Anisodus tanguticus Maxim. (Solanaceae), a traditional endangered Tibetan herb, is endemic to the Qinghai–Tibet Plateau. Here, we report the de novo assembled chloroplast (cp) genome sequences of A. tanguticus . The cp contains a pair of inverted repeated (IRa and IRb) regions of 25,881 bp that are separated by a large single copy (LSC) region  and a small single copy SSC  region. A total of 132 functional genes were annotated in the cp genome, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Moreover, 199 simple sequence repeats (SSR) and 65 repeat structures were detected. Comparative plastome analyses revealed a conserved gene order and high similarity of protein-coding sequences. The A. tanguticus cp genome exhibits contraction and expansion, which differs from Przewalskia tangutica and other related Solanaceae species. We identified 30 highly polymorphic regions, mostly belonging to intergenic spacer regions (IGS), which may be suitable for the development of robust and cost-effective markers for inferring the phylogeny of the genus Anisodus and family Solanaceae. Analysis of the Ka/Ks ratios of the Hyoscyameae tribe revealed significant positive selection exerted on the cemA, rpoC2, and clpP genes, which suggests that protein metabolism may be an important strategy for A. tanguticus and other species in Hyoscyameae in adapting to the adverse environment on the Qinghai–Tibetan Plateau. Phylogenetic analysis revealed that A. tanguticus clustered closer with Hyoscyamus niger than P. tangutica. Our results provide reliable genetic information for future exploration of the taxonomy and phylogenetic evolution of the Hyoscyameae tribe and related species. A. tanguticus is a rare and endangered Solanaceae species found on the Qinghai–Tibetan Plateau in grasslands or meadows (2200–4200 m) [A. tanguticus still relies on wild plant exploration and collection, which has led to the species becoming endangered [A. tanguticus in the Qinghai–Tibetan region should be evaluated. DNA barcoding is thought to be an effective approach for medicinal component identification and quality control of produced goods in addition to protecting consumers [A. tanguticus. Here, we assembled and de novo sequenced the whole cp genome of A. tanguticus with Illumina sequencing platforms. Additionally, cp genome annotation and structure analysis were performed and several oligonucleotide repeats, SSR, and substitutions were identified as mutation hotspots for future DNA barcoding. Further insights on A. tanguticus plastome evolution and its phylogenetic relationships have been revealed through comparative studies with reported cp genomes of Solanaceae species. Chloroplasts (cp) in plant cells are photosynthetic organelles that play a critical role in the production of essential energy and metabolites . Cps con–4200 m) . Accordi–4200 m) ,10,11 an–4200 m) ,13. Despdangered . Furtherdangered , the poponsumers ,17. Prevonsumers ,19. TherA. tanguticus were collected from Haibei station , in Qinghai province. According to the manufacturer’s instructions, a DNeasy Plant MiniKit  was utilized to isolate DNA from the fresh young leaves. Spectrophotometry and electrophoresis on a 1% (w/v) agarose gel were used to evaluate the purity of the DNA. DNA of sufficient integrity and purity DNA was used for library construction and sequencing with an IlluminaHiseq2500 . Fresh young leaves of http://dogma.ccbb.utexas.edu/, accessed on 15 March 2021), CPGAVAS, and manual corrections were used to annotate the full cp genomes [A. tanguticus). Web-based REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer/ accessed on 15 March 2021) was used for the analysis of repeat sequences, including forward, palindrome, reverse, and complement repeats. Sequence identity was set to >90% with a minimum repeat size of 30 base pairs. Micro Satellite Identification Tool (MISA) [http://downloads.fyxm.net/CodonW-76666.html accessed on 15 March 2021), only 53 protein-coding genes with lengths more than 300 bp were chosen for analysis of synonymous codon usage. The relative synonymous codon usage (RSCU) and overall codon usage were both analyzed. Trimmomatic V0.36 was used to remove adapters and poor-quality reads from the raw reads . Further genomes ,25. The  genomes . The corl (MISA)  was usedA. tanguticus