Phytophthora sojae, which causes soybean stem and root rot.Basic leucine zipper (bZIP) transcription factors are present exclusively in eukaryotes and constitute one of the largest and most diverse transcription factor families. The proteins are responsible for central developmental and physiological processes in plants, animals, and fungi, including the pathogenicity of fungal plant pathogens. However, there is limited understanding of bZIPs in oomycetes, which are fungus-like organisms in the kingdom Stramenopila. Oomycetes include many destructive plant pathogens, including the well-studied species P. sojae and four other oomycetes, two diatoms, and two fungal species were predicted using bioinformatic methods. Comparative analysis revealed expanded numbers of bZIP candidates in oomycetes, especially the Phytophthora species, due to the expansion of several novel bZIP classes whose highly conserved asparagines in basic DNA-binding regions were substituted by other residues such as cysteine. The majority of these novel bZIP classes were mostly restricted to oomycetes. The large number of novel bZIPs appears to be the result of widespread gene duplications during oomycete evolution. The majority of P. sojae bZIP candidates, including both conventional and novel bZIP classes, were predicted to contain canonical protein secondary structures. Detection of gene transcripts using digital gene expression profiling and qRT-PCR suggested that most of the candidates were not pseudogenes. The major transcriptional shifts of bZIPs occurred during the zoosporangia/zoospore/cyst and host infection stages. Several infection-associated bZIP genes were identified that were positively regulated by H2O2 exposure.Candidate bZIPs encoded in the genomes of P. sojae and other oomycetes.The identification of large classes of bZIP proteins in oomycetes with novel bZIP motif variants, that are conserved and developmentally regulated and thus presumably functional, extends our knowledge of this important family of eukaryotic transcription factors. It also lays the foundation for detailed studies of the roles of these proteins in development and infection in The online version of this article (doi:10.1186/1471-2164-14-839) contains supplementary material, which is available to authorized users. Transcription factors (TFs) are key nodes of regulatory networks that bind to the promoter regions of target genes and regulate transcription. The basic leucine zipper (bZIP) family of TFs is one of the largest and most diverse TF families found exclusively in eukaryotes. bZIPs can be identified based on the bZIP domain, which is 60 to 80 amino acids in length and consists of a basic region and a leucine zipper . The basMagnaporthe oryzae, Moatf1 is required for full virulence as it regulates the transcription of laccases and peroxidases to disrupt reactive oxygen species-mediated plant defense [Fusarium oxysporum, the bZIP HapX mediates iron homeostasis and is essential for rhizosphere competence and virulence [The bZIP family is multifunctional across species, and in fungal plant pathogens several genes play a role in regulating pathogenicity. For example, in  defense . MoAP1 i defense . In the irulence .Phytophthora infestans, has been characterized; it is known to interact with a protein kinase and is required for zoospore motility and plant infection [Oomycetes, which include many plant pathogens, are fungus-like organisms that are actually evolutionarily related to brown algae and classified in the kingdom Stramenopila . Currentnfection . Notablynfection . Althougnfection –13, systPhytophthora sojae, one of the most well-studied oomycete plant pathogens, causes stem and root rot of soybean and is responsible for serious crop losses worldwide [P. sojae genome has greatly accelerated the study of Phytophthora-host interactions [P. sojae and another eight species of oomycetes, diatoms, and fungi. In addition to the conventional bZIPs, several novel classes of bZIPs were identified, and their expansion contributed to a larger bZIP family in oomycetes, especially Phytophthora. In addition, phylogenies, secondary protein structures, and gene expression patterns were analyzed and compared between conventional and novel classes of bZIP candidates. These results provide important data for further functional studies of bZIPs and other transcription factor families, and increase our understanding of potential transcriptional regulatory mechanisms in oomycetes.orldwide . The draractions . In thisP. sojae genome, hidden Markov models (HMMs) of the bZIP domain were downloaded from the Pfam database [P. sojae protein sequences [P. sojae genome database  [P. sojae bZIP family proteins, the sequences of the bZIP domains from the 34 confirmed candidates were aligned to build a novel HMM, which was used to re-search the P. sojae protein sequences for further candidates. The process of rebuilding the HMM, confirming candidates with CDD and SMART, and then building a new HMM was repeated until no new proteins were identified. By this process, 45 bZIP TF candidates were obtained. A further 62 proteins were identified from the HMM predictions that were not confirmed by CDD or SMART   or in th3 genes) . To vali3 genes)  and the 3 genes) . Based oP. sojae genome encoded some non-conventional bZIP or bZIP-like class proteins. We hypothesized that some additional bZIP proteins remained that were not detected using the above method that was originally developed for identifying conventional bZIP domains. Therefore, to search for additional variant bZIPs, the 45 full-length protein sequences were used to individually query the P. sojae protein sequences using the blastp program with a strict e-value (1e-40) cutoff. This search produced 34 new protein matches, 23 of which (68%) were present among the 62 sequences detected by the HMM predictions but not confirmed by CDD or SMART analyses, plus another 11 that were completely novel. bZIP domains within these 34 proteins were non-conventional and thus could not be directly predicted using CD-search or SMART. However, in 26 of the 34 proteins, bZIP domain regions could be manually identified from the HMM or blast alignments based on comparisons with conventional bZIP domains. Thus, these 26 candidates were added to the previous set of 45 proteins to form a set of 71 high quality P. sojae bZIP candidates for the purposes of the next steps in the analysis. The pipeline and a summary of the prediction process are listed in Additional file P. sojae introns, e.g., most introns are between 60 and 90 bp, and begin with GT and end with AG [PsBZPc01 to PsBZPc71 . Details of these genes are provided in Additional file Among the 45 candidates, over 30% exhibited substitutions in the conserved asparagine residue within the basic region of the bZIP domain , all ognition . Of the P. sojae, the two major bZIP classes were N-R/K/Y containing 30 proteins  and C-R containing 20 proteins (28%) , all of which belonged to the C-R class , with N-R being the most frequent , two diatoms , and two fungi . Both oomycetes and diatoms belong to the kingdom Stramenopila [To survey the distribution of these novel classes of bZIP TFs in more species, the bZIP prediction pipeline, including the building and updating of species-specific HMMs, was used to predict bZIPs in four other oomycetes  was three-fold greater than that in the diatom T. pseudonana (20 candidates). Candidate numbers were compared in six classes of bZIPs, namely, N-R, C-R, R-R, V-R, N-K, and other , using the “hmmsearch” tool at the HMMER Web server [Phytophthora oomycete species hadn’t been included in UniProtKB when the searches were performed. For comparison, an alignment of N-R class domains from P. sojae, P. ramorum, and P. infestans was also used to create an HMM for querying UniProtKB. Based on a relaxed threshold , the N-R HMM found 5976 matches from 512 species. The top six species were Oryza sativa, Zea mays, Homo sapiens, Takifugu rubripes, Mus musculus, and Arabidopsis thaliana, and each species had more than 120 matches  and R-R (24%). In the non-Phytophthora species, N-R (58%) and V-R (15%) classes accounted for the majority of matches, while the remainder belonged to “other.” Only one C-R class bZIP candidate (Uniprot ID: G9MT11) was found in a non-Phytophthora species, namely the ascomycete fungus Trichoderma virens. This result confirmed that the majority of novel bZIP classes were restricted to oomycete species and that some novel classes contained multiple members.To explore whether non-N-R classes such as C-R were more widely distributed than in the nine species examined using the pipeline, we used an alignment of C-R class bZIP domains collected from b server . UniProtE-value: .0, the NE-value: .0, the NPhytophthora did not affect the sensitivity to sequences from distant species, we used the HMM of the canonical Pfam bZIP domain (ID: PF00170) to search the UniProtKB database. This HMM identified both conventional and novel classes of bZIP domains during the above predictions . In addition, we used our pipeline to catalog all bZIP members in the genomes of Tr. virens and Pe. humanus corporis. However, only the single C-R class bZIP candidate already identified was found in each case. A phylogenetic tree was constructed based on aligned bZIP domains from bZIP candidates of P. sojae, Py. ultimum, H. arabidopsidis, T. pseudonana, and M. oryzae, as well as the two novel C-R domains from non-Phytophthora species. As shown in Additional file Since the sequences of oomycete bZIP domains were unusually diverse, to ensure that seeds  from  ID: PF0070 to sea ID: PF0070 to seaP. sojae bZIPs, we constructed phylogenetic trees either from the full length P. sojae bZIPs or from the bZIP domains alone, the basic regions alone, or the leucine regions alone   show evidence for gene duplications and even in those cases the presence of orthologs of those genes in other Phytophthora or oomycete species  of the % of the P. sojae life cycle, including five free living developmental stages and five host (soybean) infection stages [P. sojae bZIP genes, the transcripts from 6 could not be detected, while the remaining 65 were detected during at least one stage  exhibited reduced transcript levels in zoospore and cyst stages. Cluster-G (18%) exhibited elevated transcript levels in zoosporangia stage and reduced transcript levels in zoospore and cyst stages. Cluster-H (18%) exhibited highly elevated transcript levels during zoospore and cyst stages. Cluster-J (25%) exhibited elevated transcript levels in germinating cysts that remained high during infection. In addition, Cluster-I with 4 members exhibited high transcript levels during cysts and germinating cysts stages. These clusters indicated that the major transcriptional shifts of bZIPs occurred during the zoosporangia/zoospore/cyst and host infection stages.Varied stage-specific expression patterns were observed. Based on hierarchical clustering (HCL), 11 gene expression clusters were obtained. As shown in Figure P. infestans, Pibzp1 was highly expressed during the mycelia stage, but repressed during the zoospore and cyst stages [P. sojae, the homologous gene PsBZPc25 had a similar expression pattern  and 2 43 genes) belonging to each gene expression cluster . PsBZPc65 (induced in germinating cysts), PsBZPc10 , and PsBZPc22 (induced in zoosporangia and 24 h post infection) also showed significantly elevated transcript levels following H2O2 treatment. These data suggest that some infection-associated bZIPs are also regulated by oxidative stress. The other bZIPs assayed either showed small but significant reductions in transcript levels following treatment  suggestporangia .P. sojae bZIPs presumably interact with promoter elements to exert regulatory effects on P. sojae physiology. Recently, several promoter elements have been identified from genome-wide analyses of genes relevant to the spore and infection stages of P. infestans[The infestans, 35. In infestans. Future P. sojae and other oomycetes.In conclusion, the identification of large classes of bZIP proteins in oomycetes with novel bZIP motif variants, that are conserved and developmentally regulated and thus presumably functional, extends our knowledge of this important family of eukaryotic transcription factors. It also lays the foundation for detailed studies of the role of these proteins in development and infection in P. sojae (v1.1), P. ramorum (v1.1), T. pseudonana (v3.0), and P. tricornutum (v2.0) protein were obtained from the DOE Joint Genome Institute (JGI) database (genome.jgi.doe.gov), P. infestans (release of 6/15/2009), F. graminearum (FG3), and M. oryzae (MG8) from the Broad institute database (http://www.broadinstitute.org), Py. ultimum  from the Pythium Genome Database (pythium.plantbiology.msu.edu), and H. arabidopsidis (v8.3) from the VBI Microbial Database (eumicrobedb.org) [Conventional HMM models of bZIP domains were downloaded from the Pfam database at pfam.sanger.ac.uk/clan/bzip . Sequencedb.org) .http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) [The “hmmbuild,” “hmmscan,” and “hmmsearch” tools in the Hmmer v3.0 package and/or its online server  were usepsb.cgi)  and SMARpsb.cgi)  were usehttp://www.drive5.com/muscle) [http://www.megasoftware.net) [P. sojae bZIPs were submitted to NPS@ (using the tool “Secondary structure consensus prediction” at npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html) [Sequence alignments were generated using MUSCLE (/muscle) . The seq/muscle) . Phyloge/muscle)  and grapare.net) . The fulns.html)  to prediP. sojae ESTs were collected from the Oomycete Transcriptomics Database (http://www.eumicrobedb.org/transcripts) [Phytophthora transcriptional