Candida albicans and Cryptococcus neoformans have revealed that acquisition of the essential macronutrient, phosphate, is essential for virulence. The phosphate sensing and acquisition pathway in fungi, known as the PHO pathway, has been extensively characterized in the model yeast Saccharomyces cerevisiae. In this review, we highlight recent advances in phosphate sensing and signaling mechanisms, and use the S. cerevisiae PHO pathway as a platform from which to compare the phosphate acquisition and storage strategies employed by several human pathogenic fungi. We also explore the multi-layered roles of phosphate acquisition in promoting fungal stress resistance to pH, cationic, and oxidative stresses, and describe emerging roles for the phosphate storage molecule polyphosphate (polyP). Finally, we summarize the recent studies supporting the necessity of phosphate acquisition in mediating the virulence of human fungal pathogens, highlighting the concept that this requirement is intimately linked to promoting resistance to host-imposed stresses. The ability of pathogenic fungi to acquire essential macro and micronutrients during infection is a well-established virulence trait. Recent studies in the major human fungal pathogens  Cryptococcus, Candida, or Aspergillus species which primarily affect immunocompromised patients. Of these species, the major fungal pathogens include Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. C. neoformans and A. fumigatus are environmental fungi, naturally found in the soil and other ecological niches, and exposure to these pathogens occurs following inhalation of airborne spores. C. neoformans is the major causative agent of cryptococcal meningitis, which has a high incidence in HIV-1/AIDS populations, and leads to approximately 180,000 deaths each year . Th. Thgpa1 C. glabrata is most closely related to the model yeast S. cerevisiae. Consistent with this, studies revealed a high degree of conservation of the PHO pathway in C. glabrata with that in S. cerevisiae [PHO4 or PHO81 in C. glabrata cells prevented the production of secreted acid phosphatases and the induction of PHO84 and GIT1 under phosphate limiting conditions, whereas deletion of PHO80 resulted in the constitutive activation of the pathway [S. cerevisiae, Pho80-Pho85-Pho81 mediated regulation of Pho4 seemingly forms the core of the PHO pathway in C. glabrata. However, in contrast to S. cerevisiae, Pho2 is largely dispensable for Pho4-dependent gene expression in C. glabrata. In addition, ectopic expression of C. glabrata PHO4 in S. cerevisiae bypassed the need for Pho2 in PHO pathway activation [C. glabrata and closely related species [C. glabrata compared to that in S. cerevisiae [C. glabrata are involved in maintaining phosphate homeostasis, including phosphate transporters such as PHO84, several acid phosphatases, the VTC1-4 genes involved in polyP synthesis, and GIT1 and GDE1 involved in obtaining phosphate from phospholipids [C. glabrata reflects an adaptation of this pathogen to particular environmental niches within the human host. Of the human fungal pathogens considered in this review, the commensal pathogen revisiae . Mutatio pathway . Thus, ativation . A recen species . This strevisiae . Remarkaholipids . The remholipids . Thus, iC. albicans, with the analogous Pho4 transcription factor being essential for growth under phosphate limiting conditions [S. cerevisiae Pho4, C. albicans Pho4 accumulates in the nucleus following phosphate starvation and regulates the induction of a number of phosphate responsive-genes [PHO84, several acid phosphatase genes, and the VTC1 and VTC3 genes required for polyP synthesis [pho4Δ cells had lower intracellular phosphate levels, no detectable polyP, and drastically reduced secreted acid phosphatase activity [C. albicans, as Pho4 regulates the induction of the GIT1-3 genes, and the GDE1 gene [S. cerevisiae [PHO84 driving the constitutive upregulation of secreted acid phosphatase activity [S. cerevisiae is also operational in C. albicans. However, as reported in C. glabrata, the homologue of Pho2, Grf10, in C. albicans is dispensable for phosphate homeostasis [C. glabrata, [C. albicans impacts on the transcription of numerous genes not involved in phosphate homeostasis [C. glabrata [C. albicans PHO pathway is also required for resistance to a diverse range of other stresses in addition to phosphate starvation [Recent studies have uncovered a similar PHO pathway in the important fungal pathogen nditions ,35. Simive-genes . These iynthesis . Consistactivity . PhosphoDE1 gene ,37,38. Srevisiae , the Phoactivity . This sueostasis . Moreovelabrata,  loss of eostasis . Althougglabrata . Moreovearvation ,40,41. SC. albicans Pho4 is regulated in response to phosphate limitation has yet to be ascertained. Sequence homology between the S. cerevisiae and C. albicans Pho4 transcription factors is largely restricted to the C-terminal DNA-binding domain [S. cerevisiae largely do not align with the C. albicans Pho4 sequence. However, the PHO81 gene encoding the Pho80-Pho85 CDK inhibitor is induced following phosphate limitation in C. albicans [S. cerevisiae. In addition, C. albicans Pho85 has been shown to complement loss of PHO85 in S. cerevisiae [PHO85 in C. albicans results in increased levels of intracellular phosphate, consistent with this kinase negatively regulating Pho4 [C. albicans Pho4 is also critical for gene expression and survival under alkaline pH conditions which, as previously reported in S. cerevisiae [How g domain  with thealbicans , suggestrevisiae , and deping Pho4 . As discrevisiae , triggerC. albicans. For example, there is emerging evidence from C. albicans and other fungal species that intracellular phosphate levels may play key roles in regulating metal homeostasis [C. albicans uncovered a previously uncharacterized role for phosphate homeostasis in regulating the target of rapamycin (TOR) pathway—the central growth control module of the cell [S. cerevisiae [C. albicans and S. cerevisiae in a mechanism involving the TORC1-stimulating GTPase, Gtr1. Thus, the TOR1 pathway monitors phosphate availability in addition to carbon and nitrogen sources.Recent work also indicates possible transcription-independent roles of the PHO pathway in eostasis , and thithe cell . This corevisiae . SpecifiC. neoformans, the study of the PHO pathway was hampered due to the difficulty in identifying candidate orthologs of Pho4 [C. neoformans Pho4 displays a higher degree of sequence homology to C. albicans Pho4, compared to the analogous transcription factor in S. cerevisiae [C. neoformans Pho4 accumulates in the nucleus following phosphate starvation and is essential for growth, and the production of secreted acid phosphatases, under phosphate limiting conditions [C. neoformans to that seen in S. cerevisiae and C. albicans. These include the high affinity phosphate transporters PHO84, PHO840, and PHO89, the VTC1 and VTC4 genes involved in polyP synthesis, the secreted acid phosphatase AHP1 (PHO2), and the GIT1 and GDE1 genes necessary for phospholipid catabolism [C. glabrata and C. albicans, C. neoformans Pho4 likely regulates additional gene targets in addition to the core PHO gene set. Pho4 was found to bind to over 110 genes in C. neoformans and loss of Pho4 impacted on the transcription of genes not directly implicated in phosphate homeostasis [In the human pathogen  of Pho4 . It was  of Pho4 , for str of Pho4 ,47. Indetabolism ,46,47. CC. neoformans, homologues of the key regulators, Pho80, Pho85, and Pho81 were identified by BLAST searches [PHO80 or harboring mutations in PHO85 were found to result in constitutive activation of the PHO pathway, as measured by secreted phosphatase activity, and C. neoformans PHO85 could complement loss of S. cerevisiae PHO85 [S. cerevisiae, Pho85-Pho80 negatively regulate Pho4 in C. neoformans, and that Pho81 counteracts this repression in phosphate limiting environments. Notably, however, differences in phosphate homeostasis mechanisms were also uncovered in this fungal pathogen. For example, in addition to PHO81, a number of other regulatory proteins were induced in C. neoformans following phosphate starvation including the CDK and cyclin genes, PHO85 and PHO80 [C. neoformans that has not been described previously. Deletion of all three phosphate transporters  is needed to impair C. neoformans growth under phosphate starvation conditions [S. cerevisiae and C. albicans upon phosphate starvation. In addition, whilst deletion of the three high affinity phosphate transporters in C. neoformans drastically impairs polyP levels [pho4Δ cells display wild-type levels of polyP in this fungal pathogen [pho4Δ mutants [C. neoformans Pho4 is also vital for survival under alkaline conditions and regulates the induction of phosphate acquisition genes in response to this stress [C. neoformans [With regard to Pho4 regulation in searches . Subsequae PHO85 . Moreoveae PHO85 . In contae PHO85 . Thus, tnd PHO80 . Moreovend PHO80 . In addinditions , whereasP levels , pho4Δ cpathogen . This co mutants ,36. Howes stress . This isoformans ,47.A. fumigatus than in the fungal pathogens described above. However, deletion of the A. fumigatus PHO80 orthologue phoBPHO80 resulted in cells that displayed constitutive secreted acid phosphatase activity, increased phosphate uptake, and increased levels of vacuolar polyP [PHO80phoB cells, including the high affinity phosphate transporters phoDPHO84 and phoEPHO89, a number of acid and alkaline phosphatases, the CDK inhibitor phoCPHO81, and the orthologue of Vtc4, vtcDVTC4 [phoBPHO80 having a similar negative role in regulating activation of PHO pathway in A. fumigatus as established in S. cerevisiae. Interestingly however, A. fumigatus cells lacking phoDPHO84 did not display the phenotypes associated with PHO84 loss in S. cerevisiae or C. albicans [C. neoformans [There is considerably less information on the PHO pathway in ar polyP . ConsistvtcDVTC4 . Collectalbicans . This isoformans , in whicS. cerevisiae is presented in A summary of the key differences in PHO pathway regulation in human pathogenic fungi compared to A number of studies have revealed the importance of intracellular phosphate levels in mediating resistance to seemingly divergent stress conditions. These are described below and summarized in S. cerevisiae illustrated that in response to alkaline pH, the PHO regulon was rapidly induced [S. cerevisiae, C. albicans, and C. neoformans, the Pho4 transcription factor accumulates in the nucleus upon the switch to alkaline growth and there is also a rapid mobilization of phosphate from vacuolar polyP phosphate reserves [+/Pi symport, the phosphate starvation state triggered by alkaline pH is most likely due to disruption of the proton gradient across the plasma membrane under pH stress. Consistent with this, C. neoformans cells grown under alkaline pH conditions in the presence of radioactive-labelled orthophosphate displayed significantly impaired uptake of phosphate compared to cells grown under acidic conditions [pho4Δ cells [+/Pi Pho89 symporter, possibly in conjunction with the Git1-Gde1 phosphate acquisition system, promotes survival. Significantly, as discussed later, many of the virulence defects associated with loss of Pho4 in C. neoformans were found to be related to impaired growth in alkaline host environments [C. albicans, the impaired serum-induced filamentation exhibited by pho4Δ cells was found to be due to the alkaline pH of serum [Transcript profiling studies in  induced . Subsequreserves ,47,49. Sreserves ,47,50. Areserves , and higreserves , relies of serum .S. cerevisiae, artificially increasing intracellular levels of phosphate (by inactivating the Pho80 negative regulator of Pho4), has profound effects on metal cation accumulation and toxicity [pho80Δ cells, together with an increased toxicity of these and many other metal cations including manganese, cobalt, zinc, and copper [A. fumigatus cells lacking the orthologous PhoDPHO80 gene also display increased sensitivity to calcium [S. cerevisiae pho80Δ cells could potentially counter the negative charge imposed by increased phosphate. Increased intracellular phosphate levels also trigger a characteristic iron starvation response in S. cerevisiae [C. neoformans, depletion of intracellular phosphate levels  also impacted on metal homeostasis, resulting in a significant accumulation of sodium cations as well as increases in zinc and iron levels [S. cerevisiae and C. neoformans illustrate that both increases and decreases in intracellular phosphate levels can stimulate increases in metal cation levels and impair the cellular resistance to such metals. In C. albicans, deletion of Pho4 significantly reduced intracellular phosphate levels and resulted in impaired resistance to a multitude of both metal and non-metal cations [pho4Δ cells were also sensitive to non-metal cations such as spermidine, indicates that the protective role of intracellular phosphate is not restricted to metals, and that a key function is in maintaining a charge balance in the cell. This is further supported by the observation that although sensitive to many metal cations, the cellular levels of such metals in pho4Δ cells are similar to that seen in wild-type cells [pho4Δ cells [C. neoformans cells lacking Pho4 were also notably sensitive to calcium cations indicating possible conservation of phosphate acquisition in promoting cation resistance [C. neoformans to CaCl2 could be rescued by supplementation of the media with phosphate [Evidence is also emerging that alterations in phosphate levels, which is the most abundant cellular anion, have wide-ranging effects on the homeostasis of biologically important metal cations . In S. ctoxicity . Dramatid copper . In this calcium . The hugrevisiae , indicatn levels . Such cen levels . Thus, s cations ,41. The pe cells . The exc4Δ cells . C. neofsistance . In supphosphate . C. albicans [C. neoformans [C. albicans pho4Δ cells, the most striking is that towards the superoxide stress-generating compound menadione [pho4Δ cells to superoxide stress as this results in a significant reduction of the activity of the copper-dependent superoxide dismutase (Sod1) enzyme [pho4Δ cells are completely rescued by supplementation of the growth medium with copper [C. albicans cells lacking Pho4, despite containing wild-type levels of intracellular copper, display significant resistance to this potentially toxic metal [CRD2 [pho4Δ cells. Interesting, such roles of phosphate homeostasis regulating copper availability do not appear to be conserved in C. neoformans [In addition to promoting resistance to alkaline pH and cationic stress, Pho4 has recently been implicated in oxidative and nitrosative stress resistance in albicans ,41, and oformans . Of the enadione ,41. Howeenadione . Instead) enzyme . Evidench copper . Moreoveic metal ,36, whicoformans , as Pho4oformans , howeverS. cerevisiae [To summarize, in addition to regulating phosphate acquisition in phosphate limiting environments, the Pho4 transcription factor mounts a similar transcriptional response to alkaline pH stress and is essential for survival under such conditions. It is also worth highlighting that the gene-expansion of Pho4 target genes in pathogenic fungi compared to that in the model yeast revisiae , may alsrevisiae , the impS. cerevisiae, C. albicans, and C. neoformans, cells lacking Vtc4 have no detectable polyP [C. neoformans cells lacking orthologues of Ppn1 and Ppx1  contained increased levels of polyP [Mobilization of phosphate from the phosphate storage molecule polyP is one of the first responses evoked in fungi in response to phosphate limiting conditions. In addition, a conserved aspect of the PHO regulon between fungal species is the upregulation of genes involved in the synthesis of polyP. As described above in le polyP ,36,44. Ile polyP ). ConsisTrypanosoma cruzi to hypo-osmotic stress results in the rapid mobilization of polyP, whereas hyper-osmotic stress triggers an increase in poly-P levels [S. cerevisiae and human pathogenic fungi which is summarized in In addition to phosphate storage, polyP has been implicated in an array of processes in prokaryotic cells , whiP levels . Here, wC. albicans, it was revealed that in addition to alkaline pH conditions, phosphate is mobilized from polyP following both hypo-osmotic stress and hyper-osmotic stress. However, the physiological significance of this is not clear, as polyP is seemingly dispensable in mediating resistance to such stresses in C. albicans [C. albicans is manganese resistance, with polyP playing an important role in the sequestration of this particular metal [C. neoformans cells either lacking polyP, or in which polyP mobilization was impaired, both displayed impaired tolerance to zinc, suggesting a role for polyP in zinc homeostasis in this fungal pathogen [C. neoformans cells [vtc4Δ C. neoformans cells could increase the rate of blood clotting [In albicans . In factar metal . Interespathogen . Such ropathogen ). Intrigns cells . In thisns cells , this maclotting .S. cerevisiae, that polyP plays a role in cell cycle progression in fungi. PolyP levels were found to drop when S. cerevisiae cells go through S-phase, and this is possibly mediated by Ppn1, as the expression levels of this polyphosphatase coincided with the observed decreases in polyP [vtc4Δ cells displayed a delay in cell cycle progression that was more pronounced in Pi limiting media, and this was due to such cells taking significantly longer to replicate their DNA [S. cerevisiae, and conceivably in other fungal cells, is the provision of enough phosphate for the upsurge of dNTP synthesis at the end of G1 phase to allow for efficient DNA replication. In an in vitro competition experiment between wild-type and vtc4Δ cells, cells lacking polyP were consistently underrepresented in the mixed population, reflecting the reduced fitness of the S. cerevisiae vtc4Δ mutant [C. albicans pho4Δ cells, which were replaced by wild-type cells when grown in mixed cultures in vitro [pho4Δ cells were unable to colonize the murine gut when inoculated simultaneously with wild-type cells [S. cerevisiae [C. albicans pho4Δ cells may be due to a lack of polyP, resulting in delays in DNA replication. This is further supported by the observation that genes upregulated in C. albicans pho4Δ cells, even under phosphate replete conditions, cluster to the functional categories of “DNA Metabolism”, “DNA Repair and Response to DNA Damage”, and “Cell Cycle” [C. neoformans triple high affinity phosphate transporter mutant, which lacked polyP, displayed an enlarged cell type indicative of possible problems with cell cycle control and delayed cell division [Evidence is emerging from studies in the model yeast in polyP . Moreoveheir DNA . Consistheir DNA . Thus, tΔ mutant . Interesin vitro . Moreovepe cells . Based orevisiae , it is il Cycle” . Furtherdivision . CollectS. cerevisiae, is the covalent attachment of polyP to target proteins as a novel post-translational modification [S. cerevisiae and explore this newfound signaling role of polyP in protein polyphosphorylation in pathogenic fungi. A further exciting role of polyP, recently identified in fication . This mofication . Such pofication . Thus, iC. albicans and C. neoformans impacts on virulence in a range of infection models. The first clue that phosphate acquisition could be important for C. albicans virulence came from the observation that the Pho4-target gene, PHO84, which encodes a high affinity phosphate transporter, is upregulated in a number of infection models [C. albicans cells lacking the major regulator of the PHO regulon, Pho4, have been shown to display attenuated virulence in both systemic and commensal animal models of infection [PHO4 were acutely sensitive to macrophage-mediated killing in one study [C. albicans survival following phagocytosis [C. albicans cationic stress resistance [A number of recent studies have shown that perturbation of phosphate acquisition in both n models ,68,69. Tn models ,70. Morenfection ,41. Morene study , but notne study  is due tocytosis , as are sistance , thus isC. albicans isolates that displayed filamentous growth on media lacking phosphate which was not recapitulated in standard clinical reference or laboratory strains [C. albicans in the Caenorhabditis elegans nematode infection model [C. elegans [pho4Δ cells [C. albicans virulence in the C. elegans infection model under phosphate replete conditions [pho4Δ cells in phosphate replete media [Interestingly, a recent study described two clinical  strains . Moreove strains . This inon model . As fila elegans , this su4Δ cells . However4Δ cells . It is anditions , thus inte media , or otheC. albicans, the indication that phosphate acquisition may be important for virulence in C. neoformans came from in vivo transcript profiling studies following macrophage uptake, which revealed that the high affinity phosphate transporter PHO84 showed the greatest level of induction (sixteen-fold) of all induced transporters [C. neoformans triple mutant lacking all three high affinity high phosphate transporters , in addition to impaired growth on phosphate lacking media also resulted in reduced formation of capsule and melanin, both of which are virulence traits in this fungal pathogen [C. neoformans virulence has been published [pho4Δ mutant displayed significantly impaired virulence, showing massively reduced fungal burdens in the brain, whereas much more minimal effects were seen with lung fungal burdens. Intriguingly, the virulence defect of pho4Δ cells was even more pronounced in a mouse dissemination model of infection. The growth rate of pho4Δ cells in lung and brain tissues, and in the blood, was compared to wild-type and reconstituted strains during infection. Remarkably, this revealed that the pho4Δ mutant was never cultured from the blood [pho4Δ cells to the brain is due to the vital role of Pho4 in promoting growth in the alkaline pH environment of the blood [pho4Δ strain. As discussed above, alkaline pH mimics a phosphate limiting environment, and thus pho4Δ-deficicient cells cannot thrive in such environments [C. neoformans triple phosphate transporter mutant could disseminate to the brain [C. neoformans virulence in both Galleria mellonella and mouse infections models, further supporting the role of phosphate acquisition in the virulence of this fungal pathogen [C. neoformans pathogenicity [C. neoformans is due to defects in phosphate homeostasis remains to be addressed.As in sporters . Followipathogen . Consistpathogen . Moreovepathogen . Most reublished . In a mohe blood . Subsequhe blood . This exronments . In thishe brain . These che brain , which mpathogen . It is agenicity ,77. HoweC. albicans and C. neoformans virulence, evidence is lacking that this is the case in A. fumigatus. Cells lacking either phoBPHO80 or phoDPHO84 did not display attenuated virulence in a murine low dose model of invasive aspergillosis [phoBPHO80 hyper-activates the PHO pathway, and evidence was presented suggesting that phoDPHO84 functions redundantly with other phosphate transporters in A. fumigatus [A. fumigatus virulence can be concluded. Whilst there is strong evidence that the PHO pathway is essential for both gillosis . Howeverumigatus . TherefoThis is an exciting time in the phosphate homeostasis field. Recent notable findings include new insight into how cytosolic phosphate levels are sensed by the binding of inositol polyphosphate signaling molecules to SPX domains, located in a multitude of phosphate homeostasis regulators. Furthermore, new roles for the phosphate storage molecule, polyP, are emerging, in addition to the newly identified post-translation modification of protein ‘polyphosphorylation’. Studies in fungi are also highlighting the importance of intracellular phosphate homeostasis in TOR signaling and mediating resistance to a range of diverse stress conditions. It is also now clear that phosphate homeostasis is an important virulence trait in human pathogenic fungi. Significantly, a recent study demonstrated the potential benefit of targeting phosphate acquisition strategies in the treatment of systemic mycoses, in that a small molecule inhibitor of Pho84 was found to potentiate the activity of the antifungals amphotericin B and micafungin . An impo


