Apophysomyces species are prevalent in tropical countries and A. variabilis is the second most frequent agent causing mucormycosis in India. Among Apophysomyces species, A. elegans, A. trapeziformis and A. variabilis are commonly incriminated in human infections. The genome sequences of A. elegans and A. trapeziformis are available in public database, but not A. variabilis. We, therefore, performed the whole genome sequence of A. variabilis to explore its genomic structure and possible genes determining the virulence of the organism.A. variabilis NCCPF 102052 was sequenced and the genomic structure of A. variabilis was compared with already available genome structures of A. elegans, A. trapeziformis and other medically important Mucorales. The total size of genome assembly of A. variabilis was 39.38 Mb with 12,764 protein-coding genes. The transposable elements (TEs) were low in Apophysomyces genome and the retrotransposon Ty3-gypsy was the common TE. Phylogenetically, Apophysomyces species were grouped closely with Phycomyces blakesleeanus. OrthoMCL analysis revealed 3025 orthologues proteins, which were common in those three pathogenic Apophysomyces species. Expansion of multiple gene families/duplication was observed in Apophysomyces genomes. Approximately 6% of Apophysomyces genes were predicted to be associated with virulence on PHIbase analysis. The virulence determinants included the protein families of CotH proteins (invasins), proteases, iron utilisation pathways, siderophores and signal transduction pathways. Serine proteases were the major group of proteases found in all Apophysomyces genomes. The carbohydrate active enzymes (CAZymes) constitute the majority of the secretory proteins.The whole genome of A. variabilis. Together with available genome sequence of A. elegans and A. trapeziformis, the study helped to indicate the possible virulence determinants of pathogenic Apophysomyces species. The presence of unique CAZymes in cell wall might be exploited in future for antifungal drug development.The present study is the maiden attempt to sequence and analyze the genomic structure of The online version of this article (10.1186/s12864-017-4136-1) contains supplementary material, which is available to authorized users. Mucoromycotina, a subdivision of Kingdom Fungi is currently placed in incertae sedis and comprises three orders namely Mucorales, Endogonales and Mortieriellales [Mucorales 14 families, 56 genera and around 225 species have been identified [Mucorales is evolving with the use of molecular techniques. Recently, Spatafora et al. proposed a phylum level phylogenetic classification of mucoralean fungi comprising of two phyla (Mucoromycota and Zoopagomycota), six subphyla, four classes, and 16 orders [Mucorales, approximately 20 species are known to cause human infections.iellales . In the entified . The traRhizopus arrhizus is the predominant (~60%) agent causing mucormycosis worldwide [Lichtheimia and Mucor species are the next group of fungi isolated from mucormycosis cases in western world [Apophysomyces species ranks second after R. arrhizus in India; around 60% of world mucormycosis cases due to Apophysomyces species are reported from this country [A. elegans was considered the only species under the genus Apophysomyces causing human infection. However, the molecular phylogenetic studies in recent years revealed Apophysomyces genus encompasses 5 species: A. elegans, A. mexicanus, A. ossiformis, A. trapeziformis and A. variabilis; A. elegans, A. trapeziformis, and A. variabilis are common human pathogenic species [A. variabilis [Apophysomyces species generally produce cutaneous and subcutaneous infection following trauma, occasional deep seated infections involving lung, paranasal sinuses and kidney have also been reported [A. variabilis is the major pathogen in India and occasional cases are due to A. elegans [A. trapeziformis has been reported from necrotising fasciitis cases among tornado victims from USA [Mucorales are known to cause severe infection and high mortality. Recently, using transcriptome and genomic approach, various pathogenic determinants have been identified in Mucorales [A. variabilis had not been sequenced and pathogenic determinants of Apophysomyces species were not clearly delineated. The present study reports the draft genome sequence of A. variabilis for the first time and compared the same with already available genomes of A. elegans and A. trapeziformis to identify the possible virulence determinants in pathogenic Apophysomyces species.orldwide , 6. Lichrn world , 8. In c country –11. Misr country . Subsequ country . A. eleg species , 15. Theriabilis . Though reported , 11, 17. elegans . A. trapfrom USA , 19. Theucorales . HoweverA. variabilis (NCCPF 102052) isolate were generated using pair-end and mate-pair libraries on Illumina platform. A. trapeziformis (B9324) and A. elegans (B7760) genomes were downloaded from NCBI genome database for the comparative analysis. The genome assembly of A. variabilis consisted of 1155 contigs and 431 scaffolds with N50 scaffold length of 647.5 kilobases (Kb) and total genome size of 39.38 Mb. Whereas, the genome sizes of A. elegans and A. trapeziformis were 38.46 Mb and 35.84 Mb respectively. The average G + C content of the three Apophysomyces genomes ranged from 41.7% to 41.9%. The genome annotation of the three Apophysomyces species was performed using AUGUSTUS, which predicted >12,000 protein coding genes with an average length ranging from 1969 to 2018 bp , followed by Lichtheimia corymbifera and Rhizopus microsporus.Protein coding genes were functionally annotated using BLASTx  from non-redundant protein database of NCBI. Approximately 88% of the genes in the genome were functionally annotated. The annotated genes were assigned to different Gene Ontology (GO) domains comprising of biological, cellular and molecular functions  proteins in their genomes.A. variabilis and A. trapeziformis and 174 tRNAs in A. elegans (Table Apophysomyces genomes contained single copy of nuclear ribonuclease P (RNase P), a ribozyme that cleaves the 5′ regions of precursor tRNAs to generate mature 5′ ends of tRNA. Like all eukaryotes, the genome of Apophysomyces species contained all five  small nuclear ribonucleo-proteins (snRNA) essential for formation of major spliceosomes. Few components of minor spliceosome  were also present in the assembly. Other essential components like U4atac and U12 snRNA were not present. A. variabilis and A. trapeziformis contained group I introns, a self splicing ribozymes. The histone 3′ UTR stem-loop was present only in A. variabilis genome and it plays a major role in transport of the histone mRNAs from nucleus to cytoplasm , M. circinelloides (2248 genes) and R. delemar (2094 genes) , electron transport chain , secreted proteases (aspartic protease), sugar transporters, CotH invasins and signal transduction pathways in all three Apophysomyces species.OrthoMCL analysis was performed for three mes Fig. . Apart flis Fig. . Apophyses) Fig. . ParalogApophysomyces species. The analysis predicted diverse protein domains, families and repeats encompassing 5.7%, 6.4% and 6.1% genes of A. variabilis, A. trapeziformis and A. elegans respectively for virulence properties of the organisms. The protein kinase domain and Ras proteins were the predominant genes associated with virulence in Apophysomyces species. CotH proteins (invasins), proteases, siderophores, CBS domains, cell wall components  and septins were the other genes determining virulence in Apophysomyces species.PHIbase tool was used to determine the possible genes associated with virulence in Mucorales, Apophysomyces genomes contain multiple copies of CotH protein (42.8 kDa protein). A. variabilis and A. trapeziformis had 17 copies of CotH genes, whereas A. elegans had 16 copies. The orthoMCL analysis revealed multiple groups of CotH paralog genes in three Apophysomyces species.Like other A. variabilis genome had 488 predicted protease gene families evaluated from MEROPS database, 468 in A. trapeziformis and 422 in A. elegans  compared to 9.2% of A. elegans and 13.6% of A. trapeziformis. Single cluster of aspartic protease paralog gene was noted in all three species. However, expansion of paralogs genes encoding aspartic protease 2 (PF13650) was observed only in A. variabilis. OrthoMCL analysis revealed the presence of transposons within the protein domain of aspartic proteases. This phenomenon suggested the possibility of aspartic protease gene insertion at multiple sites of genome. Subtilase, the major serine proteases in Apophysomyces genomes revealed multiple paralog genes; three paralog groups for A. trapeziformis, two for A. variabilis and one for A. elegans. Genes involved in iron acquisition pathways were conserved in Apophysomyces genomes (Table Apophysomyces species contained high affinity iron permease (FTR1) genes (iron acquisition from host), IucA/IucC and FhuF-like transporter (regulator for siderophore biosynthesis), heme oxygenase (utilisation of heme from blood) and ferritin (iron storage) genes. However, the expansion/duplication of iron acquisition genes  was not found in Apophysomyces genomes. Apophysomyces genomes contained multiple copies of CBS domain (iron binding and acquisition). OrthoMCL analysis did not detect any paralog gene for CBS domain.ns Table . Serine Apophysomyces genome contained glycoside hydrolases (GHs), glycosyl transferases (GTs), carbohydrate esterases (CEs), carbohydrate binding modules (CBMs) and polysaccharide lyases (PLs) genes (Table A. variabilis had 97 glycoside hydrolases (GHs) genes predominantly belonging to GH18 family (chitinases). The glycosyl transferases (GTs) were the major CAZymes found in A. variabilis. This class of enzyme largely belongs to GT2 family of chitin synthase genes, an essential component for cell wall synthesis. A. variabilis had 75 carbohydrate esterases (CEs) genes and majority consisted of CE4 family that code for chitin deacetylase, the enzyme that helps in the conversion of chitin to chitosan. A. variabilis contained other CAZymes like polysaccharide lyases (PLs) and carbohydrate binding modules (CBMs) in their genome. Apophysomyces species had similar percentage of CAZymes in their genome  genes for A. trapeziformis and 412 (3.2%) genes for A. variabilis. The predicted secretory proteins of Apophysomyces genome predominantly contained carbohydrate active enzymes. The diverse group of secreted CAZymes in Apophysomyces genome were glycoside hydrolase (GHs) that consisted of chitinases (GH 18) and lysozymes (GH 25), followed by carbohydrate esterase (CEs) that predominantly consisted of chitin deacetylase (CE4). To predict the secretory peptidases, the secretory proteins were searched against MEROPS peptidase database using BLASTp. Aspartic protease and subtilase were the major peptidases in Apophysomyces genome and those enzymes might play role in tissue destruction of the host. BLASTp analysis against lipase engineering data base revealed 9 to 14 genes coding for secretory lipase in the Apophysomyces species  genes encoding the secretory proteins were predicted for s, 435 3.