerly Penicillium) marneffei is a pathogenic, thermal dimorphic fungus that causes systemic mycosis in Southeast Asia. T. marneffei infection is characterized by fungal invasion of multiple organ systems, especially blood, bone marrow, skin, lungs, and reticuloendothelial tissues, and is highly fatal, especially when diagnosis and treatment are delayed  against CD20 and kinase inhibitors, which are being increasingly used in recent years. We report details for these 4 hematology case-patients. The study was approved by the institutional review board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster in Hong Kong.In the past 2 years, we have been alerted by 4 unprecedented cases of disseminated Patient 1 was a 56-year-old Filipino man with Waldenström macroglobulinemia, idiopathic thrombocytopenic purpura, and primary biliary cirrhosis. He had fever, night sweating, productive cough, and left facial pain for 1 week and bloody diarrhea for 2 days. He had previously received fludarabine, dexamethasone, and rituximab  for treatment of Waldenström macroglobulinemia . The idiT. marneffei. A contrast-enhanced cranial computed tomography (CT) scan showed 2 lesions (3–4-mm) with rim enhancement and perifocal edema at the right occipital and left parieto-occipital lobes. A thoracic CT scan showed 2 cavitary lesions (4–8 mm) in the right upper and lower lobes.A colonoscopy showed multiple shallow ulcers at the terminal ileum . HistoloImmunologic testing showed that the patient was negative for HIV and autoantibodies against IFN-γ. His CD3+ and CD8+ counts were within references ranges, but he had mild CD4+ lymphopenia . His fevT. marneffei. A change in antifungal treatment to intravenous amphotericin B led to defervescence and clearance of fungemia. He was given oral itraconazole as maintenance therapy. He remained well until 2 months later when he was hospitalized for deteriorating CLL complicated by neutropenic fever with multiorgan failure caused by other opportunistic infections  for refractory chronic lymphocytic leukemia (CLL) involving bone marrow . He was fections . He diedT. marneffei. His symptoms and radiologic abnormalities resolved after treatment with intravenous amphotericin B for 2 weeks, followed by oral voriconazole for 6 months.Patient 3 was a 63-year-old Chinese man with myelofibrosis and well-controlled diabetes mellitus. He had intermittent fever, right cervical lymphadenopathy, and productive cough for 4 months. He was given ruxolitinib (kinase inhibitor) 6 months before symptom onset because of transfusion-dependent myelofibrosis despite splenectomy 4 years earlier . A chestT. marneffei. He was given intravenous amphotericin B for 2 weeks, followed by oral voriconazole. He remained well at follow-up 6 months after symptom onset.Patient 4 was a 67-year-old Chinese man with acute myeloid leukemia and hypertension. He had fever and malaise for 2 days without localizing signs. He had been given sorafenib (kinase inhibitor) 8 months earlier for chemotherapy-refractory acute myeloid leukemia . His fevT. marneffei infection is an emerging complication in hematology patients receiving targeted therapies. Historically, T. marneffei infection has rarely been seen in non-AIDS patients, even in disease-endemic regions. During 1994–2014, only 3 other cases were observed in our hematology patients  are mAbs against CD20 that predominantly target B cells. Unlike T cells, the role of B cell–mediated humoral response in T. marneffei infection is poorly defined. Although case-patient 1 had mild CD4+ lymphopenia probably related to concomitant use of prednisolone and mycophenolate sodium, T. marneffei infection is rarely seen in patients with CD4+ counts >300/µL , ruxolitinib is a selective Janus kinase (JAK)-1/2 inhibitor that prevents signal transduction for type I/II cytokines, including IFN-γ, by interfering with the JAK-STAT signaling pathway. Use of ruxolitinib has been associated with opportunistic infections caused by intracellular pathogens, such as T. marneffei infection as an emerging complication in non-AIDS patients treated with targeted therapy has major public health implications. In regions to which T. marneffei infection is endemic, serologic surveillance for patients receiving targeted therapy might be useful in the early diagnosis of T. marneffei infection, as in the case of AIDS patients  credits are accepted in the US as evidence of participation in CME activities. For further information on this award, please refer to http://www.ama-assn.org/ama/pub/about-ama/awards/ama-physicians-recognition-award.page. The AMA has determined that physicians not