Cryptococcus neoformans [Extracellular vesicle (EV) release in fungi was described for the first time in 2007 in the yeast-like pathogen oformans . Since tC. neoformans EVs are rapidly disrupted by serum albumin at physiological concentrations [It remains unknown whether fungal EVs are produced in vivo, which is likely linked to the lack of protocols and molecular markers for isolation of these membranous compartments from body fluids. Vesicle properties related to their stability in tissues are also obscure. trations . This obMalassezia sympodialis-associated atopic eczema [C. neoformans suggested that EVs are produced during lung infection [Different studies provided indirect evidence supporting the hypothesis that fungal EVs are produced during infection. Sera from patients with cryptococcosis or histoplasmosis reacted with EV components ,5. In adc eczema . In vivonfection .M. sympodialis, where allergen-containing EVs induced IL-4 and TNF-α responses [C. neoformans and Candida albicans [18, a lipophilic and fluorescent stain, allowed observation of vesicle internalization by murine phagocytes and consequent cytokine production. EVs co-localized with the lipid raft marker GM1, suggesting the participation of such domains during vesicle internalization [18 labeling was restricted to the cytoplasm after 15 minutes of incubation with phagocytes. It remains unclear, however, whether fungal EVs also fuse with host cell membranes, as suggested after interaction of C. neoformans EVs with human brain microvascular endothelial cells (HBMEC) [C. albicans lacking expression of a phosphatidylserine synthase [The immunobiological activity of fungal EVs and the mechanisms by which they modulate host cell physiology have been first explored in esponses . In furtalbicans ,8, labellization ,8. Appar (HBMEC) . In this (HBMEC) . More resynthase . EV cargsynthase . Thus, pC. albicans EVs stimulated dendritic cells (DCs) to produce IL-12p40, IL-10, and TNF-α, and induced upregulation of CD86 and MHC-II [C. neoformans or C. albicans resulted in production of nitric oxide, IL-12, TGF-β, and IL-10 [C. neoformans induced a high proinflammatory response [Galleria mellonella. Treatment of larvae with EVs from C. albicans resulted in significant protection against subsequent challenges with this fungus [The molecules carried by EVs may impact antigen processing and, consequently, the immune response. d MHC-II . Treatmend IL-10 ,8. In ads fungus . These ss fungus ).Exosomes and ectosomes are major EVs produced by eukaryotic cells. Exosomes consist of small (40–100 nm) vesicles originated by invagination of the endosomal compartments membrane, which is driven by a protein complex named endosomal sorting complex required for transport (ESCRT) . This coC. neoformans [Saccharomyces cerevisiae mutants lacking expression of ESCRT machinery proteins still produced EVs, but vesicle cargo was modified in the absence of ESCRT regulators [C. neoformans [C. neoformans [MVB-like structures have been observed in oformans . Sacchargulators . Analysigulators . Groups oformans . Observaoformans . Membranoformans . EV formoformans . All theoformans ).C. neoformans, three hypotheses are suggested to explain how fungal EVs traverse the cell wall [The conclusion that fungal EVs are originated either in the cytoplasm or at the plasma membrane levels implies that, to reach the outer space, passage through polysaccharide layers in the cell wall is required. In ell wall , includiC. neoformans, vesicles interacted directly in the cell wall without any obvious trans-cell wall structures [In contrast to plants, trans-cell wall channels have not been observed in fungal cells. In fact, in ructures ,15, whicructures ,21, but C. neoformans, vesicles were observed next to areas of damaged cell walls [C. albicans, Paracoccidioides brasiliensis, Histoplasma capsulatum, S. cerevisiae, and C. neoformans . It is iewed in . The detrevisiae , C. neofoformans , P. brasliensis,  and H. cpsulatum  is in ag amidase , a cell vesicles . In Streramidase , a peptiynthesis ,25, suggynthesis . These iynthesis ,28. Tranynthesis .Since the first isolation of fungal EVs in 2007, the progress made in the field was unquestionable. It is now clear that fungal EVs are part of a general mechanism of macromolecule export that results in the extracellular release of immunologically active components with the potential to modulate host responses either in favor of infection control or of fungal dissemination. Major questions, however, remain unanswered. The currently available literature clearly points to the need of improvement of protocols supporting the generation of knowledge on the role of fungal EVs in vivo, as well as on their biogenesis pathways and mechanisms by which they reach the outer space. The availability of sophisticated microscopy tools and well-established protocols for genetic manipulation in a number of fungal pathogens supports the notion that, although still long, the way to understanding the biological roles of fungal EVs can be shortened soon.


Churg-Strauss syndrome is a systemic vasculitis accompanied by asthma, eosinophilia and involvement of various organs. It is generally considered a disease of adults with infrequent occurrence in children. We report a patient with Churg-Strauss syndrome manifesting with prominent cardiac and pulmonary involvement.A 17-year-old girl with previous history of asthma, sinusitis and anorexia nervosa. First time admitted to our Institute with a suspicion of infective endocarditis after surgical correction of scoliosis. Two years later, she was admitted for the second time due to dyspnea and hemoptysis with radiographic patchy infiltrates and a peripheral pulmonary nodule complicated by a pleural and pericardial effusions. She also presented, for the first time, purpura and papulonodular skin lesions on both legs and arms.To present clinical and laboratory investigations in Churg-Strauss syndrome in childhood.Routine laboratory tests including serum autoantibody screen, lung CT scan and histopathologic examination of skin.9/L with predominance of eosinophils (22.1%), normocytic anemia , slightly elevated ESR 45 mm/h and C-reactive protein 39.8 mg/L. Serum immunoglobulin were IgA 5.25, IgM 1.78, IgG 20.0 g/l; IgE 3000 IU/ml . Autoantibody screening was negative, ANCA were negative. Serologic tests to hepatitis B and C, HIV were negative. Serum ACE was normal. Pulmonary CT scan findings were consolidation with nodular lesions. Echocardiographic findings were mitral valve insufficiency with pericardial effusion. Electroneurography showed peripheral sensory neuropathy, while the histopathologic evaluation of skin lesions showed leukocytoclastic vasculitis.Laboratory analyses showed an increased WBC count 23.3 × 10As the corticosteroid treatment was started, the remission was accomplished in the following months. Our patient illustrates many of the typical features of Churg-Strauss syndrome. Even though, this syndome is rare in pediatric patients it is important to heighten awareness that this serious disease may affect the pediatric population.None declared.


Yersinia pestis, the causative agent of plague, is via flea bites. Non-pathogenic flea-associated bacteria may interact with Y. pestis within fleas and it is important to understand what factors govern flea-associated bacterial assemblages. Six species of fleas were collected from nine rodent species from ten Ugandan villages between October 2010 and March 2011. A total of 660,345 16S rRNA gene DNA sequences were used to characterize bacterial communities of 332 individual fleas. The DNA sequences were binned into 421 Operational Taxonomic Units (OTUs) based on 97% sequence similarity. We used beta diversity metrics to assess the effects of flea species, flea sex, rodent host species, site (i.e. village), collection date, elevation, mean annual precipitation, average monthly precipitation, and average monthly temperature on bacterial community structure. Flea species had the greatest effect on bacterial community structure with each flea species harboring unique bacterial lineages. The site (i.e. village), rodent host, flea sex, elevation, precipitation, and temperature also significantly affected bacterial community composition. Some bacterial lineages were widespread among flea species (e.g. Bartonella spp. and Wolbachia spp.), but each flea species also harbored unique bacterial lineages. Some of these lineages are not closely related to known bacterial diversity and likely represent newly discovered lineages of insect symbionts. Our finding that flea species has the greatest effect on bacterial community composition may help future investigations between Yersinia pestis and non-pathogenic flea-associated bacteria. Characterizing bacterial communities of fleas during a plague epizootic event in the future would be helpful.The vast majority of human plague cases currently occur in sub-Saharan Africa. The primary route of transmission of  Since 2000, greater than 95% of reported cases of human plague have occurred in sub-Saharan Africa . Models Yersinia pestis, the causative agent of plague, reduces the abundance of or completely eliminates specific bacterial lineages within fleas [Y. pestis [Rickettsia spp., Bartonella spp., Yersinia pestis) in wild fleas has been routine, the characterization of entire bacterial communities of wild fleas has been limited [Oropsylla hirsuta vs. Oropsylla tuberculata cynomuris) and of two more distantly related fleas  were not found to differ [In addition to flea abundance, flea-associated microbial communities may also contribute to plague transmission. in fleas , and exp. pestis . Althoug limited ,10. The o differ ,10. Howeo differ –15, but Wolbachia-positive mosquitos have suppressed rates of infection by dengue virus, Chikungunya virus, West Nile virus, and Plasmodium spp. [Wolbachia-positive mosquitos to a natural population has proven to be an effective means to decrease the number of potential vectors of human disease [Aedes aegypti midguts are significantly higher than titers in A. aegypti with wildtype microbiomes [Y. pestis: exposing ‘germ-free’ fleas to wildtype microbes increases transmission of Y. pestis [Y. pestis eliminates specific bacterial lineages within fleas [Y. pestis may also alter the ability of fleas to transmit Y. pestis and would be a novel research pursuit in the future.Symbionts of disease vectors may mediate the spread of disease through negative or positive interactions with pathogens. ium spp. –18, and  disease . The entrobiomes . These nin fleas . Flea-boIn this study, we characterized the bacterial communities of six flea species collected from nine species of rodents in March 2011 from ten sites in a plague-endemic area of Uganda. For the two most abundant flea species, we analyzed additional samples collected in October and December 2010. Due to our sampling strategy, we can test for the effects of rodent host, flea species, site, environmental conditions, and time on flea-associated bacterial communities.Xenopsylla brasiliensis and Xenopsylla cheopis were examined from the three different collection periods; Ctenophthalmus calceatus cabirus, Dinopsyllus lypusus, Stivalius torvus, and Xenopsylla nubica were examined from the March 2011 collection  was also approved by the Animal Care and Use Committee of the Division of Vector-Borne Disease at the United States Centers for Disease Control and Prevention. Fleas were collected at three times across ten sites in Uganda . Brieflyllection . Fleas aa valida .Fleas were stored in 70% ethanol upon collection. Prior to DNA extraction, individual fleas were surface-sterilized by soaking in 10% bleach for 30 seconds and then washed twice with 100% ethanol. Surface-sterilized fleas were subjected to 20 minutes of mechanical lysis using a Retsch MM301 homogenizer, and then DNA was extracted using the MO BIO PowerSoil-htp 96 Well Soil DNA Isolation Kit  with the standard protocol.We amplified the V1 and V2 hypervariable regions of the 16S rRNA gene using previously described primers: the forward primer (5’-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3’) contains the 16S rRNA gene 27f primer, the 454 Life Sciences primer B sequence, and a two-base ‘TC’ linker; the reverse primer (5′-GCCTCCCTCGCGCCATCAGNNNNNNNNNNNNCATGC TGCCTCCCGTAGGAGT-3′) contains a 12 bp error-correcting barcode, the 16S rRNA gene 338r primer, the Life Sciences primer A sequence, and a two-base ‘CA’ linker . We amplWe analyzed DNA sequence data using QIIME v1.8 . SequencAlpha diversity is a measure of diversity at a local scale ; here itFor beta diversity measurements, we rarefied the dataset to 300 (i.e. 300 DNA sequences were randomly chosen from each flea sample). Pairwise dissimilarity matrices were created in three ways using QIIME v1.8: Bray-Curtis, UniFrac, and Weighted UniFrac –31. ThesC. calceatus cabirus, a Spiroplasma species was also common . Besides the most common Bartonella lineage and the most common Wolbachia lineage, most of the common lineages tended to be dominant community members within one flea species but rare community members in other flea species. Three lineages within the Pasteurellaceae were abundant in one species, but rare or absent in other species , S. torvus, X. brasiliensis, and X. cheopis. Host, site, and collection date also significantly affected bacterial communities in Xenopsylla species, depending on the metric used to compare communities . HoweveWolbachia spp. and Bartonella spp. were commonly found in different flea species, but each flea species also harbored a unique bacterial lineage Click here for additional data file.S2 File(TXT)Click here for additional data file.S3 File(TXT)Click here for additional data file.