% genes fAscomycota and Basidiomycota. Fungi belonging to the subkingdom Mucoromycotina were closely related to Mortierellomycotina. The Mucorales were grouped in two major clusters. The three Apophysomyces species were grouped together in close association with Phycomyces blakesleeanus  and A. trapeziformis (35.84 Mb). The genome structures of three pathogenic Apophysomyces species were compared with other pathogenic Mucorales to identify the predicted virulence properties of Apophysomyces species. Apophysomyces species have more than 12,000 protein coding genes. Among Mucorales, Mucor racemosus has the maximum number of protein coding genes  with genome size of 75.32 Mbs and Umbelopsis isabellina has the least number of protein coding genes (7612 genes) with genome size of 21.87 Mbs [Rhizopus arrhizus  and Lichtheimia corymbifera  lie close to Apophysomyces species [Apophysomyces species (~1800 protein families), is also close to Rhizomucor miehei (1827 proteins) and R. arrhizus (1653 proteins) [Mucoromycotina including Apophysomyces species is closely related to Mortierellomycotina rather than Entomophthoromycotina. The phylogenetic analysis of the present study confirms the findings of super tree constructed of 9748 genes using parsimony approach [Mucorales are genetically different from Entomophthorales [The current study revealed 1.87 Mbs . The pro species . The preroteins) . Phylogeapproach . The prethorales .Ascomycetes and Basidiomycetes leads to high degree of diversity ranging from 0.02 to 29.8% of their genomes [Mucorales also have a varying percentage of transposable elements in their genomes ranging from 3.17% (Rhizomucor miehei) to 35% (P. blakesleeanus and Absidia glauca) [Apophysomyces species have lower content of TEs in their genome compared to other Mucorales. The lower content of TEs in L. corymbifera was associated with the appearance of hetereokaryon incompatibility (HET) genes and RNA interference mediated RNA degradation genes like dicer, argonaute and translation initiation factor 2C [Apophysomyces species contain multiple copies of HET genes in their genome along with RNA interference mediated genes. In comparison, R. arrhizus has 35% of TEs in their genome, which may be due to the absence of HET genes [Ascomycetes and helps in the formation of heterokaryons in sexual reproduction. The function of HET domain in Lichtheimia and Apophysomyces species is not clearly known. The genome duplication and transposon mediated gene expansion may facilitate the pathogenic mechanism of fungi [The transposable elements (TEs) in the fungal genomes play a major role in the genomic diversity and genome evolution . The pre genomes . Mucoral glauca) , 23. Theactor 2C . SimilarET genes . The HETof fungi .Saccharomycotina, an ancestry of the Dikarya [R. arrhizus [R. arrhizus, the whole genome duplication is an important phenomenon for the expansion of multiple gene families and plays significant role in pathogenesis and antifungal resistance of the organism [RNase P may attribute for whole genome duplication in R. arrhizus [L. corymbifera genome showed the presence of tandem and segmental gene duplication [RNase P was found in Apophysomyces species like L. corymbifera and indicate the possibility of tandem and segmental duplications in Apophysomyces genomes. Mucorales in general contain more duplicated genome regions (average of 4 to 13 genes) compared to other fungal genomes [Apophysomyces genomes for ABC membrane transport, major facilitator proteins, E1-E2 ATPase and other protein families that were involved in cell wall synthesis, morphogenesis, virulence mechanism and antifungal resistance.In Kingdom Fungi, the whole genome duplication has been identified in two separate group of fungi:  Dikarya , 27 and arrhizus . In R. aorganism , 28. Thearrhizus . The analication . Single  genomes . The dup genomes . MultiplApophysomyces genomes are associated with virulence of the fungi. The multiple copies of protein kinase and Ras domain in Apophysomyces genomes may play a role in pathogenesis. Protein kinase plays a major role in signal transduction pathways and regulates major cellular functions, cell cycle regulation and fungal morphogenesis [R. arrhizus. Similarly, the present analysis showed Apophysomyces genomes had multiple paralog gene clusters of protein kinase. The Ras protein regulates eukaryotic growth and differentiation, and orchestrates virulence in pathogenic fungi [The pathogen-host interaction database predicted approximately 6% of genes in ogenesis . Two-folic fungi .Mucorales is iron acquisition from the host. The mechanisms included reductive pathways consisting of high affinity iron permease (FTR1), multi-copper oxidase and ferric reductase, siderophore transporter and heme oxygenase [Mucorales to grow in the body [L. corymbifera, up-regulation of FTR1 gene, multi-copper oxidase and ferric reductase is observed under iron starvation [R. arrhizus [Mucorales. Apophysomyces genome contains all the essential components of reductive pathways of high affinity iron permease (FTR1), multi-copper oxidase and ferric reductase that help the iron acquisition from the host. Like other Mucorales, A. variabilis lacks hydroxamate siderophores (NRPS genes) in their genome. However, compensatory mechanism has been observed in L. corymbifera, in the form of upregulation of IucA/IucC family and ferric iron reductase FhuF-like transporter  during iron starvation [Apophysomyces genomes also contain genes of IucA/IucC family and ferric iron reductase FhuF-like transporter. The other modes of iron acquisition genes including heme oxygenase pathway and ferritin genes are conserved in Apophysomyces species.The most common virulence trait of xygenase –34. In nxygenase . During the body . In L. carvation . The redarrhizus . These farvation . ApophysR. arrhizus [R. arrhizus contains CBS domain (Fob1/Fob2 proteins) on their cell surface, which acts as receptor to bind ferroxamine. In deferoxamine treated mice, mutants of Fob proteins have reduced iron uptake, germination and virulence [Apophysomyces genome contains multiple copies of CBS domain, which may also play a role in the iron acquisition.Deferoxamine therapy is a risk factor for mucormycosis. Deferoxamine acts as a xenosiderophore and helps in the iron acquisition of arrhizus , 34. R. irulence . ApophysMucorales especially in patients with elevated serum iron concentrations (patients with diabetic ketoacidosis or on deferoxamine therapy) [R. arrhizus spores [Apophysomyces genome possesses multiple copies of CotH genes, which may help in angio-invasion of the fungus. Gene duplication of CotH gene families may increase the virulence properties of Apophysomyces species. Entomopthorales does not contain CotH genes and has low angio-invasive property [The angio-invasion and tissue necrosis are notable features in mucormycosis infections. The endothelial cell receptors play major role in the angio-invasion of therapy) , 34. Thes spores . CotH , closely similar to R. arrhizus (3.6%) and L. corymbifera (3.3%) [A. variabilis are serine (48.7%) and aspartic protease (34%). The number of secretory protease in Apophysomyces genome (8%) is similar to that of R. delemar (7%) [R. arrhizus, Apophysomyces species can cause severe tissue destruction in the immunocompetent host.Proteases are also considered to be important for virulence in fungi, as they are responsible for tissue destruction and necrosis in hosts. The genome of a (3.3%) . The commar (7%) . HoweverMucorales cell wall contains unique polysaccharides molecules such as chitin and chitosan, which are not seen in mammals [Apophysomyces genome contains β-1,3-glucan synthase genes (FKS1), but α-1,3-glucan synthase genes are absent. Despite the presence of FKS1 gene, the inherent resistance mechanism of Mucorales to echinocandins is not clear [Apophysomyces genomes (Table Apophysomyces contains lower amount of glycosyl hydrolases and glycosyl transferase compared to R. arrhizus and R. miehei (Table Apophysomyces species (Table . The most common CEs in Apophysomyces genomes are CE10 (carboxyl esterase) and CE4 (chitin deacetylase), but CE10 is absent in R. arrhizus and R. miehei [Mucorales. However, the functional consequences of these genes are not clear [ mammals . The Apoot clear . CAZymesot clear . The CAZes Table . Howeverei Table . The expes Table . The mos. miehei . The expot clear –22, 29.A. variabilis, a common pathogen causing mucormycosis in Indian subcontinent. The genome analysis of A. variabilis together with other two pathogenic Apophysomyces species revealed multiple gene duplications in signal transduction pathways, cell signalling molecules and virulence traits. The expansion of virulence genes such as secretory protease, CotH invasins, cell wall components (chitin synthase and chitin deacetylase) is seen in Apophysomyces genomes. The unique CAZymes responsible for the biosynthesis of cell wall polysaccharides may be future antifungal target. Further exploration of Mucorales specific cell wall components may help in understanding the pathogenesis of mucormycosis in detail.We report the maiden draft genome sequence of Apophysomyces variabilis strain NCCPF 102052 isolated from patient with necrotising fasciitis of thigh was retrieved from the National culture collection for pathogenic fungi (NCCPF), Chandigarh, India. The isolate was grown on potato dextrose agar by incubating at 35 °C for 7 days. DNA was isolated from the fresh mycelia using phenol: chloroform: isoamyl alcohol extraction protocol as described earlier [ earlier .A. variabilis NCCPF 102052 was performed using paired-end (PE)/(MP) 2*150/2*125 base pair library on Hiseq platform . The paired-end sequencing library was prepared using Illumina TruSeq Nano DNA HT library preparation kit and the mean size of the library was 645 bp. The Illumina mate- pair libraries were prepared using Cre-Lox recombination method with insert size of 3-5Kb [Apophysomyces trapeziformis  and Apophysomyces elegans  were downloaded from NCBI genome database for the comparative analysis.The whole genome sequence of of 3-5Kb . The rawof 3-5Kb  and per of 3-5Kb , 45. TheRhizopus arrhizus training set was used as species for gene prediction from both strands. Prediction of incomplete genes was allowed. Using this gene model, CDS & protein sequence was extracted. Protein coding genes were annotated using BLASTx program available from NCBI [AUGUSTUS version–3.1.01 was used for ab initio gene prediction . Rhizopurom NCBI . Best BLrom NCBI .Based onrom NCBI .PFAM annotation was performed using PFAM scan tool downloaded from PFAM database . This toTransposonPSI was used to identify diverse families of transposable elements that prevail in the genome sequences. The transposons were classified into DNA and RNA transposons .Non-coding-RNAs were identified and annotated using infernal tool of RFAM database .e value = 10^-4.Peptidase analysis was conducted using BLASTp search against MEROPS database . AnnotatCAZymes annotation was done using dbCAN database . Hmmscan. The presence of signal peptides in the protein sequence was predicted by setting the D-cut off values to “sensitive” (option eukaryotic). The parameter setup includes that secreted proteins that did not contain any transmembrane helix or the one overlapping the signal peptide within first 40 amino acids of the protein sequences. TargetP software (version 1.1) and WolfPsort (version 0.2) were used to predict protein subcellular localization. Proteins were considered secretory if subcellular localization was assigned to extracellular secretory pathway [e value = 10^-5): dbCAN was used to predict CAZymes, MEROPS for peptidase and Lipase Engineering Database for lipase prediction [The soluble secretory proteins satisfying the following parameters were analysed: a protein with N-terminal signal peptide, without the presence of trans-membrane domain, complete absence of GPI-anchor site and extracellular secretion of the proteins outside the cell , 55. Sec pathway , 59. PS- pathway . The preediction , 61.Genes responsible for possible pathogenesis and virulence mechanism were identified and annotated using PHI-base . BLASTp Apophysomyces genomes, one genome each of R. arrhizus, L. corymbifera and Rhizomucor miehei were used Gene ontology (GO) of Additional file 2: Table S1.Apophysomyces species. Table S2. Carbohydrate active enzymes (CAZymes) of Apophysomyces species. Table S3. GenBank accession numbers of whole genome sequences used in phylogenetic analysis of Apophysomyces species and orthoMCL analysis. (DOC 163 kb)Non coding RNAs in genomes of three


Hahella sp. strain CCB-MM4 is a halophilic bacterium isolated from estuarine mangrove sediment. The genome sequence of Hahella sp. CCB-MM4 provides insights into exopolysaccharide biosynthesis and the lifestyle of the bacterium thriving in a saline mangrove environment. Hahella sp. strain CCB-MM4 was isolated from soil samples obtained from Matang Mangrove Forest located in Perak, Malaysia, in September 2014  and chitin and N-acetylglucosamine utilization, as well as complex carbohydrate utilization genes. The genome data of Hahella sp. CCB-MM4 contribute to a better understanding of the biological potential of Hahella and will facilitate the uncovering of the industrial usefulness of this strain.charides , 5. Openand algL , were aland algL . Ectoineand algL , 14. EctMRYI00000000.This whole-genome shotgun project can be accessed at DDBJ/EMBL/GenBank under the accession no.