Nomascus groups consist of more than one adult female. However, these preliminary results are not well understood or incorporated into current socio-ecological theories regarding gibbon species. To better understand northern gibbons, our team has systematically studied three habituated groups of Nomascus concolor, three groups of N. nasutus, and two habituated groups of Hoolock tianxing since 2002. In this paper, we stress the challenges facing gibbons living in northern habitats and summarize their behavioral adaptations to their harsh environments. We also describe the northern gibbon social system and discuss the potential relationships between their ecology and sociality. Finally, we highlight future research questions related to northern gibbons in China.Gibbons in China represent the northernmost margin of present day gibbon species distribution (around N25 Symphalangus syndactylus) is more folivorous  living in lowland tropical forests (Gibbons (Hylobatidae) are small apes that live in tropical and subtropical forests in southeastern Asia . These athe area  through the area  and reguthe area . Both sethe area . This, ithe area . Bartletthe area  first in forests , 1999.N. concolor in Wuliang Mountain: H. tianxing in Gaoligongshan: Hoolock gibbons in India and Myanmar  . Althoug∘) , these s Myanmar . In this gibbons . It doesN. leucogenys: N. concolor: N. hainanus: N. leucogenys: N. concolor: N. concolor: N. hainanus: N. concolor and H. tianxing groups became habituated to observers in Mt. Wuliang and Mt. Gaoligong . The resulting increase in demand for cropland, together with the steel-making campaign of 1958, has led to the clearance of vast tracts of forest (Macaca mulatta and M. arctoides), gibbons were also hunted during this period  at 5.2 Gibbon habitats in these northern mountain areas are much colder than tropical forests . For exa in snow . Thus, t in snow  reportedN. concolor in China  are comparable to the home ranges of tropical gibbons , they occupy a larger total home range to satisfy their annual food requirements (Hylobates gibbons: avg. 1 300 m), except for some siamang species  and 1 160 m , they traveled short distance when dominantly feeding on leaves (N. concolor in Mt. Wuliang varies monthly from 629 m to 2 356 m (n=14 months) and daily from 300 m to 3 144 m  and daily from 345 m to 2 606 m (n=63 days) (H. tianxing group in Mt. Gaoligong has been observed to travel (on average) 3.40 h in June but 1.27 h in February  . FurtherFebruary . SeasonaH. tianxing group spent 20.12 h inactive in February, whereas the two groups of N. nasutus in Bangliang spent 20.23 h (December) and 19.69 h (January) inactive. Gibbons maintain an energy-conserving posture while sleeping or resting  during the 24 h cycle in winter , 2013. IN. hainanus: N. concolor: N. nasutus: H. pileatus: Hoolock hoolock: H. lar: Gibbons usually live in small social groups with one adult pair and 2–3 offspring . HoweverN. hainanus in Delacour’s book On the Indochinese gibbon  is required , females in the polygynous groups do not disperse and form new groups with these floating males. Two newly formed N. nasutus groups at Bangliang consisted of two adult females , but no stable group of more than one adult breeding female has been reported. A possible reason is that food patch size in the habitat of H. tianxing is smaller than that of Nomascus species  or crown size may not be accurate indicators of food availability. We highly recommend using feeding party size, feeding bout length, and number of feeding patches visited as indices to represent food availability, especially as gibbons often deplete their food patches. However, this prerequisite needs careful investigation. If these indices are validated, then detailed comparison among the foraging behaviors of gibbons living in different social systems can be made.Gibbons born in polygynous groups may develop better social skills for maintaining relationships due to their interactions with more social partners while growing up. Therefore, ontogeny may also play a role in the maintenance of polygyny. Female gorillas familiar from immaturity usually exhibit friendly relationships in adulthood . Studyin


DNA methylation plays an important role in gene expression, chromatin stability, and genetic imprinting. In mammals, DNA methylation patterns are written and regulated by DNA methyltransferases (DNMTs), including DNMT1, DNMT3A and DNMT3B. Recent emerging evidence shows that defects in DNMTs are involved in tumor transformation and progression, thus indicating that epigenetic disruptions caused by DNMT abnormalities are associated with tumorigenesis. Herein, we review the latest findings related to DNMT alterations in cancer cells and discuss the contributions of these effects to oncogenic phenotypes. DNMT3C exhibits high identity with DNMT3B, and is specialized at methylating the young retrotransposons [DNA methylation is one of the most important epigenetic modifications , playingelopment . Althougelopment , 6. Verynsposons . Beside nsposons . DNMT3L,nsposons , 5, 6. Insposons . Mice densposons , 10. Malnsposons . Thus, tDNMT3A, TET2, and IDH1, are involved in epigenetic processes [In recent years, interest in the relationship between DNA methylation and human diseases has increased. Alterations in DNA methylation patterns have been implicated in tumorigenesis in several studies –13. Owinrocesses –18 and aTumor cells typically exhibit aberrant DNA methylation patterns during malignant transformation , 19. AltBRCA1 in sporadic breast tumors [Overexpression of DNMTs  in a variety of tumors results in hypermethylation and oncogenic activation . DNMT1 ot tumors . Additiot tumors , 28. Sevt tumors . Two grot tumors , 30. Addt tumors .HOX family genes [Somatic mutations in DNMTs are the prominent features of many tumors and substantially contribute to malignant transformation . As showly genes , 37. Togde novo methyltransferases leads to lethal phenotypes [de novo methyltransferase on hematopoiesis have been evaluated through conditional knockout technology. The deletion of Dnmt3a in adult mice induces the proliferation of hematopoietic progenitors [DNMT3A may act as a tumor-suppressor gene [de novo and maintenance methylation [An in vivo mouse model with embryonically inactive DNMT3A and DNMT3B has shown that the deletion of enotypes . Recentlgenitors . On the genitors –42. Furtgenitors . Moreovesor gene , 45. Stusor gene . A lack hylation . TherefoEpigenetic disorders, which are commonly found in cancer, are attributed in part to DNMT dysfunction , 4. BecaHOXB cluster have been found in AML samples with DNMT3A mutations [DNA hypomethylation of tumor cells is the first process characterized as an epigenetic abnormality . The genutations . Althougutations .In normal somatic cells, DNA methylation occurs primarily in dinucleotides containing less CpG, whereas the CpG-enriched region is unmethylated , 50. ThrBRCA1 promoter is partially due to DNMT3B overexpression [HOX genes, are markedly enriched in DNMT3A mutant AMLs [A great deal of research has been performed to explore the mechanism of aberrant TSG methylation in tumor tissue. As expected, DNMTs have been included in the aforementioned studies. In leukemia, deletions or mutations in DNMTs often disrupt the distribution of 5-mC in the genome . Butcherpression . Using apression . Additioant AMLs .DNMT3A, as determined with Human Methylation 450 Bead Chip arrays [Notch1 and Gata3, are hypermethylated in the gene body regions [Tumor-associated DNA methylation generally occurs in the promoter regions of TSGs . Howeverp arrays . This co regions . Further regions .de novo methylation of transcribed genes [The entire epigenetic profile of the genome shows that active chromatin regions are generally characterized by acetylated histones and unmethylated DNA, whereas methylated histones associated with repressed chromatin and methylated DNA are enriched in suppressed regions . Thus, ted genes . Likewised genes . In undied genes . Two groed genes , 62. ThuDNMT3A gene have been identified in AML at a mutation rate of ~20%, and the prognosis for mutant patients is relatively poor [In recent years, mutated genes encoding a group of epigenetic modification regulators have attracted attention because of their high frequency of variation in hematological diseases . Epigeneely poor . Currentde novo methyltransferase said in coordination during methylation maintenance [As a member of the DNMT family, DNMT3A possesses the characteristic peptide structure: its catalytic domain directly binds to S-adenosyl-L-methionine (SAM) and DNA strands, and the N-terminus regulation domain is primarily involved in nuclear localization and protein interactions, in which the PWWP domain interacts with methyl lysine histones, and the PHD domain recognizes unmethylated histones. These functions serve as a signal of the histone transfer effect, thus ensuring diverse epigenetic modification . Specifintenance . Studiesntenance .DNMT3A mutations, the number of leukocytes present at diagnosis is relatively higher, and survival is comparatively shorter [DNMT3A mutation on leukemia pathogenesis  to pre-leukemic stem cells (Pre-LSCs) [RAS mutation, c-Kit variation, or FLT3-ITD abnormalities [DNMT3A mutation exerts a great influence in hematological malignancy. A variety of small molecule compounds targeting relevant epigenetic disruptions have been developed and applied in the treatment of leukemia, which also provide a comprehensive innovation for the study of pathogenesis and targeted therapy of solid tumors.Clinically, in patients with  shorter , 16. To sis Fig. . For insre-LSCs) . In addire-LSCs) , 70. DNMre-LSCs) . Additiore-LSCs) . In Dnmtmalities –43, 72. malities . In summOwing to advances in sequencing technologies, numerous gene alterations associated with epigenetics have been identified in cancer genomes. Furthermore, whole-epigenome approaches, including array-based methylation profiling and bisulfite sequencing, afford a comprehensive view of the tumor methylome, and potential mechanisms of epigenetic disruption caused by DNMT changes have been explored. However, the effects of DNMT aberrations in the promotion of tumorigenesis are not entirely clear, and novel strategies for relevant targeted therapies must be developed. Future work should focus on the elucidation of tumorigenic mechanisms induced by defective DNMTs and the production of effective therapeutic approaches.