database  and the published tag data (NCBI: GSE29651) [http://www.tm4.org/mev.html) [scripts) , and digSE29651) . Gene exev.html) .http://primer3.wi.mit.edu) and blastn was used to check the specificity of the sequence. The primers used in this study are listed in Additional file P. sojae actA gene (GeneID: 108986) was used as a reference to standardize the expression levels. Student’s t test (two tails) was performed to test the significance of differences between controls and treatments.Primers for qRT-PCR assay were designed by Primer3 Additional file 1: Table S1: Prediction pipeline statistics of bZIPs in oomycetes, diatoms, and fungi. P. sojae bZIP candidates. The lines and blocks are proportional to the sequence lengths. (JPEG 527 KB)Additional file 2: Figure S1: Predicted bZIP domains and α-helices of Additional file 3: Figure S2: Phylogenetic tree of bZIP domains from different species. The maximum-likelihood tree was constructed based on the protein sequences of the bZIP domains. Bootstrap values above 50 are shown near the tree nodes. The colors and shapes of gene labels refer to their species and bZIP domain class, respectively. Two C-R class proteins found in non-oomycete species are marked by asterisks and colored by pink. (JPEG 497 KB)P. sojae bZIP candidates. The trees were constructed using a maximum-likelihood method. Bootstrap values above 50 are shown at nodes. A, full-length proteins; B, bZIP domains; C, basic regions only; D, leucine zipper regions only. (JPEG 1000 KB)Additional file 4: Figure S3: Phylogenetic trees built from different regions of P. sojae, P. infestans, and P. ramorum. The trees were constructed using a maximum-likelihood method. Bootstrap values above 50 are shown at nodes. The colors and shapes of gene labels refer to their species and bZIP domain class, respectively. The 1:1:1 orthologous clades and the species-specific clades are respectively marked by different colored stars. (JPEG 451 KB)Additional file 5: Figure S4: Phylogenetic tree from full length bZIP candidate proteins of 2O2 treatment. Data of controls at 30 min (CK-0.5 h) were used as reference and normalized to 1.0 using P. sojae actA (GeneID: 108986) as a reference. Based on the treatment-control comparisons, asterisks indicate >1.2 fold elevation or -reductions respectively that are significant with a t-test P-value <0.05. A, Genes with reduced transcripts at one or more time-points. B, Genes that were not significantly regulated at any time-points. Histograms showing the digital gene expression profiling data from ten developmental and infection stages are shown on the right side of the H2O2 histogram for each gene. The data were averages of three independent replicates. Error bars indicate standard deviations. (JPEG 638 KB)Additional file 6: Figure S5: Relative expression levels of bZIP candidate genes following H


Some species of fungi can cause serious human diseases, particularly to immuno-compromised individuals. Opportunistic fungal infections are a leading cause of mortality, and present an emerging challenge that requires development of new and effective therapeutics. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of cellular protein translation machinery and can be targeted for discovery of novel anti-fungal agents.A. niger, C. albicans and C. neoformans. Hidden Markov Models were built for aaRSs and related proteins to search for homologues in these fungal genomes. Here, we provide a detailed and comprehensive annotation for 3 fungal genome aaRSs and their associated proteins. We delineate predicted localizations, subdomain architectures and prevalence of unusual motifs within these aaRSs. Several fungal aaRSs have unique domain appendages of unknown function e.g. A. niger AsxRS and C. neoformans TyrRS have additional domains that are absent from human homologs.Validation of aaRSs as potential drug targets in pathogenic microbes prompted us to investigate the genomic distribution of aaRSs within three fungi that infect humans – Detailed comparisons of fungal aaRSs with human homologs suggest key differences that could be exploited for specific drug targeting. Our cataloging and structural analyses provide a comprehensive foundation for experimentally dissecting fungal aaRSs that may enable development of new anti-fungal agents.The online version of this article (doi:10.1186/1471-2164-15-1069) contains supplementary material, which is available to authorized users. Fungal infections are emerging as a leading cause of human mortality in hospital settings –5. RecenAspergillus, Candida, and Crytococcus are the three most common genus of fungi associated with infectious diseases in humans [C. albicans. Candida is generally present on the skin and mucous membrane and does not cause infection, however invasive candidiasis occurs when Candida enters the bloodstream and spreads throughout the body [C. albicans infections [Aspergillus species causes second most common fungal infections in humans [A. fumigates is the most virulent species in this genus, and cases of pulmonary aspergillosis have been reported resulting from A. niger infections as well [Cryptococcus neoformans and Cryptococcus gattii[C. neoformans is present in soil and spreads through microscopic airborne fungal spores [C. neoformans infection increases the probability of re-hospitalization of AIDS patients who otherwise show positive response to anti-retroviral therapy [A. niger and C. albicans belong to Ascomycota phylum while C. neoformans belongs to Basidomycota phylum within the fungal kingdom. Genome sizes for A. niger, C. albicans, and C. neoformans are ~33 Mb (19 chromosomes), ~14 Mb (8 chromosomes), and ~19 Mb (14 chromosomes). Predicted number of ORFs (open reading frames) in A. niger, C. albicans, and C. neoformans are ~14000, ~6200, and ~6500. In addition to genome sequencing, transcriptomics analyses for these three fungi are also available [n humans –5. Candithe body . Immuno-fections . Aspergin humans . A. fumi as well . Cryptocs gattii. C. neofl spores . C. neof therapy , 12. A. vailable –15.Leishmania[Toxoplasma[C. albicans SerRS is the only full-length aaRS from these three fungal genomes for which crystal structure has been solved [A. niger, C. albicans and C. neoformans using profile-based Hidden Markov Models (HMM). In addition, homologs for aaRS-related proteins such as the editing domains (AlaX) and D-tyrosyl-deacylase (DTD), Gln and Asn amidotransferases, and aaRS-associated P43 protein have also been identified. Protein sequences of thus identified aaRSs have been comprehensively annotated in context of their domain organization. We discovered novel domain appendages in several fungal aaRSs that are absent from human homologs. Our results highlight key structural attributes of fungal aaRSs that could be exploited for drug targeting to combat mycoses in humans.Aminoacyl-tRNA synthetases (aaRSs) drive protein translational machinery by catalyzing the addition of amino acids to cognate tRNA , 17. Strishmania and Toxoxoplasma have alsn solved . Here, wA. niger and C. albicans were downloaded from their corresponding genome databases available at http://www.aspergillusgenome.org/ and http://www.candidagenome.org/ respectively. Similarly, ORF sequences for C. neoformans were retrieved from FungiDB [E. coli. The UniProt accession numbers for sequences in the dataset are given in supplementary Additional file Computationally translated open reading frames (ORFs) for  FungiDB . To anno FungiDB . Our dathttp://tcoffee.crg.cat/. Homology modeling for protein sequences was performed using Phyre2 server [All protein sequences from our dataset were individually used to perform BLAST searches against non-redundant databases available at NCBI. Top 1000 homologs from each BLAST search were saved for further analyses as we reasoned that these may effectively capture the evolutionary diversity at different positions within aaRSs, thus facilitating generation of robust HMMs. For each aaRS, HMMs were generated using the homologs identified from BLAST search. Sequence Alignment and Modeling Software System (SAM)  was used2 server  and PyMo2 server  was usedA. niger, 28 in C. albicans and 26 in C. neoformans  with 1524 residues and TrpRS (209919-mRNA) with 391 residues respectively , 7 C. albicans aaRSs that are present in two versions , and 5 C. neoformans aaRSs that are present in two versions  were predicted to (co-) localize in cytoplasm, mitochondria and nucleus  were observed in all the three fungal genomes  is frequently observed within mitochondria for synthesis of Gln-tRNAGln[A. niger and C. albicans.We also sought to catalog trans-editing domains within these fungal genomes. D-Tyrosyl-tRNATyr deacylase (DTD) catalyzes hydrolysis of bond between D-amino-acid and tRNA. These enzymes play an important role in evading toxicity resulting from accumulations of D-amino acids within the cell . Trans-eAla. Hence, examined , 51. In s Tables  and 3. Ts Tables  and 3. S-tRNAGln. In addi-tRNAGln. We addiIn some eukaryotes, a few aaRSs associate with each other and with auxillary proteins  to form multi-synthetase complex (MSC) , 54. It A. niger – 14 belonging to class I and 15 to class II. Among class I aaRSs – IleRS, LeuRS, GlnRS, ArgRS, and MetRS had N-terminal catalytic domain followed by anticodon binding domain (ABD) towards the C-terminal  lacks N-terminal RNA binding region that is present in human and other fungal homologs  and mitochondria (209919-mRNA). In case of CysRS, GluRS, MetRS, TrpRS and TyrRS the Pfam server could only annotate their catalytic domains within these sequences. However, based on the catalytic domain positioning it appears likely that their anti-codon binding domains lie towards the C-termini  displays an additional Brix domain at its C-terminus , BLAST searches for 194077-mRNA were performed against the A. niger transcriptome database located at the Broad Institute, USA. A highly significant hit was obtained spanning the fungal full-length protein sequence  domains. AlaRS, PheRS, GlyRS, HisRS, ProRS and ThrRS had catalytic domains at the N-termini of anti-codon binding domain , B (172989-mRNA) and F (173432-mRNA) were also identified  has an additional domain (DUF726) at its C-terminal  that were predicted to localize in cytoplasm (207470-mRNA) and nucleus (55404-mRNA) respectively. These enzymes catalyze the charging of both tRNAspartate . Asp-tRN-tRNAAsn. CuriousA. niger. Interestingly, A. niger P43 has GST C-terminal domain at the N-terminal of the tRNA binding region, which is different from the human counterpart. Indeed, GST_C domain is absent in human P43, however, it is present in P43 homolog from Toxoplasma gondii[T. gondii P43s  within the structurally conserved tRNA binding domain. Further, the N-termini of these proteins show poor homology amongst each other. Finally, GluRS (56891-mRNA), HisRS (51854-mRNA), and LeuRS (52554-mRNA) were observed to have ‘ELR’ motifs in their N-terminal regions  in a gondii  also has anti-codon binding domain at the C-terminal of catalytic domain. Further, cytoplasmic PheRS prototypically encompass α (orf19.2960) and β (orf19.2573) subunits  and orf19.198 (predicted cytoplasm). These two enzymes could act in tandem with two AsxRSs to generate Asn-tRNAAsn in an indirect manner. In addition, subunit components of Gln synthase were also identified – GatA (orf19.3956), GatB (orf19.2494) and GatF (orf19.4092)  shares ~35% identity with human mitochondrial GluRS (which is non-discriminating), raising the possibility of indirect synthesis of Gln-tRNAGln by the action of Gln synthase on Glu-tRNAGln. Standalone trans-editing domains like AlaX (orf19.5239) and DTD (orf19.297) were also identified  and TyrRS (CNBA1270 and CNBJ0260)  and functions . A reces Figure A. IntrigC. neoformans TyrRS part of the fusion protein suggests that the ‘ELR’ motif is likely to be solvent accessible  an interesting target for further investigation. In addition, we noted that ProRS (CNBB0150) also has an ‘ELR’ motif towards its N-terminal  and other two Gln synthases (subunit A (CNBD1400) and subunit B (CNBG4210)). We were not able to identify any homolog for GatF subunit. Interestingly, all these four amidotranferases were predicted to localize within the mitochondria Table . InteresAspergillus, Candida and Cryptococcus are three highly pathogenic fungi, particularly in immuno-compromised patients. Dissecting and understanding critical fungal protein machineries is therefore vital for establishing a base for launching new therapeutics against these pathogens. Within microbial proteomes, aminoacyl-tRNA synthetases have already been exploited for drug discovery [Leu in editing conformation is now an anti-fungal agent in the market [Treatment and prevention of opportunistic fungal infections in humans presents an emerging challenge. iscovery . Specifie market . Hence, A. niger, C. albicans, and C. neoformans there exist 8, 7, and 5 aaRSs that are encoded by two different set of genes, presumably for mitochondrial localization  maybe heterodimeric unlike its homologs in other two fungi where it is likely to be heterotrimeric. None of the aaRS in C. neoformans were predicted to localize within nucleus while 2 and 6 aaRSs in A. niger and C. albicans were predicted to have nuclear localization  schematic representation for genome and transcriptome sequencing analyses for VRS-Brix and (B) modelling for Brix domain from C. neoformans. Figure S3. Schematic representation for genome and transcriptome sequencing analyses for YRS-SAICAR fusion protein in C. neoformans. (DOCX 356 KB)Additional file 1: Table S1 and Table S2: Having the details of the dataset used for analyses and summary of fungal aaRSs with ELR motif, respectively.