Lactococcus lactis WFLU12 can enhance the growth of olive flounder and its resistance against streptococcal infection. The objective of the present study was to use comparative genomics tools to investigate genomic characteristics of strain WFLU12 and the presence of genes supporting its probiotic action using sequenced genomes of L. lactis strains. Dispensable and singleton genes of strain WFLU12 were found to be more enriched in genes associated with metabolism  than pooled dispensable and singleton genes in other L. lactis strains, reflecting WFLU12 strain-specific ecosystem origin and its ability to metabolize different energy sources. Strain WFLU12 produced antimicrobial compounds that could inhibit several bacterial fish pathogens. It possessed the nisin gene cluster (nisZBTCIPRKFEG) and genes encoding lysozyme and colicin V. However, only three other strains  harbor a complete nisin gene cluster. We also found that L. lactis WFLU12 possessed many other important functional genes involved in stress responses to the gastrointestinal tract environment, dietary energy extraction, and metabolism to support the probiotic action of this strain found in our previous study. This strongly indicates that not all L. lactis strains can be used as probiotics. This study highlights comparative genomics approaches as very useful and powerful tools to select probiotic candidates and predict their probiotic effects.Our previous study has shown that dietary supplementation with  Lactococcus lactis WFLU12 can confer farmed olive flounder protection against streptococcal infections. It can also improve fish growth by enhancing some enzyme activities (including phosphohydrolase and glycosidase) in the fish gut [Colonization of vast and various communities of microbes is necessary for nutrition uptake and well-being of fish. The intestine of fish is rich in nutrients. It provides a favorable growth environment for numerous adapted bacteria that form unique characteristics of the fish intestine ,2. Althofish gut .L. lactis strains have been published in the last decade. They might provide some important traits for various applications. For example, they have contributed to the development of fermented dairy products and potential delivery vectors for various antigens with pharmaceutical applications [L. lactis strains exhibit genome-scale diversity with a gene pool that plays a pivotal role in evolution and environmental adaptation [L. lactis isolated from different ecological sources and geographical areas showed high variability at both gene and genome levels, prompting a new classification based on ecological separation of “domesticated” and “environmental” lactococcal strains. Genome comparison between L. lactis strains isolated from dairy and plant sources has revealed that they have very flexible genomes, showing variability in terms of encoded functions of proteolysis, lactose fermentation, citrate uptake, metal ion resistance, and exopolysaccharides biosynthesis [L. lactis strains to explore niche-specific variations and genes in the gene repertoire of strain WFLU12 that might support its probiotic action.Complete genome sequences of dairy- and nondairy-ications . Numerouaptation ,9,10. Inaptation , 36 straynthesis ,13, genoL lactis WFLU12 annotated using Prokka is shown in The whole genome sequence of strain WFLU12 was obtained using the SMRT sequencing approach. Primary genome assembly was performed using ABySS version 1.5.1. Scaffolding of the primary assembly was performed using SSPACE-LongRead scaffolder version 1.0 . ResultsL. lactis subsp. lactis , suggesting that each strain might have adapted to its ecological niche. Of 2480 protein CDS in the genome of WFLU12, 75.56% were functionally assigned based on Basic Local Alignment Search Tool (BLAST) while the remaining 24.44% were hypothetical proteins. Our results are consistent with results of general genome analysis of subsp. Lactis [A total of 20 subsp. . Lactis ,18,19. Ilactis strains (L. lactis strains are diversified depending on isolation sources (dairy and plant) by analyzing the presence/absence of gene clusters related to growth on different carbohydrate substrates, biosynthesis of exopolysaccharides, stress responses , and bacterial defense mechanisms (nisin biosynthesis). Strain WFLU12 isolated from fish gut shared similarities with drain water (IO-01) and plant isolates (AI-06) of L. lactis based on both hierarchical clustering and phylogeny.Unlike the phylogenetic tree that reflects different evolution within the genome , the use of the hierarchical clustering tree could provide an overview of intra-species phenotypic variation by measuring major genetic re-arrangement events  . Based o strains . Remarkalactis strains was constituted by 5442 orthologous groups. According to Tettelin et al. [n = k × N−α. The power law coefficient within this equation is 0.302 (between 0 and 1), corresponding to the open pan-genome model [lactis and cremoris) were in a closed state, an open pan-genome model was applicable to subsp. lactis in this study. Thus, an addition of our sequenced genome will contribute to the characterization of a new gene repertoire of this subspecies.As shown in the pan-genome graph A, the sin et al. , Heap’s me model  , dispensable , and singleton genes (seen in only one of 20 strains). Consistent with 1413 core-genes outlined in a previous study for 18 strains of subsp. lactis [lactis strains assessed. Strain A12 harbored the highest number of singleton genes (221 genes), followed by strain WFLU12 with 129 genes. The genome of WFLU12 presented a longer stretch of singleton genes in the white gapped blast area compared to the other 19 strains  were more frequently found in dispensable genes of strain WFLU12 than those in pooled dispensable genes. Lastly, enrichment in singleton genes related to cell wall/membrane/envelope biogenesis in strain WFLU12 was apparent within the group of ‘cellular processes and signaling’. This might indicate the importance of such genes in the natural habitat of gut bacteria.From pan-genome analysis of strain WFLU12 , we obtaL. lactis had a variety of genes. Their gene clusters are specialized in fermentation of a wide spectrum of complex sugars  other than lactose as growth substrates in a dairy environment [L. lactis to variable substrates derived from their natural niches. In this study, a total of 117 CAZy were identified in the genome of L. lactis WFLU12. This number is similar to the number of CAZy found in other strains, ranging from 94 to 126 ; l-rhamnulose kinase ; and l-rhamnulose-1-phosphate aldolase ) involved in the metabolism of l-rhamnose [L. lactis strain possesses a gene cluster encoding enzymes for l-rhamnose metabolism. In this study, strain WFLU12 was grown on a medium containing l-rhamnose as shown in l-rhamnose isomerase has very broad specificity toward various substrates, including l-lyxose, aldoses, and ketoses. This indicates that strain WFLU12 is likely to degrade these rare carbohydrates.Unique gene and gene clusters in strain WFLU12 included sugar metabolism , cell envelope biogenesis , antagonistic activity , and many hypothetical proteins . The genrhamnose ,31,32  [L. lactis to sulfamethoxazole remains unclear [L. lactis belonged to the core genome. It is known that L. lactis WFLU12 possesses antimicrobial activity against several fish pathogens [L. lactis strains had antibacterial activity. This property is clearly advantageous for WFLU12 to compete against other microorganisms in the same habitat.Pathogenicity and antibiotic resistance islands were not present in strain WFLU12, corroborating its safety aspects. This strain was susceptible to most antibiotics assayed in this study   and gene unclear . In thisathogens . This stathogens ,42,43,44L. lactis strains might have similar abilities to survive passage through the gastrointestinal tract. Differences in these dispensable genes are shown in hierarchical clustering of gene presence/absence between L. lactis strains   . Thiolasty acids . Indeed,L. lactis has been previously described [Vitamin biosynthesis in escribed ,58,59. IL. lactis might be involved in increased metabolites such as nicotinamide and 3-hydroxybutyric acid (data not shown). Genetically, L. lactis only possesses genes involved in salvage NAD synthesis from nicotinamide (deoD), nicotinic acid , nicotinamide riboside (nadD), nicotinic acid riboside (pncB), and deamino-NAD+   . This in pathway ,61. In a pathway ,63. Simi pathway ,65. In o pathway , lack of pathway ). Thus, Genomic DNA was extracted from strain WFLU12 that was isolated from a wild olive flounder  using a http://vicbioinformatics.com/). The pipeline (based on Prokka version 1.6) included a number of features, including Prokaryote gene prediction by Prodigal v2, rRNA search using barrnap v0.2, tRNA prediction by Aragorn v1.2.36, pCDS physic-chemical properties using an in-house script, inferred proteins , signal peptide and cellular localization from signalp v4.0, conserved domains by hmmer-3, and Organism prediction using an in-house script.Using a combination of Illumina and PacBio reads, sequences were subjected to de novo Illumina/PacBio hybrid assembly and genome annotation. Scaffolds were subsequently marked using an in-house pipeline (BaseClear annotation pipeline) based on Prokka framework. Genome annotation was performed for the assembled contig or scaffold sequences using an annotation pipeline based on Prokka prokaryotic genome annotation system (This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PKRZ00000000. The version described in this paper is version PKRZ01000000.ftp://ftp.ncbi.nih.gov/genomes/) using JSpecies v1.2.1 [http://www.broadinstitute.org/cancer/software/GENE-E/).To establish the phylogenomic identity of strain WFLU12, average nucleotide identity (ANI) was calculated with phylogenetic representatives of close hitherto-sequenced lactococcal genomes obtained from NCBI bacterial genome database  and analyzed with dbCAN annotation server (http://csbl.bmb.uga.edu/dbCAN/annotate.php) [To annotate carbohydrate-active enzymes, annotated protein dataset of each strain was retrieved from the NCBI bacterial genome database (ate.php) . These rL. lactis subsp. lactis strains including strain WFLU12 was performed with EDGAR v2.2 [Comparative pan-genome analysis of hitherto-sequenced GAR v2.2 . AccordiFor functional analyses, genomic subsets within the pan-genome were subjected to the Clusters of Orthologous Group (COG) category-wise classification scheme by performing PSI-BLAST against the NCBI COG database (recently updated) in WebMGA analysis server using default parameters .p-value threshold of 1, p_adj-value threshold of 0.05) [For analysis of the cell wall in relation to adhesive genes in the genome, automatic pilus cluster was searched using LOCP v.1.0.0 with default input parameters (of 0.05) . LOCP ouL. lactis WFLU12 possessed functional genes involved in the stress response to the gastrointestinal tract environment, bacteriocin production, and dietary energy extraction and metabolism, supporting our previous study [L. lactis strains including strain WFLU12 in this study revealed niche-specific differences between strains in terms of genes and gene clusters for carbohydrate metabolism, envelope biogenesis, and bacterial defense mechanisms . This host gut-derived strain WFLU12 harbored many genes supporting its probiotic action. Our results also highlight comparative genomics as a very useful and powerful tool to select probiotic candidates and predict their effects.In this study, we found that us study . Compara