was compared with the cp genomes of Prewalskia tangutica, Scopolia parviflora, H. niger, Datura stramonium, and Nicotiana sylvestris in Solanaceae using Mauve software to identify evolutionary events such as gene loss, duplication, and rearrangement [The plastome of angement . As a reA. tanguticus were evaluated for synonymous (Ks) and non-synonymous (Ka) substitution rates in comparison with the six closely related Solanaceae species . Between the compared species, the corresponding functional protein-coding gene was separately aligned using MAFFT [The protein-coding genes of ng MAFFT . The Ka/ng MAFFT , with thng MAFFT , and theng MAFFT . Phylogeng MAFFT . Each brA. tanguticus is 155,765 nucleotides and has a quadripartite architecture. The genome consists of a pair of IRs , a large single copy (LSC) region , and a small single copy (SSC) region   has more protein-coding genes than P. tangutica (85) and H. niger (80)  . These r,487 bp) . The cp IR areas . The plager (80) . Among tger (80) . Nineteeplastome . The larA. tanguticus plastome has a total of 80,910 bp of protein-coding genes, which encodes 26,970 codons. Among these, 2867 (6%) encoded leucine, which is the most abundant amino acid, whereas only 483 (1.0%) encoded tyrosine, which is the least prevalent amino acid in the A. tanguticus cp genome. According to the codon-anticodon recognition patterns, 29 tRNAs had codons that matched all 20 amino acids . Genomic structure and gene order are conserved in these species, especially in the Hyoscyamus group  using the Mauve program [P. tangutica and H. niger cp genomes lost two genes compared with the other four species. In addition, there was no gene rearrangement in Solanaceae genomes   and non-synonymous (Ka) were parameters for assessing the rate of gene divergence in a molecular evolution study . To chartabacum) . Of the enothera , and cemenothera ,52. Our enothera ; loss ofenothera . cemA prenothera  and rpoCenothera . In Nicoenothera . Here, oA. tanguticus and to further elucidate the evolutionary relationship in the Solanaceae  with 100% bootstrap support. The three Hyoscyameae tribe species were clustered together more closely. D. stramonium, N. sylvestris, and other species were clustered outside this branch with 100% bootstrap support. These findings are consistent with previous studies [A. tanguticus grouped with H. niger when several chloroplast markers were combined [P. tangutica and S. parviflora exhibited a closer relationship in the Hyoscyameae tribe (Physochlaina. Since A. tanguticus is an endemic species that grows on the Qinghai–Tibetan Plateau, sequencing, and characterization of additional cp genomes of Hyoscyameae tribe species may provide further insight on the phylogenetic relationships and evolution among these species. The tribe Hyoscyameae is distributed throughout Eurasia and is considered as a monophyly in Solanaceae ,19. Howelanaceae . Most nolanaceae A. Moreov studies ,19,57,58combined ,19. Our ae tribe B. This mA. tanguticus. The cp genome is 152,714 bp and encodes 126 functional genes. Analysis of repeat sequences revealed that forward and palindromic repeats of 40–49 bp were more common in the Hyoscyameae tribe, and 30–39 bp reverse repeats may play a more significant role in A. tanguticus and P. tangutica cp genome rearrangement and in adapting to extreme conditions on the Qinghai–Tibetan Plateau. The A. tanguticus cp genome contained a total of 199 SSRs and can be utilized for the development of molecular markers for phylogenetic and population studies. The genome structure and gene organization were similar to three previously reported species in the Hyoscyameae tribe, while gene contraction and expansion were clearly different in the A. tanguticus cp genome. Evolutionary analysis indicated that clpP, cemA, and ropC2 showed positive selection, which suggests that protein metabolism may be an important adaptation strategy for A. tanguticus and other species in Hyoscyameae on the Qinghai–Tibetan Plateau. Phylogenetic analysis indicated that A. tanguticus has a closer relationship with P. tangutica. These results provide significant information for forthcoming studies on phylogenetic and evolutionary adaptation in A. tanguticus and its related species. High throughput sequencing was used to obtain the whole cp genome of