Candida parapsilosis is a predominant species found in nosocomial infection, particularly in hospitalized patients. The molecular epidemiology of the clinical strains of this species has not been well studied. The present study was performed with the aim of investigating the microsatellite genotyping of Candida parapsilosis among the Iranian clinical isolates.C. parapsilosis isolates that were genotyped by using a panel of six microsatellite markers.This study was conducted on 81 independent clinical C. parapsilosis isolates demonstrated 68 separate genotypes, among which 57 genotypes were observed once and the remaining 11 cases were identified for multiple times. The Simpson’s diversity index for the panel of combined six markers yielded a diversity index of 0.9951. The heterogeneity was observed among the Iranian and the Netherlands clinical C. parapsilosis isolates.The short tandem repeat (STR) typing of clinical C. parapsilosis isolates from Iran showed a high genetic diversity. It can be concluded that molecular epidemiology could be useful for screening during outbreak investigation where C. parapsilosis is involved.As the findings indicated, the clinical  Candida infection associated with non-albicans Candida (NAC) species has been increasing throughout the world [Candida parapsilosis is one of the most frequent NAC species, which causes a broad spectrum of infections from superficial to invasive candidiasis [C. parapsilosis strains is crucially important for the adoption of the appropriate treatment regarding the different antifungal susceptibility patterns of C. parapsilosis complex [C. parapsilosis has been reported to account for several cases of fungemia outbreak in hospitalized patients. Based on genotypic analysis, these outbreaks were probably due to cross-infections by health care workers [C. parapsilosis complex are indistinguishable based on their phenotypic features, molecular assays have been developed for distinguishing and genotyping these species [C. parapsilosis and other fungi [C. parapsilosis isolates from Iran. The incidence of he world , 2. Canddidiasis , 4. Thesdidiasis -8. The a complex , 10. C.  workers . Since t species , 13. Amoer fungi , 15. TheIsolate identiﬁcationC. parapsilosis clinical isolates were investigated using molecular typing method. All clinical isolates were grown for 48 h on Sabouraud glucose agar  at 30°C. Initial species identification was performed based on conventional tools using CHROMagar Candida medium  at 30°C [C. parapsilosis isolates were confirmed by the partial sequencing of the internal transcribed spacer DNA region as previously described [A total of 81  at 30°C . DNA wasescribed .Microsatellite typing C. parapsilosis. Generally, three trinucleotide repeat markers and three hexanucleotide markers were amplified in a multiplex polymerase chain reaction (PCR). In addition, each amplification primers were labelled at the 5 side with 6-carboxy-fluorescein (6-FAM), either 6-carboxy-tetramethylrhodamine (TAMRA) or hexachloro-fluorescein (HEX). The primer sequences . In the next step, the samples were boiled at 95°C for 1 min, subjected to rapid cooling at 4°C, and then injected and run onto ABI3500xL Genetic Analyzer platform  as recommended by the manufacturer. The number of repeats in each marker was achieved by comparing the relative size of each allele with those obtained by means of the reference C. parapsilosis strain CDC317. The similarities between the genotypes were visualized by constructing a minimum spanning tree (MST) using BioNumerics, version 6.0 . The discriminatory power was calculated by Simpson’s index of diversity as described previously [A panel of six short tandem repeat (STR) markers was used for typing all clinical isolates ofequences  and PCR C. parapsilosis isolates were identified from a variety of clinical specimens, including nail scraping (n=59), interdigital skin (n=8), groin (n=6), vaginal (n=2), ear discharge (n=2), skin (n=2), hand (n=1), and sputum (n=1). The STR typing of 81 C. parapsilosis isolates demonstrated 68 separate genotypes. The genetic relatedness of clinical C. parapsilosis isolates is depicted in C. parapsilosis isolates, and also two genotypes were shared among three C. parapsilosis isolates. One cluster, including three isolates , obtained from different patients of the nail samples, showed the same allelic profile at six loci in STR typing. In addition, one similar genotype was found to be related to three C. parapsilosis isolates , which were obtained from various specimens of different patients. Furthermore, the same genotype was detected to be related to two C. parapsilosis isolates  obtained from different anatomic sites. Moreover, three patients with candidiasis were sampled twice on two different days, and the same genotype was observed over the time. In C. parapsilosis isolates based on sample type analysis. The STR typing revealed a high genetic diversity in the Iranian C. parapsilosis isolates, compared to other clinical isolates from the Netherlands. Based on the MST  for the differentiation of C. parapsilosis isolates and reported this approach as a reliable method for molecular epidemiological studies [C. parapsilosis isolates. Therefore, they marked that this method has incomparable advantage over other typing methods, such as RAPD, PCR-RFLP, and internal transcribed sequence grouping [C. parapsilosis isolates by using six-marker microsatellite panel assay. As mentioned above, the majority of the clinical C. parapsilosis isolates had unique genotypes. In this regard, within the collection of 81 C. parapsilosis isolates obtained from clinical sources, 68 unique genotypes were observed. The pattern results of the STR analysis of two hexanucleotide markers revealed no variation between all C. parapsilosis isolates, except one isolate (SKCP313) that had diversity at these two loci, whereas high allelic variation was observed at other locus. In the present study, the isolation of the related genotypes of C. parapsilosis from multiple anatomical sites over time supported the evidence of an endogenous colonization. In line with our results, Diab-Elschahawi et al. reported 24% clonally related genotypes from multiple anatomical sites in patients [C. parapsilosis isolates inside and outside Iran. Recently, NAC species has became an increasing prevalence among the hospitalized patients. Some of these species exhibit decreased susceptibility to the commonly used antifungal agents , 18, 19.ironment , 21, 22.ironment -25. The  methods -28. Vari studies -31. Nota studies -34. Lasks regard . In agre studies . They algrouping . This papatients . In addiC. parapsilosis isolates were found to have a high genetic diversity. Microsatellite genotyping method could be useful for screening during outbreak investigation, especially where C. parapsilosis complex is involved.In conclusion, the Iranian


Functional classification of unigenes in each amphibian showed high accordance, with the most represented GO terms and KEGG pathways related to basic biological processes, such as binding and metabolism and immune system. As for the unigenes, GO and KEGG distributions of conserved orthologs in each species were similar, with the predominantly enriched pathways including RNA polymerase, nucleotide metabolism, and defense. The positively selected orthologs in each amphibian were also similar, which were primarily involved in stimulus response, cell metabolic, membrane, and catalytic activity. Furthermore, a total of 50 antimicrobial peptides from 26 different categories were identified in the three amphibians, and one of these showed high efficiency in inhibiting the growth of different bacteria. Our understanding of innate immune function of amphibian skin has increased basis on the immune-related unigenes, pathways, and antimicrobial peptides in amphibians.Skin as the first barrier against external invasions plays an essential role for the survival of amphibians on land. Understanding the genetic basis of skin function is significant in revealing the mechanisms underlying immunity of amphibians. In this study, we  Amphibians represent the transitional vertebrate taxon from aquatic to terrestrial life  and therDifferent from aquatic and terrestrial species, amphibian skin evolved versatile functions and complex structures to adapt to life on land . First, de novo transcriptome sequencing technology is a powerful methodology for gene function identification, comparative genomics analyses, and evolutionary biology studies, particularly for non-model species and species with large genomes [de novo transcriptome sequencing was performed for the skin from three different amphibian species, the Chinese giant salamander (Andrias davidianus), Asiatic toad (Bufo gargarizans), and Heiban frog , which inhabit different environments and vary in skin phenotypes. With functional annotation and comparative analyses of these transcriptomes, we explored the genetic basis of immune functions of the skin of amphibians.Because of serious infectious diseases and environmental degradation, amphibian species are now globally imperiled and populations are decreasing , 11. To  genomes . In thisA. davidianus, B. gargarizans, and R. Hallowell were 119,269,618, 117,350,510, and 111,152,846, respectively, which corresponded to clean bases of 17.82 G, 17.46 G, and 16.61 G, respectively . To analyze the phylogenetic relationships of the three amphibian species, a phylogenetic tree was constructed based on 1,147 single-copy orthologs using X. tropicalis as the out-group species  and 143, 155, and 142 divergent unigenes (Ka/Ks > 1) were identified in ectively . GO clasectively . The larectively . Variancectively . HoweverA. davidianus, B. gargarizans, and R. Hallowell, respectively (For positively selected genes (PSGs), 51, 61, and 52 PSGs were identified in ectively . FunctioA. davidianus, B. gargarizans, and R. Hallowell, respectively  [de novo transcriptome sequencing technology, which directly targets functional genes, is a powerful method that can overcome this limitation in genome resources, particularly for non-model species [de novo transcriptome sequencing of the skin of three amphibians, A. davidianus, B. gargarizans, and R. Hallowell, generated approximately 17 Gb of clean nucleotides for each species and identified a total of 167,064, 271,117, and 260,306 unigenes, respectively. These newly generated transcripts enrich the genome resource of amphibians and will facilitate biological research for the entire taxon.Amphibians are the transitional taxon in the evolutionary history of vertebrates. Thus, studies investigating the evolution of amphibians, particularly those that rely on molecular resources, are essential in linking the gap between aquatic and terrestrial species. However, the whole genome sequences for amphibians are now only available for the two model species  laevis) , which g species . Benefit species , 10, 11.A. davidianus belongs to the caudate group, whereas B. gargarizans and R. Hallowell are sister-groups of anurans. GO and KEGG classifications of unigenes indicated that skin functions were highly conserved across these three species. First, the GO terms related to basic functions, such as “metabolic process,” “binding,” “cell,” “cell part,” “catalytic activity,” and “cellular process,” were predominantly clustered for the three amphibians. This result is consistent with a previous study on transcriptome sequencing of skins of seven anurans [Skin is the first barrier to protect amphibians against external invasions and plays essential roles for their survival on land. Despite a wide range of amphibian species, the functions of skin are largely conserved among this taxon, including respiration, immunity, and wound repair , 18, 19.A. davidianus and the other two amphibians   . The difllowell) . This isthe skin . This rehttp://www.genome.jp/dbget-bin/www_bget?pathway:map04623). One gene in this pathway, DNA-dependent RNA polymerase III (Pol-III), was highly conserved across the three amphibians. RNA Pol-III is a cytosolic DNA sensor that plays the role of goalkeeper in protection against external invasions by converting the invasion DNA into RNA for recognition by the RNA sensor RIG-I [Despite the differences in structure and function, the innate immune functions of amphibian skins