Domestic cats enjoy an extensive veterinary medical surveillance which has described nearly 250 genetic diseases analogous to human disorders. Feline infectious agents offer powerful natural models of deadly human diseases, which include feline immunodeficiency virus, feline sarcoma virus and feline leukemia virus. A rich veterinary literature of feline disease pathogenesis and the demonstration of a highly conserved ancestral mammal genome organization make the cat genome annotation a highly informative resource that facilitates multifaceted research endeavors.% of the whole genome, 99,494 new SNVs, 8,355 new indels, 743,326 evolutionary constrained elements, and 3,182 microRNA homologues. The methylation sites study shows that 10.5% of cat genome cytosines are methylated. An assisted assembly of a European wildcat, Felis silvestris silvestris, was performed; variants between F. silvestris and F. catus genomes were derived and compared to F. catus.Here we report a preliminary annotation of the whole genome sequence of Cinnamon, a domestic cat living in Columbia , bisulfite sequencing of Boris, a male cat from St. Petersburg (Russia), and light 30× sequencing of Sylvester, a European wildcat progenitor of cat domestication. The annotation includes 21,865 protein-coding genes identified by a comparative approach, 217 loci of endogenous retrovirus-like elements, repetitive elements which comprise about 55.7The presented genome annotation extends beyond earlier ones by closing gaps of sequence that were unavoidable with previous low-coverage shotgun genome sequencing. The assembly and its annotation offer an important resource for connecting the rich veterinary and natural history of cats to genome discovery. The genome of a female Abyssinian cat  was sequenced at 1.8 × and 3.0 × whole genome shotgun (WGS) coverage at Agencourt Inc. Fca-6.2, an additional 12 × coverage of 454 reads and BAC ends was sequenced, assembled with CABOG [Here we present a genome browser, Genome Annotation Resource Fields — GARfield , which d% of the cat genome is composed of repetitive elements of familiar classes . We report more than 25 novel families of complex tandem repeat elements in the cat genome uncovered by multiple repeat detection algorithms. We searched for STR-microsatellite loci useful in population and forensic applications. Putative PCR primers for 53,710 STR loci are annotated. We also mapped known feline endogenous retroviral loci  and detected 125 kb of partial retroviral genome sequences dispersed across the cat genome. Nuclear mitochondrial (Numt) DNA pseudogenes derived from ancient transposition from cytoplasmic mitochondrial chromosomes to nuclear chromosomal positions comprise 176 kb in addition to the Lopez-Numt, a 7.8 kb element tandem-repeated 38–76 times on Chromosome D2 previously described in the 1.8× analysis of Cinnamon’s genome [Approximately 55.7F. silvestris silvestris, a wild representative of the species from which cats were domesticated approximately 10,000 years ago [F. silvestris and F. catus SNPs are reported here and both species’ genomes and their associated data have been uploaded to the GARfield genome browser (see Availability of supporting data section).The earlier 3,078,438 feline single nucleotide variants (SNVs) [ears ago . Genome % of the human ECE sequence comprising ≈3% of the cat genome. A total of 3,182 feline microRNA (miRNA) homologues were detected and mapped based upon homology to miRNA sequences from 36 species with miRNA sequence described in the miRBase database [Our annotation resolved cat homologues of 743,362 evolutionarily constrained elements (ECEs) recently identified in the human genome by alignment to 29 different mammalian genomes  and thesdatabase . Finallyhttp://garfield.dobzhanskycenter.org and the UCSC Genome Browser (http://genome.ucsc.edu), which links to a Dobzhansky Center Hub (http://public.dobzhanskycenter.ru/Hub/hub.txt) . The annotated features are available in the Genome Association Resource Fields (GARfield) genome browser Sequence and variation data is available in NCBI (SAMN02795853 for Boris the cat and SAMN02898152 for wildcat) and supporting data is also available in the GigaDB repository .ECE: Evolutionary constrained element; SNP: Single nucleotide polymorphism; SNV: Single nucleotide variant; STR: Short tandem repeat.The authors declare that they have no competing interests.F. silvestris. MMR and VAD provided the tissue and cat selection tissue for transcriptome and genome variation discovery. KN provided the tissues and sample for Cinnamon, the individual cat whose genome was assembled. SJO, AS and GT wrote the paper. GT performed gene annotation. SS designed the GARfield genome browser. GT prepared tracks of the annotated genome features. PD, AA and GT described DNA variants (SNVs and indels). AL, AK and GT annotated repetitive elements. JP designed STR primers. AM and GT detected evolutionary constrained elements. AS, AS, KPK and GT annotated endogenous retrovirus-like elements. VB, GT and NC analyzed genome methylation. PD and GT detected microRNA homologues. PD and AA searched for Numts. TMB, CA, BLG, and JQ provided the sequence and analyses of copy number variation for F. catus. PD assembled F. silvestris genome, derived SNVs from it and compared them to F. catus SNVs. All authors read and approved the final manuscript.SJO developed the overall project design. CAD, KB, CB and DG provided the sequence and analyses of Supplementary materials.Click here for file


The dramatic increase in obesity and diabetes worldwide poses a huge socioeconomic burden to healthcare systems. In type 1 diabetes, autoimmune-mediated destruction of pancreatic beta-cell results in insulin deficiency . In typeThe etiology of insulin resistance is complicated and several factors are implicated, so deciphering this multifaceted disease remains challenging, although a wide body of evidence suggests that oxidative stress, inflammation, genetic, habitual, environmental, and epigenetic factors may be involved , 11. Thuγ2 in type 2 diabetes and obesity, while M. Guclu et al. wrote a research article about the effects of combination therapy with rosiglitazone and insulin on inflammatory insults in patients with type 1 diabetes. In another related research article, Z. Yida et al. reported that cotreatment with the cholesterol lowering drug simvastatin and edible bird's nest (EBN), a traditional product commonly consumed in Asia for its nutritional value, improved insulin signaling in a rat model of high-fat diet-induced insulin resistance. Consistently, in another related study, A. Ferreira-Hermosillo et al. gave further insights on the role of inflammatory cytokines in patients with metabolic syndrome. Within the same theme of insulin resistance and metabolic syndrome, R. Adela and S. K. Banerjee wrote a review article to underscore the role of growth-differentiation-factor-15 in diabetes and related cardiovascular diseases, whereas J. Zhang et al. gave further insights on obesity and type 2 diabetes in a research article. By the same token, T.-Y. Chuang et al. reported the effects of microRNA-223 on insulin resistance by studying the adipose tissue. Finally, S. Riaz wrote an article giving novel insights on the role of Vitamin B1 on biomarkers of diabetes type 2 diabetes.This special issue will showcase a broad spectrum research and review papers addressing thematic problems associated with insulin resistance, type 1 diabetes, type 2 diabetes, and related complications. To underscore the role of insulin resistance in children, M. P. van der Aa et al. wrote a research article on the prevalence and incidence of childhood insulin resistance, while S.-H. Nam and coworkers investigated the effects of cardioankle vascular index on metabolic syndrome, a multifactorial condition characterized by insulin resistance, dyslipidemia, hyperglycemia, hypertension, and other factors. Similarly, R. Burrows et al. gave further insights for diagnosing metabolic syndrome in adolescents in a research article. In another related research article, M. Fabregat et al. investigated the genetic profile of human leukocyte antigen (HLA) alleles and non-HLA in type 2 diabetes, while P. Tiwari wrote a systematic review about the current therapeutic strategies for the management of diabetes. Given that diabetes and hypertension are characterized by elevated inflammation/oxidative stress , 12–14 aCollectively, the articles featured in this special issue cover a wide spectrum of thematic issues of great interest and would benefit a wide audience.Joseph Fomusi NdisangJoseph Fomusi NdisangSharad RastogiSharad RastogiAlfredo VannacciAlfredo Vannacci


Cyclocarya Paliurus (Bata1) Iljinskaja contains various bioactive secondary metabolites especially in leaves, such as triterpenes, flavonoids, polysaccharides and alkaloids, and its leaves are widely used as an hyperglycemic tea in China. In the present paper, we sequenced the transcriptome of the leaves and callus of Cyclocarya Paliurus using Illumina Hiseq 4000 platform. After sequencing and de novo assembly, a total of 65,654 unigenes were generated with an N50 length of 1,244bp. Among them, 35,041 (53.37%) unigenes were annotated in NCBI Non-Redundant database, 19,453 (29.63%) unigenes were classified into Gene Ontology (GO) database, and 7,259 (11.06%) unigenes were assigned to Clusters of Orthologous Group (COG) categories. Furthermore, 11,697 (17.81%) unigenes were mapped onto 335 pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG), among which 1,312 unigenes were identified to be involved in biosynthesis of secondary metabolites. In addition, a total of 11,247 putative simple sequence repeats (SSRs) were detected. This transcriptome dataset provides a comprehensive sequence resource for gene expression profiling, genetic diversity, evolution and further molecular genetics research on Cyclocarya Paliurus. Cyclocarya Paliurus (Bata1) Iljinskaja, a unique genus of Juglangdaceae, is a well-known edible and medicinal plant growing in southern China. The leaves of Cyclocarya Paliurus have been often used to produce teas with health benefits, which is named “sweat tea” for its slight sweetness [C. Paliurus exhibits various pharmacological activities such as anti-hypertensive, hypoglycemic, antioxidant and enhancement of mental efficiency [e.g. flavonoids [C. Paliurus leaves [urus Bata Iljinskaweetness . It has ficiency –7, whichavonoids , triterpavonoids , polysacavonoids  and polyavonoids . Among ts leaves , which hs leaves –14, and C. Paliurus is endangered, and there are very few populations scattered in remote forested mountains, which seriously impaired the utilization of this resource [C. Paliurus [in vitro culture of stem segment and obtained a high bud ratio, but found it was hard to root and grow into a tree [C. Paliurus have not been established yet. In order to alleviate the resource shortage, we tried to produce the C. Paliurus secondary metabolites by the plant cell culture. Our group succeeded in inducing callus and establishing the cell culture technology, and found cell culture was a promising way to yield triterpenic acids [C. Paliurus in our recent studies [However, because of the great difficulty in propagation and cultivation, resource . We invePaliurus , and achPaliurus . We studo a tree . Althougic acids , 19. Fiv studies .C. Paliurus secondary metabolites, and very limited information is available about the metabolism and related biosynthetic genes. So far, only one C. Paliurus gene named CpFPS was reported [C. Paliurus.However, there are few studies on the synthetic mechanism of reported . It’s veSalvia miltiorrhiza , Panax notoginseng [Asparagus racemosus [Cunninghamia lanceolata [Gentiana rigescens [Astragalus membranaceus [RNA-seq is a powerful tool for transcriptome analysis based on second-generation sequencing