licensed in the US who participate in this CME activity are eligible for AMA PRA Category 1 Credits™. Through agreements that the AMA has made with agencies in some countries, AMA PRA credit may be acceptable as evidence of participation in CME activities. If you are not licensed in the US, please complete the questions online, print the certificate and present it to your national medical association for review.To obtain credit, you should first read the journal article. After reading the article, you should be able to answer the following, related, multiple-choice questions. To complete the questions (with a minimum 75% passing score) and earn continuing medical education (CME) credit, please go toTalaromyces marneffei infection is correct? 1. According to the case series report by Chan and colleagues, which of the following statements about the clinical and epidemiologic characteristics of A. T. marneffei infection is the most important pathogenic thermal dimorphic fungus causing systemic mycosis in Southeast AsiaB. T. marneffei infection is typically limited to the gastrointestinal and/or urinary tractC. T. marneffei infection is usually self-limited with a good prognosis for recoveryD. T. marneffei infection is common among non-AIDS hematology patientsT. marneffei infection in non-AIDS patients with hematologic malignant neoplasms treated with targeted therapies is correct?2. Your patient is a 57-year-old Chinese man with acute myeloid leukemia and fever. According to the case series report by Chan and colleagues, which of the following statements about the recent emergence of disseminated A. The appearance of 4 cases in the past 2 years is the result of a change in the methodologies of a laboratory diagnosis of T. marneffei infectionB. The appearance of 4 cases in the past 2 years is the result of a dramatic increase in the number of hematology patients at the investigators' hospitalC. Recent emergence of disseminated T. marneffei infection is most likely because of overall immunosuppressionD. Recent emergence of disseminated T. marneffei infection is most likely because of targeted therapies, such as anti-CD20 monoclonal antibodies and kinase inhibitorsT. marneffei infection in non-AIDS patients with hematologic malignant neoplasms treated with targeted therapies would most likely be accurate? 3. According to the case series report by Chan and colleagues, which of the following statements about possible mechanisms of action underlying disseminated A. Rituximab and obinutuzumab used in 2 cases are anti-CD20 monoclonal antibodies that predominantly target T cellsB. T. marneffei infection is usually seen at CD4+ counts of more than 300/µLC. Patients with B-cell dysfunction may have impaired production of neutralizing antibodies against key virulence factors of T. marneffei D. The investigators do not suggest any role of cytokine-producing B-cells


These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements.Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs ( cis-regulatory elements, we used transgenesis to test the functions of enhancers of four genes from several species spanning the phylum Nematoda. While we found a striking degree of functional conservation among the examined cis elements, their DNA sequences lacked apparent conservation with the C. elegans orthologs. In fact, sequence similarity between C. elegans and the distantly related nematodes was no greater than would be expected by chance. Short motifs, similar to known regulatory sequences in C. elegans, can be detected in most of the cis elements. When tested, some of these sites appear to mediate regulatory function. However, they seem to have originated through motif turnover, rather than to have been preserved from a common ancestor. Our results suggest that gene regulatory networks are broadly conserved in the phylum Nematoda, but this conservation persists despite substantial reorganization of regulatory elements and could not be detected using naïve comparisons of sequence similarity.To explore the phylogenetic limits of conservation of  The recis regulatory sequences from four different nematode species in transgenic C. elegans, we sought to determine the extent of cis-regulatory conservation within this phylum.We wanted to assess the conservation of gene regulatory programs among distantly-related members of the phylum Nematoda, a group of morphologically similar worms with mostly small, vermiform bodies. This body plan is largely conserved, with numbers of certain neuronal subtypes nearly identical in even deeply diverged taxa ,15, and C. elegans host. Regulatory regions from orthologous