Dendrobium catenatum/D. officinale (here after D. catenatum), a well-known economically important traditional medicinal herb, produces a variety of bioactive metabolites including polysaccharides, alkaloids, and flavonoids with excellent pharmacological and clinical values. Although many genes associated with the biosynthesis of medicinal components have been cloned and characterized, the biosynthetic pathway, especially the downstream and regulatory pathway of major medicinal components in the herb, is far from clear. β-glucosidases (BGLUs) comprise a diverse group of enzymes that widely exist in plants and play essential functions in cell wall modification, defense response, phytohormone signaling, secondary metabolism, herbivore resistance, and scent release by hydrolyzing β-D-glycosidic bond from a carbohydrate moiety. The recent release of the chromosome-level reference genome of D. catenatum enables the characterization of gene families. Although the genome-wide analysis of the BGLU gene family has been successfully conducted in various plants, no systematic analysis is available for the D. catenatum. We previously isolated DcBGLU2 in the BGLU family as a key regulator for polysaccharide biosynthesis in D. catenatum. Yet, the exact number of DcBGLUs in the D. catenatum genome and their possible roles in bioactive compound production deserve more attention.BGLUs in active metabolites production, 22 BGLUs (DcBGLU1-22) of the glycoside hydrolase family 1 (GH1) were identified from D. catenatum genome. Protein prediction showed that most of the DcBGLUs were acidic and phylogenetic analysis classified the family into four distinct clusters. The sequence alignments revealed several conserved motifs among the DcBGLU proteins and analyses of the putative signal peptides and N-glycosylation site revealed that the majority of DcBGLU members dually targeted to the vacuole and/or chloroplast. Organ-specific expression profiles and specific responses to MeJA and MF23 were also determined. Furthermore, four DcBGLUs were selected to test their involvement in metabolism regulation. Overexpression of DcBGLU2, 6, 8, and 13 significantly increased contents of flavonoid, reducing-polysaccharide, alkaloid and soluble-polysaccharide, respectively.To investigate the role of DcBGLU genes with possible roles in medicinal metabolites production and laid a theoretical foundation for further functional characterization and molecular breeding of D. catenatum.The genome-wide systematic analysis identified candidate The online version contains supplementary material available at 10.1186/s12864-022-08840-x. Dendrobium, one of the largest genera in the family Orchidaceae with approximately 1800 species worldwide [D. catenatum is a highly prized Dendrobium species with a wealth of therapeutic functions in antitumor, anti-angiogenesis, anti-oxidation, anti-inflammation, diabetes alleviation, liver protection, stomach nourishing, body fluids supplementation, and immunity enhancement [D. catenatum are the principal medicinal part containing large amounts of polysaccharides (> 30% of dry weight) and relatively low levels of ethanol extractives (~ 4.93% of dry weight), such as flavonoids, bibenzyls, phenanthrene and fluorenone [D. catenatum, are new sources of bioactive molecules, including phenolic compounds [D. catenatum are rich with phenolic components (> 30% dry weight), while other substances such as essential and non-essential amino acids, polysaccharides, and volatile components are also found [ancement –5. The suorenone . The leaompounds , flavonoArabidopsis thaliana with 47 members (10 subfamilies), Oryza sativa with 40 members (8 subfamilies), Zea mays with 26 members (4 subfamilies), Brassica rapa with 64 members (10 subfamilies), and Medicago truncatula with 51 members (7 subfamilies) [BGLUs related to metabolite biosynthesis were cloned and characterized from several plant species, limited information is available about BGLU family in orchids. Several BGLU genes expressed in the stems of three Dendrobium species  have been identified as hub genes that are possibly involved in polysaccharides biosynthesis [Tongan vanilla, β-glucosidase has been exogenously applied on green beans to hydrolyze glucovanillin into vanillin [Cymbidium sinense, BGLU