were commonly shared, particularly the immune function of anti-microbial defense. KEGG enrichment showed that pathways related to nucleotide metabolism and immune response were significantly clustered for conserved orthologs in all the amphibians . The immor RIG-I . These rR. Hallowell contained the highest the number of AMPs representing the most families, in addition to the highest levels of expression for most of the AMPs, which might explain the wider distribution of R. Hallowell than A. davidianus and B. gargarizans.In addition to the internal anti-microbial biological processes, AMPs, which are secreted by amphibian skin, can form a natural immune defense to prevent bacterial infections , 22, 23.de novo transcriptome sequencing was conducted to explore the molecular basis underlying immune function of amphibian skins. Unigenes enriched in GO terms and KEGG pathways that were related to basic metabolism, cellular component, and immunity were most common among the three amphibians. Additionally, genes related to immune function were highly represented in both the conserved orthologs and PSGs in all the amphibians. Our study has increased understanding of the molecular basis for the immune functions of amphibian skin, and the transcriptome data set generated in this study will facilitate future molecular biology studies on amphibians.In conclusion, All the methods involving animals in this study were performed in accordance with the Laboratory Animal Management Principles of China. The Ethics Committee of Chongqing University of Arts and Sciences approved all experiments.Bufo gargarizans) and Heiban frogs  were artificial raised that purchasing from the Gaohan Aquatic Animal Husbandry Company . The Chinese giant salamanders (Andrias davidianus) were purchasing from the Chongqing Pengshuishunyu Rare Aquatic Animal Husbandry Company, which was supervised and permitted by the Chongqing fishing and fishing port supervision and management office. The permission of using rare animals was shown in A. davidianus used for sampling were four years old, and the B. gargarizans and R. nigromaculata Hallowell were two years old. The skins of A. davidianus, B. gargarizans and R. nigromaculata Hallowell were first cleaned and sterilized using 75% alcohol before sampling. The animals were released to aquatic farms after sampling. The surgery on all animals was performed under anesthesia using 500 mg/L MS-22, and approximately 1 square centimeter of skin tissue was cut from each individual. After removing the underlying tissues, the skins were frozen immediately in liquid nitrogen and stored at -80°C before RNA extraction. To eliminate individual differences in gene expression, skin from three individuals  of each species was mixed for RNA isolation. Total RNA was extracted using Trizol reagent  according to the manufacturer’s instructions. Concentrations of RNA samples were detected by Qubit 3.0 , which was followed by a quality assay using an Agilent 2100 Bioanalyzer . The strand-specific RNA-seq libraries were prepared using a NEBNext Ultra Directional RNA Library Prep Kit  according to the manufacturer’s instructions. Generated libraries were sequenced on a Hiseq 4000  platform using a paired-end run (2 × 150 bp).The Asiatic toads . Functional categories of the unigenes were determined using the BLASTX program searching against five gene databases, including the Cluster of Orthologous Groups of proteins (COG) database (https://www.ncbi.nlm.nih.gov/COG/), the NCBI non-redundant (NR) database (https://www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins/), the UniProt database (http://www.uniprot.org/), the Gene Ontology (GO) database (http://www.geneontology.org/), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/tools/kaas/) .For functional annotation of unigenes, the CDS and protein sequences of each unigene were first predicted using TransDecoder  were identified using OrthoMCL 2.0.3 software [X. tropicalis was downloaded from its genome data in bioMart . The phylogenetic tree was constructed using the concatenated sequences of all single-copy orthologs by FastTree software [For phylogenetic analysis, single-copy ortholog genes among the four species software  from thesoftware  with a mFor evolutionary analysis, the ratio of non-synonymous substitution (Ka) to synonymous substitution (Ks) of each single-copy ortholog was calculated using KaKs-Calculator software (version 2.0). Ka/Ks < 0.1  was usedP-value < 0.05[The single-copy orthologs used for phylogenetic analysis were subjected to PSG identification using KaKs-Calculator software (version 2.0) with the MYN model . Ortholoue < 0.05 were infhttp://split4.pmfst.hr/dadp/) [−5.For putative AMP identification, all assembled unigenes were aligned against the databases of anuran defense peptides (DADP) (r/dadp/)  and NR uSalmonella, Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus) were purchased from XX Company . Other strains that included Chryseobacterium, Klebsiella pneumonia (K. pneumoniae), Aeromonas hydrophila (A. hydrophila), Shewanella, and Citrobacter were isolated in our laboratory.To validate the function of putative AMPs, a common AMP among the three amphibian species was randomly selected and synthesized  for use in an antimicrobial assay. The AMP was dissolved in acetic acid solution (30%) to a final concentration of 5,120 μg/mL and then sterilized using a 0.22 μm filter membrane before use. The standard bacterial strains 5 cfu/mL) and incubated at 37°C for 18–22 h. Finally, the absorbance at 600 nm of the cultures was detected to define the MIC for each bacteria.The minimum inhibitory concentration (MIC) was determined according to the standard method of the European Committee for Antimicrobial Susceptibility Testing . BrieflyS1 FigBars with different colors represent different species.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigX. tropicalis as reference. Red dots indicate conserved orthologs. Green dots indicate divergent orthologs. Blue dots indicate orthologs under neutral selection.The KaKs values of orthologs in each species were calculated using (TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.


Clostridium perfringens is a major cause of histotoxic and intestinal infections of humans and other animals. This Gram-positive anaerobic bacterium can produce up to three sialidases named NanH, NanI, and NanJ. The role of sialidases in histotoxic infections, such as gas gangrene , remains equivocal. However, recent in vitro studies suggest that NanI may contribute to intestinal virulence by upregulating production of some toxins associated with intestinal infection, increasing the binding and activity of some of those toxins, and enhancing adherence of C. perfringens to intestinal cells. Possible contributions of NanI to intestinal colonization are further supported by observations that the C. perfringens strains causing acute food poisoning in humans often lack the nanI gene, while other C. perfringens strains causing chronic intestinal infections in humans usually carry a nanI gene. Certain sialidase inhibitors have been shown to block NanI activity and reduce C. perfringens adherence to cultured enterocyte-like cells, opening the possibility that sialidase inhibitors could be useful therapeutics against C. perfringens intestinal infections. These initial in vitro observations should be tested for their in vivo significance using animal models of intestinal infections. Clostridium perfringens is present throughout the environment, including soil, sewage, feces, foods, and the normal gastrointestinal flora of animals . ,10. C. pC. perfringens type designations correlate with disease causation, as shown in C. perfringens intestinal infections of mammalian livestock [C. perfringens produces other toxins that, while not used for typing classification, are nonetheless important for infections originating in the intestines of agriculturally-important animals. The foremost example is necrotic enteritis B (NetB) toxin, which is critical when C. perfringens causes avian necrotic enteritis in poultry [ivestock ,16,17. C poultry . C. perfringens infections, type A strains are responsible for causing most histotoxic infections. During gas gangrene, alpha toxin (CPA) plays the major role in virulence. A non-typing toxin named PFO also contributes to this disease [With respect to human  disease ,5. C. perfringens have been conclusively linked to human diseases originating in the intestines [To date, only type A and C strains of testines ,2,3,18. testines ,20. In ttestines . Pigbel testines . Their ltestines . A vacciC. perfringens type A food poisoning (FP) [C. perfringens type A FP can be much more severe and is often fatal [Although not used for typing classification, CPE is the toxin responsible for causing the gastrointestinal symptoms of ing (FP) ,22. Thising (FP) ,23. In pen fatal ,24. Studen fatal ,25. C. perfringens type A FP, which usually self-resolve within 24 h [cpe gene can be either chromosomal or plasmid-borne in type A strains, most (~70%) type A FP strains carry a chromosomal cpe gene [cpe gene [cpe-positive groups. For example, type A strains carrying a chromosomal cpe gene produce a unique small acid soluble protein (SASP4) variant that provides their spores with greater resistance to heat, cold, and chemical treatment and probably facilitates survival of these bacteria in the food environment [cpe strains and type A plasmid cpe strains, particularly with regard to sialidase gene carriage, will be discussed later. As discussed later, CPE-associated AAD cases are more severe and longer lasting than typical cases of hin 24 h ,25. Whilcpe gene . In contcpe gene . There aironment ,26,27. Ocpe-positive strains, CPE may also contribute to some cases of human EN caused by type C strains [In addition to its essential role during intestinal infections by type A  strains . However strains . Sialic acids are a carbohydrate family containing about 40 different nine-carbon relatives of neuraminic acid ,29. UndeVibrio cholerae, Streptococcus pneumoniae, and C. perfringens. Sialidases can have a nutritional function for normal flora or pathogenic bacteria [C. perfringens. Sialidases, also referred to as neuraminidases (E.C.3.2.1.18), are key enzymes that hydrolyze the α-linkage of terminal sialic acids on various sialoglycoconjugates to generate free sialic acid ,29,30,31bacteria ,29,31. Ibacteria , as willC. perfringens produces three different sialidases, which are named NanH, NanI, and NanJ [and NanJ . NanH . However, as discussed in more detail later, some C. perfringens strains produce only one or two of the three sialidases. For strains producing all three sialidases, NanI is usually responsible for ~70% of total exosialidase activity [Most activity ,34,36,37C. perfringens sialidases, a recent study constructed a series of isogenic mutants, where two of the three sialidase genes present in C. perfringens type D strain CN3718 were inactivated [Streptomyces spp., C. perfringens sialidases were found to be sensitive to p-chloromercuribenzoate, which reacts with thiol groups in proteins [C. perfringens sialidases showed different substrate preferences. NanI exhibited preferential activity in the order of α-2,3 > α-2,6 > α-2,8 sialic acid linkages and was responsible for most of the activity in CN3718 supernatants that was directed against those sialic acid linkages. NanJ showed a preference for α-2,6 > α-2,8 > α-2,3 sialic acid linkages. Finally, NanH activity was strongest for α-2,8 > α-2,3 > α-2,6 sialic acid linkages [C. perfringens sialidases work in combination to generate free sialic acid, even from complex substrates [To characterize the properties of the three ctivated . This stctivated . In thisctivated . Furtherproteins . Finallylinkages . This dibstrates . C. perfringens sialidases are encoded by chromosomal genes, although those genes are located in different regions of the chromosome [C. perfringens strains have now been sequenced. Those sequencing analyses indicated that, amongst different C. perfringens strains, the NanJ sequence shares 96% to 100% identity, the NanI sequence has 98% to 100% identity, and the NanH sequence shares 93% to 100% identity [All three romosome ,39. The identity ,38,39. C. perfringens strains vary in their patterns of sialidase production. Most