C. elegans genes driving the mCherry reporter were co-expressed as controls with the exogenous cis elements driving expression of the GFP gene. This approach allows us to isolate and compare cis-regulatory functions of the two orthologous regulatory elements in a common trans-regulatory background. Any observed differences can then be attributed to the divergence of the cis-regulatory DNA.The phylum Nematoda is comprised of animals with simple vermiform body plans and diverse life-history strategies. To look for evidence of gene regulatory conservation across this phylum, we carried out a series of enhancer-swap experiments between several distantly-related nematodes and a C. elegans [C. briggsae [Meloidogyne hapla , folade III, ,31). Finspiralis . Divergester, in ), howeveWhile our selection of species gave us unprecedented ability to test the phylogenetic limits of regulatory conservation, it also rendered reciprocal transgenesis infeasible due to the complex modes of reproduction of the parasitic species.Caenorhabditis nematodes, unc-47 [unc-25 [mec-3, is expressed in another neuronal cell type, the touch-receptor neurons, in C. elegans [C. briggsae ortholog of mec-3 has previously been shown to drive gene expression in C. elegans [elt-2, which is expressed in the endoderm [cis-regulatory elements are expressed in different cell types, and drive expression of terminal differentiation genes (unc-47 and unc-25) as well as transcription factors (mec-3 and elt-2). Where possible , as well as the head neuron RIS , and several unidentified head neurons  and two additional, extensively branched, pairs of mechanosensory neurons, FLPL/R in the head, and PVDL/R in the posterior midbody  that are also found in the C. elegans unc-47 cis element. Approximately 40% of the examined cis elements of C. briggsae and the other 3 nematodes are made up of 8-mers that are also found in the C. elegans sequence  that were identical between each reshuffled C. elegans cis element and each of the orthologs. This provided empirically derived distributions of sequence identity that could be expected solely as a result of basic nucleotide composition properties. The results for tests of 10 nucleotide blocks are shown in C. elegans sequences and the other nematode’s upstream unc-47 sequence, the number of identical motifs was plotted  in sequences preserving the single-nucleotide composition of the C. elegans cis element was 0.558, conserving dinucleotides it was 0.378, and trinucleotides it was 0.566. The probabilities of finding CACGC (the AHR-1-like motif) in these same sequences were 0.632, 0.606, 0.674, respectively. Second, these motifs were routinely found in the cis elements of the other genes we examined  to the deepest in the phylum Nematoda . This allowed us to test how regulatory information breaks down over time. Transgenic experiments were conducted in the “common garden” of C. elegans to control for the effects of trans-regulatory divergence and to focus comparisons on the cis elements .C. briggsae have not retained more sequence similarity than would be expected by chance. This finding is consistent with previous reports that suggested that conservation of cis-regulatory function does not, strictly speaking, require extended sequence conservation [cis-regulatory elements might bias discovery to only particular types of elements with highly constrained sequences [a priori clear how distant the species to be compared should be to discover cis elements of different types. Even when some short stretches of identical nucleotides are discovered between distantly-related orthologous cis elements, this should not be taken as evidence of conservation. This is because many short matches will always be found by chance, particularly in regions with biased nucleotide composition. For instance, the co-occurrence of the UNC-30 and AHR-1-like motifs upstream of unc-47 orthologs  in the elt-2 cis elements of C. elegans, C. briggsae, and T. spiralis, none of which drive expression in DVB, RIS, or AVL.Fourth, despite the lack of extended sequence conservation, for all four genes we could readily identify motifs corresponding to transcription factor binding sites previously identified as functionally important for regulation of kernels” ,72 or “ikernels” —are mainkernels” . Of courcis-regulatory sequence; if these sites arise somewhat stochastically, longer wait times increase the likelihood of new sites originating and being optimized. These new sites could diminish the strength of purifying selection acting on ancestral motifs [cis-regulatory conservation remains common among close relatives, but