Systemic Juvenile Idiopathic Arthritis (sJIA) is a disease with systemic inflammatory features as well as arthritis. Before biologic agents were available, a significant proportion of affected children suffered from poor outcomes. The Childhood Arthritis and Rheumatology Research Alliance (CARRA) Registry is a rich resource of information about pediatric rheumatic diseases including sJIA.We aimed to: (1) describe the characteristics of sJIA children in the CARRA Registry; (2) identify medication usage trends; and (3) identify subgroups at increased risk for poor outcomes.54 active study sites in the US and Canada contributed 528 sJIA patients from 2010-2013. Patients were enrolled as a cross-sectional convenience sample, not an inception cohort. Only children with a complete set of core data were included in these analyses. We tested for subgroup differences among binary and continuous variables across the groups using a chi-square test or one-way analysis of variance (ANOVA) with the null hypothesis of multiple groups being equal.435 had a complete data set at enrollment . On the whole, disease activity was low: 14.9% rash, 6.7% fever, median joint count 0, and median physician global assessment 1. Significant changes in medication usage occurred over the study period 2010-2013: DMARD and TNF inhibitor use decreased, while IL-6 inhibitor use increased. There were significant differences among racial groups with African Americans (AA) having higher mean CHAQ scores, more poor/very poor quality of life scores and poorer ACR functional class (p 0.0004), despite no differences in medication use. Current and prior biologic use was more frequent in those with longer disease duration. Children diagnosed at a younger age (< 2 years old) had more frequent biologic use and lower overall well being scores. Joint damage on imaging was reported more often in those with younger age at diagnosis (p 0.0003). 308 had at least one follow up visit, 259 of whom had visits occurring at least 3 mos from enrollment. Trends towards improvement in disease activity measures were found in these 259 patients. Of 234 children with no systemic features at baseline, 91 had active arthritis (median active joints=4). In this subset of children with persistent arthritis, there were no differences in age at onset, time to diagnosis or disease duration, but differences in medication use at baseline enrollment were noted .This study describes characteristics and medication usage of the largest sJIA cohort reported to date. Significant changes occurred in sJIA medication usage from 2010-2013, but corticosteroids are still frequently used (29.2% at enrollment). AA children have more severe disease, as do children diagnosed at a younger age. A significant proportion of children have persistent arthritis despite the use of new treatments. Further study is needed to identify predictors of persistent arthritis in order to improve treatment and outcomes in this subgroup of patients.G. Janow: None declared., L. Schanberg Grant / Research Support from: Novartis, Consultant for: UCB, Lilly, S. Setoguchi Grant / Research Support from: Novartis, E. D. Mellins* Grant / Research Support from: Novartis, Five Prime, Consultant for: Ascendant, R. Schneider* Grant / Research Support from: Novartis, Consultant for: Novartis, Roche, Y. Kimura* Grant / Research Support from: Novartis, Consultant for: Novartis.


Xylella fastidiosa is a xylem-limited bacterium in plant hosts and causes Pierce’s disease (PD) of grapevines, which differ in susceptibility according to the Vitis species (spp.). In this work we compared X. fastidiosa biofilm formation and population dynamics when cultured in xylem saps from PD-susceptible and -resistant Vitis spp. under different conditions. Behaviors in a closed-culture system were compared to those in different sap-renewal cultures that would more closely mimic the physicochemical environment encountered in planta. Significant differences in biofilm formation and growth in saps from PD-susceptible and -resistant spp. were only observed using sap renewal culture. Compared to saps from susceptible V. vinifera, those from PD-resistant V. aestivalis supported lower titers of X. fastidiosa and less biofilm and V. champinii suppressed both growth and biofilm formation, behaviors which are correlated with disease susceptibility. Furthermore, in microfluidic chambers X. fastidiosa formed thick mature biofilm with three-dimensional (3-D) structures, such as pillars and mounds, in saps from all susceptible spp. In contrast, only small aggregates of various shapes were formed in saps from four out of five of the resistant spp.; sap from the resistant spp. V. mustangensis was an exception in that it also supported thick lawns of biofilm but not the above described 3-D structures typically seen in a mature biofilm from the susceptible saps. Our findings provide not only critical technical information for future bioassays, but also suggest further understanding of PD susceptibility. Xylella fastidiosa is a xylem-limited, Gram-negative bacterium that causes devastating diseases such as Pierce’s disease (PD) in grapevines, citrus variegated chlorosis in sweet oranges, and numerous other diseases in economically important plants, including the recently identified leaf scorch and dieback disease in olive trees in Italy [X. fastidiosa [in Italy , 2. PD sstidiosa –5 and/orstidiosa .Vitis species  are particularly susceptible to PD, whereas others are resistant or tolerant  [In planta studies have demonstrated that X. fastidiosa populations are lower and biofilm is reduced in xylem vessels of certain PD-resistant Vitis spp. [X. fastidiosa mutants with altered biofilm production have been reported to produce modified disease [X. fastidiosa reduces adhesion to surfaces and mature biofilm formation and results in loss of virulence [X. fastidiosa biofilm formation in disease development is well recognized, if and how the pathogen responds to plant signals in susceptible and resistant hosts and subsequently modifies its virulence behaviors, such as biofilm formation, remains unknown. Xylem saps from different grape species have been shown to impact X. fastidiosa growth, aggregation, and biofilm formation [X. fastidiosa-caused diseases.A number of tivalis) –13. In ptis spp. , 14, 15. disease –23. For irulence ; mutatioirulence ; interfeirulence , 23. Altormation –26; howeX. fastidiosa behaviors are often studied in closed culture systems, which may influence observed phenotypes due to nutrient taxing, particularly when conducted in nutrient-poor media such as xylem sap. Under such conditions the observed behaviors may differ from those that occur in the plant xylem vessels where sap is essentially consistently renewed. We therefore evaluated X. fastidiosa phenotypes in sap culture that was systematically renewed and found that X. fastidiosa behaviors differed from those in closed (non-renewed) culture. We also identified phenotype differences when X. fastidiosa was cultured in saps from PD-susceptible and -resistant spp.; more biofilm formation was supported in susceptible saps than resistant ones, which correlates with in planta findings [X. fastidiosa in xylem sap, renewal is necessary to better assess bacterial behaviors. We also found that saps from PD-resistant V. champinii and V. aestivalis supported less X. fastidiosa biofilm development than those from V. vinifera, which correlated to growth differences in those saps.findings , 14, 15.V. vinifera Chardonnay, V. riparia, V. aestivalis and V. champinii vines at the USDA, Plant Genetic Resources Unit, Geneva, NY. Sap collection and storage procedures were as previously reported [V. vinifera Chardonnay and V. arizonica vines was provided by Dr. Andrew Walker , as well as from V. mustangensis and V. cinerea var. helleri vines provided by Dr. Mark Black . V. vinifera sap from New York was used unless otherwise indicated. Sap collections from the same species and location were pooled, filtered-sterilized with a 0.2 μm pore-membrane (Fisher Scientific), aliquoted and stored at -80°C. Aliquots were again filter-sterilized prior to use.Grapevine xylem fluid was collected in early spring from bleeding reported . AdditioX. fastidiosa strain Temecula 1 (TM1) was maintained at 28°C on PW (Periwinkle Wilt) agar [-1 of bovine serum albumin (BSA) fraction V solution (Life Technologies). The TM1 enhanced green fluorescent protein (eGFP)-tagged strain KLN59.2 [μg mL-1 kanamycin. The bacteria were stored at -80°C in modified PW broth [Wild-type  KLN59.2  was cultVitis spp. affected biofilm formation by X. fastidiosa. Cells from seven to 10-day-old X. fastidiosa cultures were collected from PW plates, acclimated to V. vinifera sap by serial daily passages in 50:50, 80:20, 90:10 and finally 100% sap:PD2 broth (vol:vol) as previously described [600 = 0.05. In each passage cells were pelleted by 10,000 x g centrifugation for five minutes, and resuspended in the next sap:PD2 broth mixtures. When in 100% sap, cells were resuspended in saps from the species listed above and adjusted to an OD600nm = 0.05. One hundred and fifty microliters of each sap-X. fastidiosa suspension was added to each well of 96-well polystyrene plates (Corning). The plates were then sealed with a Microseal ‘B’ adhesive seal (BioRad) before placing the lid and sealing the whole plate with Saran Wrap to prevent evaporation. Cultures were maintained for 10 days at 28°C with 185 rpm rotation. Plates were then carefully opened to prevent disruption of biofilms on well surfaces and rinsed three times with 200 μL per well of distilled water to remove planktonic cells and any cells loosely attached or deposited on the bottom of the well. To measure the degree of biofilm formed by remaining attached cells, 200 μL of aqueous 0.1% crystal violet was added to each well and plates were kept at room temperature (~23°C) for 20 mins. Plates were washed three times as before with water, followed by the addition of 200 μL of 1% sodium dodecyl sulfate (SDS) and agitated for five minutes. The SDS-dye solution was measured at OD600nm in a Synergy 2 plate reader (Biotek). A minimum of three biological replicates was assayed for each media condition .Initial experiments were done to determine if sap from different escribed , startinV. vinifera (PD susceptible), V. aestivalis (resistant) and V. champinii spp (resistant) as described above with some modifications. Briefly, X. fastidiosa cells were acclimated to each sap separately, allowing 24 h incubation for each passage: 50:50, 80:20, 90:10 100% sap:PD2 broth (vol:vol), before being inoculated into 100% sap of the same species in glass tubes. Each glass tube (13 x 100 mm) contained 2 mL of the sap-X. fastidiosa culture at an initial OD600 = 0.05, and was incubated at 28°C with shaking at 185 rpm. For the biofilm assays, saps were changed carefully at two, four and six-days post-inoculation without disturbing the biofilm rings: at each time point, each culture was transferred with a 5 mL pipette tip into a sterile 2.2 mL microcentrifuge tube for centrifugation at 10,000 x g for 5 mins, the supernatant was discarded and the pelleted cells were suspended in 2 mL of fresh sap of the same species in the original tube. The biofilm was quantified by crystal violet staining at day eight as previously described [To help reduce the effect of nutrient depletion on biofilm formation in closed cultures, experiments using sap renewal were