Aconitum species (belonging to the Ranunculaceae) are well known herbaceous medicinal ingredients and have great economic value in Asian countries. However, there are still limited genomic resources available for Aconitum species. In this study, we sequenced the chloroplast (cp) genomes of two Aconitum species, A. coreanum and A. carmichaelii, using the MiSeq platform. The two Aconitum chloroplast genomes were 155,880 and 157,040 bp in length, respectively, and exhibited LSC and SSC regions separated by a pair of inverted repeat regions. Both cp genomes had 38% GC content and contained 131 unique functional genes including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene order, content, and orientation of the two Aconitum cp genomes exhibited the general structure of angiosperms, and were similar to those of other Aconitum species. Comparison of the cp genome structure and gene order with that of other Aconitum species revealed general contraction and expansion of the inverted repeat regions and single copy boundary regions. Divergent regions were also identified. In phylogenetic analysis, Aconitum species positon among the Ranunculaceae was determined with other family cp genomes in the Ranunculales. We obtained a barcoding target sequence in a divergent region, ndhC–trnV, and successfully developed a SCAR (sequence characterized amplified region) marker for discrimination of A. coreanum. Our results provide useful genetic information and a specific barcode for discrimination of Aconitum species. Aconitum belongs to the Ranunculaceae and consists of approximately 400 species [A. carmichaelii and A. coreanum have been used as different herbal medicines in Korea and China, namely, Aconiti Lateralis Radix (Bu-ja in Korean and Fu-zi in Chinese) and Aconiti Koreani Tuber (Baek-bu ja in Korean and Guan-bai-fu in Chinese), respectively. In addition, the root tubers of A. kusnezoffii, A. ciliare (a synonym of A. volubile var. pubescens), and A. triphyllum  have also been used as Aconiti Kusnezoffii Tuber in both countries (Cho-oh in Korean and Cao-wu in Chinese) [A. carmichaelii have pharmacological effects such as cardiotonic action, impact on blood vessels and blood pressure, anti-arrhythmic effects, anti-inflammation, and analgesic action [A. coreanum have anti-arrhythmic, analgesic, and anti-inflammatory activities [A. kusnezoffii root tuber (Aconiti Kusnezoffii Tuber) has analgesic, cardiotonic, antioxidant, and immunological activities [Aconitum species have been used as a variety of herbal medicines. Aconitum plants also contain highly toxic components such as diester-diterpene alkaloids including aconitine, mesaconitine, and hypaconitine [Aconitum species are required because it is difficult to determine the species based on the morphological features of plants and medicinal materials.The genus  species . These gChinese) . The rooc action , whereastivities , 6. Thusconitine . Althougmatk, and rbcL have been used for the evaluation of phylogenetic relationships [psbA–trnH intergenic region showed genetic variation in 134 individuals belonging to 19 taxa of Aconitum [psbA–trnH intergenic spacer. Nineteen taxa of Aconitum were classified into ten subgroups. However, in Aconitum species, more genomic information is required for accurate identification. Although Aconitum species are important in herbal medicine, there is little genomic and sequence information available.Chloroplasts (cp) play an important role in photosynthesis and carbon fixation as well as in biosynthesis . The cp ionships , 15. In Aconitum , indicatAralia continentalis  and Angelica biserrata (Angelicae Pubescentis Radix), were clearly distinguished and tested for adulteration using a SCAR marker. Also, commercial herbal medicines were tested and were confirmed to be derived from refined herbal medicines and to be free of adulteration [The sequence characterized amplified region (SCAR) markers are valuable genetic tools for the authentication of medicinal plants and herbal medicines because they are simple, reliable, and reproducible , 17. Theteration . Therefoteration –22.de novo assembly of cp genomes from A. coreanum and A. carmichaelii and compared them with those from other Aconitum species to discover highly divergent regions. We also developed a SCAR marker, based on a sequence from a variable gene region in cp DNA, that can distinguish A. coreanum from other Aconitum species. The results will be useful for the standardization and quality control of the herbal medicine Aconiti Koreani Tuber, and could help provide important and valuable information for the verification of the evolutional traits and genetic diversity of A. coreanum and species belonging to the genus Aconitum L., as well as for the genetic engineering of these species.In this study, we completed A. carmichaelii and A. coreanum were used for next-generation sequencing (NGS) analysis, and 27 Aconitum germplasms were used for validation of the SCAR marker. DNA was extracted using a DNeasy Plant Maxi kit  according to the manufacturer’s instructions. Total libraries from A. carmichaelii and A. coreanum were generated using the MiSeq platform  by LabGenomics, Korea.Plant materials were collected from native habitats and a private farming field in Korea. A sample collection permit of the Korean Institute of Oriental Medicine was obtained from Korean National Parks Service for native habitats at June, 2011 . The leaves of de novo assembly from the low coverage whole genome sequence derived from the Phyzen pipeline (http://phyzen.com). Paired-end reads (Phred scores of 20 or more) were assembled using CLC genome assembler  with an autonomously controlled overlap size parameter of 200 to 600 bp. The principal contigs in the cp genome were retrieved from total contigs using Nucmer [Aconitum barbatum var. puberulum (KC844054) as the reference sequence. The contigs were ordered based on the results of BLASTZ analysis [A. carmichaelii and A. coreanum were obtained using OGDRAW [Cp genomes were obtained by g Nucmer  with theanalysis . Filtereanalysis  and manug OGDRAW . Codon ug OGDRAW .Aconitum species [A. carmichaelii (KY407560), A. coreanum (KU318669), A. volubile (KU556690), A. austrokoreense (KY407559), A. chiisanense (NC029829), A. pseudolaeve (KY407562), A. longecassidatum (KY407561), and A. barbatum (KC844054). The boundaries between IR and SC regions were also compared with those of eight Aconitum species: A. carmichaelii, A. coreanum, A. volubile, A. austrokoreense, A. chiisanense, A. pseudolaeve, A. longecassidatum, A. barbatum, Nicotiana tabacum (NC001879) and Thalictrum coreanum (KM206568). Tandem repeats were detected using Tandem Repeat Finder [http://pgrc.ipk-gatersleben.de/misa/misa) with a minimum number of repeats of 10, 5, 4, 3, 3 and 3 for mono-, di- tri- tetra- and hexa nucleotides, respectively, as the default parameter. IRs were detected using the Inverted Repeats Finder [mVISTA was used to analyze similarities between eight  species : A. carmnense NC0829, A. ps Finder  with defAconitum species in the Ranunculaes, 20 cp genomes of 13 Aconitum species, seven from other species of Ranunclaceae, and 8 species of five other families were used with Nicotian tabacum (NC001879) and Arabidopsis thaliana (AP000423) as the outgroup. Seventy protein-coding genes were aligned using MAFFT. . Maximum likelihood analysis was performed using MEGA6 with 1,000 bootstrap replicates [A molecular phylogenetic tree was constructed using 70 protein-coding genes from 38 species, and 36 complete cp genomes were downloaded from GenBank . To idenplicates .Aconitum species were confirmed based on mVISTA similarities, and primers were designed using Primer-BLAST (NCBI). To amplify divergent regions, about 20 ng genomic DNA was added to 20 ul PCR mixture  containing 10 pmol primers . Amplification was performed on a Pro Flex PCR system  according to the following step-cycle program: initial denaturing step at 95°C for 2 min; 35 cycles at 95°C for 40 s, 55°C for 30 s, and 72°C for 90 s; and a final extension at 72°C for 5 min. The PCR products were separated on a 2% agarose gel for 40 min at 150 V. Amplified DNA fragments were extracted from agarose gels using a Gel Extraction Kit  and subcloned into the pGEM-T Easy vector . Inserted DNA was sequenced using a DNA sequence analyzer . NGS data and Sanger sequencing data were aligned using CLUSTALW by Bioedit [A. coreanum-specific primers were designed for amplification of the SCAR marker. PCR reactions were performed with A. coreanum-specific primers. Amplification conditions were as follows: 95°C for 2 min; 35 cycles at 95°C for 30 s, 63°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. Amplified fragments were verified using 1.5% agarose gel electrophoresis.Highly divergent regions between eight  Bioedit . A. coreA. carmichaelii and A. coreanum  separated by LSC  and SSC  regions  and A. chiisanense . The total GC content was 38.13% and 37.99% for A. carmichaelii and A. coreanum, respectively, which is similar to published Aconitum species [A. carmichaelii and A. coreanum chloroplast genomes. The IR region showed a higher GC content due to the presence of four rRNA  sequences. Previous studies have reported that high GC content in IR regions is due to the presence of rRNA [Aconitum genomes were AT-rich . The chloroplast genomes of A. carmichaelii and A. coreanum encoded a total of 112 unique genes, of which 18 were duplicated in IR regions. Among the 112 genes, there were 78 protein-coding genes, four rRNA genes, and 30 tRNA genes  had a single intron and two  had two introns , 1.8% and 1.79% tRNA , and 5.8% and 5.76% rRNA . The remaining regions were non-coding sequences with intergenic spacers, introns, and pseudogenes. Two pseudogenes were identified: rps16 had one exon deletion in both A. carmichaelii and A. coreanum. The ycf1 pseudogene was located in the boundary region between IRa and SSC. The codon usage and recognition pattern of the cp genomes are summarized in A. carmichaelii and A. coreanum, respectively  and IR (two repeats) regions. The length of repeats ranged from 21 to 36 bp.Repeat sequences were abundant and diverse in the two genomes, where they play an important role in gene duplication, gene expansion, and DNA rearrangement . Repeat  genomes . The tant events , 42. We ectively . Among tA. carmichaelii and A. coreanum, respectively . The numbers of SSRs in SSC and IR regions were similar  were located exclusively in LSC regions; those of other Aconitum species extended into the IR regions. The ycf1 genes were located at the junctions of IRb/SSC and SSC/IRa border. IRb/SSC extended into ycf1 genes