P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2–99.8% and 95.2–99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/μl. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs.Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of  Plasmodium that infect humans through the bite of an infective female Anopheles mosquito. There are five different species of Plasmodium parasites that commonly cause human disease: P. falciparum, P. malaria, P. ovale, P. vivax, and P. knowlesi (zoonotic). P. falciparum is the most lethal human malaria parasite and is the most prevalent in sub-Saharan Africa [P. vivax is the most widely distributed geographically and best adapted to survive in temperate climates.Malaria is caused by protozoan parasites of the genus n Africa . P. vivaP. ovale and P. malariae mainly occur in certain areas of sub-Saharan Africa, with P. ovale occurring mainly in West Africa. P. ovale was one of the last human malaria parasites to be described. The natural distribution of P. ovale was initially thought to be restricted to sub-Saharan Africa and the islands of the western Pacific [P. ovale to also circulate in India, Bangladesh, Vietnam, and Myanmar [P. ovale infection has been considered a low-prevalence disease with limited geographic distribution, benign clinical course, and easy treatment; therefore, little attention has been paid to it [P. ovale has usually been made through microscopy. However, this can be challenging due to the fact that many P. ovale infections present with low levels of parasitemia, and therefore, highly sensitive tools may be required to detect it. In addition, mixed infections with other Plasmodium species, especially P. falciparum, might compromise the detection of P. ovale. There are no rapid diagnostic tests (RDTs) specific for P. ovale; detection of non-falciparum Plasmodium infections by RDT is limited to the use of a Pan  test. This does not allow for the discrimination of the non-falciparum infections.Both  Pacific , 3, but  Myanmar –7 Malariid to it  DiagnosiPlasmodium species has two distinct subspecies that are essentially morphologically and clinically indistinguishable, but are separated by subtle genetic dimorphisms [P. ovale curtisi (classic form) and P. ovale wallikeri (variant form) [Interestingly, this neglected orphisms , 10: P. nt form) . These tnt form)  and in Ant form) –7and theBased on the recent success in global efforts to reduce the number of malaria cases and deaths , there iMalaria elimination and control programs utilize three major diagnostic tools: antigen/antibody based RDTs, microscopy, and molecular tools (nucleic acid based tools). Microscopy remains the gold standard for diagnosis of malaria in many malaria-endemic countries. While microscopy is inexpensive, can quantify parasite burden, and can differentiate parasite species, it has several limitations. Preparation of blood smears is laborious, difficult to standardize, and the diagnosis of low parasite density is challenging and requires seasoned microscopists. Inter-user variability (a two to three-fold discrepancy) can occur in parasite quantification especially when routine training and quality management procedures are not practiced .Plasmodium–specific (pan), detecting the genus-specific aldolase and lactate dehydrogenase enzymes. The vast majority of available RDTs (90%) are specific for P. falciparum histidine-rich protein 2 (Pf HRP-2), and are therefore limited as they cannot detect other human Plasmodium species. Additionally most of these RDTs have a relatively high threshold for detecting P. ovale, around 100 parasites/μl [RDTs also have a role in case management and control programs. Some RDTs are sites/μl , 14. LasPlasmodium speciation [Given the limitations of RDTs and microscopy there is a clear need for sensitive, cost effective, and user friendly tools that can complement the current malaria diagnostics. Molecular diagnostic tools are far more sensitive for detecting malaria infections with low parasite burden while offering accurate eciation , 16. Theeciation .P. falciparum multiplex PET-PCR assay [P. ovale in one assay. We evaluated the utility of this assay using 173 clinical samples of different malaria species infections, and using well quantified P. ovale samples to estimate the limits of detection.The real-time PCR platform is advantageous for large scale screening and recently, we have shown that the photo-induced electron transfer (PET)-PCR is a convenient tool for limited resource settings due the ease of use , 19. ThiCR assay , 19. In P. vivax, P. malariae and P. ovale) were obtained from CDC’s collections previously obtained from a contractor who used American Association for the Accreditation and Assessment of Laboratory Animal Care (AAALAC) approved protocol for collection of these specimens from non-human primates (chimpanzee or monkeys).Clinical samples used in this study were anonymized samples obtained from malaria specimens routinely submitted to CDC reference diagnostic laboratory in the Division of Parasitic Diseases and Malaria for malaria diagnosis. The submission of laboratory specimens are deemed a routine surveillance activity and not a human subjects’ research activity by the CDC IRB. The authors did not have access to any identifying patient information. No human tissues were used in this study. The non-falciparum specimens  gene. In silico testing, using Geneious 9.1.4 software (http://www.geneious.com) was performed first before primers were synthesized. A BLAST search was performed to ascertain that our selected region of interest on the RBP-2 gene was specific to both P. ovale curtisi and P. ovale wallikeri  and HEX (RNase-P gene) fluorophores. The PET tag sequence and all other oligonucleotide primers for P. ovale and RNase-P gene are shown in In addition to designing uo et al . In bothPlasmodium species acquired from archived whole blood or parasites culture  at the CDC were used in this study: 2 strains of P. falciparum , 1 P. vivax , 1 P. malariae (CDC Uganda I), 4 strains of P. knowlesi , and separate blood samples of P. ovale (CDC Nigeria I strain) obtained from three different chimpanzees. The non-falciparum specimens were obtained from CDC’s collections that were obtained from a contractor who used American Association for the Accreditation and Assessment of Laboratory Animal Care (AAALAC) approved protocol for collection of these specimens from Chimpanzee or monkeys. In addition, well characterized P. ovale curtisi and P. ovale wallikeri DNA samples were kindly provided by Dr. Colin Sutherland’s lab at the London School of Hygiene and Tropical Medicine.Different P. falciparum, 36 P. vivax, 14 P. malariae, 40 P. ovale and 2 unidentified Plasmodium spp).A total of 173 anonymized clinical whole blood samples were obtained from the CDC molecular diagnostic parasitology reference laboratory . The DNA was aliquoted and stored at -20°C until used in the experiments.Plasmodium genus (FAM-labeled)/ human RNase-P (HEX-labeled) assay. This initial step detected the presence or absence of Plasmodium DNA in each sample and confirmed the successful DNA isolation from the samples. All the samples were subsequently tested using the singleplex P. ovale assay (FAM -labeled). This last step detected the presence or absence of both subspecies of P. ovale.All clinical samples were initially tested using a multiplex P. ovale) and 60°C (Plasmodium/RNase-P).All the PET-PCR assays were performed in a 20 μl reaction mix containing 2X TaqMan Environmental Master Mix 2.0 (Applied BioSystems), 250 nM of each forward and reverse primer, and 5 μl of DNA template. The reactions were performed under the following cycling parameters: initial hot-start at 95°C for 15 minutes, followed by 45 cycles of denaturation at 95°C for 10 seconds, annealing at 62°C (for The correct fluorescence channel was selected for each primer set and the cycle threshold (CT) values recorded at the end of annealing step. A cut-off CT value of 40.0 or below was used to indicate a positive result.P. falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri and P. knowlesi samples were utilized for P. ovale primer specificity testing. The specificity of the P. ovale primers was evaluated by their ability to only amplify P. ovale curtisi and P. ovale wallikeri.P. ovale PET-PCR assay, two-fold serial dilutions of three different blood samples of P. ovale (CDC Nigeria I strain) obtained from three different chimps were prepared using malaria negative whole blood starting from a parasite density of 2000 parasites/μl to 0.98 parasites/μl. The DNA was then extracted from each dilution, aliquoted, and stored until used in the experiments.To determine the limits of detection of the P. ovale PET-PCR assay was determined using 173 previously diagnosed clinical samples. The study primary investigator was blinded during experiments with these clinical samples. Samples were labelled 1–173, and only the supervising investigator was privy to master-key.The clinical sensitivity and specificity for the For calculating sensitivity and specificity, the following equations were utilized: Sensitivity = # of true positives / . Specificity = # of true negatives / .tra) gene, and the reticulocyte binding protein 2 (rbp2) gene.A total of sixteen primer sets were initially designed from selected target candidates: twelve novel primers were designed based on the 18s ribosomal RNA gene (SSU) and four primers were adapted from Miller et al , based oPlasmodium species. Primers pairs that correctly amplified P. ovale and no other species were then evaluated for their ability to amplify both the subspecies of P. ovale. Using these criteria, one primer set based on the rbp2 gene  for all the three different P. ovale preparations with a mean Ct values of 39.47, 39.98, and 39.45.Using a Ct value of 40 as the cut off, the P. ovale curtisi and P. ovale wallikeri, without amplifying any other human Plasmodium species. P. falciparum, P. vivax, P. malariae, P. knowlesi, P. ovale curtisi, and P. ovale wallikeri were utilized for primer specificity testing. Only the positive control , P. ovale curtisi, and P. ovale wallikeri were amplified , P. falciparum, P. vivax, P. malariae, P. knowlesi, and the negative control (no template control).Our goal was to develop primers that could successfully detected both P. ovale PET-PCR primers. Of the 173 clinical samples tested, the assay identified 39 of the 40 P. ovale samples. The novel P. ovale assay identified an additional P. ovale (Ct of 34.15) sample that was identified as a P. falciparum by our reference tests. The remaining non- P. ovale clinical samples did not amplify using our P. ovale specific primers. The sensitivity and specificity of our assay was calculated using a combination of a real-time PCR and nested-PCR as a reference test. A total of 173 clinical samples, consisting of different malaria species (no mixed infections) and non-malaria samples, were used to test the clinical sensitivity and specificity of the P. ovale malaria does not get as much attention as other species of Plasmodium that cause malaria in humans because of lower morbidity and mortality associated with it. Indeed, it is considered