technology, which have already made substantial contributions to our understanding of genome expression and regulation . This teoginseng , Asparagacemosus , Cunningnceolata , Gentianigescens , and Astranaceus .C. Paliurus were obtained using Illumina Hiseq 4000 platform, and a total of 65,654 assembled unigenes were generated and annotated against public protein databases followed by GO, COG and KEGG classification. Moreover, 11,247 putative simple sequence repeats (SSRs) were detected. These transcriptome data provide a valuable public genomic resource for understanding the metabolic mechanisms and facilitating the discovery of genes involved in secondary metabolism pathway and its regulatory, as well as the future gene expression profiling, functional genomic studies of C. Paliurus.In the present study, high-quality transcriptome data of the leaves and callus from C. Paliurus leaves for RNA-seq were harvested from the arboretum of Jiangxi Agricultural University in July. Calluses were induced from the leaves collected between April and May, then inoculated on fresh agar-based MS medium (Murashige and Skoog medium) [® reagent  according the manufacturer’s instructions. The RNA concentration and purity were detected by Nanodrop 2000 , and the quality of RNA was further verified by gel electrophoresis and Agilent 2100. Only high-quality RNA samples  were used to construct sequencing library.The  medium)  suppleme medium) . The colTM RNA sample preparation kit from Illumina . The poly(A) mRNA was isolated from total RNA using Oligo (dT) magnetic beads. Following purification, the mRNA was randomly cleaved into short fragments (100 to 400 bp) after adding fragmentation buffer. These short fragments were used as templates to synthesize the first-strand cDNA using reverse transcriptase and random primers. The second-strand was synthesized subsequently using a SuperScript double-stranded cDNA synthesis kit  with random hexamer primers (Illumina). The cDNA fragments were purified and resolved with EB buffer for end repair and A-tailing addition, and then connected with paired-end adapters. After PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles, the cDNA target fragments of 200–300 bp were size-selected to establish the cDNA library on 2% Low Range Ultra Agarose. After quantification by TBS380, the paired-end RNA-seq sequencing library was sequenced from the 5' to 3' ends using an Illumina Hiseq 4000 platform with the 2×151 bp paired-end read module.RNA-seq transcriptome library was prepared from 5μg of total RNA using TruSeqhttp://trinityrnaseq.sourceforge.net/, Version number: trinityrnaseq, release-20140413) without reference genome [The raw paired-end reads, which were transformed by the Base Calling into sequence data, were cleaned to high-quality reads by removing the joint sequences and adaptor sequences, reads containing more than 10% N rate, and trimming the low-quality reads  from the 3' end of the sequence and the raw reads with an average length less than 30bp. Then, de novo transcriptome assembly was conducted using software Trinity  with the accession numbers (GEUI00000000).De novo assembly was carried out according to the established method , which whttp://www.ncbi.nlm.nih.gov/), Swissprot (http://www.ebi.ac.uk/uniprot/), Pfam (http://pfam.sanger.ac.uk/), String(http://string-db.org/) with identity set at >30% and a cutoff E-value of 10−5, and annotated and classified on Gene Ontology (http://www.geneontology.org/), Clusters of Orthologous Group (http://www.ncbi.nlm.nih.gov/COG/), and the KEGG pathway (http://www.genome.jp/kegg/) with a threshold E-value of 10−5. The aligning results were used to identify the sequence direction and to predict the coding regions. If the aligning results from different databases were conflicted with each other, a priority order of alignments from Nr, SwissPort, KEGG, GO and COG was followed. Based on the Nr annotations, the Blast2GO program was used to obtain GO annotations according to biological process, molecular function and cellular component [C. Paliurus were extracted from Plant Transcription Factor Database (PlantTFDB), and unigenes were mapped to them using Blastn program.All assembled unigenes were aligned with BLASTX program for homology searches against publicly available protein databases Non-redundant (omponent . The unihttp://pgrc.ipkgatersleben.de/misa/) with the default parameters. The parameters were adjusted for identification of perfect di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide, and hexa-nucleotide motifs with a minimum of 6, 5, 5, 5, and 5 repeats, respectively. Primer pairs were designed using Primer 3.0.The unigenes were scanned for microsatellites using the MISA software  unigenes were annotated in Nr database  database, the String and SwissPort protein database, the Gene Ontology (GO) database, the Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database using the BLASTX program with an E-value cut-off of 10database . Beyond database . Furtherdatabase . The simdatabase . Additiodatabase .C. Paliurus unigenes were retrieved using Blast2GO [Cyclocarya Paliurus. Under the Cellular Component classification, the unigenes were mainly related to “cell”  and “cell part” , followed by “organelle”  and “membrane” . Only a few unigenes were assigned to extracellular matrix, extracellular matrix part and collagen trimer. In the Molecular Function category, the majority were assigned to “binding”  and “catalytic activity”  prominently. The above-mentioned findings were similar to the recent report of Chinese Chive transcriptome functional annotation [Gene Ontology (GO) is an international standardized gene functional classification system. The GO terms for Blast2GO . A totalnotation .COG is a database which is widely used to predict and classify functional genes. Every protein in the COG database is assumed to be evolved from an ancestor, and the whole database is built on coding proteins with complete genomes as well as system evolution relationships of bacteria, algae, and eukaryotes , 37. OutC. Paliurus were presented in C. Paliurus leaves and callus. The above mapped information with KEGG pathway database would be very useful for future researches on gene function and its regulatory mechanism of C. Paliurus.The KEGG pathway database provides a wealth of information on molecular interaction and reaction networks t o further understand the biological functions of unigenes. The mapped results indicated that 11,697 (17.81%) unigenes were predominantly annotated with Enzyme Commission (EC) numbers and divided into five branches according to the metabolic pathway  Metabol. It was http://planttfdb.cbi.pku.edu.cn) in this paper  play critical roles in plant growth, bioactive component synthesis and gene expression regulation, especially in the secondary metabolism regulation. Plants show various TFs expression patterns when growing in different environments or facing stress, which further significantly affect the synthesis of secondary metabolites –40. Theris paper . Among ttabolism , 42, esptabolism , alkaloitabolism , 45, andtabolism . The expC. Paliurus leaves ranged from 0.73 to 4.73%, depending on the growing region, harvest time, determination method and so on [C. Paliurus leaves [C. Paliurus  is one of the biggest transcription factor families in plant. This family regulates not only the plant growth and developmental processes including formation of trichome and light signal transduction, but also the stress responses and secondary metabolism , 57. Plabidopsis , OsRa-c bidopsis  and TcJAbidopsis . Furthereracea L –65.Zea mays, which regulated the synthesis of anthocyanin with the synergy of R/B proten family [Arabidopsis thaliana [C. Paliurus. Therefore, more study remains to be done to identify the MYB genes from C. Paliurusfor studying the regulation of flavonoid biosynthesis.It was reported that there were approximately 197 and 155 MYB genes in Arabidopsis and rice, respectively . The firn family . Grotewon family . Accordin family , maize Pn family  and PAP1thaliana , were inC. Paliurus, providing critical information for further regulation study on the gene expression and secondary metabolism. We have established the cell suspension culture of C. Paliurus to produce the bioactive components such as flavonoids, triterpenoids and polysaccharides, and the above mentioned transcription factor information will be helpful to further develop and improve our current approach to achieve a higher yield of these compounds by gene expression regulation. Therefore, we can use genetic engineering for improving plant flavonoid secondary metabolic pathways to effectively increase the content of secondary metabolites by studying the transcription factors involved in flavonoid secondary metabolism, and understanding the mechanisms of plant secondary metabolic regulation.In the present paper, we found many transcription factors involved in flavonoid metabolism of C. Paliurus were scanned to explore the SSR profiles using MISA software and the results were shown in C. Paliurus and its closely related species.SSRs, or microsatellites are important molecular markers for genetics and biology researches, including gene mapping, genetic diversity assessment, comparative genomics, and molecular breeding. In this paper, 65,654 assembled unigene sequences from C. Paliurus is a well-known edible and medicinal plant, but no genomic information is available yet. In this paper, the transcriptome of C. Paliurus leaves and callus without a reference genome was analyzed using Illumina Hiseq 4000 platform. A total of 65,654 assembled unigenes were generated. The annotated unigenes were functionally classified in the GO, COG, and KEGG databases. Moreover, the putative simple sequence repeats (SSRs) were detected. To our knowledge, this is the first attempt to de novo assemble the whole transcriptome of C. Paliurus. This study provided not only a comprehensive enough coverage for gene cloning, expression, and functional analysis, but also a valuable public platform to understand the biosynthesis and regulation of secondary metabolites in C. Paliurus, especially those important bioactive components.