in all Aconitum species except for A. barbatum. The length of ѱycf1 ranged from 1,215 to 1,395 bp in Aconitum species. The ycf1 gene was embedded at the IRa/SSC border. The trnH genes were all located in LSC regions, 17–114 bp away from the IRa/LSC boundary  and Arabidopsis thaliana (AP000423), were set as outgroups. Maximum likelihood (ML) analysis was performed with 1,000 bootstrap replicates. The reconstructed phylogeny showed that 21 nodes had bootstrap values >95%, and 19 of these had bootstrap values of 100%. The clusters consisting of 38 species, including 21 Aconitum species, were well supported in six families; Ranunculaceae, Berberidaceae, Menispermaceae, Lardizabalacea, Euptrleaceae, and Papaveraceae in Ranunculales. Ranunculaceae and Berberidaceae are the closest families according to the APG III system of flowering plant classification [Aconitum species were strongly clustered, and M. saniculifilia and T. coreanum were closer than other species in Ranunculaceae. Twenty-one Aconitum species formed a clade as two subgenera, Aconitum subgen. Aconitum and Aconitum subgen. Lycoctonum. A. barbatum clustered with A. longecassidatum and A. pseudolaveve in the subgenus Lycoctonum [Aconitum subgen. Aconitum, A. ciliare (NC031420), A. carmichaelii (KY407560), A. ciliare (KT820666), A. jaluense (KT820668), A. japonicum (KT820670), A. kusnezoffii (NC031422), and A. carmichaelii (NC030761) clustered monophyletically (subclade A) with a bootstrap value of 100%. Another monophyletic subclade (subclade B) also formed a cluster and included A. volubile (KU556690), A. jaluense (KT820669), A. austrokoreense (KY407559), with a weak bootstrap value of 62%. A. kusnezoffii (KT964696) clustered paraphyletically with subclade A and B. In addition, A. jaluense was included in both subclades (subclade A and B)  and A. jaluense (KT820668) (A. kusnezoffii (KT964696) and A. jaluense (KT820669) ..Aconitumcoctonum . In AconT820668)  and A. kT820669) , respectA. kusnezoffii and A. jaluense were unchanged in the Aconitum subgen. Aconitum  clustered monophyletically  having different accessions from the same species did not clearly cluster monophyletically  and A. coreanum (KU318669) showed a polyphyletic relationship in Aconitum subgen. Aconitum , whereas no PCR product was observed for other Aconitum species  in Aconitum germplasms. We observed several nucleotide substitutions that could be used to identify A. coreanum and A. kiriensis from other Aconitum species using comparative analysis of entire sequences of the nrDNA-ITS regions. However, the number of nucleotide substitutions in the nrDNA-ITS regions was not enough to develop a SCAR marker, and matK gene sequences did not show any sequences differences similar to those found in the psbA-trnH region. Therefore, we conducted further comparative analysis of complete cp genome sequences to overcome the limitations of the universal DNA barcode used previously to distinguish Aconitum species. Since Aconitum cp genomes have high similarity (97.36–99.9%), it was difficult to develop species-specific markers for each Aconitum species. Although plants of Aconitum species show morphological differences in their areal parts, identification of the botanical origin of the dried tubers used in herbal medicines is very difficult. In this study, we developed an A. coreanum-specific SCAR marker using ten Aconitum taxa. However, further study will be required to distinguish between different Aconiti herbal medicines employing Aconitum species and to identify the botanical origins at the species level for quality control purposes. The SCAR marker developed in this study will help authenticate rapidly and accurately herbal medicines, and determine whether they have been adulterated or not.In a previous study, 19  species . HoweverA. coreanum and A. carmichaelii belonging to the Ranunculaceae family. The two Aconitum species had slightly different genome sizes, a feature shared by other Aconitum species. Furthermore, the distributions and locations of SSRs were determined. The cp genome sequences were compared with those of six other Aconitum species. The most divergent regions were found in the trnK-UUU–trnQ-UUG, trnS-GCU–trnG-GCC, petN–psbM, ndhC–trnV-UAC, ycf4–cemA, rpl18–rpl20, and trnT–psbD non-coding regions, and in the ycf1-, ycf2-, rpl20-, and rpoc2-coding regions. A phylogenetic analysis was performed on the 20 cp genomes of 13 Aconitum species with the cp genomes of five other families within the Ranunculales. We also developed a SCAR marker for A. coreanum using a divergent region of the Aconitum cp genome. These results provide useful information for phylogenetic studies on the Ranunculaceae and the biological systematics of Aconitum L., as well as for the conservation biology of A. coreanum. Furthermore, the SCAR maker could be useful in distinguishing A. coreanum from other Aconitum species, particularly with respect to the authentication of herbal medicines.In this paper, we obtained the complete chloroplast sequences of S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.


Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55 Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis’s capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree. Hevea brasiliensis Muell. Arg, the Pará rubber tree, or most commonly known as the rubber tree, is a medium to large monoecious and cross-pollinated tropical tree belonging to the family Euphorbiaceae (spurge)H. brasiliensis, Euphorbiaceae includes a number of plants with economic importance, such as cassava (Manihot esculenta), the castor oil plant (Ricinus communis) and Barbados nut (Jatropha curcas). The genus Hevea is comprised of eleven speciesH. brasiliensis, Hevea bethamiana, Hevea camporum, Hevea camargoana, Hevea guianensis, Hevea microphylla, Hevea nitida, Hevea pauciflora, Hevea paludosa, Hevea rigidifolia and Hevea spruceana34Hevea species are diploid (2n = 2x = 36) with the exception of triploid H. guianensis (2n = 2x = 54) and the existence of one genotype of H. pauciflora with 2n = 2x = 186Hevea behaves as a diploidHevea has an amphidiploid origin stabilized during the course of evolutionH. brasiliensis yields an economically viable amount of latex that makes up most (99%) of the world’s natural rubber production. In 2014, global natural rubber production was estimated at 12 million tons with a corresponding value of 22 billion US dollars (http://www.lgm.gov.my).H. brasiliensis seedlings to AsiaH. brasiliensis is a deciduous tree that grows to 30–40 m tall and can live up to 100 years in the wildcis-configuration. Natural rubber differs from synthetic rubber in polymer length and side chain composition, which contribute to its superior performance properties in elasticity, resilience and efficient heat dispersion. Hence, despite accounting for less than 50% of total rubber consumption, natural rubber is still indispensable for heavy-duty tires and high-performance engineering components.The rubber tree is indigenous to the Amazon basin in South America, but is now widely planted in Southeast Asia, especially in Malaysia, Indonesia and Thailand. The domestication of rubber trees outside Brazil occurred more than 100 years ago with the transfer of H. brasiliensis, rubber is synthesized on the surface of rubber particles suspended in the latex of laticifers or specialized parenchyma cellsH. brasiliensis clone RRIM 600, a secondary clone originally developed from clones Tijr 1 and PB 86, is the most widely planted clone in Malaysia and Thailand13Phytophthora spp. (http://www.rubberboard.org.in).In H. brasiliensis rubber production and other desirable traits have been progressing slowly. A major hurdle for crop improvement of H. brasiliensis is the lack of a comprehensive whole-genome overview and therefore, a holistic knowledge of the latex biosynthetic process. A previous attempt to assemble the H. brasiliensis genome produced a fragmented draft using 43-fold sequence dataH. brasiliensis is imperative for discovery of the precise pathways and genes responsible for desirable agronomic traits. Here, we report a more complete H. brasiliensis genome assembly that allows genomic comparison with other sequenced plant genomes and cap analysis gene expression (CAGE) studies on tissue-specific transcription of genes, particularly those related to rubber biosynthesis. Our effort unraveled genome-level insights on the evolutionary relationship of H. brasiliensis with other Euphorbiaceae species. Most importantly, our analysis reveals possible contributors to H. brasiliensis’s capacity to produce high levels of latex and alternative means of transcriptional regulation for rubber biosynthesis.Despite its economic importance, past attempts in genetic analyses to improve H. brasiliensis was constructed using the whole-genome shotgun sequencing approach utilizing both Illumina and PacBio reads. A total of 288.84 Gb (~134-fold coverage) of raw sequencing data was generated using the Illumina platform from both paired-end and mate-pair libraries with different insert sizes J. curcas (33.7%)M. esculenta (34.86%)The genome assembly of rt sizes . The genH. brasiliensis to the genome assembly. Of the 51,631 H. brasiliensis ESTs available from NCBI, 96.8% were detected in the assembly .The completeness of the assembly was evaluated by aligning EST sequences of assembly . The assH. brasiliensis was analyzed by aligning H. brasiliensis scaffolds against itself using reciprocal BLAST matches. The 90 pairs of scaffolds that contained the largest number of paralogous gene pairs were selected and visualized in a dotplot , of which ~11.7% and ~43.6% are Copia and Gypsy elements, respectively. In contrast, DNA transposons only represent ~1.2% of the nuclear DNA. Closer inspection revealed that the 3.71 ratio of Gypsy to Copia LTR-retrotransposons in the rubber genome is similar to that of the sorghum genome (3.66)Transposable elements represent major constituents of plant genomes. A combination of d genome . This led genome . The notab initio gene predictions, homology-based searching and transcriptome alignments was used for genome annotation. Protein sequences from the M. esculenta, R. communis, J. curcas, P. trichorpca and Viridiplantae datasets from Swiss-Prot were used as homology-based evidence. Assembled contigs of RNA-seq reads from leaf, latex and bark tissuesH. brasiliensis genome maintains more genes associated with ‘binding’ and ‘catalytic activity’ subcategories than other genomes  and dicotyledons , sharing a total of 8,627 common gene families  revealed that a large cluster of 12,406 gene families is shared among the four