by some to be “the benign malaria,” with less complicated course (4). However there have been many well documented case reports of severe complications associated with P. ovale such as acute respiratory distress syndrome (ARDS), acute renal failure (ARF) and splenic infarction [P. ovale infections represent up to 8% of imported malaria cases in Italy (mainly from West Africa) [P. ovale infections imported to the US, Spain, Singapore, and Malaysia from endemic regions [farction –24. Glob Africa) . There h regions –29.P. ovale infections often time have low parasite burdens [P. ovale need to be able to detect low parasite densities. In our study, we successfully designed PET-PCR primers for the detection of both subspecies of P. ovale with good clinical sensitivity (97.5%) and specificity (99.2%). These primers also had good limits of detection, detecting up to 1 parasites per μl. This limit of detection is within the recommended WHO requirements for a nucleic acid tests (2 parasites/μl). The limit of detection of our assay is better than what has been described for microscopy and RDTs [P. ovale infections might go undetected [P. falciparum sample (by our reference tests) as P. ovale (Ct of 34.15). It is possible that this is a true P. ovale sample mixed with P. falciparum and that our novel primers picked it up correctly or that it was a false positive result. Unfortunately, we were not able to retest this sample as we ran out of DNA from that particular clinical sample. With the success of global malaria elimination initiatives, infections with the more common human malaria parasites  have decreased significantly [P. ovale and P. malariae) may increase. Williams et al, demonstrated changing malaria epidemiology, with increasing P. knowlesi incidence following the control of P. falciparum and P. vivax in Sabah, Malaysia [P. ovale circulates, effective malaria control strategies may cause a similar phenomenon leading to increasing P. ovale infection rates. Therefore, the success of any malaria control program requires that effective diagnostic tools are readily available for the detection of any human Plasmodium species, and especially when presented at low parasite densities to allow their accurate detection and prompt treatment. The described P. ovale PET-PCR assay will contribute towards this program. burdens . Therefoand RDTs  being 50detected . Our assMalaysia . In regiP. ovale samples identified in this study into P. ovale curtisi or P. ovale wallikeri either by sequencing or by using primers specific to these subspecies. However, our primer design was aimed at developing primers capable of detecting the two subspecies in the same assay. We were able to demonstrate that the novel primers we developed detect both P. ovale curtisi and P. ovale wallikeri using the known P. ovale curtisi and P. ovale wallikeri DNA kindly provided to us by Dr. Colin Sutherland.There are some limitations to our study. Firstly, we performed retrospective analysis of archived samples available to us from our CDC reference laboratory. It would have been ideal to perform a similar study prospectively. Secondly, the limitation of the achieved samples, all of mono-infections, did not allow us to test the sensitivity and specificity of our primers in mixed infections know to occasionally occur. Finally, we did not further classify the P. ovale. It would be helpful to conduct further evaluation in the field to further validate this assay for large scale future field use.In summary, we have designed specific and sensitive PET-PCR primers that are capable of detecting submicroscopic levels of both the subspecies of


Leishmania kDNA and hybridisation, we were able to identify L. (Viannia) subgenus in 12 out of 173 sandflies captured in the municipality of Rio Branco, Acre state, revealing a positivity of 6.94%. By sequencing the Leishmania 234 bp-hsp70 amplified products from positive samples, infection by L. (V.) braziliensis was confirmed in five sandflies: one Evandromyia saulensis, three Trichophoromyia auraensis and one Pressatia sp. The finding of L. (Viannia) DNA in two Ev. saulensis corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time in Brazil, Th. auraensis and Pressatia sp. infected by L. (Viannia) parasites.Studies on the sandfly fauna to evaluate natural infection indexes are still limited in the Brazilian Amazon, a region with an increasing incidence of cutaneous leishmaniasis. Here, by using a multiplex polymerase chain reaction directed to  Leishmania species with a significant diversity of parasites found in the Amazon Basin (Leishmania (Viannia) braziliensis (Vianna 1911) is the primary widespread etiologic agent of ACL in the country  braziliensis by Nyssomyia whitmani (Antunes & Coutinho 1939), L. (V.) lainsoni  by Trichophoromyia ubiquitalis (Mangabeira 1942) and the transmission of L. (V.) guyanensis (Floch 1954) by Nyssomyia umbratilis (Ward & Fraiha 1977) , characterised by single or multiple skin lesions, has been recorded in all Brazilian states and is undergoing territorial expansion, revealing changes in its epidemiological profile (MS/SVS 2010). In Brazil, the disease is caused by seven on Basin . Except  country . In an e country . RegardiLeishmania spp. in non-blood-fed female sandflies captured in Rio Branco municipality, Acre state, from April 2011 to April 2012, using five HP light traps  directed to Leishmania kDNA  biotinylated probe  females fed on rabbit blood containing 2 × 105L. braziliensis/mL) controls were included. In two out of 173 samples, Leishmania kDNA product (120 bp) was visualised on stained agarose gel. Following hybridisation, the sensitivity increased and ten other samples revealed positive signals, beyond confirming the presence of L. (Viannia) DNA in both sandflies previously detected by gel electrophoresis . Thus, 12 out of 173 sandflies were positive, resulting in a detection rate of 6.94% for the finding of parasite DNA. The two positive samples first detected by PCR and agarose gel were related to forest area in the Bosque district , and corresponded to Th. auraensis (Mangabeira 1942) and Pressatia sp. (Mangabeira 1942). For the ten positive samples identified after hybridisation, two corresponded to Evandromyia saulensis (Floch & Abonnenc 1944) captured in the forest area of Chico Mendes Municipal Park . The remaining eight samples were identified as Th. auraensis from forest area of Bosque district (Area II). We did not find positive insects in the Moreno Maia settlement Leishmaninia kDNA  and to tandflies . The amped probe   obtained from representative strains of different species of Leishmania circulating in Brazil that broadly covers the L. (Viannia) diversity  was used as another source of comparison. For this, we performed clustering analysis to check similarity between samples.DNA samples from the 12 positive female sandflies were submitted to parasite species identification by amplifying the ments da . This spncing da . At leassoftware . Speciessoftware  by compahsp70C PCR and sequencing to identify among L. (Viannia) spp. found in the 12-sandfly DNA extracts, revealed five of them as L. (V.) braziliensis , based on highly stable polymorphisms presented among L. (V.) braziliensis DNA sequences, separating it from other species (da Pressatia sp. (the only positive one) and Th. auraensis (three out of nine positive) collected in Area II, and Ev. saulensis (one out of two positive) from Area I. BLAST analysis revealed only one Th. auraensis specimen identical to L. (V.) braziliensis with 100% identity and E-value of 6.00E-75. The other four samples also matched with L. braziliensis, although with lower identity values varying between 95% and 99%, presenting E-values below 10 (the default threshold) . The DNA fragment used was short (234 bp) but informative to distinguish between L. (Viannia), regarding the current taxonomy for the subgenus, due to polymorphisms detected for each species within this fragment .The results of the 234 bp-ecies da . These sgment da . In our Th. auraensis was the most frequent species collected (243/456 - 53.3%) and nine specimens were find harboring parasite kDNA out of the 96 non-fed blood Th. auraensis females analysed (9.4%). These results contrast with previous work performed in Acre, where this species comprised 19% of all captured sandflies  lainsoni and L. (V.) braziliensis, using a FRET-based real time PCR , this Amazon Region presents a large variety of species, as observed in other areas of primary forest . Th. aurandflies . Curiousandflies . In the time PCR . Other ipophilic , althougpophilic . These fLeishmania DNA found in the present study refer to Ev. saulensis (two positive out of nine submitted to molecular diagnosis - 22.2%) that constituted 3.5% of the total sandfly species collected, followed by Pressatia sp.  representing 3.3% of all species found in the study area. For the genus Evandromyia (Mangabeira 1941), there have been reports of Ev. cortelezzii (Brethes 1923) and Ev. sallesi  naturally infected with L. (L.) infantum in Minas Gerais, Brazil  braziliensis in the municipality of Belo Horizonte, Minas Gerais . Pressatia sp., as other sandflies, are too similar to allow species discrimination based on morphological characteristics and may correspond to subpopulations in the early stages of speciation that have not yet evolved morphological differences (Ev. saulensis and Pressatia sp.The other positive samples for L. (Viannia) DNA in two Ev. saulensis with the confirmation of L. braziliensis in one specimen, as far as we know, corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time, in Brazil, the identification of Th. auraensis and Pressatia sp. infected by L. (Viannia) parasites , corroborating previous investigation carried out in Peru. The role of these species as vectors of parasites responsible for ACL remains to be established for better understanding the risk of New World tegumentary leishmaniasis transmission in the Neotropics.In general, the finding of