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Bemisia tabaci is a genetically diverse complex with multiple cryptic species, and some are the most destructive invasive pests of many ornamentals and crops worldwide. Encarsia sophia is an autoparasitoid wasp that demonstrated high efficiency as bio-control agent of whiteflies. However, the immune mechanism of B. tabaci parasitization by E. sophia is unknown. In order to investigate immune response of B. tabaci to E. Sophia parasitization, the transcriptome of E. sophia parasitized B. tabaci nymph was sequenced by Illumina sequencing. De novo assembly generated 393,063 unigenes with average length of 616 bp, in which 46,406 unigenes  were successfully mapped. Parasitization by E. sophia had significant effects on the transcriptome profile of B. tabaci nymph. A total of 1482 genes were significantly differentially expressed, of which 852 genes were up-regulated and 630 genes were down-regulated. These genes were mainly involved in immune response, development, metabolism and host signaling pathways. At least 52 genes were found to be involved in the host immune response, 33 genes were involved in the development process, and 29 genes were involved in host metabolism. Taken together, the assembled and annotated transcriptome sequences provided a valuable genomic resource for further understanding the molecular mechanism of immune response of B. tabaci parasitization by E. sophia.The whitefly  Bemisia tabaci (Hemiptera: Aleyrodidae), is well known as a worldwide invasive pest and may cause severe damage to various vegetables by feeding on phloem sap and transmitting many viruses [B. tabaci [B. tabaci has demonstrated a remarkable resistance to many groups of chemical insecticides [B. tabaci. Parasitoid or parasitoid–produced regulatory molecules can be used to improve conventional pest control strategies.The whitefly  viruses . It is a viruses . B and Q viruses . There a. tabaci –12. Overcticides –16. Due Encarsia sophia is one of the specific parasitoids of Aleyrodidae species and has been used as efficacious classic biological control agents in many regions [B. tabaci, especially the third and fourth instar nymphs [E. sophia prefers to lay male eggs in the host parasitized by the heterogeneous wasp. When E. sophia and other kinds of wasps are raised or released together, the antecedent colonizers should inhibit the colonization of followers [E. sophia has strong plasticity adaption abilities[Endoparasitoids have been identified as very important natural enemies of various arthropods, and could be used as biological control agents–19. Hyme regions . It can r nymphs . The femr nymphs . Homogenollowers . Previouabilities.However, the relationships between endoparasitoids and their hosts are complicated and involve long-term co-evolution. Many studies have investigated parasitoid biological characteristics, chemical communication, phylogenetic co-evolution, and physiological responses . An incrDrosophila melanogaster and its parasitoid wasp Asobara tabida (Hymenoptera: Braconidae) is a well-studied system. Most genes associated with insect immunity appeared to be differentially expressed after wasp parasitized [Plutella xylostella, Chilo suppressalis, Tenebrio molitor and Octodonta nipae [Eretmocerus mundus may parasitize B. tabaci and induce the specific transcription of functional genes related to immune responses in the host [B. tabaci parasitization by E. sophia has not yet been explored. In this study, we used deep sequencing to explore B. tabaci response to E. sophia parasitization. Our results demonstrate that immune- and metabolic-related genes that are differentially expressed in parasitized versus non-parasitized B. tabaci nymph.RNA-Seq is widely used to obtain transcriptomes of the organism, tissue, or organ, to identify genes that were regulated under certain conditions, and to reveal the regulatory mechanisms in different organisms –39. In rasitized . Most trta nipae –44. A prthe host . HoweverBemesia tabaci was obtained from the greenhouse at the Beijing Academy of Agriculture and Forestry. All experimental populations were derived from one pairs of newly emerged B. tabaci female and male. In our laboratory, the B. tabaci was reared on cotton plants (Zhong-mian-suo 49) in insect proof cages at 26 ± 1°C, and with a photoperiod of 15L: 9D. The purity of the cultures was monitored every three to five generations using the random amplified polymorphic DNA-polymerase chain reaction technique with COI gene [E. sophia was obtained from the greenhouse at Beijing Academy of Agriculture and Forestry. All whitefly instar nymph stages were provided as hosts to E. sophia. Then approximate fifty E. sophia  individuals were released into cages to breed and the newly emerged female and male as parents for five generations breeding.The biotype Q of COI gene . E. sophrd or early 4th instar, they were transferred in culture dish with a piece of cotton leaf, whose petiol were wrapped into soggy cotton, and then the mated E. sophia was released into B. tabaci rearing cage for parasitization. Sixty paired E. sophia were released into one culture dish. Wasp E. sophia were removed after 2 hours parasitization. The first group of samples was collected at 24-hr after parasitization (24AP). At this time period, the parasitoids were at the egg stage in which the embryo had formed and gradually began to move. The brown substance in the egg began to accumulate and chorion had appeared. In other words, the parasitoid possessed immune regulation ability, but the ability was not strong at the egg stage. Therefore, we could identify the immune defense response of the host against the parasitoid. The second sampling period was 72-hr after parasitization (72AP) when the wasps reach larval stage move around and absorb nutrition from the host. At this time, E. sophia may start to regulate host development and metabolism to finish their own development in whiteflies. Each treatment and control had three replicates.Thirty pairs of whiteflies were fed on cotton leaf in a micro insect cage and the fresh cotton leaf were provided every 24 hours. When they had reached later 3™ reagent  according to the manufacturer’s instruction and treated with DNaseI. The concentration and integrity of RNA sample were determined using 2100 Bioanalyzer (Agilent Technologies). The first- and second- strand cDNA synthesis, end reparation, addition of “A” bases to 3' ends, ligation of adapters at the end of DNA fragments, and PCR amplification. The cDNA library was qualified and quantified with an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-time PCR system, respectively, and then sequenced using the Illumina HiSeq™2000 platform at the Beijing Genomics Institute .Total RNA was extracted from all nymph samples using TRIzolhttp://www.ncbi.nlm.nih.gov/sra) with the following accession numbers: SRR1909644 (24AP), SRR1909651(72AP), SRR1909652 (CK-24AP), and SRR1909653 (CK-72AP). All the open reading frames (ORF) of unigene in B. tabaci were identified. If a unigene had many ORFs, we selected the longest one.In order to obtain clean reads, the low quality and adapter-polluted reads were removed from raw data. The good quality reads were assembled using Trinity and assehttp://blast.ncbi.nlm.nih.gov/Blast.cgi), Swiss-Prot (http://expasy.org/tools/blast), Kyoto Encyclopedia of Genes and Genome  databases with an E-value cut-off of 10-5. Gene Ontology (GO) annotation of unigenes was analyzed using the Blast2Go software [B. tabaci and E. sophina genome sequences, we selected eight transcriptome datasets of B. tabaci from the NCBI database, and try to utilize the annotation that were the most closely related to B. tabaci gene in the parasitized library.The unigenes were used for BlastX search and annotation against the NCBI non-redundant (nr)  correction), a q-value less than 0.05 or the absolute value of fold change greater than 2 represented a significant difference between the treatment and the control.In order to find all the differentially expressed genes, the same FPKM (Fragments Per Kilobase per Million fragments) value of unigene was first calculated for the treatment and control groups . The resβ-actin gene was used for normalization. The four RNA samples represented nymphs at 24AP and 72AP, and their respective control (non-parasitized nymphs) at the same developmental stages.The quantitative real-time PCR technique was used to verify the reliability of the deep sequencing. Nine differentially expressed genes were randomly selected. The st Strand cDNA Synthesis Kit (TaKaRa) with oligo (dT)18 as primer following the manufacture’s protocols. The reaction system consisted of 10 μl of SYBR Green, 0.4 μl of ROX, 2 μl of diluted cDNA, 0.4 μl of each primer and 6.8 μl of distilled water. The reactions were loaded on the CFX96™ Real-Time PCR Detection System  under the following conditions: 50°C for 2 min; 95°C for 2 min; and 40 cycles of 95°C for 10s, 60°C for 15s, and 72°C for 20s, followed by melting curve generation (68°C to 95°C). Data analysis was performed by one-way ANOVA following by Tukey’s test using SPSS software.First-strand cDNA was synthesized from the total RNA (1.2 μg) by using PrimeScripTM 1E. sophia parasitization regulated B. tabaci development, immune-response and the differences in regulatory mechanisms between E. sophia egg- and larvae-stages. Approximately, 35 million and 39 million reads were generated from non-parasitized and parasitized B. tabaci nymphs at 24AP, respectively, and 31million and 37 million reads were from non-parasitized and parasitized B. tabaci nymphs at 72AP, respectively. De novo assembly produced 292,696 B. tabaci unigenes with an average size of 616 bp. Of these unigenes, 35.96% were between 200 and 300bp, 27.43% were between 300 and 500bp, 22.65% were between 500 and 1000bp and 13.96% had nucleotide lengths above 1000bp  were successfully mapped. In order to predicate protein function, the unigenes were further given a gene ontology (GO) classification and subjected to KEGG pathway analysis. A total of 35,688 unigenes were annotated and assigned to GO terms, which consisted of three main categories: biological process, cellular component and molecular function. A total of 11,993 unigenes were categorized as cellular components, 12,102 unigenes were grouped under the molecular function, and 11,593 unigenes under biological processes. KEGG pathway analysis indicated that there were 4,721 unigenes assigned to different pathways in which translation, signal transduction, neurodegenerative diseases, infectious diseases, and endocrine system were the main B. tabaci pathways after E. sophia parasitizzation.For functional annotation, the 292,696 unigenes were aligned to the GenBank protein databases with a cut-off E-value of 10B. tabaci, of which 852 genes were differentially up-regulated and 630 genes were differentially down-regulated  were between two- to six-fold higher than at the egg stage (24 AP). Only a few genes changed more than six-fold  at 72AP in RNA-seq analysis. Quantitative RT-PCR analysis  significantly reduces oxidative damage [et al. also discovered that the transcriptional levels of Tenebrio molitor superoxide dismutases were up-regulated following bacterial infection or parasitization by Scleroderma guani [SODC, PERO, and CATA were suppressed two-to four-fold at 72AP, but did not show significant changes at 24AP. At 72AP, the E. sophia reaches the larval stage and the damage to host becomes worse than that of egg stage. The decrease in the transcription of protective enzymes showed the parasitoid immune suppressive strategy.Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) are three common enzymes in organisms. Organisms produce reactive oxygen species (ROS) under environmental stresses, which is cytotoxic to cells. However, the organism utilizes these protective enzymes to eliminate redundant ROS and protect themselves from damage . When Tre damage . Zhu et ma guani . In our et al.