strains produce all three sialidases, with NanI usually being responsible for most of the sialidase activity in culture supernatants of those C. perfringens strains [C. perfringens growth since some strains of this bacterium naturally lack the nanI gene [nanI gene is consistently absent from the type A FP strains carrying a chromosomal cpe gene, as well as the genetically related type C darmbrand strains [nanI gene is carried by most plasmid cpe-carrying type A AAD strains, cpe-negative type A normal human intestinal flora strains, and type C pig-bel strains [C. perfringens, it is not surprising that exosialidase activity is typically significantly lower for naturally nanI-negative strains compared to strains carrying a nanI gene [As true for their toxin production,  strains ,36. Howecpe gene lack the nanJ gene, as well as the nanI gene. However, those typical FP strains do carry the nanH gene. It should be noted that occasional strains of C. perfringens besides the type A chromosomal cpe FP strains or type C darmbrand strains also lack a sialidase gene. For example, Strain 13 is a cpe-negative type A strain that can cause gas gangrene yet it lacks the nanH gene [Most type A FP strains with a chromosomal anH gene ,40. C. perfringens is complicated . This regulatory RNA then modulates sialidase gene expression [nanI and nanJ sialidase gene expression by a putative response regulator, referred to as ReeR [nanJ and nanI expression, possibly via a proposed sensor kinase named RevS. RevR increases nanJ expression, but negatively regulates nanI expression [codY null mutant of C. perfringens type D strain CN3718, our group showed that CodY represses NanJ and NanH production, although it does not affect NanI production [The regulation of sialidase production by plicated . A numbepression . Rather pression . ReeS is as ReeR . In contpression . Finallyoduction . C. perfringens sialidase genes. However, gel mobility shift assays have demonstrated high affinity binding of a purified protein named NanR to DNA from the promoter region of the nanI gene [E. coli, Vibrio vulnifcus, and Staphylococcus aureus, this result suggests that NanR may be involved in nanI expression regulation [C. perfringens [No proven regulator has yet been identified that directly modulates sialidase production by binding to the promoters of anI gene . Since Ngulation ,40,47. CnanI and nanJ genes, primer extension analyses identified three or two putative transcription start sites, respectively [nanI and nanJ. This multiplicity of promoters may provide one explanation for why so many different regulators control sialidase expression, although (as mentioned) detailed understanding of regulator binding to these promoter sequences is currently lacking [For the ectively . These p lacking . C. perfringens was actually the first bacterium demonstrated to be capable of utilizing a sialic acid (Neu5Ac) as a carbon source [C. perfringens when growing in a semi-defined medium [C. perfringens obtain nutrients in vivo by releasing sialic acid from glycolipids or glycoproteins on the host cell surface or in mucus. Interestingly, contact with Caco-2 cells was shown to upregulate the expression of NanI [Only a limited number of bacterial species, mainly those having a close association with the sialic acid-rich environment of the host, can utilize Neu5Ac . C. perfn source . This red medium . It was d medium . This ef of NanI , which cC. perfringens can use as carbon sources and for energy production , which can be hydrated to Neu5Ac. The three pneumococcal sialidases share a common catalytic mechanism up to the final product formation step, and all three sialidases are implicated in pathogenesis, including colonization, and are potential drug targets [S. pneumoniae then takes up and metabolizes sialic acid using a similar pathway as present in C. perfringens [Sialidases can also promote in vivo growth and colonization of bacterial pathogens . For ext targets . S. pneufringens .C. perfringens is unclear. One study demonstrated that a nanJ and nanI double null mutant of C. perfringens strain 13 remains fully virulent in the mouse myonecrosis model [C. perfringens in muscle. However, as noted in that study [The contribution of sialidases to growth and colonization during extraintestinal infections caused by is model . This reat study , this reE. coli in the gastrointestinal system [V. cholerae plays a significant role in both in vitro and in vivo colonization and growth [V. cholerae sialidase enhances the binding and uptake of cholera toxin [V. vulnificus, a Nan utilization system is also important for bacterial colonization and growth in the intestines [Clostridium sordellii adhesion and enhances Non-TcsL mediated cytotoxicity [C. difficile uses sialic acid generated by sialidases of other bacteria to grow and persist in the intestines [Increasing evidence indicates that sialidases and sialic acid often play significant roles in growth and colonization of the intestines by bacterial pathogens ,49,52,53l system . Similarra toxin . In V. vtestines . The siatestines ,51. C. perfringens with Caco-2 cells increases the production of this enzyme [C. perfringens possesses a sialic acid utilization system, similar NanI-generated sialic acid in the intestines should be useful for in vivo growth of this bacterium.As mentioned earlier, NanI may contribute to in vivo growth. Contact of s enzyme  and NanIs enzyme . Since CC. perfringens toxins [nanI gene in C. perfringens type A strain 13 caused a slight increase in supernatant activities of alpha toxin and perfringolysin O (PFO), which are contributors to gas gangrene [nanI gene in type D strain CN3718 reduced ETX toxin production in vitro and that either genetic or physical complementation could recover this ETX production [codY and ccpA transcript levels, suggesting a model whereby NanI generates sialic acid release in the intestines and that free sialic acid then alerts a type D strain of its presence in the intestines, making it worthwhile for this bacterium to upregulate ETX production to induce disease. Earlier studies demonstrated that NanI production also impacts the production of several s toxins ,37,57. SC. perfringens sialidase on toxin action, particularly on those toxins important for intestinal infections. An exception is ETX, where conclusions from early studies had been contradictory. One early study [nanI, nanJ, or nanH single null mutants, a nanI/nanJ double null mutant, and a triple sialidase null mutant, to study possible sialidase enhancement of ETX action [C. perfringens toxins active during intestinal infections is not yet clear. There have been only limited studies to date on the effects of ly study  reportedly study  reportedX action . ResultsX action  conclusiC. perfringens adherence to host cells and tissues [C. perfringens in vivo remain unclear. However, some strains produce a collagen adhesion protein (CNA), and/or fibronectin binding proteins  that have been implicated in adhesion and colonization [Only limited studies have addressed the mechanism of  tissues ,61,62. Fnization ,61,62,63C. perfringens intestinal adherence [Sialidases are known to contribute to the ability of some bacterial pathogens to adhere to host cells, tissues, or mucosal surfaces in the airways or intestines . A recendherence . That stdherence  demonstrdherence . Interesdherence . codY null mutant shows 50% less adherence to Caco-2 cells compared to wild-type CN3718, despite this mutant producing the same levels of NanI as the wild-type strain [C. perfringens to more easily bind to the still unidentified receptor(s) on the enterocyte-cell surface. However, NanI itself does not appear to be a significant adhesin since the CN3718 e strain . This reC. perfringens adherence to host cells via both nonspecific and specific mechanisms. Sialic acids, typically present at the distal ends of carbohydrate chains, are negatively charged carbohydrates [C. perfringens sialidases may lead to barrier disruption and may increase access for C. perfringens so they can adhere [C. perfringens colonization. A C. perfringens sialidase (not specified) has, in fact, been shown to affect barrier resistance in some, but not all, host cells [C. perfringens adherence. As mentioned, C. perfringens CN3718 exhibits much greater adhesion for certain mammalian cells, such as Caco-2 and HT-29 intestinal cell lines, than for cell lines of nonintestinal origin [C. perfringens intestinal infections only bind to and affect certain cells. Those observations suggest that NanI can also have specific effects in promoting C. perfringens adherence and toxin binding. This could involve specific effects of NanI sialidase on modifying host cell surface adhesins/toxin receptors and/or trimming back nearby molecules to better unmask the adhesin or toxin receptor on host cell surfaces. Sialidases may enhance hydrates ,30. In an adhere . Therefost cells . Howeverl origin . SimilarC. perfringens causes diseases originating in the intestines, its secreted proteins come into contact with host proteases, such as trypsin, that are present in the intestinal lumen. While some C. perfringens proteins, e.g., CPB, are highly sensitive to intestinal proteases [C. perfringens protein is ETX, which is initially produced as an inactive prototoxin and then activated by intestinal trypsin, chymotrypsin, and carboxypeptidases that remove N- and C-terminus residues from the prototoxin [C. perfringens intestinal diseases. When roteases , other pototoxin . Similarototoxin . Interesototoxin ,34. The ototoxin . CollectC. perfringens adherence and toxin binding, typical type A FP strains, i.e., those with a chromosomal cpe gene, do not carry the nanI gene. Furthermore, those typical FP strains possess very low exosialidase activities [nanI gene. When a nanI null mutant of a type A AAD strain was characterized, this nanI null mutant was shown to enter the sporulation cycle earlier and to produce more CPE than its wild-type parent. This finding supports the dispensability of NanI for the typical FP strains, which cause an acute disease. During the acute FP, NanI would not be needed for nutritional purposes or to enhance bacteria adherence or colonization since the typical FP strains are ingested in very large numbers in contaminated food, then quickly sporulate in the intestines to produce CPE, induce diarrhea via that CPE, and exit from the intestines via the flushing action of diarrhea  different toxins [As described above, sialidases may offer several contributions to t toxins ,3. ThoseTannerella forsythia, which causes periodontitis, by two- to three-fold [Sialidase inhibitors have proven to be efficacious for the treatment of some infectious disease . The mosree-fold . C. perfringens [Streptomyces spp. culture, is an unusual 6-acetamido-3-piperidinecarboxylate [Vibrio cholerae, Arthrobacter ureafaciens, C. perfringens, and sheep liver [Two classic sialidase inhibitors, i.e., Siastatin B (SB) and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA), have been tested against fringens ,57,69,70C. perfringens CN3718 mutants ENanJ, ENanI, or ENanH, which contain natural levels of only NanJ, NanI, or NanH, respectively [50s for these inhibitors are shown in A recent study measured the effects of SB and NADNA on sialidase activities of overnight culture supernatants of ectively . NanI waC. perfringens AAD strain F4969 to Caco-2 cells can be reduced by either SB or NADNA [C. perfringens type D strain CN3718. Consistent with those results, CN3718 cultures grown in the presence of the SB inhibitor also exhibited substantially reduced culture sialidase activity and ETX production. However, the NADNA sialidase inhibitor did not inhibit sialidase activity in CN3718 cultures, even at very high doses of the inhibitor. In the presence of NADNA, CN3718 cultures still possessed strong sialidase activity and also made the same amounts of ETX as wild-type CN3718 grown in the absence of any sialidase inhibitor [C. perfringens infections.It was also shown that the adherence of or NADNA . In addinhibitor . CollectC. perfringens virulence have, correctly, focused on toxin contributions. Only a few in vivo studies have thus far examined the role of exoenzymes in C. perfringens pathogenesis and their results have been equivocal. However, those studies examined the possible role of sialidases in gas gangrene. Recent in vitro studies suggest that sialidases, particularly NanI, may be important contributors to C. perfringens intestinal infections. Those contributions could include upregulated production of some toxins relevant to intestinal infections, enhanced binding and activity of some of those toxins, increased adherence of C. perfringens to host cells, and possibly generation of substrates for growth and metabolism [C. perfringens virulence. Most studies of tabolism ,34,36,57C. perfringens infections originating in the intestines [C. perfringens sialidases may be helpful for those studies. In vitro studies also suggest that some sialidase inhibitors might be useful therapeutics for treating testines . This po