New genes are constantly formed, sometimes from non-genic sequences, creating what is referred to as de novo genes. Since the total number of genes remains relatively steady, gene deaths likely balance out new births. In metazoan genomes, microRNAs (miRs) genes, small and non-coding, account for the bulk of functional de novo genes and are particularly suited to the investigation of gene death.Drosophila-specific de novo miRNA (mir-977) that may be facing impending death. Strikingly, after this testis-specific gene is deleted from D. melanogaster, most components of male fitness increase, rather than decrease as had been expected. These components include male viability, fertility and males’ ability to repress female re-mating. Given that mir-977 has a negative fitness effect in D. melanogaster, this de novo gene with an adaptive history for over 60 Myrs may be facing elimination. In some other species where mir-977 is not found, gene death may have already happened.In this study, we discover a The surprising result suggests that de novo genes, constantly rising and falling during evolution, may often be transiently adaptive and then purged from the genome.The online version of this article (10.1186/s12864-018-4755-1) contains supplementary material, which is available to authorized users. Perhaps the most surprising insight to emerge from evolutionary genomics is that while genome sizes fluctuate over several orders of magnitude, gene content remains stable over long evolutionary periods. Given this observation, it is tempting to assume that new genes seldom arise in genomes. However, numerous studies, beginning with  and otheA common form of gene death is akin to “Dead On Arrival” transposons , 19, i.eDrosophila, evolved conservatively for millions of years, but whose evolution sped up in more recent lineages. Although it is tempting to think that these miRNA loci are undergoing gene death, it is also possible that they are adaptively evolving after a shift in their function. To test these possibilities, we deleted one of these miRNAs from the genome and tested the effect of the knockout on a number of fitness components. We found no evidence of a novel function, but abundant reasons to believe that the deletion genotype is more fit that the functional copy, suggesting that this gene is indeed undergoing elimination. Our results suggest that gene death is an important component of genome evolution that can be studied by combining sequence and functional approaches.To study the processes that lead to the death of established new genes, it would be very informative to capture genes as they are being eliminated from the genome. The difficulty is that it is hard to predict from sequence data alone when a gene is on its way out. In a previous survey  we foundmir-977 is based on the phylogenetic history of the entire cluster shown in Fig. Culicinae) genomes as outgroup  togethetinually  . We nowmir-977 locus from the miR-972 cluster, we examined its nucleotide substitution rates among Drosophila lineages . The seed sequence in its mature product is almost identical between the melanogaster and virilis groups. However, younger Drosophila lineages show much faster evolution. Indeed, while the conservation statistic is 0.616 between D. virilis and D. melanogaster, it is 1.096 between D. simulans and D. melanogaster [P < 0.05, Fisher’s exact test). Furthermore, this gene was altogether lost in the D. pseudoobscura lineage [Focusing on the ges Fig. . This X-nogaster  .The 1.8 lineage .mir-977 have also diverged among species. Using public small miRNA-seq data  technology . This apnce Fig. . We thenuct Fig. , while emir-977 deletion in a defined genetic background, we are in a position to assay its fitness effects. Since mir-977 is expressed exclusively in testes, we focused on measuring male fitness components. The same w1118 background is used in the experiments of the mir-977 KO flies and in the wildtype control.Having established a precise mir-977− males. Strikingly, we observed a significant increase  of male fertility in deletion males  compared to control, while slightly, and not statistically significantly, decreasing egg hatchability . It thus appears that the male fertility benefit of the mir-977 deficiency is entirely due to an increase in ovulation induction.To further tease apart individual male fertility components , we counted egg production and egg hatchability separately after mating. Deleting mir-977 is X-linked, it seems plausible that it could exert a fitness effect on the X chromosome at the expense of the Y. We therefore asked whether there is sex-linked meiotic drive associated with this new miRNA [miR977− or wild type males to w1118, mir-977+ females. We then compared the relative abundance of female and male progeny. Their ratio reflects distortion in sex chromosome transmission. We found a slight, but statistically insignificant, sex ratio distortion effect  of mir-977 deletion . Since mir-977 expression is confined to males, its deletion should not affect females. To test this, we counted female survival in the same experiment. We found no significant difference between control and mir-977− females . After 2.5 days, these mated females were presented with reference males for a second mating (Stage II) and their mating rates at stage I and II were measured . Thus, we again see a beneficial effect of mir-977 elimination on male mating success.To assay mating success, ured see ; Fig. 3dmir-977. This microRNA originated not long prior to the origin of the Drosophila clade 60 Myrs ago. While it then evolved conservatively on the deeper branches, it has recently been lost in the D. pseudoobscura lineage and has been evolving fast in the D. melanogaster subgroup. To further probe the possible fate of mir-977 in D. melanogaster, we created a complete knockout of this gene and assayed the effects of the deletion on male fitness. Interestingly, we see a consistent increase in performance of the deficiency across male fitness components, most notably fertility, viability and prevention of female re-mating.To investigate the potential fates of genes that have originated de novo relatively recently in evolutionary time, we focused on mir-977 can be deleterious under some circumstances. However, given the much faster evolution of this gene in younger Drosophila lineages and its complete loss in D. pseudoobscura, impending loss of mir-977 in D. melanogaster appears to be a reasonable expectation. If so, our results suggest that rather than becoming non-functional, this miRNA has turned actively deleterious to male reproductive functions . However, within the tight cluster of miRNA genes in the neighborhood of mir-977, most such deletions may delete (parts of) the neighboring genes and are thus deleterious. Second, given the advantage of the right deletion, its spread in the population would be rapid and the polymorphism of a null mir-977 mutation would last only briefly. In contrast, an observed polymorphism is more likely the indication of a fitness-neutral mutation slowly drifting in the population [Drosophila Population Genomics Project [mir-977 polymorphism. Third, this gene has indeed been eliminated at least twice independently in Drosophila , we didmir-977 to the testes transcriptome? Ecological factors can have changed, rendering its effects deleterious in the new environments. Alternatively, or perhaps additionally, the transcriptional regulatory network topology may have been re-arranged during evolution. Detailed transcriptome studies in multiple species with mir-977 present or absent may answer these questions.Our results suggest a general approach to studying novel gene death. Clearly, sequence evolution alone is insufficient to predict gene fate. However, adding experimental results can yield additional insights. What has happened to shift the fitness contribution of The constant rises and falls of de novo genes suggest that de novo genes do play a role in adaptation. Interestingly, the transient nature of the adaptive functions almost guarantee the controversy surrounding them.w1118 and FM7c (NO.12246)) were obtained from the Bloomington Stock center. The reference line for mating success assays was w1118/Y; miniwhite-UASeGFP/miniwhite-UASeGFP, a red-eyed strain with an insertion of mini-white at the 51D position on chromosome 2 via the PhiC31 site-specific chromosomal integration system. The crossing scheme for mir-977 mutant generation is shown in Fig. Flies were raised in standard media at 25 °C under a 12:12 h light/dark cycle. Stocks . qRT-PCR of miRNAs was conducted using stem-loop reverse transcription  followedCTmethod . 2S RNA mir-977 sequences were retrieved from the current Release 21 of miRBase (http://www.mirbase.org) [http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) [Mature ase.org) . Secondaold.cgi) .D. erecta, since only male whole-body data are available for this species). The data were retrieved from the GEO database ,GSM1165053 for D. simulans, GSM1357621 for D. erecta, and GSM5486109 for D. virilis). miRNA precursors in D. melanogaster were retrieved from miRBase [D. simulans, D. erecta, and D. virilis were obtained from [D. simulans, D. erecta, and D.virilis were supplemented with data from [Expression analyses were based on miRNA deep sequence data, using small RNA libraries from testes  to estimate egg production  and egg hatchability . Twelve replicate crosses were set up for each genotype  of male fertility, each three to five-day-old mir-977 KO or control males (M/Y) to w1118 females (w1118/w1118). In the F1 progeny for each cross, the ratio of w1118/M females to w1118/Y males reflects the distortion in sex chromosome transmission of the mir-977+ or mir-977− chromosome. Twenty replicate crosses were set up for each genotype  to hemizygous mutant or control males (M/Y). The number of M/Y in F1 male progeny in these crosses reflects male viability of control or mir-977 KO males compared to balancer males. As a confirmatory experiment, survival of M/M females reflects female viability of control or mir-977 KO females compared to balancer females. We counted 350 to 500 offspring of each sex and genotype in this assay.Viability assays followed the protocol described in Chen et al. , we crosw1118 female was mated to either a control or a mir-977 KO male (Stage I); after 2.5 days, these mated females were presented with reference males for a second mating (Stage II). Both control and mir-977 KO males were white-eyed but reference males were red-eyed. Mating rates at stages I and II can then be inferred from the eye color of F1 progeny of each mating. F0 females with white-eyed progeny suggest successful fertilization at stage I; F0 females with both white-eyed and red-eyed progeny suggest successful fertilization at stage II. Stage I and II mating rates reflect male mating ability and female receptivity. Forty replicate crosses were set up for each genotype.To survey mating success , each Additional file 1:Figure S1. Independent deletion confirms male fertility increase due to mir-977 deletion. Figure S2. Lifecycle of new gene. Table S1.mir-977 expression in Drosophila species. Table S2. Expression variation of mir-977 in testes across 5 lines of D. melanogaster. Table S3. Male fertility of mir-977 KO. Table S4. Stimulating ovulation and sperm quality of mir-977 KO. Table S5. Meiotic drive of mir-977 KO. Table S6. Viability of mir-977 KO. Table S7. Mating success of mir-977 KO. Table S8. Primers used in this study. Table S9. TALEN pairs design for mir-977. (PPTX 277 kb)Additional file 2:mir-977’s phenotypic effect. (PDF 414 kb)Supplementary Text 1.The choice of genetic background. Supplementary Text 2. Discussion about the off-target effect. Supplementary Text 3. Possible mechanism of Additional file 3:mir-977 KO. Detail components: male fertility (related to Fig. Raw data of male fitness components for


A 41-year-old male presented at Emergency Department (ED) with right flank pain associated with hematuria for 3 days. Patient had a previous history of nephrolithiasis. The physical examination and blood tests were normal. Urine analyses showed haematuria > 1.000.000/μL. After clinical evaluation, a computer tomography (CT) showed right ureteral dilatation caused by a 5 mm proximal stone and a distal intraluminal mass of 8 cm in length. In this setting, an ureteroscopic biopsy was performed and revealed a large polypoid lesion histologically suggestive of fibroepithelial polyp. Due to technical difficulties  and after discussing all available minimally invasive options, we opted for a laparoscopic approach. Instead of ureterectomy of the affected segment of the ureter, as classically performed, we proceeded with an ureterotomy, blunt dissection of the tumor and ureterolithotomy, with complete removal of the mass. This approach did not require ureteral anastomosis and the ureteral dilatation facilitated its primary closure. No complications occurred, even after 3 years of follow-up. Available at: http://www.intbrazjurol.com.br/video-section/20160471-pazeto_et_al/Int Braz J Urol. 2017; 43 (Video #19): 1195-1195