[P. xylostella larvae. We also found most of the cytochrome P450 genes were highly expressed after being parasitized by E. sophia. Some genes were over-expressed at 24AP and others were over-expressed at 72AP.In our study, some genes involved in insecticide resistance or detoxification were found to be differentially expressed under parasitization. Although these genes have no direct connection with defense against parasitoids attacking, they can be regarded as a stress response caused by parasitoid secretions. Takeda et al. confirmeD. melanogaster and Anopheles albimanus, and are involved in B. tabaci development. In addition, heat shock 70 kDa protein cognate 3 was found to participate in the immune response. Therefore, we deduced that heat shock protein families can defend the host from damage by participating in the immune response and B. tabaci development.Heat shock proteins (HSPs) are recognized as a family of highly conserved chaperones which respond to all kinds of environmental stress factors, such as heat, toxins, UV radiation, and invading pathogens by protecting protein from misfolding and denaturation . We idenAphidiu servi parasitized Acyrthosiphon pisum late-stage nymph stopped growth [B. tabaci late nymphs parasitized by Encarsia bimaculata also stop growing [Encarsia formosa parasitized Trialeurodes vaporariorum Westwood nymph, the wasp didn’t molt to until host nymph reached to last instar [The parasitoids complete their development by absorbing the host’s hemolymph and tissues. However, the development of the parasitoid and the host are synchronous. A previous study found that d growth . B. taba growing . After Et instar . In ordet instar , while, t instar ,70.P. xylostella after parasitization by D. semiclausum [et al. [C. suppressalis after C. chiilonis parasitization. Based on our transcriptome data, parasitization by E. sophia led to JHEH1 up-regulation at 72AP (Juvenile hormone epoxide hydrolases (JHEHs) have been identified as regulatory proteins in the catabolism of juvenile hormones ,72. A priclausum . However [et al.  discover at 72AP  & Fig 4.SCD1, was induced by insulin, but inhibited by leptin [SCD in B. tabaci nymph were up-regulated 6.69 and 4.52 times, respectively  is an endoplasmic reticulum enzyme that catalyzes the biosynthesis of monounsaturated FA from saturated FA . SCD inay leptin . We founectively . Furthersurvival . Environsurvival . We founE.sophia infection influenced carbohydrate, lipid, and energy metabolism in the host. Some studies have found that trehalose content changed after parasitization [+-transporting ATPase were significantly enhanced. Cytochrome c oxidase is involved in ATP synthesis as a terminase of the mitochondrial inner membrane respiratory chain [tization ,79. In try chain . However+ [+-ATPases at 72AP than at 24AP. This suggests that E. sophia parasitization of B. tabaci involved increased energy consumption. The host was regulated to produce more energy to supply to the parasitoid. Visser et al. found most wasps lacked a lipid synthesis mechanism and could not accumulate energy [There are three types of ion transporting ATPases: P-type, V-type, and F-type. In organisms, their main function is to synthesize ATP and transport H+ . There we energy . TherefoB. tabaci in response to E. sophia parasitization using RNAseq. The most of differentially expressed genes of B. tabaci after parasization have potential roles in immunity, development and metabolism to meet parasitoids needs. The transcriptome profiles provided a basis for future research in elucidate the host-parasitoid interaction. In addition, the identified immune-, development and detoxification–related genes may be target for B. tabaci control.In summary, our study first presented comprehensive transcriptome profiles of


Chrysoporthe austroafricana , C. cubensis  and C. deuterocubensis  were determined. Additionally, the mitochondrial genome of another member of the Cryphonectriaceae, namely Cryphonectria parasitica , was retrieved and annotated for comparative purposes. These genomes showed high levels of synteny, especially in regions including genes involved in oxidative phosphorylation and electron transfer, unique open reading frames (uORFs), ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), as well as intron positions. Comparative analyses revealed signatures of duplication events, intron number and length variation, and varying intronic ORFs which highlighted the genetic diversity of mt genomes among the Cryphonectriaceae. These mt genomes showed remarkable size polymorphism. The size polymorphism in the mt genomes of these closely related Chrysoporthe species was attributed to the varying number and length of introns, coding sequences and to a lesser extent, intergenic sequences. Compared to publicly available fungal mt genomes, the C. austroafricana mt genome is the second largest in the Ascomycetes thus far.In this study, the complete mitochondrial (mt) genomes of  Chrysoporthe species are economically important pathogens of Eucalyptus species and other Myrtales [Chrysoporthe austroafricana [Chrysoporthe cubensis [Chrysoporthe inopina [Chrysoporthe hodgesiana [Chrysoporthe doradensis [Chrysoporthe deuterocubensis [Chrysoporthe zambiensis and Chrysoporthe syzigiicola [Cryphonectriaceae, which also includes Cryphonectria [Chrysoporthe, the relationship among the members of this genus remains largely unresolved [Myrtales , 2. Specafricana , Chrysopcubensis , 5, Chry inopina , Chrysopdgesiana , Chrysopradensis , Chrysopcubensis , Chrysopigiicola . These sonectria , 10. Altresolved , 9.http://www.ncbi.nlm.nih.gov/genome/browse/?report=5). The availability of these genomic resources has enhanced their usage in evolutionary biology [Mitochondrial (mt) sequences have been used to determine and/or confirm phylogenetic relationships, especially in cases where nuclear genes have not accumulated sufficient phylogenetic signal, to clarify conflicting phylogenies , 12. Thi biology –22.atp6, atp8 and atp9), three subunits of cytochrome oxidase , apocytochrome b (cob) and seven subunits of the nicotinamide adenine dinucleotide ubiquinone oxidoreductase subunits . Untranslated genes include the small subunit ribosomal RNA (rns) and the large subunit ribosomal RNA (rnl), as well as a varying number of transfer-RNA (tRNA) genes [Fungal mt genomes are circular A+T rich sequences that usually contain a set of 14 conserved genes that encode proteins involved in oxidative phosphorylation (OXPHOS) and electron transport , 23. TheA) genes , 17. AddA) genes –27. LineA) genes –31.Hanseniaspora uvarum [Sclerotinia borealis [The diversity of mt genomes is evident among Ascomycetes, where the smallest mt genome is 18,512 bp for a uvarum  and the borealis . These sborealis , 34–36. borealis , 22 and borealis .Fungal mt introns encode various homing endonuclease genes (HEGs) , 37–39. Chrysoporthe species have not yet been determined. The aim of this study was therefore, to provide insights into the structure, organization and source of variability in the mt genomes of these closely related species. Additionally, availability of these genomes also provides invaluable resources for evolutionary studies. For this reason, the complete mt genomes of C. austroafricana, C. cubensis and C. deuterocubensis were assembled and annotated. Also, for comparative purposes, the mt genome for C. parasitica was fully annotated. Comparisons were focused on genome organization, gene order and gene content of these four mt genomes.Despite the increasing number, and potential applications, of mtDNA in systems biology and evolutionary studies , 42, 43,C. austroafricana was sequenced as part of a whole genome sequencing project [C. cubensis and C. deuterocubensis mt genome sequences were obtained from subsequent whole genome sequencing projects . The C. parasitica mt genome sequence was obtained from the Joint Genome Institute (JGI) database (http://genome.jgi.doe.gov/Crypa2/Crypa2.download.html). After assembly of the C. austroafricana, C. cubensis and C. deuterocubensis nuclear genomes, the mt genome of C. parasitica was used to identify conserved mt gene regions by reference assembly in CLC Genomics Workbench v 7.0.1 . Identified mt sequences were further confirmed by BLASTn [The mt genome of  project , 45. Addy BLASTn  searchesy BLASTn  and PRICy BLASTn  softwarey BLASTn  where coC. austroafricana, C. cubensis and C. deuterocubensis were annotated using the MFANNOT tool  with default settings. The C. parasitica mt genome was annotated via BLASTn searches using previously published C. parasitica mt genes deposited in GenBank, and completed using the MFANNOT tool. Additionally, open reading frames (ORFs) were predicted in CLC Genomics Workbench v 7.0.1 and compared to those predicted by MFANNOT. ORFs coding for hypothetical proteins were identified by BLASTP against the NCBI with a cut-off of 50% sequence identity. Introns and tRNA genes were characterized using the online program RNAweasel . ORFs encoding HEGs were identified based on their conserved domains through similarity searches in the Pfam (http://pfam.xfam.org/) and InterPro (http://www.ebi.ac.uk/interpro/) protein domain databases, as well as the NCBI (BLASTP) [The mt genomes of (BLASTP) .http://www.bioinformatics.org/sms2/codon_usage.html) with the fungal mt genetic code 4. To identify the presence of direct sequence repeats and inverted sequence repeats in intergenic regions of the mt genomes, REPFIND (http://zlab.bu.edu/repfind/) and EMBOSS [Gene order among the four mt genomes was determined by comparing pairwise BLASTN results using genoPlotR . Furtherd EMBOSS  were useAll phylogenetic analyses in this study were performed using maximum likelihood (ML) method implemented in RAxML . SequencC. austroafricana (GenBank accession no. KT380883), C. cubensis (GenBank accession no. KT380885), C. deuterocubensis (GenBank accession no. KT380884) and C. parasitica (Genbank accession no. KT428651) are circular mtDNA molecules of 190,834 bp, 89,084 bp, 124,412 bp, and 158,902 bp in size, respectively  could be identified. Additionally, the small subunit RNA (rns), large subunit RNA (rnl) and RNA subunit of mt RNase P (rnpB) genes were identified in all four species .All fourteen expected mt protein coding genes  had TAG as the stop codon C. austroafricana, C. deuterocubensis and C. parasitica, respectively. The most frequently used codons in the OXPHOS genes were those that code for hydrophobic amino acids, including Leu (TTA), Ile (ATA) and Phe (TTT), accounting for approximately 10%, 6% and 5% of all codons in all four species , unlike in C. deuterocubensis and C. parasitica where only two tRNA-Arg genes, with anticodons TCT and ACG, were observed. Most anticodons in the tRNA genes had a T or A nucleotide at the third position. Some amino acid codons in the mt genomes lacked corresponding tRNA genes , C. deuterocubensis (intron 3) and C. parasitica (intron 2). Overall, only 17 introns could be considered orthologous based on sequence identity and insertion point in the exonic sequence. Only three introns located in the cox1, cob and cox2 genes were orthologous in all the four mt genomes analysed. Orthologous introns were mostly identified in the mt genomes of the closely related Chrysoporthe species compared to distantly related C. parasitica. BLAST analysis of the identified introns against NCBI GenBank database revealed sequence identities with sequences from other fungal mt genomes , these ORFs seem to be expressed (data not shown). 15, 6, 9 and 17 of these intron encoded ORFs in C. austroafricana, C. cubensis, C. deuterocubensis and C. parasitica respectively were in frame and did not have typical start codons. In the mt genomes of C. austroafricana, C. deuterocubensis and C. parasitica, 8, 2 and 4 introns respectively had two ORFs that encoded either LAGLIDADG or GIY-YIG type HEGs, or both ORFs encoded the same type of HEG  was located immediately after the cob gene in all three mt genomes and was 600 bp in size. Sequence alignment of these three ORFs revealed sequence identities of up to 99%. BLASTP searches of this unique ORF in the NCBI protein database did not reveal sequence alignments with high sequence coverage or identity. Searches for functional domains in Pfam and InterPro protein databases were also unsuccessful for this ORFs.In the mt genomes of C. austroafricana being the largest at 190,834 bp and C. cubensis the smallest at 89,084 bp. The mt genomes with more introns for example 35 and 32 in C. austroafricana and C. parasitica respectively had larger mt genomes compared to C. cubensis and C. deuterocubensis which had far fewer 14 and 19 . Similarly, the C. cubensis, C. deuterocubensis and C. parasitica cox1 gene had 6, 6 and 8 introns, respectively, and this was the largest gene in all of these mt genomes  was located in the C. austroafricana mt genome (intron 2 of the nad2 gene), while the shortest intron was 492 bp located in the cox3 gene of C. parasitica mt genome.Size polymorphism is a prominent feature of fungal mt genomes. Several factors have been associated with size variations including number and size invading introns and expanding intergenic regions. The mt genomes analysed in this study exhibited great size variation with 4 and 19 . In C. a genomes . The eff genomes . Among tC. austroafricana, C. cubensis, C. deuterocubensis and C. parasitica respectively , 38.39% , 45.45%  and 48.30%) . The length of sequences in the region covering the ribosomal RNAs was largest in C. austroafricana and smallest in C. cubensis , 20,458 bp (22.96%), 33,238 bp (26.72%) and 63,373 bp (39.88%) in ectively . The percubensis . To detecubensis . This reC. austroafricana, C. cubensis and C. deuterocubensis were determined. This is the first report of complete mt genome sequences in the genus Chrysoporthe. The sizes of these genomes were within the range of other fungal mt genomes [C. austroafricana, C. cubensis, C. deuterocubensis and C. parasitica, which were 190,834 bp, 89 084 bp, 124 412 bp, and 158 902 bp in size, respectively. Notably, compared to all mt genomes in the NCBI organelle database, the C. austroafricana mt genome is the second largest fungal mt genome to date after that of Sclerotinia borealis [C. austroafricana, C. cubensis, C. deuterocubensis and C. parasitica seem plastic, containing only 40%, 34%, 35% and 42% protein CS, respectively. Large numbers of introns contributed up to 28% of the total genome size, while the remaining portion of noncoding sequence was comprised of highly variable intergenic regions.In this study the mt genome sequences of  genomes , 33. TheChrysoporthe austroafricana, C. cubensis and C. deuterocubensis are closely related species [cox1 gene of C. austroafricana, which was the largest reservoir of 11 introns in all four mt genomes. Comparing the cox1 gene of C. austroafricana to those of C. cubensis, C. deuterocubensis and C. parasitica, the C. austroafricana cox1 gene was more than double in size due to additional introns. This observation was not limited to cox1 genes, but to all other mt genes harbouring these introns. Long stretches of intergenic sequences also seemed to influence the mt genome sizes of these species. In other fungi, these regions harbour ORFs that have been associated with genome size variations [ species , 8, 9. H species , 59, 60. species , 61, 62.riations .C. austroafricana, C. cubensis, C. deuterocubensis and C. parasitica have highly similar gene content. These genomes encode fourteen genes associated with oxidative phosphorylation and electron transport in fungi. Also, genes encoding the rns, rnl, and tRNA genes, were contained in these mt genome sequences. This was consistent with previously described mt genome sequences [rnpB gene that was located in a similar location, immediately before the small subunit ribosomal RNA. BLAST searches for this gene in GenBank did not yield many hits which could be either because it was not identified in the fungal mt genomes. ORFs identified in the Chrysoporthe species mt genomes analysed contained a putative rps5/maturase fusion protein similar to that of previously identified in C. parasitica. Phylogenetic analysis however showed that these rps5/maturase like ORFs grouped together with rps3/HEG fusion proteins. The rps3 gene is commonly identified in other Sordariomycetes [The mt genome sequences of equences , 25. Theomycetes , 24, 42.Sordariomycetes, a comparable level of synteny was observed, especially with regard to genes known to occur in pairs, such as nad4L-nad5, cox3-nad6, nad1-nad4, nad2-nad3, atp8-atp6 and cob-cox1 [Sordariomycetes [C. austroafricana mt genome was due to duplicated tRNAs that were absent in the mt genomes of C. cubensis, C. deuterocubensis and C. parasitica.Gene order in the four mt genomes investigated in the current study was highly conserved, despite the considerable size differences. This was in agreement with previous observations that mt genome size variations do not affect gene order . When cocob-cox1 , 22. A homycetes , 15, 42.nad2 gene in the mt genomes of C. austroafricana, C. cubensis and C. deuterocubensis. This duplication could not be identified in the C. parasitica mt genome, suggesting that it could have occured after the divergence of Chrysoporthe and Cryphonectria. The high sequence identity (between 97–99%) of this duplication in all three genomes of C. austroafricana, C. cubensis and C. deuterocubensis, combined with the high level of synteny around the duplicated gene region, suggests a single duplication event in the most recent common ancestor, which was then transmitted vertically when these species diverged. Notably, the sequences flanking the 5’ region of this ORF included a duplicate tRNA-M gene, which also flanks the 5’ region of the nad2 gene. The occurrence of these duplications in regions around tRNAs is in agreement with studies that have associated tRNA genes with editing, excision and integration capabilities in mt genomes and are also linked to horizontal gene transfer events [Results of this study revealed duplications of some mt genome regions. One such duplication was a 2,718 bp ORF with sequences homologous to the r events .atp8 gene was predicted in the mt genome of C. deuterocubensis and C. parasitica between cox2 and atp9. The absence of this duplication in C. cubensis and C. austroafricana suggests independent evolutionary processes involving either gain or loss after the divergence of these species. The ORFs homologous to nad3 in C. austroafricana and C. parasitica but absent in C. cubensis and C. deuterocubensis could be due to subsequent loss after these species diverged. The presence of these duplications is consistent with what has been reported for Sclerotinia borealis [Fusarium species [An ORF with partially duplicated sequences of borealis  where th species  contribuCryphonectriaceae mt genomes showed known diversity of mitochondrial introns [C. austroafricana and C. parasitica. Intron content showed remarkable diversity especially within the cox1, cob, nad1, nad2 and nad5 genes. Differences in intron content can occur in species and even subspecies groups [Cryphonectriaceae seems to be a common feature with potential influence in mt genome size variations.Fungal mt introns show extensive diversity even among species in the same genus , 24, 25. introns , 39, 62.s groups , 64, 65.Chrysoporthe mt genomes, some of which encoded similar HEGs and had similar insertion points. Introns that lacked sequence similarities could be indicative of independent evolutionary histories involving multiple acquisitions [Chrysoporthe species mt genomes homologous to those of the C. parasitica mt genome was consistent with the notion that introns are either fixed or lost after divergence events [In fungal mt genomes, intron acquisition can occur via both vertical and horizontal transmission, and appears to occur at homologous gene positions , 66, 67.isitions , 36. Thee events , 68.Cryphonectriaceae mt genomes. This, coupled with the low sequence identities (< 45%) of HEGs within each mt genome seems to rule out intragenomic proliferation, indicating that these genes could have diverse origins. Similarly, low sequence identities were observed between intron encoded and free standing HEGs within and between these mt genomes. Therefore, acquisition of these genes seems to have independent evolutionary origins which was then followed by the accumulation of mutations [Fungal mt introns contain ORFs that encode HEGs of the LAGLIDADG or GIY-YIG families , 39, 69.utations , 70.Chrysoporthe spp. This uORF (orf199) was 600 bp long in all three mt genomes, and was located immediately after the cob gene. Comparative analysis using both amino acid and nucleotide sequences could not reveal significant BLAST matches, thus the origin and function of this uORF could not be identified. However, this uORF seems to have been transmitted vertically among the Chrysoporthe spp. from the most recent common ancestor and could potentially be used as a marker for this lineage.In other fungal mt genomes, unique ORFs (uORFs) have been identified and are implicated in genome size expansion , 42. OneC. austroafricana, C. cubensis, C. deuterocubensis and C. parasitica revealed introns and coding sequences as the primary source of genome size variation. Coding sequences included sequences from the 14 OXPHOS genes, intronic ORFs and free standing ORFs. Intergenic sequences seemed to contribute more in the size variation between C. austroafricana and C. parasitica. Also, introns did not seem to contribute much between these two mt genomes which could be attributed to the high number of introns whose size difference was the least compared to C. cubensis and C. deuterocubensis. Generally, the size of the mt genomes seemed to drastically increase with increasing number of introns and coding sequences.Analysis of factors hugely contributing to size polymorphism in the mt genomes of C. austroafricana, C. cubensis, and C. deuterocubensis. The C. austroafricana genome was the second largest fungal mt genome to date, and the large size could be attributed to the numbers and sizes of invading introns varying size of coding sequences and intergenic regions. Despite the size polymorphism, results from comparative analyses of these genomes indicated a conserved gene order and direction of transcription. From all analysed introns, only a few introns were orthologous, while the rest seem to have been acquired independently. The general lack of high sequence identity between proteins sequences of HEGs and uORFs identified in this study but high sequence identity when compared to protein sequences in NCBI GenBank suggest possible acquisition via horizontal transfer. However, further evolutionary analysis is required for these genes. These genomes present invaluable resources for future studies focusing on the evolution and population biology of Chrysoporthe species.In this study we were able to determine the mt genome sequences of S1 Figrns) and dark red; small sub-unit of ribosomal RNA (rnl).Rnpb genes were mannualy retrieved from annotated fungal mt genomes publicly available in GenBank. Phylogenetic analysis was performed using maximum likelihood method implemented in RAxML with HKY model of selection. Branch support was calculated using 1000 bootstrap replicates. Green boxes depict atp6 gene, red; small sub-unit of ribosomal RNA ((PDF)Click here for additional data file.S2 FigC. austroafricana, C. cubensis and C. deuterocubensis which show sequence similarity to C. parasitica S5 ribosomal protein/maturase fusion protein. Sequences used in this phylogeny were retrieved from GenBank using BLAST. The LG+G model of substitution was used. Branch support for was calculated using the bootstrap method with 1000 replicates.Phylogenetic analysis of orf540, orf551 and orf551 from (PDF)Click here for additional data file.S3 Figrnpb gene of Chrysoporthe austroafricana grown in complete and minimal media.The graph shows the average expression for genes that are involved in oxidative phosphorylation and electron transport and the (PDF)Click here for additional data file.S4 FigIntronic ORFs are depicted by yellow arrowed boxes on introns of genes where found and are labelled according to the gene and intron position. Intergenic ORFs are also depicted by yellow arrowed boxes and are labelled with “IN” prefix.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableChrysoporthe austroafricana, C. cubensis, C. deuterocubensis and Cryphonectria parasitica.Comparison of codon usage and tRNAs for the 14 genes involved in oxidative phosphorylation and electron transport in the mitochondrial genomes of (PDF)Click here for additional data file.S3 TableThe best hits for each query (intron) is shown. Default BLAST parameters were used. The prefix CA, CC, CD and CP is added to the intron names for clarity.(PDF)Click here for additional data file.S4 TableResults from all possible comparisons were calculated. Only Blast hits with a threshold of over 50% alignment coverage, 50% sequence identity and E-value of 0.00005 are shown.(PDF)Click here for additional data file.S5 TableHEG type were annotated using the NCBI Conserved Domain Database (CDD).(PDF)Click here for additional data file.