Candida albicans and other emerging non-albicans spp. has been insufficiently studied. This study aims to compare the activity of CSS and MR-impregnated CVCs against the yeasts most frequently isolated from CLABSIs.Whenever the rate of central line-associated bloodstream infections (CLABSIs) remains high even after the implementation of preventive strategies, the use of chlorhexidine/silver sulfadiazine (CSS) or minocycline/rifampin (MR)-impregnated central venous catheters (CVCs) is currently recommended. Nevertheless, the efficacy of such CVCs against C. albicans, C. glabrata and C. parapsilosis sensu stricto were used. Segments of standard polyurethane, MR and second-generation CSS-CVCs were tested. The biofilm metabolic activity was measured by a semi-quantitative XTT reduction assay.For biofilm formation assays, type strains and clinical isolates of Candida spp., with inhibition ranging from 60% to 100%. The MR catheter segments promoted C. albicans and C. parapsilosis biofilm formation and exhibited an inconspicuous effect against C. glabrata.CSS catheter segments significantly reduced the biofilm metabolic activity by all tested Among the recommended antimicrobial CVCs, CSS-CVCs proved to be superior in the inhibition of biofilm formation by the most frequent yeasts causing CLABSIs. Data from this in vitro study may suggest that patients at high risk for invasive candidosis could benefit from the use of CSS-CVCs. Candida spp. represented a total of 8.2% [Although there are some signs of a possible decline in the incidence of central line-associated bloodstream infections (CLABSIs) , it cert of 8.2% .Candida spp. are the fourth frequent causative pathogen of CLABSIs [Candida cells may adhere to the CVC surface and produce extracellular polymers, providing a structural matrix that will further facilitate adhesion [Candida cells display an increased resistance to antifungal agents and, from such strategic anchorage point, yeasts may disperse into the bloodstream, leading to serious infection [Globally,  CLABSIs  and cont CLABSIs , with an CLABSIs . Throughadhesion . In suchnfection –9.C. albicans, C. parapsilosis and C. glabrata [albicans spp. has been noticed in the past years [Despite being a heterogeneous group of organisms, most of the invasive infections are caused by glabrata , 11. Risglabrata , 13. Morst years –16. Thisst years .Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, S. epidermidis and Candida albicans [albicans species, has not been fully addressed. Moreover, if effectiveness of MR-CVCs against Candida has been previously demonstrated [Recommendations for CLABSI prevention issued by the US Centers for Disease Control and Prevention (CDC) target, for example, the education of healthcare personnel, hand hygiene and aseptic techniques for insertion and care of central venous catheters (CVCs) . Even thalbicans  and MR-Calbicans . In spitnstrated , 21, somnstrated –24.Candida infection, whenever other preventive strategies have failed.Therefore, the aim of this manuscript is to compare and clarify the efficacy of the recommended CSS and MR-impregnated CVCs against the most frequent yeasts isolated from CLABSIs, in order to help clinicians to make the best option for patients at high risk of invasive C. albicans, C. glabrata and C. parapsilosis sensu stricto previously collected from patients admitted at a tertiary university hospital and identified using the Vitek 2 system  (Table Type strains belonging to the American Type Culture Collection (ATCC) or to the Centraalbureau voor Schimmelcultures (CBS) were used, along with clinical isolates of e) Table .Table 1MYeast strains were kept frozen in yeast peptone dextrose medium (YPD)  supplemented with 40% glycerol at −70 °C until testing. For each experiment, microorganisms were subcultured twice on Sabouraud agar , at 35 °C, during 24 h to assess the purity of the culture and its viability.Candida strains were grown overnight in Sabouraud broth at 37 °C and 180 rpm. Afterwards, yeast cells were harvested by centrifugation, washed with PBS and standardized to 1 × 106 cells/mL in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with L-glutamine and buffered with MOPS acid (Sigma-Aldrich).Three different central venous catheters were used as substrate for the biofilm formation assays: standard uncoated polyurethane double-lumen central venous catheters , minocycline-rifampin impregnated central venous catheters  and second-generation chlorhexidine/silver sulfadiazine-impregnated central venous catheters . For biofilm assays, 1-cm segments of each catheter were used. For each experimental condition, 3 catheter segments were used and experiments were performed twice, in different days.6 cells/mL) [To evaluate biofilm formation in the catheters, 2 mL of standardized yeast suspensions (1 × 10ells/mL)  were addells/mL) . Brieflyp value smaller than 0.05. All statistical analysis was performed using the SPSS software (v. 20.0).Biofilm formation, in different time points, was evaluated with One-Way ANOVA .Candida species , at all studied time points .CLABSIs remain a global medical concern and further work on preventive measures must definitely be pursued in order to reduce its incidence. Hence, when CLABSI rate is high, the CDC strongly recommends the implementation of a strategy that should include the education of healthcare personnel responsible for catheter insertion and maintenance, the use of maximal sterile barrier precautions and skin antisepsis using >0.5% chlorhexidine preparation with alcohol during CVC insertion. Whenever after successful implementation of such comprehensive strategy the CLABSI rate is not decreasing, the CDC equally recommends the use of CSS or MR-impregnated CVCs in patients whose catheter is expected to remain in place >5 days, since such catheters can decrease the risk for CLABSI and thus result in lower hospital final costs .Compared to standard non-coated catheters, first-generation CSS-CVCs  did reduce the risk for CLABSI , 27. RegC. albicans [C. albicans and C. parapsilosis strains, but also promoted biofilm formation by both yeasts, most markedly in the case of C. albicans. Regarding C. glabrata, the MR-CVC segments showed an inconspicuous effect, with a slight reduction in biofilm formation only at some time points. Using another in vitro model and considering C. albicans, Gaonkar et al. found similar results with increased adherence of the yeast to MR-CVCs compared to control CVCs [Candida [Candida spp., even though the analyzed studies were underpowered to detect such difference. In vivo increased colonization of MR-CVCs by Candida spp. may result from the exposure of both surfaces of the catheter to a higher and continuous bacterial challenge from the hub and the insertion site [MR-CVCs  were associated with lower rates of CLABSI when compared with first-generation CSS-CVCs . Since talbicans , 36 and rol CVCs . Further[Candida . Neverthion site . Issues ion site , 40, bution site .Candida biofilms when exposed to CSS and MR-impregnated CVCs [C. albicans and C. parapsilosis adherence, but also that the use of MR-CVC increased the colonization by Candida spp. [In our study, a validated XTT reduction assay was used to evaluate the susceptibility of ted CVCs . One limted CVCs . Howeverida spp. .Candida spp., the proportion of colonized MR-CVCs was greater than that of colonized standard CVCs [In a meta-analysis conducted by Novikov et al. in hospitalized patients comparing the incidence of specific bacterial and fungal species colonizing antimicrobial CVCs and standard CVCs, it was found that antimicrobial CVCs in clinical use may become colonized with distinct microorganisms probably in relation to their antimicrobial spectrum of activity. In the particular case of ard CVCs . Hence, Instead of using clinical risk factors to tailor the selection of the best CVC for a given patient, alternatives are being studied targeting a wider antimicrobial spectrum per catheter. This is the case of the catheter impregnated with chlorhexidine (CHX) and MR, as proposed by Raad et al.: CHX-MR CVC may be more effective in completely inhibiting the biofilm formation by resistant bacteria and fungi, with prolonged antimicrobial activity . NeverthCandida activity comparing to MR-CVCs. CSS-CVCs exhibited a strong antibiofilm activity against C. albicans and, moreover, against the emerging C. glabrata and C. parapsilosis. This study further documented not only the lack of antibiofilm activity of MR-CVCs against the tested yeasts, but also the promotion of biofilm formation in in vitro conditions, particularly of C. albicans. Therefore, following current CDC recommendations to use antimicrobial impregnated CVCs whenever CLABSI rate remains high and regarding patients at serious risk of invasive Candida infection, the use of CSS-CVCs may be suggested.In this biofilm formation experiment, CSS-CVCs had a superior anti-


Arachis hypogaea L.), an important leguminous crop, is widely cultivated in tropical and subtropical regions. Peanut is an allotetraploid, having A and B subgenomes that maybe have originated in its diploid progenitors Arachis duranensis (A-genome) and Arachis ipaensis (B-genome), respectively. We previously sequenced the former and here present the draft genome of the latter, expanding our knowledge of the unique biology of Arachis. The assembled genome of A. ipaensis is ~1.39 Gb with 39,704 predicted protein-encoding genes. A gene family analysis revealed that the FAR1 family may be involved in regulating peanut special fruit development. Genomic evolutionary analyses estimated that the two progenitors diverged ~3.3 million years ago and suggested that A. ipaensis experienced a whole-genome duplication event after the divergence of Glycine max. We identified a set of disease resistance-related genes and candidate genes for biological nitrogen fixation. In particular, two and four homologous genes that may be involved in the regulation of nodule development were obtained from A. ipaensis and A. duranensis, respectively. We outline a comprehensive network involved in drought adaptation. Additionally, we analyzed the metabolic pathways involved in oil biosynthesis and found genes related to fatty acid and triacylglycerol synthesis. Importantly, three new FAD2 homologous genes were identified from A. ipaensis and one was completely homologous at the amino acid level with FAD2 from A. hypogaea. The availability of the A. ipaensis and A. duranensis genomic assemblies will advance our knowledge of the peanut genome.Peanut ( Arachis hypogaea L.) is a grain legume and oilseed crop that is an important source of vegetable oil and protein. It is widely cultivated in tropical and subtropical regions. In Africa and some Asia countries, peanut is more prevalent than any other leguminous crop, including soybean. With an annual production of ~46 million tons and a remarkable 45–56% oil content, it plays a key role in daily human nutrition. Moreover, peanut oil is important to human health owing to its rich nutritional elements, such as oleic acid, linoleic acid, resveratrol, fiber, and vitamins , with an AABB genomic constitution   and highly repetitive content (64%) makes the assembly of the peanut genome sequence very challenging , was sequenced using a shotgun approach on the Illumina HiSeq2500 platform  and Zea mays , greater than that of C. arietinum , and substantially lower than those of G. max  and Medicago truncatula  (Supplementary File A. ipaensis genes have homologous gene models in the TrEMBL (99.82%) and Interpro (71.29%) databases , followed by