Euphorbiaceae genomes , as well as the more distantly diverged species , was evaluated , abnormal leaf fall (Phytopthora spp.), powdery mildew (Oidium heveae), Corneyspora leaf fall (Cornyespora cassiicola), pink disease  and white root disease (Rigidoporus spp.)H. brasiliensis genome will provide a basis for improvement of disease resistance.Disease resistance is one of the most important traits identified as a target for rubber plant breeding. The rubber tree is susceptible to several fungal diseases, including South American leaf blight (H. brasiliensis suggests an enhanced ability to detect and defend against pathogen invasion in latex-producing H. brasiliensis. These R genes found in the H. brasiliensis genome belong to the nucleotide-binding site (NBS) resistance genes and constitute about 0.57% of all H. brasiliensis genes. The NBS-encoding genes can be classified into multiple subfamilies, including 186 NBS-leucine-rich repeat (NBS-LRR), 45 coiled-coil (CC)-NBS-LRR, 35 CC-NBS, 15 Toll interleukin-1 receptor-NBS-LRR (TIR-NBS-LRR) and 4 TIR-NBS. Some of these disease resistance-related genes were found organized in clusters in the H. brasiliensis genome. In particular, scaffold284, scaffold589 and scaffold2863 were found to contain clusters containing four, six and four disease resistance-related genes, respectively , ethylene and jasmonic acid (JA). Nonexpressor of PR genes 1 (NPR1) and its paralogues, NPR3 and NPR4, are known to function as SA receptors36caffolds . In contH. brasiliensis is a high molecular weight biopolymer that is made up of cis-isoprene units derived from isopentenyl diphosophate (IPP). The biosynthesis of IPP proceeds via two distinct routes: the cytoplasmic mevalonate pathway (MVA) and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway  was observed in H. brasiliensis latex and leavesH. brasiliensis genome has a larger number of MVA and MEP pathway genes than A. thaliana (58 versus 33). For example, DXP synthase, catalyzing the first step in the MEP pathway, has expanded from three genes in A. thaliana to nine in H. brasiliensis. All of the enzymes involved in the MVA and MEP pathways are encoded by multiple genes in H. brasiliensis, indicating the expansion of these gene families in this genome. The expansion of MVA and MEP pathway genes in H. brasiliensis meets the need for its formation of isoprenoids.Natural rubber from  pathway . In the H. brasiliensis  activityH. brasiliensis, the entire CPT gene family comprising seven members (designated as CPT1-7) was identified. Based on the phylogenetic analysis of CPT sequences from H. brasiliensis and 14 other plant species, five of the seven H. brasiliensis CPT genes (CPT1-5) form a subgroup iliensis . The polsubgroup . A membecis-polyisoprene productionH. brasiliensis, REF and SRPP are encoded by nine and eight genes, respectively, which are the largest numbers reported in any plant genome (H. brasiliensis genome correlates with its capacity to produce high levels of latex. A cis-prenyltransferase-like (CPTL) protein that was suggested to be involved in rubber biosynthesis in lettuce and dandelion46H. brasiliensis genome. Among the annotated rubber biosynthesis-related genes, some are found in gene clusters  and small rubber particle (SRPP), have been shown to participate in t genome . This siclusters . For exaH. brasiliensis genes, we analyzed the transcription start sites (TSSs) of genes expressed in different tissues by CAGE analysis. This method enables high-throughput gene expression analysis and the precise mapping of TSSs to the genomeH. brasiliensis genome allowed us to identify 2,948,134 potential TSSs. The CAGE data obtained from biological replicates derived from different tissues showed high correlation, indicating the reproducibility of the analysis  have different TSSs depending on the tissue. The observation of multiple TSSs from the CAGE analysis provides invaluable resources for identifying alternative promoters that cannot be obtained by other transcriptomic methods.The expression profiles of rubber biosynthesis-related genes including CPTs, CPTL, SRPPs and REFs in different tissues were analyzed to investigate the genetic basis underlying latex production . It was The expression profiles of MVA and MEP pathway genes were also examined to provide insights into the formation of the intermediates associated with rubber biosynthesis. CAGE analysis revealed high expression levels of MVA pathway genes in latex, as compared to leaf and bark . One memH. brasiliensis genome was characterized as high in repeat content and heterozygosity, and with a large genome size that poses technical challenges in assembling its genome. Using a combination of a high coverage Illumina paired-end, mate-pair and long PacBio reads, the H. brasiliensis RRIM 600 genome was assembled. The final assembly spans 1.55 Gb and represents more than 93.7% of the transcriptome sequences14212223H. brasiliensis genome assembly is much improved in continuity, allowing for comparative analyses with other plant genomes and genome-wide characterization of protein-coding genes.The H. brasiliensis and other members of Euphorbiaceae including M. esculenta, R. communis and J. curcas1819H. brasiliensis gene blocks showed one-to-one, one-to-two and one-to-three synteny relationships with J. curcas, M. esculenta and R. communis, respectively. When compared with other Euphorbiaceae genomes, our analysis revealed that H. brasiliensis has the highest number of disease resistance-related genes. The expanded inventory of these genes could conceivably play a role in resistance to abiotic stresses or diseases caused by fungal pathogens. Our genomic and CAGE-seq analyses offer some insights into the biological features that are unique to H. brasiliensis, particularly those that are involved in rubber biosynthesis. The H. brasiliensis genome contains one of the richest set of rubber biosynthesis-related genes including CPTs, REFs and SRPPs. The expansion of these gene families and the high expression of these genes in latex could be an important contributor to high latex production in H. brasiliensis. Interestingly, some of these rubber biosynthesis-related genes are organized in clusters in the genome, indicating coordinated evolution and expression for latex production. Expression profiles of rubber biosynthesis-related genes analyzed using CAGE analysis showed that some of these genes exhibit tissue-specific TSSs. Although further studies are required to confirm these observations, these results reveal alternative means of transcriptional regulation besides changes in mRNA abundance. Detailed analyses on the expression of these rubber biosynthesis-related genes and their complex transcriptional control provide clues to understanding the unique properties of the rubber tree. The whole-genome sequence information and CAGE-seq data are valuable resources for use in the improvement of the economically important traits of the rubber tree.Comparative genomics analysis clearly indicates conserved synteny between H. brasiliensis clone RRIM 600 using the Qiagen DNeasy Plant Mini kit  following the manufacturer’s instructions. A 500 bp insert size paired-end library was prepared using the Illumina TruSeq DNA Sample Preparation Kit version 2 . Mate-pair libraries with insert sizes of ~3 Kb, 5 Kb, 7 Kb and 10 Kb were prepared according to the Illumina Nextera Mate Pair Sample Prep Kit . Sequencing was performed on the Hiseq 2500 and Miseq systems . One PacBio library with a 10 Kb insert size was sequenced with 100 SMRT cells using P5-C3 chemistry. PacBio sequencing was performed on RS II  at the Yale Center for Genomic Analysis in West Haven, CT, USA.High-quality genomic DNA was extracted from young leaves of H. brasiliensis clone RRIM 600. Leaf and bark samples were obtained from young rubber plants; latex was collected from mature plants growing at the Centre for Chemical Biology, Penang, Malaysia. Fresh tissues from leaves and bark were flash-frozen in liquid nitrogen and ground to a fine powder. Latex samples were added to the extraction buffer directly. High-quality total RNAs were extracted using the CTAB-LiCl reagent followed by further TRI Reagent®  treatmentTotal RNAs were isolated from leaf, bark and latex of k-mer auto-extensione-value cutoff of 1e-20. Hits that comprised > 90% identity and > 85% coverage were considered mapped markers. Quality of the genome assembly was assessed by mapping all available H. brasiliensis EST sequences downloaded from NCBI and published transcriptome data from leaf, latex and bark tissues14212223Raw sequencing reads from each library  were prede novo identification of repeat families in the genome sequences.Repeat sequences in the genome assembly were identified using RepeatMasker (version 4.0.5)ab initio gene predictions, homology-based searching and transcriptome alignment. Augustus (version 3.0.3)de novo gene prediction. Protein sequences from M. esculenta, R. communis, J. curcas, P. trichorpca and the Viridiplantae dataset from Swiss-Prot were used as homology-based evidence. RNA-seq reads from leaf, latex and bark tissuese-value of 1e-5. GO terms were assigned to the annotated genes using the Blast2GO pipeline (version 3.1.3)e-value cutoff of 1e-10. Transcription factors in H. brasiliensis were identified and classified into families using the iTAK pipeline (http://bioinfo.bti.cornell.edu/tool/itak).Gene models were predicted using the Maker-P pipeline (version 2.31.8)31H. brasiliensis, A. thaliana, O. sativa, S. lycopersicum, V. vinifera, M. esculenta, R. communis and J. curcas was used to identify gene clusters. All-against-all BLASTP analysis of all protein sequences was performed with an e-value cutoff of 1e-5. OrthoMCL (version 2.0.9)Ks) and non-synonymous (Ka) substitution rates for orthologous gene pairs in H. brasiliensis and Euphorbiaceae members were computed using the KaKs_Calculator tool (version 2.0)H. brasiliensis’s scaffolds that have more than 15 gene syntenic regions against all four organisms examined  were visualized using Circos (version 0.67–7).The non-redundant protein dataset from H. brasiliensis were identified using HMMER (version 3.1)NBS genes in Accession codes: The H. brasiliensis genome assembly, genome and CAGE sequencing data were deposited at DDBJ/EMBL/GenBank BioProject under accession PRJDB4387. The rubber tree genome database is accessible at http://plant.psc.riken.jp/cgi-bin/gb2/gbrowse/rubber.How to cite this article: Lau, N.-S. et al. The rubber tree genome shows expansion of gene family associated with rubber biosynthesis. Sci. Rep. 6, 28594; doi: 10.1038/srep28594 (2016).