Orbicella faveolata. Samples were collected during a warm water thermal anomaly, coral bleaching event and Caribbean yellow band disease outbreak in 2010 in Puerto Rico. Multiplex sequencing of RNA on the Illumina GAIIx platform and de novo transcriptome assembly by Trinity produced 70,745,177 raw short-sequence reads and 32,463 O. faveolata transcripts, respectively. The reference transcriptome was annotated with gene ontologies, mapped to KEGG pathways, and a predicted proteome of 20,488 sequences was generated. Protein families and signaling pathways that are essential in the regulation of innate immunity across Phyla were investigated in-depth. Results were used to develop models of evolutionarily conserved Wnt, Notch, Rig-like receptor, Nod-like receptor, and Dicer signaling. O. faveolata is a coral species that has been studied widely under climate-driven stress and disease, and the present investigation provides new data on the genes that putatively regulate its immune system.Climate change-driven coral disease outbreaks have led to widespread declines in coral populations. Early work on coral genomics established that corals have a complex innate immune system, and whole-transcriptome gene expression studies have revealed mechanisms by which the coral immune system responds to stress and disease. The present investigation expands bioinformatic data available to study coral molecular physiology through the assembly and annotation of a reference transcriptome of the Caribbean reef-building coral,  Coral reefs are experiencing a dramatic decline in coral cover and reef biodiversity, reports of which have been documented as early as the 1970s . ClimateOrbicella faveolata is an important Caribbean and Atlantic reef-building coral. It has experienced recent population declines and is classified as a threatened species under the federal endangered species act (Next Generation Sequencing (NGS) technologies promise to reveal the genetic mechanisms that control the coral immune system on a whole-genome and whole-transcriptome scale. The versatility of NGS allows for the analysis of samples collected in situ and can thus be used to study physiological responses to natural disease and climate stress events. Several investigations have used NGS to identify putative immunity genes that are differentially expressed under stress and disease . Orbicelcies act . In partcies act . To bettcies act .O. faveolata through a warm water thermal anomaly and bleaching event in 2010 in Puerto Rico. The present RNA-Seq-based investigation sampled Caribbean Yellow Band-Diseased (CYBD), bleached and asymptomatic colonies of O. faveolata during the same event. A reference transcriptome was assembled, annotated, and translated into a predicted proteome. Protein families and signaling pathways that were represented in the transcriptome but that have not been studied previously in the context of coral immune responses to stress and disease were selected for in-depth analysis. Phylogenetic analysis uncovered novel homologues of the Wnt protein family in the O. faveolata transcriptome, the signaling pathway of which is involved in immune cell differentiation and migration. Domain architectures for novel O. faveolata Dicer-like proteins, function in small RNA expression and antiviral immunity, are compared to putative homologues conserved across phyla. Finally, coral-specific Nod-like receptor, Rig-like receptor and Notch signaling pathways are illustrated to support future research on the study intracellular pathogen sensing and wound healing in corals. The results of this work expand current bioinformatic resources available for O. faveolata and present an in-depth analysis of evolutionarily conserved gene sets involved in the regulation of coral innate immunity.Most recently, O. faveolata affected by multiple environmental stressors that are known to induce innate immune responses (2) from four colonies were collected on a single dive at 10 m depth in October 2010 on Media Luna reef in La Parguera, Puerto Rico . Samples were collected under a permit issued by the Department of Natural Resources of Puerto Rico to the Department of Marine Sciences at the University of Puerto Rico at Mayaguez. Reefs in this region experienced ten degree-heating weeks at the time of sample collection. Degree-heating week is a remote sensing metric that estimates accumulated thermal stress in corals during sea surface temperature anomalies . RNA was eluted from the column with nuclease-free water, and RNA quality and concentration were verified by 2% denaturing agarose gel electrophoresis and a NanoDrop spectrophotometer , respectively.The area of each tissue sample was estimated from photographs scaled with millimeter precision using ImageJ software . The ratPRJNA236103 and accession numbers for each sample are reported in O. faveolata transcriptome using 59,114,519 processed sequence reads and default parameters  according to standard protocols of the genomic core facility at the Scripps Research Institute in Ft. Pierce, Florida, USA. All samples were sequenced in multiplex on two lanes of an Illumina flow cell. Raw sequence reads from each sample are available on the NCBI Short Read Archive under Bioproject rameters .Symbiodinium spp. (O. faveolata expression sequence tags (EST) , Acropora digitifera mRNA sequences    . The complete genome of N. vectensis and whole transcriptome of Symbiodinium were used as training sets  , Nematosof 1E-3) , and Hydof 1E-3) . Transcrary file . Sequencing sets . Sequencary file . A prediary file . The preary file .O. faveolata Expressed Sequence Tags (ESTs) in NCBI. O. faveolata ESTs have been submitted to NCBI by various sources. The sequences downloaded and used here are provided as a supplementary file  using default settings for the KEGG Automatic Annotation Server (KAAS) with a minimum BLAST score of 60  terms and protein domain identities to 20,913 (64%) sequences with a maximum E-value of 1E-3 . A summaidarians .O. faveolata, an in-depth comparison to the  and low-density Lipoprotein Receptor (LRP), that transduce signals to control the expression of target genes required for development in a β-catenin-dependent and independent manner . Componevulgaris , from whvulgaris . Thereforiptomes . Howeverillepora  and Acrogitifera , and the pallida . Twelve s genome . These scriptome . Seven und Wnt11 . The extNematostella, Hydra, and Acropora , activates MAPK signaling cascades through MyD88-dependent phosphorylation of Jun-Kinase (JNK). JNK in turn activates Jun and leads to the expression of pro-survival factors, such as Bcl-2 . PRR-induced signals are transmitted by cytosolic kinases, such Mitogen Activated Protein Kinase (MAPK). MAPKs make up a highly conserved family of protein kinases that regulate immune signaling pathways . Signalsas Bcl-2 . In O. fl stress . MechaniO. faveolata transcriptome.Host cells can also detect PAMPs associated with intracellular pathogens by Nod-Like Receptors (NLRs) and Rig-Like Receptors (RLRs) . NLRs deHydra, Nematostella, Acropora, Amphimedon  domains. LRR domains are also hallmarks of membrane-bound TLRs, however, NLRs are located exclusively in the cytoplasm . After pphimedon . In the tivation . Therefoathogens .To date, interactions between the coral host and associated microbes have largely focused on coral-fungal and coral-bacterial interactions . HoweverO. faveolata transcriptomes  and Small Interfering RNA (siRNA) pathways. They have only recently gained attention in corals, and they may have conserved roles in antiviral immunity . The preriptomes . The dicriptomes . RLRs hariptomes . Homologhordates , but theO. faveolata. Putative immunity genes in the coral transcriptome were identified by annotation with gene ontologies and KEGG orthologies. It is well established that Wnt-like proteins are important in coral larval development, but their role in immune cell development and migration remains largely uninvestigated. Phylogenetic analysis of Wnt-like predicted proteins sequences revealed seven novel family members in the O. faveolata transcriptome, which can be used to guide future research on their function in coral innate immunity. Components of the Notch, Nod-like, Dicer-like, and Rig-like signaling pathways reveal possible mechanisms for host cell communication, wound healing, intracellular bacterial and viral recognition. The results of the present investigation provide new data to advance the field of coral innate immunity, which has the ultimate goal of understanding the biological mechanisms that confer resistance or susceptibility of corals to climate-driven stress events and disease outbreaks.The present investigation expands the bioinformatic resources available for the Caribbean reef-building coral, 10.7717/peerj.1616/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.1616/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.1616/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.1616/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.1616/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.1616/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.1616/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.1616/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.1616/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.1616/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.1616/supp-11Supplemental Information 11O. faveolata components were identified by BLASTp alignments to human components of the KEGG Wnt pathway.Click here for additional data file.10.7717/peerj.1616/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.1616/supp-13Supplemental Information 13Click here for additional data file.