genes are upregulated during ovule development, when β-glucosidase-mediated hydrolysis of cellulose may occur [BGLU genes are also significantly downregulated in C. sinense leaves, which may be an important reason for decreased starch content and abnormal sugar metabolism in the chloroplast [In D. catenatum, BGLU genes are significantly upregulated in symbiotically germinated seeds [BGLU genes associated with the glutathione metabolism pathway are upregulated in response to cadmium stress [In Dendrobium crumenatum, a significant increase in β-glucosidase activity is observed during floral bud development [Many of the secondary metabolites are glucosylated to increase their solubility and stability, and activation of the glucosylated compounds is mediated by enzymes called β-glucosidases (BGLUs) . BGLUs aamilies) , 14. Foramilies) . Althougynthesis . In Tongvanillin . In Cymbay occur . Fifteenoroplast . In D. ced seeds , and BGLm stress . In Dendelopment .Dendrobium plants [D. catenatum [Usually, the accumulation of medicinal metabolites, especially alkaloids, are low across tissues, making them hard to meet the threshold for drug-making . Althougm plants . Neverthatenatum , 26 makeD. catenatum [DcBGLU2 as a key regulator for polysaccharide accumulation in response to phytohormone treatments [DcBGLUs in D. catenatum genome and their corresponding roles in medicinal compounds production deserve more attention. The present study identified 22 candidate GH1 DcBGLUs members through a genome-wide analysis. Then their sequence feature, molecular phylogenetic relationship, conserved motif, gene structure, chromosomal localization, and cis-elements were characterized. Furthermore, the dynamic expression patterns in three more tissues were examined based on our and other published RNA-seq data. These results provide valuable information on the DcBGLU family in D. catenatum and lay a foundation for further exploring its function in plant metabolism.Previously, we sequenced the genome of atenatum  and repoeatments . HoweverD. catenatum were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/genome/?term=JSDN00000000) under the accession code JSDN00000000 [D. catenatum BGLU candidates of the GH1 family (DcBGLUs), the hidden Markov model (HMM) profile of Glyco_hydro_1 (PF00232) from the Pfam database (http://pfam.xfam.org/) were downloaded and searched against the BGLU domain in D. catenatum protein sequence data using the HMMER software . After removing repeated or incomplete proteins, the remaining potential candidates were double-checked using the SMART (http://smart.embl-heidelberg.de/) and CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) databases. The gene identifiers used in this study are listed in the Table Shttps://web.expasy.org/protparam/) tool. The subcellular localization of each BGLU protein was predicted using the online ProtComp v. 9.0 server (http://www.softberry.com). The signal sequences were predicted using the SignalP  tool, and the N-glycosylation sites were detected by the NetNGlyc 1.0 server .The genome sequences and protein data of the 00000000 . To idenA. thaliana and D. catenatum were aligned using MEGA X [https://www.evolgenius.info/evolview/).To classify and investigate phylogenetic relationships of BGLUs, the protein sequences from g MEGA X . The phyDcBGLU genes was analyzed using the WebScipio server (https://www.webscipio.org/) [http://bioinformatics.psb.ugent.be/webtools/plantcare/html) was used to identify potential cis-elements in the 2000-bp gene promotors.The exon/intron organization of io.org/) . The conio.org/) . At the D. catenatum leaves transiently transformed with BGLU-OE plasmids, were homogenized in liquid nitrogen and then subjected to total RNA isolation using TRIzol reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. RNA integrity was checked on a 1.5% agarose gel. Subsequently, 1.0 mg total RNA was reverse transcribed into first strand cDNA with a Prime Script™ reagent Kit with gDNA Eraser , according to the manufacturer’s instructions. The cDNAs were subjected to a quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using SYBR Green Premix Kit  in the ABI PRISM 7500 Fluorescent Quantitative PCR System (Thermo Fisher Scientific). All the primers used were listed in Table SActin as