Rickettsia species in Madagascar, human and small mammal samples from an urban and a rural area, and their associated fleas were tested.Rickettsiae are obligate intracellular bacteria responsible for many febrile syndromes around the world, including in sub-Saharan Africa. Vectors of these pathogens include ticks, lice, mites and fleas. In order to assess exposure to flea-associated Rattus rattus out of 86 small mammals presented antibodies against TGR. Out of 117 fleas collected from small mammals, Rickettsia typhi, a TGR, was detected in 26 Xenopsylla cheopis (24%) collected from rodents of an urban area (n = 107), while two of these urban X. cheopis (2%) were positive for Rickettsia felis, a SFGR. R. felis DNA was also detected in eight (31%) out of 26 Pulex irritans fleas.Anti-typhus group (TGR)- and anti-spotted fever group rickettsiae (SFGR)-specific IgG were detected in 24 (39%) and 21 (34%) of 62 human serum samples, respectively, using indirect ELISAs, with six individuals seropositive for both. Only two (2%) Rickettsia pathogens, R. typhi and R. felis, are present near or in homes. Although our results are from a single district, they demonstrate that rickettsiae should be considered as potential agents of undifferentiated fever in Madagascar.The general population in Madagascar are exposed to rickettsiae, and two flea-associated  Rickettsia africae, a SFGR, in Amblyomma ticks collected from cattle  were placed inside 15 and 20 houses in the rural and urban areas, respectively. Where possible a further BTS trap was set in the immediate vicinity of houses , Maryland, USA, were used as TGR and SFGR ELISA antigen, respectively. Coating and blocking buffer was PBS-X powder  dissolved within ultra-pure water . Peroxidase labelled anti-human IgG , anti-rat IgG peroxidase-conjugate  and protein-A peroxidase (Sigma) were used as secondary antibodies for human, rat and shrew sera, respectively. Secondary antibody dilutions were chosen to optimise the performance of the ELISAs according to the difference in absorbance  of negative and positive controls. Anti-human secondary antibodies dilutions were 1:4000 for TGR and 1:3000 for SFGR ELISA, while anti-rat conjugate and protein-A conjugate were diluted at 1:2000. The absorbance was read at 405 nm using ELx800 spectrophotometer  after 15 min of incubation. On each plate, one positive control and three negative controls were included. The positive controls for TGR and SFGR (human and mouse) were provided by NMRC. The human negative controls for TGR and SFGR were pools of Malagasy human sera confirmed negative for TGR and SFGR at the NMRC. The negative rodent controls were obtained from black rats born in captivity at Institut Pasteur de Madagascar. A sample was considered positive when the ratio (net OD/mean three negative controls + 3SD) ≥ 2. Positive samples were subsequently included in a titration ELISA with four-fold dilution of the sera from 1:100 to 1:6400 to determine the individuals’ antibody titer.To assess previous exposure to eviously  with sliFlea species were identified using a dissecting microscope using morphological keys for all fleas described in Madagascar . DNA wasn = 117) and one flea per house in Miandrarivo (n = 26) were screened for the presence of Rickettsia spp. using the genus-specific real-time PCR assay with RKND03F (5′-GTG AAT GAA AGA TTA CAC TAT TTA T-3′), RKND03R (5′-GTA TCT TAG CAA TCA TTC TAA TAG C-3′) and RKND03P (5′-FAM-CT ATT ATG CTT GCG GCT GTC GGT TC-TAM RA-3′), based on the gltA gene  or SFGR . Evidence of a difference in seroprevalence for SFGR between males and females approached significance , females 19% (n = 23); P = 0.052). No such pattern was observed for TGR ; P = 0.42). Titers of anti-SFGR- and anti-TGR-specific IgG detected in human sera ranged from 100 to 6400. The geometric mean titers were 673 for anti-TGR IgG and 635 for anti-SFGR antibodies.Overall 39 and 34% of human serum samples tested positive for IgG against TGR and SFGR respectively Table . There wR. rattus were > 6400. No rodent from Ambarivatry presented with antibodies to TGR, and anti-SFGR IgG were not detected in small mammal sera from either area.Two rat samples from the 46 small mammals collected in the Andrefanigara were positive for anti-TGR antibodies based on the ratio method Table . The titX. cheopis fleas tested from 19 rats and one mouse caught in Andrefanigara (n = 107), 24% were positive for R. typhi DNA and 2% for R. felis DNA. Fleas positive for R. typhi DNA were collected from eight rats, and for these rats the proportion of positive fleas ranged from 7% (n = 15 fleas) to 100% , with a median of 90%, indicating a very clumped distribution of infected fleas. Rats from houses and their immediate vicinity were not more likely to carry infected fleas  than those from outdoor lines around vegetation . Of the P. irritans fleas tested from Miandrarivo, 31% (n = 26) were positive for R. felis DNA.Results of rickettsial DNA detection in fleas from Tsiroanomandidy are shown in Table Rickettsia spp. ompB sequences were obtained from 6 P. irritans from Miandrarivo and 6 X. cheopis from Andrefanigara (GenBank accession numbers KX090272–KX090283). These sequencing data were consistent with results from the species-specific qPCR assays and revealed four variants of R. felis and a single variant of R. typhi  [X. cheopis fleas, and Tsiroanomandidy district has a particularly high plague incidence [X. cheopis fleas may explain the high exposure rate to TGR. A study in Malang, Indonesia, a city with a history of plague, reported similarly high anti-TGR IgG seroprevalences, with a significant increase from rural to suburban to urban population  [X. cheopis fleas in the urban site and a lack of evidence of infection in the few X. cheopis sampled at the rural site. Clearly, our flea data represented a single time point, whilst seroprevalence in the human population reflected exposure rates over time, and more data are needed to assess urban–rural differences in flea abundance, flea infection rates and anti-TGR IgG seroprevalence in the human population.A previous study of apparently healthy participants in seven African countries that used an indirect immunofluorescent antibody assay (IFA) found seroprevalences of antibodies against .5–3.1%) . These r.5–3.1%)  study mancidence . Thus hictively) . We founR. felis may be a primary source of these cross-reactions [R. felis reacted with SFGR antigens, but not with TGR antigens [Anti-TGR and SFGR IgG were detected in six human sera. Thus 25% of samples that reacted to TGR antigens also reacted to SFGR antigens. This is a similar proportion to the findings reported in the Dupont et al.  study ofeactions . Howeverantigens . Thus, oRickettsia sp. Apart from R. typhi, the TGR also includes R. prowazekii. As this louse-borne pathogen is most common in Africa in cooler, high altitude areas [R. typhi in fleas, we believe exposure to R. typhi is more likely. A range of SFGR are known to occur in Africa, with most of these transmitted by ticks [. In the Dupont et al. [R. conorii antigens by IFA were used in a western blot against R. conorii and R. africae (another SFGR group member). The results indicated that the majority of sera reacted most strongly against R. africae [R. conorii, Rhipicephalus spp., are found throughout Africa, the prevalence of antibodies to SFGR appeared highest in areas where Amblyomma ticks (commonly associated with cattle) are found. This led Dupont et al. [R. africae rather than R. conorii. However, since this study, several studies have found that R. felis may be an important cause of febrile disease in Africa [R. africae in Amblyomma ticks collected from cattle in Madagascar but, as described above, a low seroprevalence of anti-SFGR antibodies in pregnant women using an IFA test [R. africae antigen with strong positive samples from the original test indicated titres suggestive of previous R. africae infection. However, reactivity to R. felis antigens was not assessed. It was not, therefore, currently possible to ascertain the proportion of the population exposed to R. africae, R. felis or other SFGR species and further work is needed to confirm the identity of SFGR infecting the Malagasy population.Whilst the group-specific ELISA tests are useful for an epidemiological serosurvey, the gold standard for diagnostics is IFA, followed by western blot, as this is more specific and can provide information on the most likely infecting de areas , 26 and by ticks , and ourt et al.  study, a africae . Moreovet et al.  to specun Africa , 20, 28.Rickettsia spp. in the general population of Tsiroanomandidy district, rickettsioses may be under-diagnosed as causes of undifferentiated fever, as had been reported in studies of febrile patients in Kenya, Tanzania and Senegal [R. typhi and R. felis to cause disease in humans. Similar studies are needed to determine the role of rickettsial agents in febrile syndromes in Madagascar.Awareness of rickettsioses amongst clinicians in Madagascar is low. Given the high seroprevalences of antibodies to  Senegal , 29, 30.X. cheopis is likely to be a major vector for murine typhus in Madagascar, as has been found elsewhere [Rickettsia felis is most commonly associated with the cat flea, Ctenocephalides felis [X. cheopis [R. felis-specific qPCR reported R. felis in P. irritans [R. felis-specific qPCR assay as used in this study detected rickettsial DNA in P. irritans fleas, further testing revealed that this was actually detection of a new Rickettsia sp., Rickettsia asembonensis, which is closely related to R. felis but not known to cause disease in humans [R. felis ompB sequence from P. irritans in Madagascar confirmed that this flea species can carry R. felis. However, the epidemiological cycle of R. felis in Africa remained unclear, with R. felis detected in a number of other arthropods, including mosquitoes and bed bugs [Our results indicate that lsewhere . Rickettes felis , 31. How cheopis –34. A stirritans . More ren humans , 36. Ourbed bugs .X. cheopis collected from rats, seroprevalence of rat antibodies against Rickettsia spp. in this site was surprisingly low, with only two R. rattus out of 84 positive with anti-TGR IgG. One of the two seropositive rats carried no fleas, whilst the second carried 13 X. cheopis fleas, one of these fleas was positive for R. felis by qPCR. Our seroprevalence compares with high seroprevalence detected in several other studies. Antibodies to R. typhi were detected in 23.7% of rodents in Thailand [Rattus captured in Jayapura, Indonesia [R. rattus do play a role in Madagascar given their known reservoir capacity [R. typhi positive fleas feeding on them.Although we detected rickettsial DNA in Thailand , 11% of ndonesia  and 35.9ndonesia . Our lowndonesia , it seemcapacity  and the R. typhi and R. felis in fleas. We also confirm that the human flea, P. irritans, can be infected with R. felis, a Rickettsia species recently highlighted as an agent of febrile disease elsewhere in Africa. Further testing of human sera and flea samples from other districts is currently being conducted, and will provide more detail on the prevalence, distribution and risk of Rickettsia infections.We found evidence here of exposure to both TGR and SFGR pathogens in the general human population, suggesting that rickettsioses should be considered as potential causes of undifferentiated fever in Madagascar. Molecular evidence demonstrates the presence of