RAS  and BRAF were determined by Sanger sequencing. Tumor mismatch repair proteins and MSI status were examined using immunohistochemistry and polymerase chain reaction, respectively. The clinical value of these abnormalities was statistically analyzed, and prognostic value of different treatment regimens was also evaluated. Among 461 mCRC patients, mutations in RAS, BRAF, and MSI-high (MSI-H) status were observed in 45.3% (209/461), 5.6% (26/461), and 6.5% (30/461) of cases, respectively. Brain metastasis and high carcinoembryonic antigen level were highly correlated with KRAS mutation (P = 0.011 and P < 0.001), and tumors from females or located in the right colon tended to harbor BRAF mutation (P = 0.039 and P = 0.001). RAS/BRAF mutations may predict brain and/or lung metastases. Although neither clinical nor prognostic importance of MSI status was identified in our study, KRAS and BRAF mutations were demonstrated to be independent prognostic factors for overall survival and progression-free survival. Besides, in wild-type group, patients treated with chemotherapy plus targeted therapy exhibited the most favorable prognosis. Therefore, RAS/BRAF mutations may serve as indicators for prognosis and treatment options in mCRC.To investigate whether mutation profiling and microsatellite instability (MSI) status were associated with clinicopathological features and the prognosis in metastatic colorectal cancer (mCRC), mutations in  Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the fourth most frequent cause of cancer-associated mortality worldwide . PreviouEGFR) is an imperative molecular target in mCRC  exhibited a better predictive value compared with single RAS/BRAF mutations  or C-MET expression   .RAS/BRAF mutations were significantly shortened than those of cases with all wild-type. Particularly, cases exhibiting BRAF mutations had the worst prognosis (median OS and PFS: 12.8 months and 8.6 months), instead the any-other-RAS-mutated group had longer median OS and PFS (25.9 months and 21.6 months) than the other two mutational groups  of the enrolled patients had demised during the follow-up period. The median follow-up period was 24.3 months , while 24 (5.2%) patients lost to follow-up. The potential influence of gene mutations and MSI status on survival was assessed with the Kaplan-Meier method. It was concluded that OS and PFS for patients with  Figures . HoweverKRAS or BRAF mutation emerged as an independent risk factor for OS [hazard ratio (HR): 1.826, 95% confidence interval (CI): 1.361–2.450, P < 0.001 and HR: 4.798, CI: 2.989–7.700, P < 0.001; P < 0.001 and HR: 3.864, CI: 2.375–6.287, P < 0.001). In brief, our findings revealed that RAS/BRAF mutations played an essential role in patients' survival.Furthermore, clinical value of various prognostic factors was estimated using Cox proportional hazards model. As confirmed by multivariate analyses, RAS/BRAF mutant group, different treatment regimens showed no significant difference on OS and PFS  received oxaliplatin-based or irinotecan-based chemotherapy, including 159 cases treated with chemotherapy alone, 118 combined with surgery, and 169 combined with targeted therapies . FurtherEGFR agents. Data showed that the disease control rate (DCR) was 72.9% (35/48), with no patient for complete response (CR), 11 patients for partial response (PR), and 24 cases for stable disease (SD) for the first response evaluation at 3 months. In addition, 4 subjects with gene mutations also received cetuximab treatment (1 with BRAF mutation and 3 with KRAS exon 4 mutation), but DCR was 0.0% (0/4). Thus, the DCR and the response rate (including CR and PR) of wild-type patients were relatively better than those of cases with RAS/BRAF mutations (72.9% versus 0.0% and 22.9% versus 0.0%), although no statistical significance was attained.Among wild-type participants, 48 were treated by chemotherapy plus anti-RAS/BRAF mutations and the MSI status in human mCRC has not previously been comprehensively elucidated.As a pathologically and clinically heterogeneous malignancy, CRC presented high aggressiveness and an accompanying worse prognosis on account of its aggressive nature. Despite the complexity of carcinogenesis, the discovery of extensive molecular markers for CRC has attracted special interests. As a result, gene detection has been attached to important connections with CRC evaluation and targeted therapy. However, the predictive and prognostic value of RAS/BRAF abnormalities emerged early in CRC tumorigenesis [KRAS mutations and rare NRAS or HRAS mutation, BRAF mutation showed relative intratumoral homogeneity [RAS/BRAF were not mutually exclusive, although the finding conflicted with several studies from other populations [RAS/BRAF mutations could coexisted. Similarly, our results corroborated the fact. Given increasing data on mutation profiling was accumulated, associations among RAS/BRAF genes will be further expounded.Based on our data, the prevalence of gene mutations or the MSI-H status was in line with previous publications , 22–24. igenesis , and tumigenesis . Differeogeneity , 27. In ulations , 29. Oneulations  proposedRAS/BRAF mutations and MSI status, and results revealed that RAS or BRAF mutation possessed clinical significance in promoting the development and metastasis of mCRC. In brief, KRAS mutations may be important indicators to identify subsets with increased CEA level and brain metastases. The viewpoints were partially different from literatures published, in which KRAS mutations were related to older age, differentiation degree, and later clinical stage [NRAS or HRAS mutations remained controversial due to their rarity. A recent CRC study [NRAS mutations were found to be tilted to the right colon and MSI-L cancers. Nevertheless, no clinical relevance of NRAS mutations was observed in our research; HRAS mutation was too rare to further explore. Recently, Zhang et al. [BRAF mutations were observed more frequently in the right colon and female patients, which supported the conclusions of our study. Particularly, no significant association was found between the MSI status and RAS/BRAF mutations, albeit a recent report [BRAF mutations. This bias might be caused by the limited data and the different detection techniques.Moreover, we characterized al stage , 29, 30.RC study  proposedg et al.  reportedt report  showed tRAS/BRAF, which are driver mutations and represent the principle aspect of gene abnormalities in CRC [RAS/BRAF mutations and MSI status. Numerous experimental model systems have confirmed that RAS/BRAF abnormalities contributed to cell invasion and apoptosis suppression during metastatic cascade, which may bring about organ involvement and tumor progression [KRAS exon 2-mutated CRC patients exhibited an obvious propensity for lung metastases. Similar results have also been described by Morris et al. [RAS/BRAF mutations harbored the trend towards lung metastases. Here, our data for the first time revealed that RAS/BRAF mutations were significant predictors for higher risk of brain metastases, followed by lung metastases, suggesting its value in distinguishing CRC with highly aggressive behavior from low metastatic ability. Thus, the emergence of RAS/BRAF mutations provided powerful insight into the complexity of tumor foci genotype and gained useful clues for treatment option.The initiation and progression of CRC are a multistep process accompanied by inactivation of tumor suppressors and accumulation of gene mutations, especially somatic changes in s in CRC . Anothergression , 32. In gression , KRAS exs et al. , in whicUnfortunately, when it came to the MSI status, neither predictive nor prognostic relevance was observed in mCRC. This phenomenon was concordant with studies issued . But forKRAS was regarded as an adverse predictors for disease-specific survival more early in 1990 [RAS/BRAF aberrations in CRC has spurred much more attention. In agreement with previous series [BRAF mutation, suffered inferior prognosis compared with wild-type counterparts. Interestingly, cases carrying NRAS mutations showed relatively better survival than those with other RAS mutations. Besides that, as the National Comprehensive Cancer Network (NCCN) recommends targeted therapies for mCRC patients, our analysis suggested that chemotherapy combined with targeted therapy could remarkably improve the prognosis of wild-type patients. Importantly, bevacizumab had been considered as an independent prognostic factor according to our data, which accorded with some meta-analyses and randomized controlled trials [RAS/BRAF mutations were emphasized to be predictive biomarkers of resistance to therapies against EGFR, and only wild-type CRC patients may gain survival benefit from cetuximab and panitumumab.Mutation in  in 1990 . Not unts series , 34, ourd trials , 37. MeaOwing to the retrospective nature, there have been inevitably selection bias in our outcomes. Firstly, some participants and their medical record documentation may be lost to follow-up, especially for those who were not hospitalized after first-line chemotherapy. Secondly, the patients were heterogeneous and selected according to availability of molecular detection, which limited the data analyses. Therefore, more prospective studies are required to confirm our conclusions.RAS/BRAF mutations may serve as significant predictors of malignant behavior. Accordingly, radiological diagnosis combined with gene detection may help to evaluate the prognosis of novel CRC cases and devised optimal individualized medicine in the future.Altogether,