the internal control.Approximately 300 mg of D. catenatum (GSE155403 and SRP150489) [D. huoshanense (SRP122499) [Dendrobium nobile (PRJNA338366) [DcBGLUs and the corresponding orthologue genes were screened across four more tissues, mainly leaves, stems, roots, and protocorm-like body (PLBs), and under MF23 treatment. All the raw SRA reads were transformed into fastq format, filtered, aligned, assembled, and estimated for expression levels. The results were visualized using heatmap generated from the TBtools [Based on the transcriptome data from P150489) , 32, D. P122499) , and DenA338366) , the exp TBtools .DcBGLU2, DcBGLU6, DcBGLU8, and DcBGLU13 were amplified using D. catenatum cDNA template with the primer sets listed in Table SD. catenatum leaves following an Agrobacterium-mediated method [The full-length coding sequences of d method   were harvested, grounded in liquid nitrogen, and used for extraction of compounds. The isolated compounds were determined by using a spectrophotometer (BeckmanCoulter DU730).Contents of reducing-polysaccharides were determined following Tonukari et al.’s methods . ContentActin from D. catenatum was used as the internal control. The 2-ΔΔCt method was used to calculate relative gene expression. Statistical significance is defined as follows: ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (Student’s t-test).For qRT-PCR gene expression analysis, D. catenatum genome make it easier to conduct genome-wide identification of genes. A total of 22 candidate DcBGLU genes of the GH1 family were identified in the D. catenatum genome after strict HMMER screening and domain confirmation, and they were designated as DcBGLU1 to DcBGLU22 following the LOC accession numbers in the NCBI gene bank  of the predicted proteins varied widely from 5.18 (DcBGLU19) to 8.30 (DcBGLU13). DcBGLU5, 13, and 17 were basic proteins, DcBGLU1 and DcBGLU21 were neutral proteins, and the rest were acidic proteins , AtII (6 members), and AtIII (11 members) contain members only from A. thaliana, suggesting that gene deletion could occur during the evolution of D. catenatum.Multiple sequence alignment was conducted to further classify and characterize DcBGLU proteins, and results showed high conservation among the members Fig. S. Due to are Fig. . The resTo identify the conserved functional motifs in DcBGLU proteins, we searched using the MEME online tool and identified ten motifs. These conserved motifs possessed 14 to 82 amino acids, and the number of motifs varied from two to ten. The results showed that more than half of the members (13/22) possessed all these ten motifs and that motif two and nine were the two most conserved ones widely present in DcBGLU proteins  to some having up to 27 exons (DcBGLU18), including five with ten or less exons and thirteen with 11-13 exons  elements, were detected  found in the promoters of DcBGLU genes  and chromosome 13 with three members . Interestingly, DcBGLU5, 6 and 22, which belonging to the same subgroup I on the same chromosome 13, tended to cluster together, whereas the other family members were clustered separately.The genomic distribution of mes Fig. b. Most oBGLUs are well documented to play essential roles in response to developmental and environmental stimuli. Nevertheless, the functions of BGLUs in D. catenatum are far from clear. In the present study, four RNA-seq datasets of D. catenatum (GSE155403 and SRP150489) [D. huoshanense (SRP122499) [D. nobile (PRJNA338366) [MF23  , 32, D. P122499) , and D. A338366)  were retDcBGLUs in eight tissues  from RNA-seq datasets (PRJNA348403) [DcBGLUs showed different transcriptional patterns across various tissues , suggesting their potential role in pigmentation and scent release. For DcBGLU10, 11, 12, 14, 17, and 21, they tended to be more highly expressed in white roots and green root tips than in other tissues. Additionally, the DcBGLUs classified in the same cluster did not always have the same expression pattern. For example, compared with the members of DcBGLU2, 9, 10 and 14 in cluster IV, DcBGLU8 and 18 from the same clade were more highly expressed in leaves. These results indicated that the expression pattern of DcBGLU genes was diverse and tissue-specific.Because of the close relevance between gene expression and function, the expression