Antimicrobials are critical to contemporary high-intensity beef production. Many different antimicrobials are approved for beef cattle, and are used judiciously for animal welfare, and controversially, to promote growth and feed efficiency. Antimicrobial administration provides a powerful selective pressure that acts on the microbial community, selecting for resistance gene determinants and antimicrobial-resistant bacteria resident in the bovine flora. The bovine microbiota includes many harmless bacteria, but also opportunistic pathogens that may acquire and propagate resistance genes within the microbial community via horizontal gene transfer. Antimicrobial-resistant bovine pathogens can also complicate the prevention and treatment of infectious diseases in beef feedlots, threatening the efficiency of the beef production system. Likewise, the transmission of antimicrobial resistance genes to bovine-associated human pathogens is a potential public health concern. This review outlines current antimicrobial use practices pertaining to beef production, and explores the frequency of antimicrobial resistance in major bovine pathogens. The effect of antimicrobials on the composition of the bovine microbiota is examined, as are the effects on the beef production resistome. Antimicrobial resistance is further explored within the context of the wider beef production continuum, with emphasis on antimicrobial resistance genes in the food chain, and risk to the human population. The emergence of antimicrobial resistance in bacterial pathogens is a serious global issue. Antimicrobial use in livestock, aquaculture, pets, crops, and humans selects for antimicrobial-resistant (AMR) bacteria that reside in agricultural and clinical biomes. Besides pathogens, AMR bacteria include many harmless and beneficial microbes acting as a genetic reservoir of AMR gene determinants , whiWorldwide, beef production is the third largest meat industry , behind swine and poultry [Antimicrobials are used in beef cattle for the therapeutic treatment of infections caused by bacteria or other microbes. Cattle can be afflicted by a variety of endemic infectious diseases, which may exist ubiquitously in the ranching environment . EndemicAntimicrobial use in cattle is unavoidable for the treatment of infections for which vaccines, bacterins, or alternate therapies are not available. A prevalent, controversial practice involves antimicrobials used in non-therapeutic applications. Judicious antimicrobial use typically requires that diseased cattle are treated individually to maximize therapeutic efficacy and reduce the spread of AMR, but entire herds are often dosed with in-feed antimicrobials. This is the typical administration route for practices such as (I) prophylaxis, (II) metaphylaxis, and (III) growth promotion. These practices are described by inconsistent and often agenda-driven terminology. For example, prophylaxis and metaphylaxis are considered therapeutic uses by the American Veterinary Medical Association and the US Food and Drug Administration (FDA) , 9, but Although prophylaxis/metaphylaxis may be a more judicious use of antimicrobials than growth promotion, growth promotion is often a benefit of either treatment. For example, antimicrobial treatment and prevention of cattle liver abscesses simultaneously provides prophylactic/metaphylactic therapy and growth promotion. Liver abscesses occur frequently in cattle, and are common in feedlots, where high-energy grain-based diets can cause acidosis, leading to ruminal lesions that predispose cattle to hepatic disease caused by invasive bacteria . Cattle Antimicrobial usage data is scarce: most countries do not survey or collect usage data, and cattle producers and pharmaceutical companies have little incentive to report such information. Where usage data exists, typically in high-income countries, it takes the form of volume sales data rather than actual usage. The caveat of antimicrobial sales and distribution data is that it does not accurately indicate how or if antimicrobials were used. In a global analysis of antimicrobial usage, Van Boeckel et al.  estimateData for antimicrobial usage by animal type is not routinely available, such that the proportion and type of antimicrobials sold exclusively for use in cattle is largely unknown or estimated. Some information can be gleaned from country data where specific antimicrobial formulations with indicated routes of administration  are provided for specific livestock Fig. . HoweverOne method to improve antimicrobial usage estimate by species is to take into account (I) the size of the animal population (demographics), and (II) the average theoretical weight of the animal species at time of treatment (physiology). This is the population correction unit (PCU), and is used in the UK Veterinary Medicines Directorate UK-VARSS report , the EU Eimeria spp. infestations [In some countries, the majority of antimicrobials manufactured or sold are used in food animals rather than in human medicine  vs. ~3,290 t for humans (2012) , 25; EU:stations . In the 14 vs. ~3stations . Besidesstations , a policstations . SignifiCampylobacter, Salmonella, E. coli and Enterococcus spp. These species are typically used as ‘indicators’ of AMR in production animals as they (I) are of importance in human disease, (II) are relatively easy to culture, (III) can be isolated from healthy animals, and (IV) have established AMR minimum inhibitory concentration (MIC) breakpoints (for human infections). To reiterate, for several of the bacterial species discussed below, the designation of “resistant” or “sensitive” is often author-determined because clear criteria have not been established by relevant standardization bodies, such as the Clinical Laboratory Standards Institute (CLSI), and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Surveillance programs monitoring AMR in beef production are typically constrained to human enteropathogens and sentinel AMR indicator species, but independent research from many countries gives rough estimates of AMR in cattle pathogens. Several recent studies have found strong correlations between the level of use of specific antimicrobials and the level of resistance observed [Much focus on AMR in food animals concerns the hazards for human health, but AMR is also a veterinary problem. Knowledge about resistance in exclusively bovine pathogens is also exceptionally poor compared to that of bovine zoonotic enteric pathogens, such as observed , 29.Scientific literature pertaining to AMR in pathogens of significance to beef production was reviewed, and the median percent resistance of 16 different pathogens to antimicrobials was collected from 58 scientific reports  is the most frequent and economically important of the primary cattle diseases . Approxint agent , Pasteurus somni , 93. H. with BRD . These uwith BRD . Typing iversity , suggestiversity , 96. In iversity . Clinicaiversity .M. haemolytica (4 × 107 CFU), Lhermie et al. [M. haemolytica persisted after this high-dose, a higher risk for AMR development may have been created by a practice thought to be more judicious than mass medication [M. haemolytica in beef cattle, compared to substantial reductions after therapy with a single subcutaneous injection of tilmicosin  or tulathromycin  [M. haemolytica from pen mates, highlighting the importance of bacterial transmission in the dissemination of AMR [M. haemolytica collected from healthy cattle vs. cattle with clinical BRD. In that study, tetracycline resistance (18%) was the most prevalent resistance phenotype [M. haemolytica and P. multocida can also be recovered from diseased antimicrobial non-treated cattle. Via the pan-European VetPath susceptibility monitoring program, de Jong et al. [M. haemolytica  were resistant to tetracycline, and 5.7, 3.5 and 0.4% of P. multocida  were resistant to tetracycline, spectinomycin, and florfenicol, respectively [In calves experimentally infected with e et al.  demonstrdication . Thus, a5 mg/kg) . Antimicn of AMR . Furthern of AMR  found thhenotype . Resistag et al.  analyzedectively .M. haemolytica to 6 antimicrobial classes including ceftiofur, danofloxacin and enrofloxacin, florfenicol, oxytetracycline, spectinomycin, tilmicosin and tulathromycin. They found that in 2009, ~5% of isolates were resistant to 5 or more antimicrobials as compared to ~35% in 2011 [M. haemolytica isolates resistant to oxytetracycline were 3.5-fold more likely to be resistant to 1 or more antimicrobials, compared to non-oxytetracycline-resistant isolates [P. multocida and H. somni. Klima et al. [M. haemolytica, P. multocida and H. somni from BRD mortalities, and determined that 72% of M. haemolytica and 50% of P. multocida isolates exhibited AMR. Surprisingly, 30% of M. haemolytica and 12.5% of P. multocida were resistant to >7 antimicrobial classes, including aminoglycosides, penicillins, fluoroquinolones, lincosamides, macrolides, pleuromutilins, and tetracyclines [M. haemolytica populations, suggesting that a clonal population was not responsible for this observation [MDR has also been reported in BRD agents. Lubbers et al.  evaluate in 2011 . M. haemisolates . MDR hasa et al.  isolatedcyclines . The MDRervation . MDR waservation . These eMh1, was recently detected in M. haemolytica strain 42548 by Eidam et al. that carried resistance to aminoglycosides , tetracyclines (tet(H) gene), and sulfonamides (sul2 gene) [Mh1 has a size of 92 ,345 bp, harbors ~107 genes, and shares a high degree of similarity with ICEPmu1, an ~82 kb element identified in P. multocida that encodes ~88 genes [Pmu1 is depicted in Fig. Pmu1 integrates into a