is mostly absent among more distantly-related species.In this study we aimed to understand how the patterns of divergence of gene regulatory mechanisms between closely related species scale up over long evolutionary times. Models have predicted  that regl motifs . On shorl motifs . As the cis element, whether it is flexible [cis-regulatory elements may provide clues to their evolution [Naturally, the rates of divergence and motif turnover are different for different genes. An important factor determining the rate of evolution could be the organization of a flexible ,78 or coflexible , a billbflexible ,81. Modeflexible . Better volution ,82,83.C. elegans as a convenient and reliable experimental system for testing the functions of putative regulatory elements from nematode species, many of them parasites of major economic and medical significance, that are not amenable to transgenic studies [C. elegans has been a genetic model system for decades means that the wealth of information about gene regulation in this species could be leveraged into hypothesis-driven investigation of non-model organisms.Practically, our results advocate the use of  studies . Furthercis elements. By the measures of sequence conservation we applied, including alignment-free methods, M. hapla does not have appreciably greater sequence similarity to C. elegans than does T. spiralis. Nevertheless, M. hapla cis elements of all four tested genes drive more consistent and correct expression in transgenic C. elegans than elements from T. spiralis do. This means that some sequence properties were retained to a greater extent by the more closely related species. Identification of these properties would lead to a better understanding of function and evolution of gene regulatory elements.As discussed above, functionally conserved sequences can retain no more sequence conservation than would be expected by chance. Indeed, motifs that mediate functional conservation, namely transcription factor binding sites, are short enough that they would be likely to be found by chance in sequences of the lengths of these C. briggsae, M. hapla, B. malayi, and T. spiralis were identified as best tblastn/blastx matches with the C. elegans protein sequence. For C. briggsae, B. malayi, and T. spiralis, the genome browser on Wormbase was used. For M. hapla, the genome browser at www.hapla.org was used. Forward primers were designed proximal to the next upstream gene, or failing that the 5’-most part of the contig on which the orthologous coding sequence was found. Reverse primers were selected to make in-frame translational fusions with GFP in the 5’-most part of the gene with protein coding sequence similarity with C. elegans. The only cases in which this was not possible were B. malayi and T. spiralis elt-2, in which protein-coding conservation started deep in the protein-coding sequence, and the fusions were generated in the first exon. A previous study of elt-2 from a parasitic nematode, the less divergent Haemonchus contortus [C. elegans, the H. contortus protein retained function when expressed transgenically in C. elegans by a C. elegans heat shock promoter, so this increases our confidence that these can be elt-2 orthologs despite coding sequence divergence. In all cases, the start codon of the ortholog was included in the fusion. To generate reporter transgenes, upstream non-coding sequences were PCR amplified from genomic DNA and cloned upstream of GFP into the Fire vector pPD95.75, or upstream of mCherry (for C. elegans genes), which was inserted in place of GFP in a modified vector pPD95.75 [elt-2 transgenes carried a nuclear localization signal upstream of GFP or mCherry. Prior to injection, all transgenes were sequenced to ensure accuracy.Orthologous genes from ontortus , found tpPD95.75 . elt-2 tC. briggsae; 10 ng/μL for the other species) promoter::GFP plasmid, 5 or 10 ng/μL promoter::mCherry plasmid, 5 ng/μL pha-1 rescue transgene, 100 ng/μL salmon sperm DNA) into temperature-sensitive C. elegans pha-1(e2123) strain [We injected a mixture (5 ng/μL (for ) strain . Transfo) strain ,37 thoroC. elegans-DNA-driven reporter  positive cells that also express GFP . Of the several UNC-30 motifs in the M. hapla unc-47 element, we selected for mutagenesis the longest extended match to the C. elegans sequence: aTAATCCcc  strand). This motif was mutagenized to aTAGGCGac (changes highlighted). Of the several matches to the AHR-1-like motif in the T. spiralis unc-47 element, the motif selected for mutagenesis was a palindromic sequence (CACGCGTG), which matches two overlapping instances of the AHR-1-like motif (one on each