conducted with saps from escribed . A closeX. fastidiosa specific RST31/33 primers [To measure bacterial populations in the culture, saps were changed the same way as described above. That is every two days bacterial cells (both planktonic and biofilm) in each glass tube were collected and centrifuged, followed by discarding the supernatant and adding fresh sap to the pelleted cells. Cells in each tube were then dispersed by extensive repetitive pipetting before immediately serially diluted and plated onto PWG (Periwinkle Wilt Gelrite) plates at days zero, four, and eight. Dispersal of cells was essential to break up aggregates as thoroughly as possible so that individual colonies on plates could be generated from single cells. The plates were incubated at 28°C for approximately 14 days and bacterial colonies were counted. Representative colonies were confirmed by PCR with  primers . Four reμL min-1 with a syringe pump , corresponding to a flow speed of ~1.250 μm min-1. Cells were acclimated to V. vinifera sap as described in the biofilm assays in 96-well plates, and approximately 100 μL of cells at OD600nm = 0.05 were introduced in each channel and observed for up to 10 days. Phenotypes were evaluated in each sap type at least three times.Microfluidic chambers were fabricated as previously described . The devV. vinifera or V. champinii) was filled with the same sap type plus PI at a final concentration of 2 μg mL-1 and continuous flow was resumed. After an additional six days (at day 10 from the beginning of the experiment) images were captured. Live KLN59.2 cells were detected through fluorescence of the eGFP and dead cells were detected through fluorescence of PI. In merged images dead cells devoid of eGFP appear only red (as in the V. champinii samples) whereas dead cells that still retained eGFP fluorescence appear yellow (as some in the V. vinifera samples). The experiment was performed with sap from each Vitis spp. at least three times. Scanning electron microscopy was performed as previously described [To determine the presence of live and dead cells, KLN59.2 cells  were anaescribed .p value less than 0.01 was considered to be significant.All statistical analyses were performed using the R program. To compare the mean biofilms or populations between treatments, a linear mixed effects model was used to include fixed effects of sap type, culturing, and an interaction between sap type and culturing and random effects to capture variability between the experiments. Post hoc comparisons between the treatments were performed using Tukey’s HSD (honest significance difference) to correct for multiple comparisons. A residual analysis was performed to assess the validity of the assumptions of normality and homogeneous variances. A V. champinii sap, supported less biofilm formation than other saps (p > 0.1), when including or excluding V. champinii in the analysis.As an initial screen, we assessed biofilm experiments when cells were in closed culture conditions in 96-well plates. The results indicated that saps vary with regard to biofilm formation and one of the resistant saps, her saps . When coV. vinifera, V. aestivalis, and V. champinii as the representatives of PD-susceptible and resistant spp., respectively, and examined biofilm formation when saps were renewed every two days in glass tubes. Results were compared to biofilm formation in the same saps in glass tubes under closed culture. For the closed culture, X. fastidiosa formed similar levels of biofilm in V. vinifera and V. aestivalis (p > 0.1) and more biofilm in V. champinii (p < 0.01) eight days post-inoculation , and more than seven times greater than that of V. champinii (p < 0.001). When the two culturing conditions were compared within each sap, significantly more biofilm was formed in the renewed as compared to closed culture in V. vinifera (p < 0.001), whereas the biofilm remained unchanged between the two culture systems (p = 0.01) in V. aestivalis and was reduced when sap was renewed compared to closed culture for V. champinii (p < 0.001). Taken together, data in X. fastidiosa biofilm formation and 2) biofilm formation differences in saps from PD-susceptible V. vinifera versus resistant V. aestivalis and V. champinii are most pronounced in sap renewed culture.To exclude the possibly that nutrient depletion might have an effect in the observed phenotypes, we then chose saps from culation . In contX. fastidiosa replicates and spreads, and once a threshold population level is reached, cells switch from a motile to a sessile state and form aggregates and biofilms, leading to the clogging of xylem vessels and disease [X. fastidiosa virulence-associated behaviors, such as biofilm formation, can be regulated in a cell density-dependent manner (i.e. via quorum-sensing) [X. fastidiosa formed significantly more biofilm in V. vinifera as compared to V. aestivalis or V. champinii in sap renewal cultures, we hypothesized that bacterial growth would be greater in V. vinifera sap.After initial entry into the xylem  disease . Therefosensing) , and thuX. fastidiosa cells were grown in the same way as they were for biofilm experiments shown in V. vinifera (~ 2.0 log units) sap was significantly less than those in V. aestivalis  and V. champinii saps . From day four to day eight, although populations in V. vinifera and V. aestivalis saps both increased, the average population level in V. vinifera was significantly higher by day eight (~ 3.5 log units) than that in V. aestivalis (p < 0.01). X. fastidiosa remained undetectable in V. champinii sap at both days four and eight. These data indicate that by growing X. fastidiosa in sap renewal culture a significantly greater population is achieved when cells are in V. vinifera sap as compared to saps from both resistant Vitis spp., and also that sap from V. champinii has an inhibitory effect on X. fastidiosa growth.To assess growth, X. fastidiosa aggregation and twitching motility behaviors [Since sap renewal culture had a significant effect on bacterial population dynamics and biofilm formation, we also examined culturing with continuous renewal in microfluidic chambers . This teehaviors , 34. FloV. vinifera sap, individual X. fastidiosa cells were observed attaching to the chamber wall surfaces and some cell aggregates were visible on day one , and were unable to form large aggregates or a lawn of cells at day ten. This result was consistent with the renewal growth and biofilm results and shows that direct acclimation in V. champinii or to V. vinifera prior to V. champinii , three-dimensional structures such as pillars and mounds were observed, and some of these structures extended the entire height (160 μm) of the channel  or star-shaped aggregates (V. arizonica) ten days post-inoculation  during the initial stage even with culture renewal  is critical for determining X. fastidiosa behaviors in different grape saps that might not be seen in closed culture. renewal . This coPseudomonas aeruginosa, carbon sources impact biofilm structures with amino acids or citrates promoting formation of flat, uniform biofilms, while glucose promotes formation of large cell aggregates in structured biofilms [2+ increases P. fluorescens initial attachment to surface and also changes subsequent biofilm development and structure [Overall, the differences in biofilm formation between susceptible and resistant saps seem to be well explained by cell growth, that is, susceptible sap supports more growth and biofilm than the saps from resistant species, at least for the three saps tested in the sap replenishment assay. Beyond the need of a threshold population size to initiate the development of a 3-D biofilm structure, other factors such as nutrient source and cation concentration also impact biofilm structure development. For example, in biofilms ; Mg2+ intructure . FurtherV. aestivalis and V. champinii. Fritschi et al. classified V. champinii and V. aestivalis as belonging to separate groups based on X. fastidiosa cell population dynamics in planta, as cells in V. champinii spiked in population but leveled in growth while growth was more supported in V. aestivalis over time [V. mustangensis, a PD-resistant spp., supports the formation of a thick lawn of cells in the chambers. However, the cells failed to form the 3-D structures such as pillars and mounds typically seen in a mature biofilm formed in the saps from susceptible spp. such as V. vinifera. Interestingly V. mustangensis is a known reservoir for X. fastidiosa that supports cell growth [X. fastidiosa in different saps or biochemical analysis of sap components, will help to better understand the difference among different resistant spp.While saps from PD-susceptible and -resistant spp. fall into two separate categories, our findings suggest that within saps from PD-resistant spp. there is a complex spectrum of responses, as observed for the different growth and biofilm formation supported by saps from ver time . In addil growth , 51, indl growth , 11. FurX. fastidiosa reaches a threshold population to initiate quorum-sensing regulation resulting in formation of biofilms that can clog xylem vessels and contribute to PD [V. champinii and V. aestivalis do not support viable populations equivalent to PD-susceptible V. vinifera sap. Reduced populations of viable cells may fail to reach necessary threshold levels to trigger quorum sensing regulated responses, including biofilm formation [et al. found that sap from PD-resistant V. rotundifolia is both less nutritious than V. vinifera sap and contains compounds involved in host defense against X. fastidiosa [Our findings are consistent with a model whereby te to PD , 52. Howormation , 54. Redstidiosa , indicat


Anaplasma phagocytophilum and Anaplasma platys, and ehrlichiosis, caused by Ehrlichia chaffeensis, Ehrlichia ewingii, the "Panola Mountain Ehrlichia" and Ehrlichia muris-like pathogens have been identified as emerging tick borne infectious diseases in dogs and human patients. Persistent intravascular infection with these bacteria is well documented in dogs, but is less well documented in human beings.Anaplasmosis, caused by Serology and PCR targeting multiple microbial genes, followed by DNA sequencing, was used to test sequential blood samples. Tissue culture isolation was attempted in two laboratories.A. platys, E. chaffeensis, and E. ewingii DNA was amplified from two Anaplasma and Ehrlichia seronegative family members and their dog, all lacking typical symptoms of anaplasmosis or ehrlichiosis. Following treatment with doxycycline, the dog and mother were Anaplasma and Ehrlichia spp. PCR negative.A. platys and Ehrlichia spp. in two humans and their dog. Diagnosticians and clinicians should consider the potential for co-infections due to these tick borne organisms.Sequential PCR testing provided molecular evidence supporting intravascular persistence of  Anaplasma phagocytophilum and A. platys, and ehrlichiosis, caused by Ehrlichia canis, E. chaffeensis, E. ewingii, E. muris and the “Panola Mountain Ehrlichia”, are emerging infectious diseases affecting dogs and human patients in North America and other parts of the world [. Among various animal species, including humans, Anaplasma and Ehrlichia spp. can induce acute, self-limiting or fatal infections, while persistent non-clinical infections often occur in dogs [. Following tick transmission, dogs can remain infected with A. phagocytophilum, A. platys, E. canis, E. chaffeensis, and