Cajanus cajan (67.4%), and least similar to those of gramineous crops, such as Sorghum italica (33.53%) and S. bicolor (34.51%)  genes were identified in 58 families, which was equal to or slightly higher than of the numbers found in O. sativa and A. thaliana, much higher than in L. japonicus but lower than in G. max and Glycine soja  at different growth stages in A. thaliana (Supplementary File Ka)/synonymous substitutions per synonymous site (Ks) analyses of phyB in A. duranensis and G. max showed evidence of positive selection     , for which 80,218 SSR primers were designed (Supplementary File Arachis magna and Arachis batizocoi may have more abundant genetic diversity than the diploid A species A. duranensis when compared with the reference A. ipaensis (ICG_8206) genome assembly. The geographical origin of Arachis indicated that the distribution of A. duranensis is more extensive and also closer to that of A. ipaensis which has only one known location of origin, than A. magna   and L. japonicus (~0.73)  and A. ipaensis (~0.95)  at 44.3 Mya and that of A. ipaensis and C. arietinum (Ks = ~0.64) at 52.5 Mya.The ion Lna, . Distribd Figure . The A. Ka/Ks (ω) and Ks between the orthologs of A. duranensis and A. ipaensis formed three clusters, such as Ks = 0–0.3, 0.5–1.5, and >1.5, and the ω values decreased as the Ks values increased  play key roles in the responses of plants to various pathogens. The R genes can be classified into various subfamilies based on the present of different domain, such as CC-NB-LRR, TIR-NB-LRR, ser/thr-LRR, Kin-LRR, and others  and A. duranensis (XP_015956675.1 and XP_015963325.1)  Figure . Interes) Figure . The GO A. duranensis. One gene is homologous with MtNSP2 and PsSYM7  that are homologous to the TF genes of the GRAS family in A. hypogaea L.) is a typical upland crop in tropical, subtropical, and warm temperate climates. Drought adaptation plays a central role in their growth and development. During drought stress, TFs, such as MYB, MYB-related, NAC, WRKY, bZIP, and ERF, are involved in numerous physiological responses /chaperones are important defense mechanism members against abiotic stresses, such as drought, salinity and extreme temperatures  gene family is involved in the regulation of stomatal density and distribution to adjust for drought stress by modulating the apertures of these pores flanked by two guard cells  and rice  (Supplementary File http://lipids.plantbiology.msu.edu/), 116 nonredundant homologs potentially involve in lipid biosynthesis were obtained in A. ipaensis (Supplementary File FATA and FATB), respectively, the two key enzymes leading to the synthesis of fatty acid, were identified. Moreover, multiple copies or isoforms of some key genes, such as FAD2, LACS, and KAS, involved in triacylglycerol synthesis were also detected in the A. ipaensis genome  were identified in A. ipaensis  were involved in the fatty acid biosynthesis process and that the proteins encoded by the three genes were subcomponents of the endoplasmic reticulum membrane. They had similarity three-dimensional structures, which was supported by the predicted protein subcellular localization  between A. ipaensis and A. thaliana as assessed by the branch-site likelihood ratio test, indicating positive selection during domestication (Supplementary File FAD2 (2), KASIII (2), and FATB (6) homologs with high Ka/Ks values . Peanut (A. hypogaea L.) is an allotetraploid , probably derived from a single recent hybridization event between A. duranensis  and A. ipaensis   then introduced to Crops Research Institute-Guangdong Academy of Agricultural Sciences (China) was used. Although cytogenetic, phylogeographic and molecular evidence showed that the accession of A. ipaensis K30076 was the most probable B-genome donor for A. hypogaea  was the most closely related to A. hypogaea  was sequenced by Illumina HiSeq2500 platform. Total genomic DNA was isolated from the etiolated unopened young leaves of 20-day-old plants cultivated in dark chamber according to a modified CTAB procedure .SOAPdenovo2 (version 2.04.4) .The gene coverage of the assembled genome was comprehensively evaluated using available public transcript sequence tags or expressed sequence tags. Core eukaryotic genes identified by CEGMA v.2.3 . The predicted genes were used to BLAST search against the PlantTFDB . The FAR1 motif was predicted using the Multiple Expectation Maximization for Motif Elicitation (MEME)/Motif Alignment and Search Tool (MAST) system (http://meme-suite.org/) .To identify TFs in A. ipaensis genome assembly to against the Rfam databese (version 12.1) , Tendem Repeats Finder (TRF) . The transposable elements (TEs) were classified as described without consideration of the gaps in the genome assembly  was used to mine SSRs in A. ipaensis genome, and primer 3 v3.0 was used for primer design  with default parameters, respectively.Reads from five re-sequenced genotypes including two A-genome genotypes (ICG_8123 and ICG_8138) and three B-genome genotypes  were used to identify genome SNP and InDel variations . Ks values of the homologs within collinearity blocks were calculated using the perl script, add_ka_and_ks_to_collinearity.pl included in MCScanX package, and the median of Ks values was considered to be a representative of the collinearity blocks.Syntenic blocks between the genomes of A. ipaensis and other plant species as a TBLASTN query to against the PRG datebase with an E-value of 1e-10 as cut-off. Amino acid sequences of all NBS-LRR genes from A. ipaensis, A. duranensis, and A. thaliana were aligned to construct phylogenetic tree using MEGA v6.0 with automatic bootstrap criteria (Maximum Likelihood) . The PredictProtein was used to perform GO terms, protein-protein and protein-DNA binding sites and sub-cellular localization . Genes involved in symbiotic nitrogen fixation associated with nodule development and AON process were obtained by comparison with orthologous genes in other legumes using multiple protein sequence alignment in COBALT (Arabidopsis were obtained from the Arabidopsis Lipid Gene Database (Mekhedov) (http://lipids.plantbiology.msu.edu/). All the Arabidopsis lipid genes (81) in the database were used to TBLASTN search against the A. ipaensis genome with a cutoff E-value of 1e-50. Finally, a total of 116 non-redundant oil biosynthesis genes were obtained in A. ipaensis. Multiple amino acid sequence alignment of FAD2 homologs was performed using the COBALT . The PredictProtein and SWISS-MODEL was used to integrate GO terms, protein binding sites, sub-cellular localization and protein tertiary structure, respectively . Hydrophobicity and hydrophilicity regions were predicted using ProtScale (Gasteiger et al., Signal peptide analysis of the XP_007162321.1-D2 was predicted using SignalP 4.1 Server with default parameter (Petersen et al., XQL, and XC designed the experiments and managed the project. QL, HFL, and YH performed the research. QL, SW, XYL, GYZ, SL, HL, and HYL analyzed the data. QL wrote the manuscript with the help of GQZ, ZL, and RV. All authors read and approved the final manuscript.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.


Urogenital schistosomiasis is a widely contracted parasitic helminth infection often associated with haematological abnormalities. We investigated the relationship between the haematological profile and the intensity of schistosomiasis among children in the Yeji district. Schistosoma haematobium (S. haematobium) infected and 50 noninfected controls aged 6–17 years matched for age and sex were recruited into the study. Blood and urine samples were collected and haematological profile and presence of S. haematobium eggs were assessed using standard protocols. A total of 100 participants comprising 50P < 0.0001), haematocrit (HCT) (P < 0.0001), mean cell volume (MCV) (P = 0.0053), mean cell haemoglobin (MCH) (P < 0.0001), and mean cell haemoglobin concentration (MCHC) (P = 0.005) levels were reduced in cases compared to controls. Mixed cell percentage (MXD) (P = 0.018) and red blood cell distribution width (RDW-CV) (P = 0.012) were significantly elevated among cases as compared to controls. Haematuria was a clinical characteristic of heavy infection. Haemoglobin (HGB) ( S. haematobium infection creates an imbalance in the haematological profile. We found low HGB, HCT, MCV, MCH, and MCHC levels coupled with increased % MXD count and RDW-CV. Also, low MCV, MCH, and MCHC and high % MXD count are independently associated with S. haematobium infection among our study participants. S. haematobium infection usually occur in school age children resulting in increased morbidity and mortality among affected populations. Majority of S. haematobium infections are asymptomatic; therefore persons living in low resource areas only visit healthcare facilities after complications have developed , MCH < 27 [OR = 8.81 ], and MXD > 13 [OR = 2.50 ].Multivariate logistic regressions of factors associated with Schistosoma haematobium is one of the major neglected tropical diseases causing public health and socioeconomic problems and affecting about 700 million people worldwide [ S. haematobium infection among school age children in the Yeji municipality, Ghana. Our results showed that children with S. haematobium infections had low HGB, HCT, MCV, MCH, and MCHC. Total WBC and WBC differential count (MXD and RDW-CV) were higher among S. haematobium infected children. We also report that low MCV, MCH, and high % MXD counts are independently associated with S. haematobium infections.Urogenital schistosomiasis caused byorldwide . We asse S. haematobium and HGB concentration has been studied extensively [ S. haematobium infection were more anaemic than the healthy controls  [ S. haematobium infected children with low HGB who did not recover until six weeks after treatment in Kenya [Interestingly, HGB levels were reported to be within normal ranges among school aged children infected withNigeria) . There hin Kenya . In thisin Kenya . S. haematobium infected children compared to control. This agrees with previous studies [ haematobium in the bladder wall to reduce the uptake of iron during its pathogenesis through consumption of blood by the schistosomes or extrusion of schistosomal ova which causes urethra and bladder irritation [We also report RBC indices  below normal range in studies , 17 whic studies . It is aritation . S. haematobium infection [Leukocytosis and eosinophilia have been implicated in the intensity ofnfection . Consistnfection .Interestingly, a smaller percentage of children had heavy infections and were more anaemic than those with light infections . However Schistosoma infected individuals advance the effect of schistosomiasis through its pathogenesis [ S. haematobium infection among school aged children ( S. haematobium infections were 5.57, 8.81, 2.07, and 2.50 times at risk of developing low MCV, MCH, and MCHC and high MXD, respectively. Previous work [ Schistosoma infections. A significant rise in absolute eosinophil count is an independent risk factor for exacerbating the pathogenic effect of schistosomiasis [Microcythaemia and hypochromia due to reduced MCV and MCHC inogenesis . Multivachildren . Our finous work  affirms somiasis .The insignificance in the pattern of the infection albeit higher prevalence observed could be due to our small sample size. Clearly a follow-up study with a larger sample size prospectively would better assess the role of this infection on haematological profile. This study design would allow a better understanding of the profile over time and consequently give a true picture of the degree of exposure and the associated risk factors. Also, our findings may be limited by our inability to assess serum iron and ferritin levels among our study participants. S. haematobium infection creates an imbalance in the haematological profile. Among our study participants, we observed low HGB, HCT, MCV, MCH, and MCHC levels coupled with increased % MXD count and RDW-CV. We also report that low MCV, MCH, and MCHC as well as high % MXD count are independently associated with S. haematobium infection among our study participants. Treatment coupled with nutritional intervention, health education, and feeding programs should be considered to manage the deleterious effect of S. haematobium infections.We conclude that