E. coli as the etiologic agent of necrotizing fasciitis is a rare occurrence. A 66-year-old woman underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy. She rapidly developed necrotizing fasciitis which led to her death 68 hours following surgery. An E. coli strain was isolated from blood and fascia cultures. DNA microarray revealed the presence of 20 virulence genes.Necrotizing fasciitis is a serious disease characterized by the necrosis of the subcutaneous tissues and fascia. Streptococcus pyogenes [ Staphylococcus aureus [ Streptococcus, S. aureus, Enterobacteriaceae, and some anaerobes [ E. coli is a rare cause of necrotizing fasciitis.Necrotizing fasciitis is a severe disease characterized by the necrosis of the subcutaneous tissues and fascia . Despitepyogenes , Staphyls aureus , or a minaerobes –6. E. co E. coli strain. Its virulence gene complement was determined by DNA microarray.Herein, we report a fatal case of necrotizing fasciitis caused by a highly virulent9/L . At 48 h after surgery, the patient was fully conscious and alert and felt no unusual pain but her temperature was 38.3°C, blood pressure 80/40 mmHg, and heart rate 130 beats/min. An abdominal ultrasound did not reveal anything specific. Upon examination, grey subcutaneous lesions on both hips and buttocks and on the vulva were noted. She was quickly transferred to the operating room where necrotizing fasciitis covering the abdomen, the thorax, the back, and the groins was confirmed. Surgical debridement was initiated but stopped after three hours given the very large extent of the necrotized tissues. She received clindamycin, ceftriaxone, and immunoglobulins before surgical debridement; fluconazole and vancomycin were added after debridement. The patient died 16 hours later, 68 hours following the TAH-BSO.A 66-year-old woman with a prior history of lipectomy and bowel obstruction underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy (TAH-BSO) consecutive to postmenopausal uterine leiomyomata. The complete blood count on admission was within normal range. She received 1 g of cefazolin immediately after surgery and 2 g of cefazolin was added 8 h and 16 h postoperatively. Bruises around the vulva were first noted 18 h following surgery. A bruise on the abdomen and reduced urine flow and oliguria were noted 26 h after surgery despite extensive intravenous rehydration. The patient was afebrile with a blood pressure of 100/80 mmHg and heart rate of 100 beats/min. A new hematological analysis revealed leukocytosis with a white cell count of 13.8 × 10 E. coli by API 20E  with 99.9% certainty and designated LSPQ A134697 . Somatic and flagellar serotypes were identified as O rough:H7, respectively. An antibiotic resistance analysis showed the bacterium to be susceptible to the 14 antibiotics including cefazolin, ceftriaxone, cefotaxime, ceftazidime, cefoxitin, cefotetan, ertapenem, meropenem, imipenem, gentamicin, tobramycin, amikacin, colistin, and ciprofloxacin.Both blood and fascia cultures revealed the presence of a Gram-negative bacillus. It was identified as E. coli LSPQ A134697 genome was further analyzed using an oligonucleotide microarray capable of detecting 189 E. coli virulence genes, according to Bruant et al. [ cnf1 , hlyA (α-hemolysin), hra (heat resistant agglutinin), and vat (vacuolating autotransporter toxin), six adhesins, fimA (subunit of type 1 fimbriae), fimH (adhesin), sfa (S fimbriae), papA , papC (pilus assembly), and papG (specific pilus tip adhesin), one capsule synthesis, kpsMTII (group II capsular polysaccharide synthesis), two siderophores, iroN (catecholate siderophore) and fyuA (Yersinia siderophore receptor), one invasin, ibeA (virulence factor), and six other activities, chuA (heme transport), iss , ompA and ompT (outer membrane proteins A and T (proteases)), traT (surface exclusion), and yjaA (stress-response protein). Conversely, other known virulence genes such as cnf2 , afa , faeG , f17A , cdt , iuc (aerobactin synthesis), iutA (outer membrane receptor protein), and stx1 and stx2 (Shiga toxins I and II) were not detected.Thet et al. . It reve E. coli is a rare occurrence. Chen et al. [ E. coli was found in 2 out of 126 (1.6%) patients with necrotizing fasciitis caused by a single etiologic agent. Necrotizing fasciitis caused by E. coli has been reported from chronically ill patients [ E. coli strains isolated from skin and soft tissue infections, Petkovšek et al. [ cnf1 and hlyA were present in 32 and 30% of the isolates, respectively, and that only 4% of the strains harbored eight or more virulence factors. Recently, Grimaldi et al. [ E. coli. The isolate was capable of producing the Cnf1 toxin and at least nine other virulence factors. Shaked et al. [ E. coli necrotizing fasciitis. All seven patients died during hospitalization, three of them during the first 48 h. The cnf1 gene was found in the E. coli strains from two of the latter three cases. It was not researched in the other four cases. No other virulence factors were researched.Necrotizing fasciitis caused byn et al.  showed tpatients , 24 or ipatients , 26 but k et al.  showed ti et al.  reportedd et al.  reported E. coli-induced necrotizing fasciitis. It spread through the soft tissues ultimately leading to the patient's death, 50 hours following the first apparition of bruises, despite aggressive antibiotic treatment and the bacterium being sensitive to the antibiotics used. DNA microarray revealed the presence of four toxin genes, cnf1, characteristic of necrotoxigenic E. coli (NTEC), a pathotype of extraintestinal pathogenic E. coli (ExPEC), hlyA, hra, and vat. Six fimbrial adhesins and pili revealed here are cell-surface components that facilitate adherence to other cells, an essential step in pathogenesis in addition to 10 additional virulence genes described in We reported here a very rapidly evolving case of an E. coli LSPQ A134697 have been identified in ExPEC strains, most notably in uropathogenic E. coli (UPEC), the causative agent of the vast majority of urinary tract infections, with the exception of the product of the chuA gene, present in enterohemorrhagic E. coli (EHEC). Together, these virulence factors cover a wide range of activities in toxicity, attachment, invasion, immune suppression, bacterial cell viability, and iron scavenging. The unusually large complement of virulence genes found here identifies this E. coli strain as a potentially very aggressive pathogen capable of surviving the host defense mechanisms. Whether some of these virulence factors, alone, or in various combinations with other factors could also provide some novel mechanisms of resistance to the antibiotics used is unknown and worth further investigating.Various subsets of the virulence factors present in E. coli. It lays the foundations for a better understanding of the various mechanisms involved in the very high pathogenicity of this particular and deadly E. coli strain. However, risk factors and interactions with the host can also play a crucial role in infection development and its rapid progression. In this study, the origin of this E. coli strain remains unknown. We are now in the process of determining the nucleotide sequence of its genome. It will be compared with other E. coli genome sequences, including UPEC and non-UPEC strains isolated from humans and animals.The current work was the first to report such a large complement of virulence factors in a fatal case of necrotizing fasciitis caused by


Clostridium sporogenes DSM 795 is the type strain of the species Clostridium sporogenes, first described by Metchnikoff in 1908. It is a Gram-positive, rod-shaped, anaerobic bacterium isolated from human faeces and belongs to the proteolytic branch of clostridia. C. sporogenes attracts special interest because of its potential use in a bacterial therapy for certain cancer types.C. sporogenes is closely related to C. botulinum. The genome of C. sporogenes DSM 795 consists of a circular chromosome of 4.1 Mb with an overall GC content of 27.81 mol% harboring 3,744 protein-coding genes, and 80 RNAs.Genome sequencing and annotation revealed several gene clusters coding for proteins involved in anaerobic degradation of amino acids, such as glycine and betaine via Stickland reaction. Genome comparison showed that The online version of this article (doi:10.1186/s40793-015-0016-y) contains supplementary material, which is available to authorized users. C. sporogenes was isolated from human faeces ) as well as the Sec-specific tRNA (trnU [dark lilac]) as shown in Figure C. sporogenes show identical organization to those identified in C. botulinum and show only slightly differences to those found in E. acidaminophilum [C. botulinum. The glycine reductase cluster of C. sporogenes lacks the second copy of grdA [orange] in comparison to E. acidaminophilum, but contains two paralogs for thioredoxin reductases of the thioredoxin system [blue tones]. These two paralogs could also be identified in the cluster found in C. botulinum. In C. sporogenes the gene cluster coding for the betaine reductase (grdRIH [brown tones]) is much shorter than E. acidaminophilum’s cluster, as genes coding for thioredoxin (trxA [light blue]) and thioredoxin reductase (trxB [dark blue]) as well as the two genes coding for the C-subunit of the reductase (grdCD [green tones]) are not present. The 32.5 kb comprising cluster of S. ovata includes genes coding for the glycine-specific subunit (grdEB [red tones]), genes coding for the betaine-specific subunit (grdIH [brown tones]), two copies of genes coding for the substrate-unspecific subunit C (grdCD [green tones]), and two copies coding for the thioredoxin and a thioredoxin reductase (trxAB [blue tones]). All these genes show identical clustering as identified in the genome of C. sporogenes. The genes coding for proteins necessary for the selenocysteine-incorporation show a different arrangement as identified in C. sporogenes,C. botulinum and E. acidaminophilum, where these genes are organized in a selABCtrnU operon [lilac tones] [grdBE [red tones]) show high sequence homology to genes coding for betaine-specific subunit (grdIH brown tones]).In contrast to sa ovata , in whicnophilum , whereasc tones] . It is aC. sporogenes is able to produce solvents such as ethanol and butanol [C. sporogenesDSM 795 harbors the complete set of genes necessary for glycolysis  as well as aldehyde dehydrogenase and several bifunctional aldehyde-alcohol dehydrogenases, essential for ethanol production. Genes coding for key enzymes of butanol fermentation, such as butyryl-CoA dehydrogenase, acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, several alcohol dehydrogenases, acetate kinase, phosphate acetyltransferase, two copies of butyrate kinase, two copies of phosphate butyryltransferase as well as three copies of formate acetyltransferase are also present . C. botulinum is classified into seven serotypes (A to G) according to the neurotoxin antigenic specificity [C. sporogenes species (ATCC 15579 and PA3679) and available representatives of all serotypes of C. botulinum, except for serotype G, were chosen and retrieved from NCBI  . This number is nearly identical to the quantity of orthologs  found between C. botulinumATCC 3502 and C. botulinum E3 str. Alaska E43. We identified 163, 148, and 125 paralogs in C. sporogenesDSM 795, C. botulinumATCC 3502, and C. botulinum E3 str. Alaska E43, respectively; but these genes were not included into analysis.Phylogenetic analysis based on 16S rDNA revealed that C. sporogenesDSM 795 and C. botulinumATCC 3502 revealed that both organisms have 2,739 orthologs in common, with 818 singletons in C. sporogenesDSM 795 and 672 singletons in C. botulinumATCC 3502. The most important difference between both strains is the presence of the botulinum neurotoxin (BoNT/A) gene cluster in C. botulinumATCC 3502 and its absence in C. sporogenesDSM 795 . As shown in Figure s system  is inserC. sporogenes are closely related to neurotoxin producer C. botulinum. This study presents an overview of physiological, morphological, and genomic characteristics of the type strain C. sporogenesDSM 795. Detailed insight into its proteolytic metabolism was gained on genomic level. Also, the ability of C. sporogenes to produce solvents such as ethanol and butanol was linked to a set of genes and compared to other solventogenic clostridia. Genome comparison of C. sporogenesDSM 795 with two other sequenced strains of this species revealed high similarity. C. sporogenesDSM 795 was also compared at the genomic level with two strains of the close relative C. botulinum.Members of the non-toxic species


Bacillus thuringiensis and Bacillus cereus are two important species in B. cereus group. The intensive study of these strains at the molecular level and construction of genetically modified bacteria requires the development of efficient genetic tools. To insert genes into or delete genes from bacterial chromosomes, marker-less manipulation methods were employed.B. thuringiensis and B. cereus strains that does not leave selection markers. Our approach takes advantage of the relaxase Mob02281 encoded by plasmid pBMB0228 from Bacillus thuringiensis. In addition to its mobilization function, this Mob protein can mediate recombination between oriT sites. The Mob02281 mobilization module was associated with a spectinomycin-resistance gene to form a Mob-Spc cassette, which was flanked by the core 24-bp oriT sequences from pBMB0228. A strain in which the wild-type chromosome was replaced with the modified copy containing the Mob-Spc cassette at the target locus was obtained via homologous recombination. Thus, the spectinomycin-resistance gene can be used to screen for Mob-Spc cassette integration mutants. Recombination between the two oriT sequences mediated by Mob02281, encoded by the Mob-Spc cassette, resulted in the excision of the Mob-Spc cassette, producing the desired chromosomal alteration without introducing unwanted selection markers. We used this system to generate an in-frame deletion of a target gene in B. thuringiensis as well as a gene located in an operon of B. cereus. Moreover, we demonstrated that this system can be used to introduce a single gene or an expression cassette of interest in B. thuringiensis.We present a novel genetic manipulation method for oriT recombination system provides an efficient method for unmarked genetic manipulation and for constructing genetically modified bacteria of B. thuringiensis and B. cereus. Our method extends the available genetic tools for B. thuringiensis and B. cereus strains.The Mob/The online version of this article (doi:10.1186/s12934-016-0492-9) contains supplementary material, which is available to authorized users. Bacillus cereus group currently includes eight closely related species: B. anthracis, B. cereus, B. thuringiensis, B. cytotoxicus, B. mycoides, B. pseudomycoides, B. weihenstephanensis and B. toyenensis + 397.2046 (±20 ppm) was extracted to detect ZmA in each sample [High-resolution liquid chromatography mass spectrometry (LC–MS) was performed using an Agilent 1260 LC device  equipped with a Ch sample , 38.