E. ewingii for months to years, prior to immunological or therapeutic elimination of the infection or the development of chronic debilitating disease manifestations [. Dogs are natural hosts for A. platys, E. canis and potentially E. ewingii, but are considered opportunistic hosts for other Anaplasma and Ehrlichia spp. [. Persistent E. chaffeensis[E. canis[A. platys DNA was PCR amplified from blood samples collected from a veterinarian one month apart [Anaplasma and Ehrlichia human blood borne infections may become more common.Anaplasmosis, caused by he world -3. Caninhe world -6. Among in dogs -9. Follostations -9. Dogs hia spp. . Persistaffeensis,11 and E[E. canis,13 humanth apart . With thBartonella sp. research study. At time points spanning a six-month period, blood, buffy coat and inoculated cell cultures from members of the household, tested by PCR and sequencing, identified combinations of A. platys and Ehrlichia species. Anaplasma platys, E. chaffeensis, and E. ewingii DNA was amplified and sequenced from the dog, the physician and her daughter’s blood, but not from two other household members.The data in this manuscript was generated after a physician requested to be entered into a Bartonella sp. bacteremia in various patient populations. In 2008, the woman had developed intermittent subcutaneous edema and mildly increased liver enzyme activities. Her 16-year-old daughter, adopted from China at 6 months of age, had been healthy until 2008, after which she developed upper body muscle pain requiring treatment by a physical therapist. Between 2008 and 2011, both mother and daughter were examined by several specialist physicians; neither had symptoms or hematological abnormalities (thrombocytopenia) consistent with anaplasmosis or ehrlichiosis; and both were HIV negative.In September 2011, a 57-year-old-female physician requested to be entered into an IRB approved research study , investigating the prevalence of E. chaffeensis and E. ewingii. Due to an acute illness, the puppy was hospitalized hours after purchase; pneumonia was diagnosed radiographically and an extended hospitalization period was required to effectively treat the pneumonia. Subsequently, aggressive behavior was observed, resulting in occasional bites of the mother and daughter but not the other household members. In 2012, the dog was diagnosed with microvascular dysplasia, a congenital, developmental abnormality common in small breed dogs, that can contribute to hepatic dysfunction and behavioral abnormalities, potentially explaining the tendency for the dog to bite.In 2008 the family purchased a 15-week-old male Papillon from a Chicago pet store. The puppy originated from a Missouri breeding facility, a region endemic for tick borne , three sample sets were collected within a 7-day period to enhance detection of Bartonella spp. DNA using the BAPGM platform. Using blood aseptically collected on Monday, Wednesday and Friday , (daughter in January and February 2012) and (dog in December 2011 and January 2012), Bartonella spp. serology and BAPGM  enrichment blood culture testing for Bartonella spp. was performed, according to previously published protocols [.In accordance with a prior study , three srotocols ,16.E. chaffeensis DNA from the mother’s blood in September 2011, aseptically obtained ethylenediaminetetraacetic acid (EDTA)-anti-coagulated whole blood was processed in two formats (whole blood and buffy coat cells) in an effort to increase Ehrlichia sp. PCR sensitivity. Blood samples  and  were sent concurrently to the NCSU-IPRL and UTMB for parallel tissue culture isolation attempts.After the initial PCR amplification of Anaplasma and Ehrlichia 16S rRNA gene conventional PCR (cPCR) assays were used to test whole blood, buffy coat and cell culture supernatant fractions prepared from the mother, father, daughter, grandmother and dog [16S rRNA gene [Ehrlichia canis DNA as a positive control. Subsequently, newly introduced or derived (sodb) PCR assays were used by a blinded IPRL researcher to target the A. platys p44 gene, E. chaffeensis and E. ewingii p28 genes, and Ehrlichia spp. sodb gene in blood and cell culture supernatants. Primers and cPCR conditions are provided in Table Genomic DNA was extracted using either the QIAsymphony SP  or the DNeasy Blood & Tissue Kit  as per manufacturer recommendations. Each DNA extraction process included several negative control non-infected blood samples. To avoid DNA contamination of samples, DNA extraction and PCR sample preparation were performed in a room separate from the PCR amplification and gel analysis rooms, with a unidirectional work flow. Previously described  and dog ,18. AmplRNA gene  using EhAmplicons were sequenced directly or cloned into plasmid pGEM-T Easy Vector System Promega®  by GENEWIZ Inc. . Sequences were aligned and compared with GenBank sequences using AlignX software .E. chaffeensis DNA from the mother’s blood, her physician requested E. chaffeensis serology from a commercial laboratory ; otherwise human serological testing was performed at the Rickettsial Disease Laboratory, UTMB. Serum specimens from all family members from multiple collection dates were serially diluted (twofold) from 1:64 to 1:2048 and tested for E. chaffeensis IgG class antibodies by ImmunoFluorescent assay (IFA) with Alexa Fluor 488 goat-anti-human secondary antibody (dilution 1:1000). An IFA titer of 1:64 or higher was considered positive. E. chaffeensis Western immunoblot was performed by SDS-PAGE using NUPAGE NoVEX 4-12% polyacrylamide gels . Separated proteins transferred to nitrocellulose membrane using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell  were blocked overnight with 5% nonfat dry milk (Bio-Rad) in 1X Tris-buffered saline (TBS) before incubation with serum samples diluted (1:100) in 5% nonfat dry milk in 1X TBS overnight at room temperature. Blots were washed thoroughly then incubated with alkaline phosphatase labeled anti-human IgG (γ chain-specific) secondary antibodies and color reactions developed with BCIP/NBT phosphatase substrate . Serum from the dog was tested for Ehrlichia antibodies by IFA in the IPRL, using antigens derived from canine monocytic DH82 cultures infected with E. canis and E. chaffeensis and for Anaplasma or Ehrlichia antibodies using the Snap® 4Dx®,  [After amplification of , Maine) ,20.2 flasks containing DH82 and IES6 tick cells (IPRL) or DH82, RF/6A, Vero, HEL299 and C6/36 cells (UTMB) and incubated at 37°C in 5% CO2. Negative control flasks were maintained in parallel. Cytology (Diff Quik or Gimenez staining) and PCR were sequentially performed.Parallel isolation attempts were made using white blood cell pellets (WBC) into various cell lines at NCSU-IPRL and UTMB. EDTA anti-coagulated whole blood from each individual was lysed with either ACK lysis buffer or hypotonic saline and centrifuged at UTMB or centrifuged in two stages at IPRL to prepare WBC pellets which were suspended in medium and dispersed into 25 cmThe family resided in suburban Chicago. There were no forested areas that might contain tick-infested deer for at least 1 mile in any direction. The mother did not recall any tick infestations in the home before or after purchasing the puppy and no family member reported a history of tick attachment. Occasional raccoon, skunk, opossum, coyote and other small mammals and birds were seen in the neighborhood. The mother and daughter were the primary caregivers for the dog and the only family members to have experienced consistent dog contact and occasional bites.Bartonella spp. antibodies were not detected in the mother, daughter or dog’s serum specimens, and at all testing time points, BAPGM enrichment blood culture platform specimens (4 PCRs/sample/date of collection) were Bartonella sp. PCR negative. Thus, there was no serological or PCR evidence to support Bartonella spp. exposure or infection.Based upon sequential testing, 16S GEP primers that amplify both Anaplasma and Ehrlichia species, Ehrlichia chaffeensis 16S rDNA was cPCR amplified and sequenced from one of three September 2011 blood specimens (Table 16S rDNA sequence shared 99.7% (359/360 base pair) similarity with E. chaffeensis strain Arkansas CP000236  ehrlichiemia was documented in the dog, daughter and mother  was amplified and sequenced from the dog and mother’s blood. Amplified Anaplasma and Ehrlichia DNA sequences shared between the dog, daughter and mother were identical.Beginning with the mother’s December 2011 specimens, DNA was independently extracted from whole blood and WBC fractions from each family member in an effort to enhance cPCR sensitivity. By sequential testing, 16S rDNA PCR results, other Anaplasma and Ehrlichia spp. genes were targeted, using freshly extracted, stored, whole blood and WBC preparations. The initial A. platys, E. chaffeensis and E. ewingii DNA amplification results were confirmed by cPCR amplification and DNA sequencing of the p44, p28, and sodb genes ; however, at UTMB, five sera collected between September 2011 and March 2012 (mother) and February and March (daughter) were not E. chaffeensis seroreactive at a 1:64 dilution by IFA testing. Western immunoblotting also did not identify Ehrlichia spp. antibodies. When tested by the IPRL in January 2012, the dog was not E. canis, Babesia canis, Babesia gibsoni, Bartonella henselae, Bartonella vinsonii subsp. berkhoffii, Leishmania infantum, or Rickettsia rickettsii IFA seroreactive and was also ELISA negative for Dirofilaria immitis antigen and Anaplasma spp., Borrelia burgdorferi C6 peptide, and Ehrlichia spp. antibodies.When tested by a commercial laboratory in February 2012, the mother was reportedly Anaplasma/Ehrlichia GEP 16S cPCR amplified E. ewingii and A. platys from the dog, daughter and mother’s IPRL DH82 cultures. E. chaffeensis DNA  was amplified from daughter and mother’s UTMB RF/6A cultures. The dog and mother’s E. chaffeensis 16S rRNA, sodb sequences and A. platys p44 sequences from cell culture were 100% identical to sequences previously amplifed from blood, serum or WBC specimens. The dog’s March 2012 WBC inoculated DH82 (pid 8) and IES6 (pid 23) cell cultures yielded 16S rRNA and sodb E. canis DNA.Organism isolation efforts at both NCSU and UTMB generated Diff Quik or Gimenez staining indications of intracellular bacteria or morulae, but did not result in successful isolation or evidence of organism amplification during 8–10 weeks in culture. Structures consistent with morulae were observed in the dog, daughter and mother’s inoculated cell cultures, but not in the father, grandmother or un-inoculated control cell cultures. In May 2012, the mother was treated with doxycycline 100 mg PO BID for 15 days and the dog was treated with doxycycline 5 mg/kg every 12 hours for 5 weeks. When retested in June 2012, blood and tissue culture cPCR results were negative and morulae-like structures were not visualized. The daughter was not treated.A. platys, E. chaffeensis and E. ewingii DNA in the blood of a dog and two family members. Specifically, during the six month study period, E. chaffeensis, E. ewingii, and A. platys DNA was amplified and sequenced from the mother’s blood and/or cell culture supernatant specimens at 5, 3, and 2 time points, respectively. Anaplasma and Ehrlichia gene targets not routinely assayed in our laboratory confirmed the initial identification of pathogen DNA in all three family members. Furthermore, the partial gene sequences amplified between family