chromosomal copy of tRNALeu [tetR-tet(H) genes), streptomycin (strA and strB), streptomycin/spectinomycin (aadA25), gentamicin (aadB), kanamycin/neomycin (aphA1), phenicols (floR), sulfonamides (sul2), macrolides/lincosamides (erm(42) gene) or tilmicosin/tulathromycin (msr(E)-mph(E) genes) [Pmu1 was shown to conjugatively transfer in vivo into recipient P. multocida, M. haemolytica and E. coli at frequencies of 1.4 × 10−4, 1.0 × 10−5 and 2.9 × 10−6 respectively [E. coli transconjugants demonstrated up to 64-fold higher MIC values for florfenicol, suggesting better functional activity of FloR in E. coli [blaOXA-2) was also present, and conferred greater ampicillin resistance in E. coli harboring ICEPmu1 [Pmu1 resistance genes may not be indigenous to Pasteurellaceae, acquisition of AMR determinants from Enterobacteriaceae is likely [Pmu1 and ICEMh1 were isolated from feedlot BRD cases in Nebraska in 2005 and Pennsylvania in 2007, respectively [A putative ICE, designated ICEl2 gene) , 104. IC tRNALeu . Eleven ) genes) . ICEPmu1ectively . E. coli E. coli . A β-lac ICEPmu1 . As manys likely . ICEPmu1ectively . There iM. haemolytica and P. multocida, high-level (MIC ≥ 64 mg/L) macrolide resistance has been attributed to mutations in the multicopy 23S rRNA genes  [M. haemolytica and P. multocida 23S rRNAs at position A2058 [erm(42), which appears to have been disseminated among the Pasterellaceae [floR florfenicol resistance gene in M. haemolytica, Katsuda et al. [P. multocida  and Dichelobacter nodosus . Collectively, these findings reveal the importance and diversity of AMR and HGT mechanisms in BRD pathogens.Besides HGT via MGEs, AMR determinants arise spontaneously via mutation. In some isolates of  A2059G) . Resistaon A2058 . Methylaellaceae . Plasmida et al.  identifiFusobacterium necrophorum, an anaerobic rumen bacterium, is the major etiological agent isolated from condemned livers, followed closely by Trueperella pyogenes [F. necrophorum is an opportunistic pathogen also associated with calf diphtheria and foot rot [T. pyogenes in mixed culture with F. necrophorum was higher in abscesses from tylosin-fed cattle (53% vs. 10% in the non-tylosin fed cattle). In contrast, the incidence of F. necrophorum was higher in cattle that were not fed tylosin (61%), as compared to those that were (33%). No differences in tylosin susceptibility between isolates from antimicrobial-free or tylosin-exposed cattle were identified [Fusobacterium spp. isolated from humans is also relatively rare [T. pyogenes is of greater concern, due to the versatility of the bacterium as a cause of liver, skin, joint, and visceral abscesses, and roles in mastitis and abortion [erm(X) or an erm(B) gene similar to that found on the Enterococcus faecalis MDR plasmid pRE25 [T. pyogenes isolates, of which 27 were derived from cattle, and identified erm(X) as the most prevalent tylosin resistance determinant. An erm(X) tylosin and tetracycline tet(33) resistance plasmid, pAP2, was also identified [T. pyogenes may of greater significance in bovine mastitis as compared to liver abscesses [Liver abscesses in beef cattle result from aggressive grain-feeding, and represent an economic liability. Liver abscess incidence in North American feedlot cattle ranges from 12 to 32% [pyogenes . Althougfoot rot . In a 2-foot rot  found thentified . AMR in ely rare , 113, suabortion . Tylosinid pRE25 , 116. Thbscesses , 118.Moraxella bovis and bovoculi. The disease is common worldwide in cattle, transmitted by flies, and if untreated, may result in ulceration and cornea rupture. In the US, only oxytetracycline and tulathromyin are approved for the treatment of bovine keratoconjunctivitis, although penicillin may be used in other countries. In a study of 32 Moraxella spp. isolated from cattle and sheep, Maboni et al. [Moraxella bovoculi, Mb58069. It was found to be resistant to florfenicol, oxytetracycline, sulfonamides, and displayed intermediate resistance to macrolides. Ten AMR determinants were co-located on a >27 kb genomic island [Moraxella bovis may also enhance antimicrobial resistance. Prieto et al. [Moraxella bovis readily forms biofilms, increasing resistance to ampicillin, chloramphenicol, gentamicin, and oxtetracycline by 256-, 1,024-, 512-, and 1,024-fold as compared to when this bacterium grows planktonically [Infectious bovine keratoconjunctivitis is a painful ocular disease caused primarily by non-self-limiting infections with i et al.  found thi et al.  publishec island . The bioo et al.  found thonically  Thus, anBrucella abortus (Brucellosis), Coxiella burnetti (Q fever), and Leptospira spp. (Leptospirosus); (II) bovine pneumonia agents: Mycoplasma mycoides subsp. mycoides small colony type (Contagious bovine pleuropneumonia), and Mycobacterium bovis (Bovine tuberculosis); and (III) enteritis agents: Mycobacterium avium subsp. paratuberculosis (Johne’s disease), and Bacillus anthracis (Anthrax) [Mycobacterium spp.); (II) the pathogen resides in an antimicrobial-exclusive intracellular niche that renders antimicrobial therapy impractical (e.g. Brucella abortus and Coxiella burnetti); or (III) a secreted toxin causes pathology (e.g. Bacillus anthracis). Control of outbreaks of these diseases rarely involves antimicrobial therapy and relies on animal segregation, herd control, or depopulation [Many countries maintain registries of notifiable diseases associated with zoonotic, unvaccinable, highly infectious, economy-damaging, or largely untreatable pathogens. For cattle, notifiable diseases include (I) abortive agents: Anthrax) . Althougpulation .Mycobacterium bovis have been performed because of the role of M. bovis in human tuberculosis (TB). Although it can infect many species, the main reservoir of M. bovis is cattle, and transmission to humans is primarily via contact with infected animals and drinking unpasteurized milk [M. tuberculosis and M. bovis collected over 15 yr, Bobadilla-del Valle et al. [M. bovis. Susceptibility testing to first-line anti-TB drugs revealed that 10.9% of M. bovis were streptomycin-resistant, and 7.6% were MDR (isoniazid- and rifampin-resistant). The aminoglycoside streptomycin is approved for use in cattle against aerobic Gram-negatives such as enteritis-causing E. coli and Salmonella spp. [M. bovis appears to be rare in developed countries, but may occur more frequently in developing countries [AMR susceptibility tests of human clinical isolates of zed milk . In cline et al.  found thlla spp. . Bovine-ountries , 126.E. coli reservoirs, with most strains harmless commensals. Some E. coli, particularly invasive and enterohemorrhagic E. coli (EHEC) cause septicemia in neonatal calves, but are primarily pathogenic to humans. E. coli strains from bovines and other food production animals serve as indicators of AMR prevalence in Gram-negative bacterial populations, thus sentinel ‘generic’ E. coli help establish and track the persistence of AMR genes in environments affected by beef production and other human activities. For example, in a recent survey of AMR in E. coli from Nebraska cattle feedlot runoff catchment ponds and the effluent of municipal wastewater treatment plants, Agga et al. [E. coli resistant to 3rd generation cephalosporins and trimethoprim/sulfamethoxazole were found at equivalent high-frequency (>70% of E. coli isolates) in both livestock and municipal wastewater environments [Cattle are a et al.  found thronments .E. coli susceptibility testing. Cottell et al. [E. coli originating from 88 steers that were treated with ceftiofur and/or chlortetracycline in an experimental US feedlot. The ESBL blaCTX-M-32, was detected in cefoxatime-resistant E. coli in 29 animals, and was found to be present on a self-transmissible IncN-family plasmid , trimethoprim/sulfamethoxazole (76%), nalidixic acid (47%), at least one aminoglycoside (76%), chloramphenicol (65%) and ciprofloxacin (41%). The authors suggested that slaughter hygiene prevented the transmission of ESBLs into the food chain [E. coli O157:H7 was investigated in 510 fecal, hide, carcass, and raw meat samples from 4 beef slaughterhouses in China. STEC was detected in 1.4% of fecal and hide sample, but not in pre- and post-evisceration carcasses, nor in raw meat samples, with all isolates sensitive to 16 relevant antimicrobials [E. coli resistant to 3rd-generation cephalosporins or trimethoprim/sulfamethoxazole, Schmidt et al. [E. coli at various sites along the beef processing continuum. The prevalence of cephalosporin-resistant and trimethoprim/sulfamethoxazole-resistant E. coli in fecal samples at processing was 75 and 95%, respectively. Prevalence in pre-evisceration carcasses was 3 and 33%, and resistant isolates were only found in 0.5% of final carcasses, and no isolates were associated with the final striploin product. All cephalosporin-resistant E. coli isolated were resistant to ampicillin, ceftiofur, and ceftriaxone, and 64% of isolates harbored blaCMY, conferring additional resistance to clavulanate/amoxicillin and cefoxitin [Extended-spectrum β-lactamases (ESBLs) that inactivate newer cephalosporins are a major focus of sentinel l et al.  evaluateiewed in ). In Gered farms . In a Swed farms  screenedod chain . Similarcrobials . During crobials , 134. Int et al.  determinefoxitin . These rSalmonella spp.  are frequent laboratory-confirmed infectious agents of gastroenteritis. Although the enteritis is usually self-limiting, invasive S. enterica spp. infections often require antimicrobial therapy. Cattle are infected/colonized by many Salmonella species, and ground beef is a vehicle of Salmonella transmission, implicated in 45% of outbreaks linked to beef [S. enterica serotypes Dublin and Newport are associated with bovine salmonellosis, and adult cattle may carry and shed Salmonella asymptomatically for many years. In humans, serotype Dublin has the highest proportion of invasive infections resulting in hospitalization and mortality [Salmonella is a risk to human health. In North America, MDR Salmonella are on average resistant to 7 antimicrobials [Salmonella (and other enteropathogens) are collected from humans, animals, and retail meat for the National Antimicrobial Resistance Monitoring System (NARMS) [Salmonella was isolated from 7.9% of beef cattle, and in 0.9% of ground beef samples [rd generation cephalosporin) in bovine-origin serotype Dublin increased from 0 to 86% between 1996 and 2013 [Non-typhoidal  to beef . In cattortality . Due to crobials . In the  (NARMS) . In 2013 samples . MDR , streptomycin (24.3%), tetracycline (20.9%), erythromycin (4.3%), and gentamicin (2.6%) [fections . Mostly atically . Susceptabortion , 142. Infloxacin . In a Jaesistant . In a Sws et al.  found ths et al.  examinedn (2.6%) .Enterococcus spp. are ubiquitous Firmicutes in the healthy intestinal microbiota of both humans and cattle, and indicate fecal contamination. Most Enterococcus spp. are not foodborne pathogens, nor are they bovine pathogens [Enterococcus faecalis and faecium may cause life-threatening human infections, such as UTIs and meningitis. Control of enterococci infections is complicated by high-level MDR [E. faecalis transferred gentamicin resistance plasmids to transplanted human flora in a BALB/c mouse model [Enterococcus were recovered from ~90% of cattle, and ~80% of retail ground beef tested. The incidence of MDR  in both E. faecium and faecalis was lower in cecal isolates from beef cattle  than in cecal samples from chickens  or turkeys  [Enterococcus typically focus on the emergence of resistance to vancomycin— an antimicrobial used in the treatment of MRSA and other Gram-positive infections [Enterococcus spp. in the United States or Canada [E. faecium NARMS isolates were found to be quinupristin/dalfopristin-resistant [Enterococcus spp. in the beef production environment have been studied mainly for their presumptive importance as AMR determinant sentinels/reservoirs.athogens . Despiteevel MDR . Enterocevel MDR , 148. E.se model . The US ctively) . Other sfections , 150. Var Canada , 151, buesistant . Overall10 microbes/ml; rumen fluid [Prevotella, Butyrivibrio, Ruminococcus, as well as many unclassified organisms [Firmicutes and Bacteroidetes, but also contains Proteobacteria and human enteropathogens, which are shed in feces [Cattle house a dense (>10en fluid ) consorten fluid . Differeen fluid , 155. Carganisms , 157. Otrganisms , 159. Thrganisms . The fecin feces , 162. CoUnlike in humans and experimental animal models, there is currently limited information concerning the effect of antimicrobials on the bovine microbiota/resistome. However, much work describes the effect of therapeutic and sub-therapeutic administration of antimicrobials on the prevalence of specific bacteria in bovines. These studies typically involve antimicrobial administration to a controlled animal cohort, followed by culture-dependent collection of an organism-of-interest for susceptibility testing. These approaches provide a biased snapshot of microbiome changes. Newer methods include culture-independent collection of metagenomic DNA for detection and quantitation of specific AMR genes by PCR-based methodology, or for high-throughput sequencing and functional AMR gene annotation Table . There aPereira et al.  characteblaCMY-2 and blaCTX-M (ceftiofur resistance), tet(A) and tet(B) (tetracycline resistance), and 16S rRNA genes in fecal community DNA from the pens of each treated cohort. Pens where all cattle were treated with ceftiofur had greater numbers of blaCMY-2 and blaCTX-M ceftiofur resistance determinants than single-animal treatment pens [blaCMY-2 and blaCTX-M gene copies compared to cattle in pens that did not receive chlortetracycline. In contrast, tetracycline AMR gene prevalence decreased in pens where all cattle received ceftiofur compared to pens where only one animal received ceftiofur [Kanwar et al.  recentlyent pens . Chlorteeftiofur . The auteftiofur .cfxA2 and cfxA3 were most abundant, and have previously been associated with Prevotella—a common rumen microbe [Utilizing advanced total community metagenomic sequencing, Chambers et al.  examined microbe . Ceftiof microbe , allowin microbe . This woE. coli in the beef production continuum. With respect to treated and non-treated cattle, E. coli was collected from live-animal feces, hides, intestinal digesta, carcasses, and ground beef. Animals fed chlortetracycline/sulfamethezine harbored more tetracycline-resistant E. coli than non-treated animals (50.9% vs. 12.6%), but there were no differences in the prevalence or profile of AMR E. coli between treatments in the hide, carcass or ground beef samples [E. coli can enter the food chain at slaughter regardless of AGP administration [E. coli, in agreement with similar studies using the same antimicrobials [Enterococcus spp. harboring erm(B) and/or msrC (a macrolide/streptogramin efflux pump gene) [Enterococcus decreased in the period between when cattle entered and exited the feedlot, and that the AMR Enteroccocus were derived from strains present in the intestinal microbiota before tylosin administration [Sub-therapeutic antimicrobial administration is one of the most controversial beef production practices with many studies exploring this topic in the context of AMR development. Alexander et al.  investig samples . To the stration . Sub-thecrobials . Anothermp gene) . The autstration . SelectiM. haemolytica and Enterococcus, respectively. Forty beef steers were injected once with tilmicosin  or tulathromycin  or fed sub-therapeutic tylosin  continuously over 28 d. Therapeutic tilmicosin and tulathromycin decreased nasopharyngeal carriage of M. haemolytica: at the beginning of the trial, 60% of the steers tested positive for M. haemolytica, at 7 d post- injection, none of the steers treated with tilmicosin harbored M. haemolytica, and only one steer treated with tulathromycin was positive for M. haemolytica. Sub-therapeutic tylosin had no effect on nasopharyngeal carriage, and tylosin-exposed M. haemolytica isolates did not acquire macrolide resistance. In contrast, a significant proportion of the bystander Enterococcus acquired erm(B) erythromycin resistance following treatment with either injectable tilmicosin or tulathromycin, or in-feed tylosin, and were 76-fold more likely to be erythromycin-resistant than those recovered from non-antimicrobial-treated steers. Catry et al. [E. coli and Pasteurellaceae, respectively. Narrow spectrum penicillins were the most frequently administered parenteral antimicrobials, often in combination with an aminoglycoside, such as neomycin or dihydrostreptomycin [E. coli, 20.6% were resistant to least one antimicrobial. The most frequent resistance patterns were ampicillin-tetracycline-streptomycin (15.9%), tetracycline-streptomycin (11.4%), and ampicillin-streptomycin (9.8%) [P. multocida isolates and 42 M. haemolytica isolates originating from the nasal cavity, the predominant resistance found was to the aminoglycoside spectinomycin [Given the importance of antimicrobials in the treatment of BRD agents, much research examines the effect of antimicrobial treatment on AMR development in BRD bacteria. Investigated the effects of therapeutic and sub-therapeutic macrolide administration on the nasopharyngeal and enteric microbiota, with specific focus on y et al.  correlatptomycin . Among rn (9.8%) . Among 2inomycin . The autinomycin . Others inomycin , 174, buinomycin , 175.Pmu1  was elevated following heavy metal supplementation [4; 10 or 100 mg/kg in-feed), Amachawadi et al. [Enterococcus spp. to determine if elevated copper supplementation co-selects for macrolide resistance. The transferable copper resistance gene tcrB was identified in 8.5% of Enterococcus from elevated copper- and tylosin-fed cattle, compared to copper alone (4.5%), tylosin alone (3.5%), or the low copper/no tylosin control (2.0%) [tcrB-positive isolates proved to be E. faecium, and interestingly, all tcrB-positive isolates harbored tetracycline tet(M) and erythromycin resistance erm(B) determinants [Cattle also receive trace mineral supplements that include elements with AGP activity. Some heavy metals, such as zinc, manganese, and copper may be given as salt-mixes, injected, or administered in slow-release ruminal capsules . Copper mu1 Fig.  encodes entation . In a sti et al.  investigl (2.0%) , 182. Alrminants . The autrminants . Thus, hThe primary concern relating to antimicrobials in agriculture is the potential for AMR determinants to expand and spread via the food chain. Although urban lifestyles rarely bring people into direct contact with livestock, the animal production continuum extensively connects with numerous industries, infrastructure, and ecologies. For example, manure from antimicrobial-treated animals may be applied to crops, or waste from farms may drain into rivers, reservoirs, and wastewater treatment plants. In the US, cattle produce between 0.86 and 6.4 million t of manure daily . AMR canNoyes et al.  examinedProteobacteria were most-prevalent in anaerobic and aerobic sludge accounting for 35.95 and 58.36% of annotated reads, respectively, followed by Firmicutes  [Metagenomics have also proved useful in the examination of AMR genes found in wastewater treatment plants associated with tanneries and slaughterhouses. Wastewater treatment plants are thought to be HGT hotspots because of high bacterial diversity and density , 190. Wactively) . Concernctively) . MDR effctively) . The autctively)  examinedctively) . The AMRctively) , 193. ThE. coli-based libraries (containing 25.9 Gb of DNA) were screened for resistance to kanamycin, chloramphenicol, tetracycline, and the β-lactams carbenicillin (penicillin) and ceftazidime . Of 87 AMR E. coli clones with genes conferring resistance to at least one of the antimicrobials tested, 80 carried unique AMR genes, suggesting that the cow microbiome harbors AMR genes that are unique or unidentified elsewhere. A novel clade of chloramphenicol acetyltransferases was also described [Firmicutes were predominant (50% of sequenced clones), followed by Bacteroidetes (23%) and Proteobacteria (14%) [E. coli clones with >5 putative genes with predicted AMR or MGE functions [Sequence-based metagenomic AMR gene profiling is also limited to those genes with similarity to already known AMR genes, and metagenomic shotgun read lengths present difficulties for the characterization of the AMR genomic context. Functional metagenomic library-based approaches have proved to be complementary in the identification, quantification, and characterization of novel resistance determinants. Wichmann et al.  examinedescribed . Flankinia (14%) . Anotherunctions . Thus, lunctions .mcr-1) in E. coli isolates from poultry, swine, and infected humans [S. enterica serotype Newport, which confers resistance to tetracycline, ampicillin, and carbenicillin [Pmu1 and ICEMh1 found in P. multocida and M. haemolytica, respectively [Given the ubiquity of AMR determinants in bovine and other microbial communities, it is difficult to appraise the relative risk any particular determinant presents for the likelihood of transfer into a human