strand). This sequence was mutagenized to CACAAGTG, changing the CACGC sequence on the (+) strand to CACAA and on the (-) strand to CACTT.We tested the functions of motifs corresponding to consensus sequences of binding sites of UNC-30  (TAATCC)DpnI to selectively digest the wild-type plasmid template. A second PCR reaction was performed, amplifying the mutagenized cis element and some flanking vector sequence. This PCR product was purified and digested for directional cloning back into the expression vector. Mutations were verified by sequencing before microinjection.All mutations were introduced by PCR with overlapping, opposite-facing primers carrying the mutant sequence. Primers were used to amplify plasmid DNA carrying the wild-type sequence. Following PCR, the reaction was digested with the methylation sensitive restriction enzyme C. elegans carrying transgenes were grown with abundant food. Worms of appropriate stages were selected. These were immobilized on agar slides with 10 mM sodium azide in M9 buffer. The slides were examined on a Leica DM5000B compound microscope under 400-fold magnification, except in Mixed-stage populations of cis-regulatory elements. Finally, GFP fluorescence in the gut is a known site of off-target expression [GFP transgenes carrying the other nematode’s cis elements alone (without a C. elegans mCherry control), and results were consistent  were mapped back to the orthologous sequence, and the total amount of the sequence covered by blocks of conservation of different sizes is plotted in Motifs matching between C. elegans elements to their orthologs were calculated by generating 1000 reshuffled replicates of the C. elegans sequence. Replicates were generated using single, di-, and tri-nucleotide sampling from the C. elegans sequence. Each replicate was compared to each ortholog and scored for similarity in windows of different sizes. The distributions of these similarity scores were plotted  did not change the pattern, but rather the amount and consistency of expression. If the same principle holds for the genes examined here, the conserved patterns we detected represent the cell types where the foreign cis elements are truly active in C. elegans, but the expression levels could be overestimated. The fact that in most instances only subsets of the overall pattern were conserved suggests that artificially higher expression levels were not solely responsible for the conserved expression patterns we detected.We used multicopy extrachromosomal transgenes, which could have made the detected levels of expression higher and less consistent than what would have been produced by single-copy transgenes. In previous work ,37 we diC. elegans cis element and its orthologs—could be due to cis-regulatory changes (in the function of the donor element), trans-regulatory changes (in the function of transcription factor(s) in C. elegans), or due to the experimental combination of the two. In addition, endogenous expression patterns may have diverged between C. elegans and other species. For technical reasons, it is difficult to determine endogenous patterns of gene expression in divergent parasitic nematodes used in this study. It is even more difficult to generate transgenic animals in these species. These technical limitations make it essentially impossible to assess divergence in endogenous expression patterns or to disentangle their causes . For these reasons, we focused on enumerating similarities, rather than differences in expression. Our tests actually underestimate the extent of regulatory conservation, because a failure of a cis element from a distant nematode when tested in C. elegans may reflect a genuine divergence in cis-regulation in that species that was compensated in trans, therefore maintaining the same overall expression pattern.Any apparent divergence—i.e. incongruence between the pattern driven by the S1 FigC. elegans unc-47 regulatory sequence drives expression of mCherry in all transgenic strains; (A) M. hapla, (B) B. malayi, (C) T. spiralis unc-47 regulatory sequences drive expression of GFP. (D) T. spiralis unc-47 element drives expression of GFP in RIS and DVB. Animals photographed at 400x magnification. Images are mosaics of single animals.(A-C) (PDF)Click here for additional data file.S2 FigC. elegans unc-25 regulatory sequence drives expression of mCherry in all transgenic strains; (A) M. hapla, (B) B. malayi, (C) T. spiralis unc-25 regulatory sequences drive expression of GFP. Animals photographed at 400x magnification. Images are mosaics of single animals.