members were identical, suggesting they were infected with the same pathogen genotypes. Despite repeated molecular documentation of these bacteria, Ehrlichia spp. IFA antibody titers and Western immunobloting were negative at UTMB and the dog was not Anaplasma or Ehrlichia seroreactive at NCSU-IPRL. As A. platys and E. ewingii have not been successfully cultivated in cell culture, IFA serological assays were not available for testing purposes. Whether Anaplasma and Ehrlichia spp. co-infection altered the expected humoral immune response, whether assay antigens were not well matched with the infecting genotypes or whether anergy played a role in seronegativity remains unknown. Similar comparative correlations between canine and human vector-borne infections can be found in the literature. Persistent infections, spanning months to years, with A. platys, E. chaffeensis and E. ewingii, have been frequently reported in dogs [. In contrast, there is limited evidence supporting persistent human E. chaffeensis infections [E. canis isolate, thus infection does not infer protective immunity in dogs [E. chaffeensis has also been reported in a liver transplant patient [E. chaffeensis and Rickettsia rickettsii co-infection was reported in a 44-year-old man [A. platys and E. canis co-infections in dogs [This study provided PCR amplification and sequencing evidence supporting the persistence of  in dogs ,7,18,21.fections ,11. Base in dogs . Re-infe patient . In conj patient  however,-old man . Experim16S rDNA PCR results from the mother and subsequently the daughter and dog, other gene targets were amplified and sequenced, isolation was attempted in two laboratories, and two investigators tested samples independently and at different time points. Because microbial-specific genes were targeted, the same PCR assays are applicable for testing human and veterinary patient populations and vectors for the presence of bacterial DNA. PCR has limitations. False negative results occur when testing samples with low template concentrations and selective amplification of the predominant organism can occur in patients co-infected with genetically similar organisms. By comparing PCR results from different sample sources , the assays used in this study may not have been sensitive enough to consistently detect these bacteria within blood specimens collected during the same week (data not shown), illustrating the need to enhance the sensitivity of Anaplasma and Ehrlichia PCR, particularly when patient samples contain DNA sequences of genetically related organisms. DNA carryover or amplicon contamination within a laboratory may result in false positives [Anaplasma and Ehrlichia sp. DNA, and both the dog and mother became PCR negative after treatment with doxycycline, a finding that supports infection with viable organisms.To confirm and validate the initial, unexpected ositives ,26. ImpoA. platys[A. platys infection was published in the English literature. There is substantial epidemiological support for Rhipicephalus sanguineus (the brown dog or kennel tick) as the vector and the dog as the primary reservoir host for A. platys and E. canis[R. sanguineus seemed more likely than exposure to A. americanum, which is a field tick that will feed on numerous animal species [E. chaffeensis DNA was amplified from 56% of R. sanguineus obtained from dogs and puppies housed in a kennel in Cameroon, a country where both canine and human infection with E. chaffeensis has been previously reported [E. ewingii by R. sanguineus were not successful [A. platys, E. chaffeensis and E. ewingii infection in the pet and the family members and E. canis in the dog remains unknown; however, this puppy originated from a kennel in a highly endemic state for tick transmission of E. chaffeensis and E. ewingii by A. americanum[A. platys and E. canis are transmitted by a tick (R. sanguineus) that infests dogs in kennels.Nearly two decades ago investigators in Venezuela described inclusions in human platelets, a subset of which were ultrastructurally consistent with A. platys,28. UntiA. platys, no subs E. canis. As the  species . Althougreported . In contccessful . The soumericanum,29 and AA. platys and Ehrlichia spp. DNA was documented in an unusual familial cluster involving a dog and two family members. Future studies should determine whether repeated documentation of DNA of these organisms reflects ongoing infection and whether there are medical consequences associated with the persistence of DNA of these organisms. To guide testing and treatment decisions, sequential PCR testing of blood, WBC or cell culture-enhanced samples may increase DNA-detection sensitivity.In summary, BAPGM: Bartonella alpha proteobacteria growth medium; EDTA: Ethylenediaminetetraacetic acid.Bartonella species infection in animals and humans. All other authors have no potential conflict. In the context of this manuscript, none of the authors disclose any conflicts of interest.In conjunction with Dr. Sushama Sontakke and North Carolina State University, Dr. Breitschwerdt holds U.S. Patent No. 7,115,385; Media and Methods for cultivation of microorganisms, which was issued October 3, 2006. He is the chief scientific officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of EBB coordinated communications and generated the initial draft manuscript. BCH, BAQ, and RGM performed tissue culture isolation and PCR testing at North Carolina State University. TBS, LSB, and DHB performed tissue culture isolation and serological testing at the University of Texas Medical Branch at Galveston. All authors contributed to the content of the manuscript and all authors reviewed the final submission.


Natural compounds have been used to develop drugs for many decades. Vast diversities and minimum side effects make natural compounds a good source for drug development. However, the composition and concentrations of natural compounds can vary. Despite this inconsistency, half of the Food and Drug Administration (FDA)-approved pharmaceuticals are natural compounds or their derivatives. Therefore, it is essential to continuously investigate natural compounds as sources of new pharmaceuticals. This review provides comprehensive information and analysis on natural compounds from plants (phytogenic compounds) that may serve as anti-obesity and/or anti-diabetes therapeutics. Our growing understanding and further exploration of the mechanisms of action of the phytogenic compounds may afford opportunities for development of therapeutic interventions in metabolic diseases. Living organisms found in nature have chemical compounds or substances collectively termed natural compounds . The varNatural compounds have been a good source of new pharmaceuticals for a long time. Approximately 50% of the drugs approved by the Food and Drug Administration (FDA) are phytogenic compounds or derivations. Natural compounds have been crucial in drug development ,9. MorphPharmaceutical companies use high throughput screening (HTS) for drug development ,8. EnzymIsolating bioactive compounds from raw sources is another problem when dealing with phytogenic compounds ,8. NaturDespite different symptoms, obesity and diabetes have some similarities. A large percentage of obese patients have insulin resistance . Insulin2 [2 is considered as normal and between 25 and 30 kg/m2 is categorized as overweight. Obesity can lead to various chronic diseases, such as hypertension, type 2 diabetes (T2DM), hyperlipidemia, and coronary artery disease.Obesity is an increasing global problem, especially in developed countries. The World Health Organization (WHO) defines an obese individual as someone whose body mass index (BMI) is over 30 kg/m2 . Under 2ob gene, which encodes leptin, is dependent on the amount of body fat. When mutation occurs in this gene, leptin regulation is disturbed. The FTO (fat mass and obesity associated) gene, whose function remains unknown, can contribute to a 3–5 kg weight difference in obese individuals according to its DNA-sequence variations [FTO variation is related to BMI and implies level of obesity from childhood to adulthood [The causes of obesity are diverse but generally there are four main factors: the amount of food/drink intake, physical activity (exercise), genetic factors , and medriations . Also, Fdulthood . People dulthood . In thisdulthood  and thyrdulthood ; howeverSeveral methods are used to treat obesity. One approach is to suppress appetite to decrease food intake . Many faThe hallmark of diabetes is hyperglycemia caused by a defect in insulin secretion and/or insulin action . ChronicDiabetes can be classified into various groups, but most common are type 1 and 2. Type 1 diabetes (T1DM) is caused by insufficient insulin secretion due to autoimmune destruction of β-cells. Only 5%–10% of diabetic patients have T1DM. T2DM is a consequence of insulin resistance and lack of appropriate response to hyperglycemia. Most (90%–95%) of diabetic patients have T2DM. Some diabetic patients are able to manage their blood glucose level with exercise, proper diet, and oral glucose-lowering agent depending on their condition. These diabetic patients do not require external insulin to survive. On the other hand, those with severe β-cell damage cannot manage their blood glucose level with simple exercise and diet. They need regular injections of insulin to survive .Most T2DM patients do not need exogenous insulin to manage their blood glucose level. This constitutes non-insulin dependent diabetes (otherwise known as adult-onset diabetes). Many T2DM patients are obese. Many of those who do not need exogenous insulin have increased body fat in the abdomen. Often it is undiagnosed for many years because hyperglycemia advances slowly and it is difficult for the patient to notice the symptoms of diabetes .Treatment for T2DM is continuously being developed. The most common pharmaceutical prescribed for T2DM is metformin ,29. MetfPhytogenic compounds have been used to treat various diseases for decades ,9. ThousPanaxginseng (Asian ginseng) and Panax quinquefolium (American ginseng) contain many ginsenosides. Ginsenoside Rb1 has many therapeutic actions on the glucose metabolism and lipid metabolism. When ginsenoside Rb1 (10 mg/kg) was intraperitoneally injected to normal diet fed mice and high fat diet (HFD) fed mice groups for 3 weeks, it reduced body weight, total food intake, fat contents, serum leptin, and serum nitric oxide equal to or lower than the normal diet group [P. ginseng and they also have a role in inhibiting pancreatic lipase  and body weight (11.6%) significantly. In addition, body temperature (2.8%) and energy expenditure (35%) increased significantly in ob/ob mice [The concentrations of ginsenoside Re, Rb2, and Rd in han root . Among than root . Extract/ob mice .Hoodia gordonii is a thirst quencher [H. gordonii and H. pilifera. When P57AS3 was injected intracerebroventricularly into the third ventricle, 24 h food intake was reduced by 40%–60% [H. gordonii could regulate food intake and energy homeostasis. When glycosides 1 and 2 isolated from dried stems of H. gordonii were gavaged orally at 6.25–50 mg/kg to rats for 8 days, food intake and body mass were decreased at all doses compared with the control group [H. gordonii has effects on diverse factors such as mitochondrial carnitine palmitoyltransferase-1 (CPT-1), thyroid hormones, NPY, and insulin like growth factor-1 (IGF-1) [H. gordonii, food intake and the level of NPY were