pathogen and clinical therapy failure. Confounding the issue are AMR determinants that are expressed or silent in different hosts, as well as AMR determinants akin to housekeeping genes . For thed humans , 201. A nicillin . This canicillin . The nexectively , 104. ElS. enterica serotype Typhimurium phage type DT104, which may express resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (resistance-type ACSSuT) [Salmonella genomic island 1 (SGI1). The MDR region harbors Class I integrons—genetic elements capable of consolidating multiple AMR gene cassettes [An example of risk and source determination may be given by the long-term global epidemics of ground beef-associated MDR  ACSSuT) –205. In assettes . Integroassettes . Althougassettes , 209 chaE. coli and Salmonella outbreaks have been associated with beef [Linking on-farm antimicrobial use to human infection is difficult. While antimicrobial usage evidently selects for drug-resistant organisms, there is a gap in knowledge connecting usage to the flow of AMR determinants from the bovine microbiota to outbreaks of human AMR diseases. To bridge this gap, a number of studies compared outbreak clinical isolates to animal isolates taken at similar times from nearby locations –212. Typith beef –215, butith beef . This reith beef ). Dairy-ith beef , 217. Diith beef ).Table 2As in most environments, AMR determinants exist ubiquitously in the beef production biome, regardless of antimicrobial exposure. Nevertheless, the use of antimicrobials for bovine welfare and growth promotion contributes selective pressure that increases the abundance of AMR genes and their host bacteria, and promotes the genesis and dissemination of MDR organisms. The presence or absence of connections between AMR in bovine microbial populations to human health threats are likely to become clearer with the increasing application of whole-genome sequencing and metagenomic resistomics. The role of MGEs in AMR propagation is likely to be an important focus for understanding the impact of veterinary antimicrobials. Future investigations may validate mitigation strategies, such as the separation of antimicrobials for use in beef cattle from those used in humans. Proper and judicious use of antimicrobials will help prolong the usefulness of both clinical and veterinary antimicrobials, but ever-increasing usage of antimicrobials in food-animal production suggests that microbes will only continue to acquire resistance. Of particular concern for cattle are the MDR BRD agents: in the future, respiratory infections may become untreatable with current antimicrobials. On a positive note, several studies reveal that adequate hygiene and appropriate treatment at slaughterhouse and wastewater treatment facilities are efficacious at reducing or eliminating transmission of AMR organisms and genes. Thus, such procedures and facilities should be explored further, and promoted in deficient areas of food-animal production.The literature search was conducted from January to March 2016 via Google Scholar and PubMed. Recent (2012-present) studies that described AMR or usage in context with beef production, bovine pathogens, commensal bacteria, metagenomics, the resistome, and cattle were included. Older reports, or studies referring to dairy operations were excluded, except for where beef production information was sparse.A literature search was conducted for AMR in bovine pathogens. Journal articles (–88, 2000


Many diseases have been demonstrated to associate with acute or chronic inflammation, such as rheumatoid arthritis, Alzheimer's disease, depression, Kawasaki disease, and even cancer. However, treatment options for inflammatory diseases have been limited to anti-inflammatory medications  or steroid hormones. Complementary and alternative medicines (CAMs) provide natural and effective protection for those who suffer from inflammatory diseases. This special issue is focused on the evidence-based research of CAMs for treatment of inflammatory diseases. Astragalus membranaceus (AM) and Angelica sinensis (AS) at a ratio of 5 : 1, helps relieve the inflammatory response through reducing IgE levels and cytokine levels. S. Park et al. showed that pomegranate peel extract (PPE) could alleviate inflammatory reactions, including production of reactive oxygen species (ROS) and expression and secretion of inflammatory cytokines.Inflammation occurs in response to various cellular stresses including infection, irradiation, or chemical or physical injury. One of the major responses during inflammation is the release of cytokines and/or chemokines. Y. Y. Choi et al. showed that Dangguibohyul-Tang (DBT), a herbal formula composed ofκB pathways. T. Mao et al. showed that Qingchang Wenzhong Decoction (QCWZD) ameliorates dextran sulphate sodium- (DSS-) induced ulcerative colitis (UC) in rats by downregulating the IP10/CXCR3 axis-mediated inflammatory response. D. Wang et al. proved that Yupingfeng Pulvis (HFBP) alleviates the inflammation in the lung tissue of mice by reducing the proportion of Th17 cells and increasing the proportion of Treg cells in bronchoalveolar lavage fluid.Studies on the signaling pathways provide the anti-inflammatory mechanism of CAMs. M. Gu et al. demonstrated that genistin is cardioprotective due to its antioxidant and anti-inflammatory activities via P2X7/NF-Using human umbilical vein endothelial cells, E. S. Choi et al. showed that Samul-Tang  protects vascular endothelium from inflammation and might be used as a promising vascular protective drug. G. O. L. Carapeba et al. used animal models to evaluate the treatment effect of intra-articular hyaluronic acid in 4 dogs with osteoarthritis associated with hip dysplasia compared to traditional conservative treatment.Hyeonggaeyeongyo-tang (HYT) is an ancient formula of oriental medicine traditionally used to treat rhinitis; however, clinical evidence has not yet been established. M. Kim et al. showed that HYT improved nasal symptoms and quality of life in patients with allergic rhinitis and nonallergic rhinitis. This is the first clinical study to evaluate the use of HYT to treat patients with rhinitis. X. Lu et al. showed that Sequential treatments with Tongsai and Bufei Yishen Granules during acute exacerbation of chronic obstructive pulmonary disease- (AECOPD-) risk window (RW) periods can reduce inflammatory response and improve pulmonary function and shorten the recovery courses of AEs.E. S. B. Barroqueiro et al. showed that babassu mesocarp extract (EE) has specific antimicrobial activity in vitro and has an important antiseptic effect in vivo possibly due to the antimicrobial and immunomodulatory activity. N. J. S. Lopez et al. evaluated the extrusion process as an alternative for improving the biological potential of sorghum bran: phenolic compounds and antiradical and anti-inflammatory capacity. The extrusion process increased total phenol content in sorghum bran, which positively affected antiradical capacity.In this issue, C.-Y. Cheng et al. reviewed the anti-inflammatory effects of traditional Chinese medicines against ischemic injury in in vivo models of cerebral ischemia. TCMs provide neuroprotective effects through downregulating ischemia-induced microglial activation and expression of proinflammatory cytokines, enzymes, and transcription factors.These studies provide new insights to treat inflammatory diseases and help us understand better about the mechanism and function of CAMs.Xiang LiuXiang LiuYing-Ju LinYing-Ju LinYong ChengYong Cheng


P. atlantica subsp. Kurdica, with the local name of Baneh, is a wild medicinal plant which grows in Kurdistan, Iran. The identification of resistance gene analogs holds great promise for the development of resistant cultivars. A PCR approach with degenerate primers designed according to conserved NBS-LRR (nucleotide binding site-leucine rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from P. atlantica subsp. Kurdica. A DNA fragment of the expected 500-bp size was amplified. The nucleotide sequence of this amplicon was obtained through sequencing and the predicted amino acid sequence compared to the amino acid sequences of known R-genes revealed significant sequence similarity. Alignment of the deduced amino acid sequence of P. atlantica subsp. Kurdica resistance gene analog (RGA) showed strong identity, ranging from 68% to 77%, to the non-toll interleukin receptor (non-TIR) R-gene subfamily from other plants. A P-loop motif (GMMGGEGKTT), a conserved and hydrophobic motif GLPLAL, a kinase-2a motif (LLVLDDV), when replaced by IAVFDDI in PAKRGA1 and a kinase-3a (FGPGSRIII) were presented in all RGA. A phylogenetic tree, based on the deduced amino-acid sequences of PAKRGA1 and RGAs from different species indicated that they were separated in two clusters, PAKRGA1 being on cluster II. The isolated NBS analogs can be eventually used as guidelines to isolate numerous R-genes in Pistachio. Pistacia) is a genus of the tropical Anacardiaceae family plants. Three Pistacia species grow naturally in Iran, including P. vera Linnaeus, P. khinjuk and P. atlantica Desf. A large population of wild P. atlantica subsp. Kurdica trees, locally known as Baneh, grow across Kurdistan province, Iran. P. atlantica subsp. Kurdica is used for medicine, food and industrial purposes. Native people grind and mix baneh nuts with other nuts. The gum is also used in the production of chewing gum . In the present study, we isolated a genomic RGA of the NBS-LRR type from  length   . Percenaccuracy . A tryptP. atalatica subsp. Kurdica and other species identified their relatedness with known R-genes. Similar to previous reports [Pistacia NBS analogs are required to gain better knowledge about the RGAs in Pistacia and to draw further conclusions.The phylogenetic analysis of NBS analogs identified their group based on similarity. The phylogenetic tree based on neighbor joining using the percentage of identity of the deduced amino acid sequences of  reports , the phePistacia species, P. vera L., P. khinjuk stocks P. eurycarpa Yalt. (P. atlantica subsp. Kurdica Zoh.) and P. atlantica Dsef. originate from Iran [Pistacia atlantica and P. khinjuk are the rootstocks most resistant to root-knot nematode and drought. It is thus possible that P. atlantica subsp. Kurdica have large numbers of resistance genes. Using identified P. atlantica subsp. Kurdica RGA, different primer sets can be designed to analyze Pistacia’s wild relatives, to discover their novel genomic resources and to improve their genetic composition.Four important rom Iran . Pistaci