RNF213/Mysterin has been identified as a susceptibility gene for moyamoya disease, a cerebrovascular disease characterized by occlusive lesions in the circle of Willis. The p.R4810K (rs112735431) variant is a founder polymorphism that is strongly associated with moyamoya disease in East Asia. Many non-p.R4810K rare variants of RNF213 have been identified in white moyamoya disease patients, although the ethnic mutations have not been investigated in this population. In the present study, we screened for RNF213 variants in 19 Slovakian and Czech moyamoya disease patients. A total of 69 RNF213 coding exons were directly sequenced in 18 probands and one relative who suffered from moyamoya disease in Slovakia and the Czech Republic. We previously reported one proband harboring RNF213 p.D4013N. Results from the present study identified four rare variants other than p.D4013N  in four of the patients. P.V4146A was determined to be a novel de novo mutation, and p.R4019C and p.E4042K were identified as double mutations inherited on the same allele. P.W4677L, found in two moyamoya disease patients and an unaffected subject in the same pedigree, was a rare single nucleotide polymorphism. Functional analysis showed that RNF213 p.D4013N, p.R4019C and p.V4146A-transfected human umbilical vein endothelial cells displayed significant lowered migration, and RNF213 p.V4146A significantly reduced tube formation, indicating that these are disease-causing mutations. Results from the present study identified RNF213 rare variants in 22.2% (4/18 probands) of Slovakian and Czech moyamoya disease patients, confirming that RNF213 may also be a major causative gene in a relative large population of white patients. RNF213/Mysterin was identified as a susceptibility gene for MMD, and its p.R4810K variant (rs112735431) has been shown to be a founder polymorphism that is strongly associated with MMD in East Asia [RNF213 rare variants other than p.R4810K have been identified in MMD patients in ethnically diverse populations, including Asians, whites, and Hispanics, while p.R4810K is absent in non-Asian populations [RNF213 rare variants in MMD patients.Moyamoya disease (MMD) is a progressive cerebrovascular disease characterized by bilateral stenoses of the arteries around the circle of Willis with prominent arterial collateral circulation –3. Recent rs11273431 has bulations . These rRNF213 rare variants in 19 white Slovakian and Czech MMD patients. Results revealed four rare variants, including a novel de novo mutation and a haplotype carrying two mutations.In present study, we screened RNF213 p.D4013N was previously reported by our group [Eighteen Slovakian or Czech probands and one relative with MMD  were recRNF213 using previously described primers [RNF213 p.V4146A locus . The markers were genotyped using ABI Prism Linkage Mapping Set .Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit . Direct sequencing was performed on 69 coding exons of  primers . The amiRNF213 exon 43 and 44, including RNF213 p.R4109C and p.E4042K in II-1 in Family 2 . Randomly selected colonies were prepared for sequencing.Cloning of the Family 2 , was perhttp://www.1000genomes.org/) and the Exome Variant Server (http://evs.gs.washington.edu/EVS/). The effect of the variants on protein function was assayed using two prediction algorithms: Polyphen2 (http:/genetics.bwh.harvard.edu/pph2) and SIFT (http://sift.bii.a-star.edu.sg/). Variant homology was determined using the protein BLAST search engine (http://blast.ncbi.nlm.nih.gov/Blast.cgi).Minor allele frequency (MAF) of variants in the European population was investigated using two variant databases: the 1000 Genomes Project  and Pfu Turbo DNA polymerase . The generated constructs were confirmed by sequencing.us study . The p.DRNF213  plasmids were transfected into 4x105 cells of HUVECs using lipofectamine 3000 (Life Technologies) respectively. The transfected cells were seeded into culture insert , and after 12 hours, the insert was removed to test cell migration. After 8 h incubation, digital images of wound narrowing  were obtained. For tube formation assay, transfected HUVECs  were seeded onto Geltex LDEV Free matrix (Life Technologies) with μ-slide angiogenesis (Ibidi), and after 15h incubation digital images of the formed tubes were captured. To quantify, the area of re-endothelialization (migration assay) or the area and total length of the tubes, and the number of tube branches (tube formation assay) were calculated using ImageJ software .Migration and tube formation were assessed as described previously . Brieflyt-test. Values of P < 0.05 were considered statistically significant.Results are presented as mean ± SD. The number of samples is provided in the figure legends. Statistical tests were performed using unpaired Student’s RNF213 rare variants identified in the present study  are shown in nt study  are descA 21-year-old white female was admitted complaining of a 2-day history of headache, visual problems, and right-hand clumsiness in March 2015. Neurological examination disclosed the presence of severe anomic aphasia, agraphia, semantic memory deficits, right-sided hemianopsia, and visual alexia. Initial native brain computed tomography (CT) showed acute cerebral infarction in the left parietal lobe . Acute cDuplex ultrasound revealed thickening of the carotid bulb in both internal carotid arteries (ICAs) . TranscrWhen analyzing the patient’s previous medical history, we discovered repeated collapses and short periods of muscle tone loss (“drop attacks”), which were initially reported at 9 years of age. These episodes were provoked by various stimuli  and were interpreted as epileptic seizures. At 14 years of age, the patient was admitted to the hospital and MRI was performed. The MRI/MRA images revealed typical findings suggestive of MMD . HoweverRepeated electroencephalograms confirmed epileptiform changes provoked by hyperventilation and photostimulation. Electromyography verified positivity for neurogenic tetania. Valproate was introduced to the treatment strategy. Headache episodes have been mentioned in subsequent years. In June 2014, the patient was admitted to the hospital owing to a sudden onset of fever accompanied by headache and photophobia. Brain CT and CSF examination were interpreted as normal. Laboratory tests and serological findings were negative, and echocardiography and abdominal sonography showed normal results.The patient has three healthy siblings, and both parents are healthy. We performed complex MRI/MRA and sonographic examinations, including duplex sonography and TCCS in all family members without any definite pathological findings.A white female patient suffered from an acute ischemic stroke in the right hemisphere presenting with a severe left-sided hemiparesis at the age of 19 years . After iA white female suffered from repeated stroke and underwent extra-intracranial (EC-IC) bypass with encephalo-duro-angio-myo-synangiosis (EDAMS) on the left side at 31 years of age. She also had a history of arterial hypertension, idiopathic thrombocytopenic purpura, sideropenic anemia, and mild hypercholesterolemia. Repeated percutaneous transluminal angioplasty was performed in both renal arteries, the right subclavian artery, and the mesenteric superior artery between the ages of 38 and 43 years. Cerebral angiography, which was performed at 40 years of age, showed left ICA occlusion with collateral blood flow through the left EC-IC bypass, the anterior communicating artery, and the left posterior communicating artery, as well as stenosis of the right ICA (in the siphon) and aneurysmatic dilation of the top of basilar artery . Additionally, mild post-ischemic changes in the right paraventricular localization were found on brain MRI, which was performed at the same age. One year later, ultrasonographic examination revealed collateral blood flow to the left cerebral hemisphere and the left ophthalmic artery, as well as severe stenosis in the renal arteries. The patient only suffered from intermittent headaches during the follow-up period, and her current neurological status is stable.A white female, the daughter of the II-2 case, developed severe paresis of the left lower limb with gradual regression at the age of 5 years (April 1994). Identical symptoms occurred also in June 1994, and brain CT revealed narrower lateral ventricles. Although she was treated with immunosuppressants (prednisone and azathioprine), repeated paresis occurred during treatment. The patient was diagnosed with arterial hypertension. In December 1994, angiography was performed with normal findings in the renal arteries but resulted in a MMD diagnosis. In March 1995, the patient underwent EDAMS surgery on both sides. In addition to anti-hypertensive medication, anti-platelet therapy was administered. In May 1996, carotid angiography showed a normal post-operative finding. In March 1997, brain CT revealed mild asymmetry of the lateral ventricles; echocardiography revealed mild septal hypertrophy. In January 2000, MRA was performed with normal findings. Depending on the actual blood pressure values, the anti-hypertensive therapy was altered accordingly. In June 2006, ultrasonographic examination verified MMD ; echocardiography showed normal findings. Doppler examination of renal arteries was repeated in December 2007 with normal findings. The patient only suffered from intermittent headaches during the follow-up period; otherwise her current neurological status remains stable. History of Idiopathic thrombocytopenic purpura was absent.RNF213 was performed in 18 probands and one relative with MMD. RNF213 exonic variants found in these MMD patients were shown in RNF213 p.V4146A (T>C), was identified in II-2 of Family 1. However, direct sequencing in the healthy parents, one sister, and two brothers  revealed the wild-type genotype of p.V4146A, and haplotype mapping using microsatellite genetic markers around p.V4146A confirmed parentages and siblingship. Taken together, these data indicate that the mutation is a de novo mutation. II-1 from Family 2 harbored two RNF213 rare variants, including p.R4019C (C>T), which was previously found in white MMD patients [Exon sequencing of RNF213 in mammals , and MAFs of p.W4677L were 1.49% and 1.86% in two European variant databases . P.V4146 mammals . Arginin mammals . Conserv mammals .de novo mutation. Furthermore, both pD4013N and p.R4019C are located in the RING finger domain, which binds E2 and their substrate to function as E3 ligase, while p.V4146A is outside of this domain   . In the l vector . RNF213 observed . Migratiin vitro . Thus, wficantly .de novo mutation, which had not been previously reported. The case harboring p.V4146A was the third reported MMD case with a de novo mutation; the previous described variants were p.K4115del [RNF213 in ECs [RNF213 variants with low penetrance such as p.W4677L are needed.In the present study, we identified four rare variants, p.V4146A, p.R4019C, p.E4042K, and W4677L, in four Slovakian or Czech patients with MMD. P.V4146A was demonstrated to be a K4115del  and p.S4K4115del . Double K4115del . InteresK4115del . AnotherRNF213 variants induced inhibited angiogenesis in ECs. This lower angiogenesis phenotype was reported to be caused by RNF213 p.R4810K [RNF213 variants on angiogenesis. Furthermore, these results strongly support the concept that reduced angiogenesis plays an important role in MMD etiology.Functional analysis of p.D4013N, which was previously identified in a Czech MMD family [p.R4810K ,9, indicRNF213 rare variants identified in the present study were located on the locus corresponding to the region from the RING-finger domain to the C-terminus of the RNF213 protein. This was consistent with characteristics previously described in RNF213 rare variants in Asian and white MMD patients  and was demonstrated to have a strong association with the disease [RNF213 revealed non-R4810K RNF213 rare variants in 22.2% (4/18 probands) of Slovakian or Czech MMD probands (RNF213-associated MMD may be present in a relatively large population of non-Asian MMD cases. This concept was supported by a recent report showing a relatively high frequency of non-R4810K RNF213 rare variants (10.6% (10/94 probands)) in non-Asian MMD patients from North America [Genetic epidemiological studies have demonstrated that  disease . Our preprobands , raisingRNF213 may also be a major causative gene in a relative large population of white patients. Routine screening should be performed for RNF213 rare variants in MMD patients regardless of ethnic background. This could be useful for a definitive MMD diagnosis and also serve to provide a better understanding of MMD etiology.The present study suggests that S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.S8 Fig(DOCX)Click here for additional data file.S9 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.