decreased dose-dependently. CPT-1 and thyroid hormones such as Tri-iodothyronine (T3) and thyroxine (T4) were increased. CPT-1 is a fatty acid oxidation enzyme that participates in lipid metabolism and the increase of CPT-1 indicates enhanced fatty acid oxidation. Elevation of T3 and T4 indicates an increase in energy expenditure [H. gordonii may participate in suppressing appetite, increasing energy expenditure and lipid metabolism, and promoting carbohydrate metabolism.quencher ; however 40%–60% . P57AS3 ol group . Also, H (IGF-1) . When maenditure  and mainenditure . Thus, HBerries are well known for their antioxidant effect. Blueberry has been suggested to improve cardiovascular defects and to have antioxidant anti-obesity and anti-diabetic effects ,41,85. BEnergy expenditure includes thermogenesis, physical activity, and obligatory energy expenditure . There aNelumbo nucifera has been used as treatment for many diseases for millennia. Each part of N. nucifera has different therapeutic effects that include relief of fever, inflammatory skin conditions, and bleeding disorders [N. nucifera leaves was treated to HFD-induced obesity mice for five weeks, it decreased activities of α-amylase and lipase, and increased lipid metabolism and expression of UCP3 mRNA in C2C12 myotubes [N. nucifera include 11 types of flavonoids: eudesmane sesquiterpene, 13 kinds of megastigmanes, and eight forms of alkaloids [P. ginseng, N. nucifera is involved in lipid metabolism in addition to energy expenditure  is one of the major catechins in sinensis . When EGsinensis . EGCG trsinensis , EGCG losinensis , EGCG reN. nucifera leaves using ethanol can inhibit pancreatic lipase, α-glucosidase, α-amylase, and hypolipidemic effects [N. nucifera leaf flavonoids (NLF). NLF decreased TG level in normal rats and decreased the levels of TC and TG in acute hyperlipidemic mice dose-dependently. By inhibiting lipid absorption in the liver, NLF can ameliorate the level of steatosis [N. nucifera have a strong ability to lower lipid levels. Rhizome of N. nucifera is composed of polyphenolic compounds and can alleviate hepatic steatosis in db/db mice [N. nucifera polyphenol from its root suppresses hepatic lipogenesis through decreasing activities of FAS and malic enzyme, which is related to lipogenesis, but not CPT. Seeds of N. nucifera contain polyphenolics and flavonoids. N. nucifera seed ethanol extract (NSEE) may inhibit the differentiation of human pre-adipocytes into adipocytes [N. nucifera contain flavonoids and polyphenolics, these compounds may have a similar role in suppressing lipogenesis.Flavonoids extracted from  effects . In the teatosis . Leaves /db mice . N. nuciipocytes . When NSWild blueberries contain anthocyanin, which is an antioxidant polyphenol . When a Cinnamomum. Cinnamon extract improves insulin resistance and lipid metabolism by activating of PPAR-α and PPAR-γ [db/db mice were gavaged with cinnamon powder dissolved in water, fasting glucose level, free fatty acid, LDL cholesterol, and AST (aspartate aminotransferase) levels decreased because of the activation of PPARs. In ob/ob mice, cinnamon extract improved insulin sensitivity, insulin-stimulated locomotors activity, and glucose tolerance while insulin secretion was not changed [® twice per day for 12 weeks.Cinnamon is produced from bark of d PPAR-γ . Target  changed . Water-s changed . In the Concerning obesity, licorice flavonoids may suppress the accumulation of abdominal white adipose tissues and body weight gain in HFD-induced obese C57BL/6J mice . When thMomordica charantia is a tropical plant commonly found in India, Asia, East Africa, and South America. It is also known as bitter melon [er melon . Bitter er melon . Bitter er melon . FAS, acer melon , and reder melon . Taken tThe role of carbohydrate metabolism in the development of obesity is not clearly understood, but many studies show evidence of a relationship between carbohydrate metabolism and obesity. Comparing conventional diet  group and Atkins diet  groups, the Atkins diet group showed a greater weight loss for 6 months in a clinical study . The dow13CO2 were measured for carbohydrate absorption. Volunteers were orally administrated an extract of black (0.1 g), green (0.1 g), and mulberry (0.1 g) teas in each meal. Participants consumed meals containing lipid and carbohydrate or meals containing only lipid without carbohydrate. Meals containing lipid, carbohydrate and tea extracts increased breath-hydrogen concentration, indicating malabsorption of carbohydrate. On the other hand, no changes were evident in the carbohydrate-free meals group with or without tea extract. This data implies that compounds within tea extract may have an inhibitory effect on carbohydrate absorption.The extract of black, green, and mulberry teas may interrupt carbohydrate absorption by inhibiting α-amylase , α-glucoSoy has many beneficial effects for obesity and diabetes . Chow suTrigonella foenum graecum is a leguminous herb widely available in India, Mediterranean, and North Africa. Seeds of fenugreek are used as a spice in India and it is used to make bread with wheat and maize flour. Furthermore, it has been used for medicinal purpose for many years. When fenugreek seeds were administered to diabetic patients, blood glucose level was reduced and plasma insulin level was increased [ncreased . Anotherncreased . Researcncreased . This rencreased .Not only has cinnamon been used as natural food preservative and spice, but it has been used in traditional medicine for treating rheumatism, wounds, diarrhea, headache, and colds . In addi in vitro study with rat adipocytes suggests that cinnamon regulates insulin signaling [Anignaling ; this stignaling . In thisignaling . The resN-terminal kinase (JNK) phosphorylation were increased in dioscorea polysaccharide treated subjects [Dioscorea is a tropical plant renowned for its anti-diabetic effects ,119. Inssubjects . These rsubjects . This hesubjects . This inS. vaccinii [y mice, both acutely and chronically. Both acute and chronic administration groups showed reduction of blood glucose level compared to the control. PPAR has multiple functions, which includes metabolism of carbohydrate and lipid [Blueberry improves insulin resistance. Freeze-dried whole blueberry powder supplemented HFD was fed to mice for 8 weeks . Resultsvaccinii . The fernd lipid . The blund lipid . In addind lipid . This mand lipid . Overallnd lipid ,41,42.Glycyrrhiza can be used to treat diabetes. Chemically synthesized amorfrutins was administered to HFD-induced obese mice [Glycyrrhiza was administered orally to KKAy mice [Glycyrrhiza extract binds to PPAR-γ [Glycyrrhiza can improve insulin sensitivity and hyperglycemia.Amorfrutins extracted from ese mice . Blood gese mice . EthanolKAy mice . Blood go PPAR-γ ,68. Out o PPAR-γ ,123. ThiErvatamia microphylla. It has anti-diabetic effects. Blood glucose level was reduced when conophylline was administered orally to streptozotocin (STZ)-induced diabetic rat [in vivo experiment with Goto-Kakizaki rats showed that conophylline reduced blood glucose level and increased plasma insulin level [Conophylline is a compound extracted from etic rat . Conophyetic rat . A studyetic rat . Furtherin level . These sAnoectochilus roxburghii, commonly known as jewel orchid. Oral administration of kinsenoside to STZ-induced hyperglycemic rats reduced blood glucose level [Kinsenoside is a compound of se level . Plasma Papaya is a tropical fruit available in numerous countries. This fruit has been used in traditional medicine for a long time. Extract of papaya decreased blood glucose level in STZ-induced diabetic mice . AccordiSilybum marianum. Silymarin was administered orally to alloxan-induced diabetic rats [Silymarin is a mixture of several flavonoids extracted from tic rats . These rtic rats . Therefotic rats . Particitic rats . Silymartic rats . These sNymphaea stellata is Egyptian lotus. Chloroform extract of N. stellata flower was orally administered to STZ-induced diabetic rats [N. stellata extract increased the plasma insulin level. Further investigation with immunohistochemistry indicates increase in pancreatic β-cells mass [N. stellata could potentially regenerate pancreatic β-cells, which could be developed into a treatment for diabetes.The common name for tic rats . Blood glls mass . This inA few studies suggest that capsaicin has an anti-diabetic effect. When capsaicin was administered to Zucker diabetic fatty rats, a reduction of blood glucose level and higher plasma insulin levels was evident compared to the control group . HFD-indM. charantia was administered intraperitoneally to STZ-induced diabetic mice [M. charantia treated group showed reduced blood glucose level compared to the diabetic control group. A human clinical study was performed with 18 maturity-onset diabetes patients [M. charantia or vehicle was given to the patients 30 min prior to measuring blood glucose level. Patients who took M. charantia juice showed lower blood glucose level compared to the vehicle control group. These results indicate that M. charantia is able to lower blood glucose in a diabetes mice model and in patients.Bitter melon has long been known to have an anti-diabetic effect. Aqueous extract of tic mice . The M. patients . Juice oVitis vinifera extract [V. vinifera inhibited the activity of glycogen phosphorylase b in a HepG2 cell line [V. vinifera can reduce high blood and hepatic glucose levels in T2DM patients.Resveratrol is one of the polyphenols existing in  extract . Resvera extract ,48. Extrell line . Glycogeell line . Thus suObesity and diabetes have some common links and are connected closely to each other. The term “diabesity” was coined in the 1970s to emphasize the strong relationship between obesity and diabetes . HoweverVaccicum spp. improves insulin resistance [V.angustifolium (wild blueberry) also contains anthocyanins that improve dyslipidemia by regulating genes related to lipid metabolism [sistance ,100, supsistance , and regsistance . Anthocysistance . In additabolism . Anthocytabolism  and reduCapsicum induces thermogenesis by activating BAT [Capsicum.ting BAT ,127. Whiting BAT . As a reGlycyrrhiza affects both obesity and diabetes. This phytogenic compound can improve hyperglycemia [Glycyrrhiza extract increased β-oxidation and decreased acetyl-CoA synthesis [Glycyrrhiza extract can reduce body fat [Glycyrrhiza has the potential to attenuate symptoms of obesity and diabetes.It is clear that extract of glycemia . Glycyrrynthesis . Clinicabody fat ,102,69. M. charantia extract can improve hyperglycemia [M. charantia extract can suppress adipocyte hypertrophy and reduce lipogenic gene expression, such as expression of FAS, ACC-1, and LPL [M. charantia extract reduces SREBP-1c and resistin [M. charantia may act as an inhibitor of lipogenesis and lipolysis stimulator. M. charantia extract could reduce hyperlipidemia and hyperglycemia.glycemia ,59 and rglycemia . M. char and LPL . In addiresistin . Thus, MCinnamomum extract can lower hyperglycemia [Cinnamomum extract seems to reduce body fat composition [Cinnamomum and its relation to insulin [glycemia ,50,99. Iposition ,100. Thu insulin , and theIn this review, phytogenic compounds that affect obesity and diabetes have been discussed. While a simultaneous treatment for obesity and diabetes has been explored, no treatment is available. Obesity and diabetes shares some similarities that include inflammation and insulin resistance .Lepob/J and B6.BSK(D)-Lepdb/J mice, are needed. This mouse model has the physiology of both obesity and diabetes. Thus, it would be interesting to test if the phytogenic compounds mentioned in Due to unique characteristics of phytogenic compounds and similarities of obesity and diabetes, some phytogenic compounds may be used to develop treatment for both diseases. For example, extract of cinnamon can improve hyperglycemia and reduce body fat composition. Further investigations would provide support for the potential of cinnamon. Studies referred to in