(A-C) (PDF)Click here for additional data file.S3 FigC. elegans mec3 regulatory sequence drives expression of mCherry in all transgenic strains; (A) C. briggsae, (B) M. hapla, (C) B. malayi, (D) T. spiralis mec-3 regulatory sequences drive expression of GFP. Animals photographed at 400x magnification. Images are mosaics of single animals.(A-D) (PDF)Click here for additional data file.S4 FigB. malayi mec-3 regulatory sequence drives expression of GFP. Animal photographed at 400x magnification, ventral cord at bottom. Image is a mosaic of single animals. (B) 1000x magnification of animal with ventral side up. (C) 1000x magnification of animal with ventral side up, vulva at center.</SI_Caption>(A) (PDF)Click here for additional data file.S5 FigC. elegans unc-25 regulatory sequence drives expression of mCherry in all transgenic strains; (A) C. briggsae, (B) M. hapla, (C) B. malayi elt-2 regulatory sequences drive expression of GFP. Stages shown are 8E, comma, and pretzel embryonic stages, with L1 larval stage below. Animals photographed at 400x magnification.</SI_Caption>(A-C) (PDF)Click here for additional data file.S6 FigAll blocks of sequence identity in window sizes shown for each comparison with positions within the upstream non-coding sequence.(DOCX)Click here for additional data file.S7 FigC. elegans and orthologous unc-25 upstream sequences. All blocks of sequence identity in window sizes shown for each comparison with positions within the upstream non-coding sequence.Motifs with identity between (DOCX)Click here for additional data file.S8 FigC. elegans and orthologous mec-3 upstream sequences. All blocks of sequence identity in window sizes shown for each comparison with positions within the upstream non-coding sequence.Motifs with identity between (DOCX)Click here for additional data file.S9 FigC. elegans and orthologous elt-2 upstream sequences. All blocks of sequence identity in window sizes shown for each comparison with positions within the upstream non-coding sequence.Motifs with identity between (DOCX)Click here for additional data file.S10 Figunc-25, mec-3, and elt-2. The number of identical 10 nt blocks between each of the nematode relatives and the C. elegans upstream sequence of the same genes shown in red, on histograms showing the number of predicted 10 nt matches between the relatives’ sequences and 1000 reshuffled C. elegans sequences that preserve tri-nucleotide frequencies.Graphs showing the proportion of sequence similarity over different window sizes for (PDF)Click here for additional data file.S11 Figunc-25 sequences fused to mCherry (C. elegans) or GFP  near the translation start site (bent arrow), or further downstream. Exons are thick black boxes, introns are gray lines. UNC-30 (box arrow) consensus motifs are shown above, in uppercase letters; conserved flanking nucleotides shown in lowercase. Locations of motifs relative to the endogenous translation start site are indicated. Daggers denote binding sites found to be bound by UNC-30. See Cartoons depicting all orthologous upstream (PDF)Click here for additional data file.S12 Figmec-3 sequences fused to GFP near the translation start site (bent arrow) or further downstream. Exons are thick black boxes, introns are gray lines. UNC-86 (triangle) and MEC-3 (fletched arrow) consensus motifs are shown above. Locations of motifs relative to the endogenous translation start site are indicated. Several distal motifs are omitted from C. elegans, C. briggsae, and B. malayi. See Cartoons depicting the all orthologous upstream (PDF)Click here for additional data file.S13 Figelt-2 sequences fused to GFP near the translation start site (bent arrow). Gut-enriched extended GATA motif (sideways heart) and generic GATA motif  are shown above. Locations of motifs relative to the endogenous translation start site are indicated. See SI_Caption>Cartoons depicting the all orthologous upstream (PDF)Click here for additional data file.S14 FigM. hapla unc-47 element drives expression of GFP. C. elegans unc-47 regulatory sequence drives expression of mCherry. Images are mosaics of single animals. Mutant T. spiralis unc-47 regulatory element drives expression of GFP, but not consistently in RIS (B) or DVB (C). μ denotes mutated unc-47 cis elements. C. elegans unc-47 regulatory sequence drives expression of mCherry in these cells. All animals photographed at 400x magnification.(A) Mutant (PDF)Click here for additional data file.S15 FigM. hapla unc-47::GFP is expressed in several D-type neurons of the ventral cord. (B) T. spiralis unc-47::GFP is expressed in RIS and DVB. (C) M. hapla mec-3::GFP is expressed in the head neuron FLP.(A) (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.