Cochliobolus lunatus in the past decade. Potato (Solanum tuberosum L) farms were routinely surveyed for brown-to-black leaf spot disease caused by C. lunatus. A biphasic gene data set was assembled and databases were mined for reported hosts of C. lunatus in the last decade. The placement of five virulent strains of C. lunatus causing foliar necrosis of potato was studied with microscopic and phylogenetic tools. Analysis of morphology showed intraspecific variations in stromatic tissues among the virulent strains causing foliar necrosis of potato. A maximum likelihood inference based on GPDH locus separated C. lunatus strains into subclusters and revealed the emergence of unclustered strains. The evolving nutritional requirement of C. lunatus in the last decade is exhibited by the invasion of vertebrates, invertebrates, dicots, and monocots. Our results contribute towards a better understanding of the host-range dynamics of C. lunatus and provide useful implications on the threat posed to the environment when C. lunatus is used as a mycoherbicide.We undertook an investigation to advance understanding of the host-range dynamics and biocontrol implications of  Cochliobolus species have caused plant disease disaster such as the southern leaf corn blight epidemic of 1970s in the United States of America [Exserohilum turcicum) and corn head smut (Sporisorium reilianum) in northern China in the 1990s [ Cochliobolus species often cause diseases to several plant families including Alliaceae, Anacardiaceae, Araceae, Euphorbiaceae, Fabaceae, Malvaceae, Rutaceae, Zingiberaceae and Solanaceae [Race specifiche 1990s , 3, and he 1990s , 5. In tlanaceae .Cochliobolus lunatus [ C. lunatus includes plant species, namely, Cynodon sp., Oryza sp., Pennisetum sp., Saccharum sp., Sorghum sp., Triticum sp., and Zea sp. [ C. lunatus was suggested to be located mainly in Australia, Brazil, Guinea, India, Cameroon, Columbia, Ecuador, Fiji, Gambia, Guadalcanal, Malaysia, Nigeria, Pakistan, Papua New Guinea, Sierra Leone, Sri Lanka, Sudan, Tanzania, Thailand, and USA [http://www.tifton.uga.edu/fat/disfunt2.htm). The proposed geographical circumscription and putative hosts of C. lunatus have not been updated. lunatus  and rela lunatus –13. The  Zea sp. . Geograp and USA  but not C. lunatus has emerged in the last decade as a virulent and destructive pathogen [ C. lunatus successfully thrives on important crops such as rice (Oryza sativa L.), wheat (Triticum aestivum), cassava (Manihot esculenta), sorghum (Sorghum bicolor), Hymenaches species, strawberry (Fragaria × ananassa), Amaranthus species, and potato [ C. lunatus solely endemic to the outlined geographical locations? If no, has C. lunatus gained hosts and new geographical zones in the last decade? The aims of this study were (1) to determine the interrelatedness of 5 virulent strains of C. lunatus causing foliar necrosis of potato using morphological descriptors coupled with phylogenetic tools and (2) to establish the current host-range diversity of C. lunatus in the last decade.pathogen , 16. Remd potato . Decadesd potato  pioneeriRoutine survey was performed in potato plantations of Burdwan District , West Bengal, India, during the winter months of December to March of 2010, 2011, and 2012. Mainly potato cv. Kufri Jyoti is farmed in Burdwan District. The area receives an average annual rainfall of 1173–1442 mm and temperature of 10–20°C during potato farming season. Potato plants showing brown-to-black leaf spot disease previously described [ glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene using C. lunatus (GenBank accession number Gb X58718) sequence. Amplification of GPDH was performed as follows. The PCR mix contained 11 ng genomic DNA, 5 μL Green GoTaq reaction buffer , 0.2 mM each of deoxyribonucleoside triphosphate (dNTP), 0.2 μM of each primer, and 1.1 U of GoTaq DNA polymerase in a total reaction volume of 25 μL in triplicates . The quality of the amplicon was checked by performing agarose gel electrophoresis. The PCR products were purified and sequenced. Sequences were assigned to molecular species based on 98–100% sequence similarity threshold in the GenBank with the following accession numbers: JX512810, JX512809, JX907827, JX477595, and JX907828, respectively, for rDNA. GPDH sequences have been submitted in DNA Data Bank of Japan (DDBJ) as accessions AB859034, AB859035, AB859036, AB859037, and AB859038, respectively.The genomic DNA was isolated from fungal isolates grown in potato dextrose broth (PDB) . Approximately 100 mg of mycelia mat was disrupted in the presence of TRI-reagent  using mortar and pestle containing 2 mg/mL proteinase K  following the manufacturer instructions. The quality and quantity of the DNA were determined using a 1% agarose gel electrophoresis and a nanodrop spectrophotometer , respectively. For molecular identification, the partial sequence of 5.8S rDNA, complete internal transcribed spacer 2 region (ITS2), and partial 28S rDNA region were amplified as previously described . To disthttp://hcv.lanl.gov/content/sequence/ELIMDUPES/elimdupes.html). Sequence alignment was performed using Muscle program [Using GenBank BLAST search tool, a studied set of rDNA sequences deposited in the last decade was collected based on the information associated with the sequences such as GC content, length >250 bp), and geographic origin of host. Importantly, records with 100% sequence similarity from the same host and geographical coordinates were removed. Unique sequence sets were screened using ElimDupes  produced greyish-brown mycelium and cylindrical clavated fived-celled conidia void of stromatic tissue  locus which had been shown to be effective in resolving Cochliobolus species in phylogenetic inference [ GPDH locus  were found. Based on TN93 + G + I substitution model [ GPDH locus discriminated the five strains of C. lunatus causing foliar necrosis of potato with strong bootstrap support ≥81% ( C. lunatus type isolates (The low bootstrap support ≤61%) generated in rDNA ML tree . Other C. lunatus new hosts reported [Herein, the term host-range diversity described the group of different hosts on whichivanesan  in 1987 ivanesan  account.t decade . New hoswheeleri , geograpreported –40 in th C. lunatus observed in the last decade  close proximity to host is prerequisite for pathogens to jump from a natural host to a new host [ C. lunatus profusely produced melanized colonizing hyphae during invasion in potato [Although studies have shed light on specific aspects ofize crop , virulenize crop , secretoize crop , and heaium spp. , the nutnew host , (2) thenew host , 45, andnew host . Importanew host , 47, 48.n potato  and non-n potato , to supp C. lunatus gain access to hosts and not how C. lunatus spreads in a given biota and prevails as an environmental hazard. C. lunatus had extensively been used as mycoherbicide formulations in the past decade [ Helminthosporium gramineum and C. lunatus, to generate a strain with high potential to produce conidia, phytotoxin ophiobolin, and improved potential to control rice weed. Introduction of genetically manipulated strains and unmodified strains of C. lunatus could have hazardous implications to the environment. This is because, in some cases, C. lunatus failed to control the targeted weeds but caused severe damages in economically important crops in the same biota. For instance, C. lunatus isolated from barnyardgrass and used as mycoherbicide failed to control competitive weeds in rice fields but severely damaged bean varieties [ C. lunatus exhibits a temperature-dependent virulence [Nonetheless, the above-mentioned factors seem more likely to be limited in explaining howt decade –13. Remat decade  fused tharieties . Nevertharieties . C. lunairulence , 44 and  C. lunatus have coevolved with their different hosts translated by their different placement in ML tree inference and speciation in their nutritional requirements  versus (I), (II), and (III),  C. lunatus have invaded strawberry [ Mimusops elengi [ Amaranthus spinosus [ Grewia optiva [ Clerodendrum indicum [ C. lunatus.Owing to the divergence in evolution (s Tables  and 2. Irawberry , Mimusops elengi , Amarantspinosus , Grewia a optiva , Clerode indicum , and pot indicum , 48. Not C. lunatus colony, conidia size, conidia colour, conidia texture, and the presence or absence of stromata should be regarded as prominent acquired adaptational traits. These characteristic traits were not consistent between the five strains causing foliar necrosis of potato but provided indicators for generic circumscription. Phenotypic intraspecific variations can obscure placement of Cochliobolus species and make correlation to phylogeny difficult. As shown, C. lunatus have considerable ecological and economic importance being a highly successful colonizer in monocots, dicots, vertebrates, and invertebrates. On this basis, the purpose as a biocontrol agent is overshadowed by its virulent and indiscriminate destructive potential in the ecosystem. For this reason, we suggest that the use of C. lunatus as mycoherbicide should be stopped.From an evolutionary viewpoint, the variations observed in


Klebsiella pneumoniae to carbapenems is mainly associated with acquired carbapenem-hydrolyzing β-lactamases [K. pneumoniae are the K. pneumoniae carbapenemases (KPCs) [KPCblais crucial for infection control measures and appropriate choice of antimicrobial therapy [KPC blain strains of Klebsiella pneumoniae.Resistance of ctamases . These βctamases ,2. The m therapy . In DourThe samples were collected during February-May/2012 from patients attended at University hospital, Dourados/MS and identified by classical bacteriological methods. The strains were most frequently obtained from the urinary tract, vaginal secretion, nasal and rectal swab. Antibiograms were realized by using the disk-diffusion method on Mueller-Hinton agar (Bio-Rad Laboratories), and susceptibility break points were determined as previously described and interpreted as recommended by the Clinical and Laboratory Standards Institute . All strK. pneumoniae carbapenemase from patients were isolated. Among the wards, those that had a higher incidence of samples were recovered from intensive care units (ICUs) of the hospital, probably due to immune deficiency of patients, submitted to invasive therapeutic procedures. The strains identified as producing carbapenemases were evaluated by PCR amplification using primers specific for KPC blagene. Five K. pneumoniae carbapenemase strains were positive in PCR. Eighteen strains were positive to modified Hodge test, but were PCR negative. This profile difference could be due to the presence of other classes of carbapenemases. Thus, these eighteen strains of K. pneumoniae carbapenemase need to be tested for the presence of β-lactamases such as IMP, VIM, OXA, and NDM-1.From October 2012 to April 2013, 26 strains of