profiles of A348403)  were anaDcBGLU2 was closely related to reducing-polysaccharide production [D. catenatum leaves. Upregulated expression was detected in the infiltrated leaves 6 hours later . Besides, we also tested the possible functions of the members from the other two clusters, DcBGLU6 from cluster I and DcBGLU13 from cluster II, in medicinal metabolites production. Overexpression of DcBGLU6 greatly induced accumulation of reducing-polysaccharides and flavonoids, and slightly but significantly increased accumulation of alkaloids. Meanwhile, overexpression of DcBGLU13 enhanced the production of soluble-polysaccharides  and flavonoids, but slightly reduced alkaloids . These results reflected the diversified functions of DcBGLUs in regulating medicinal metabolites accumulation in D. catenatum.As our previous study suggested that the expression of oduction , we thuster Fig. a, with aion Fig. b. Besidedes Fig. c and flaids Fig. d, e. TheDendrobium orchids are highly prized medicinal herbs that have been widely used for many years. The plant contains various bioactive components, including polysaccharides, flavonoids, and alkaloids [Dendrobium species to reveal the putative genes and pathways involved in active metabolites biosynthesis [BGLU members in different plant species has been performed with the rapid advancements of whole-genome sequencing technologies. Compared with the other plant species, the number of CH1-BGLU genes in Orchidaceae  is much smaller, suggesting that there may be gene loss or pseudogenization in the process of evolution [D. catenatum, a systematic analysis of BGLU family members has not been available despite being one of the most important medicinal orchids and the importance of BGLU genes in plant secondary metabolism. The recent availability of high-quality annotated reference genome [D. catenatum. This study, to our knowledge, provides the first report concerning the systematic analysis and functional role of BGLU family genes from D. catenatum on secondary metabolism. This study identified 22 full-length BGLU genes from the D. catenatum genome, 16 of which were unevenly distributed on 12 chromosomes. We also analyzed the phylogenetic relations of DcBGLUs and evaluated gene expression patterns in different tissues and under biotic stress. In addition, correlation analysis between gene expression and metabolites content verified four candidate genes involved in active metabolites biosynthesis. Overall, the current study comprehensively investigated and presented the BGLU genes in D. catenatum. This endeavor will be beneficial for the in-depth exploration of biological functions of the BGLU gene family and provide potential targets for molecular breeding.lkaloids . Severalynthesis , 41, 42.ynthesis . Glucosiynthesis , 12. Recvolution . Neverthe genome  providesDcBGLU genes throughout the D. catenatum genome and characterized them using multi-sequence alignment and phylogenetic analysis. The results showed that the DcBGLUs shared high sequence similarity and conserved domain during evolution. DcBGLUs were mainly targeted into cytosol, chloroplast, and vacuole, where they can access the physiological substrates for catalysis. Besides, most DcBGLUs contain at least one predicted N-glycosylation site, corresponding to the estimation that many of DcBGLUs might hydrolyze their substrates through secretory pathways.The current study identified 22 D. catenatum [cis-elements are widely distributed in most of DcBGLU promoters (16/22), suggesting that DcBGLUs might partially mediate MeJA-induced metabolites accumulation. Salicylic acid (SA) is another stress signaling molecule that can be used as an elicitor to promote the biosynthesis of plant secondary metabolites [DcBGLU members, including DcBGLU1, 5, 6, 11, 13, and 18, have SA-responsive elements, implying their possible roles in elicitor-induced active metabolites accumulation.Jasmonate (JA) is a plant-specific signaling molecule broadly associated with the biosynthesis of various secondary metabolites. The exogenous application of methyl jasmonate (MeJA) has been frequently used to manipulate the production of polysaccharides  and alkaatenatum . In the abolites . Both Meabolites , 48. TheCatharanthus roseus [Leonurus japonicas) [Ceratocystis fimbriata infection can enhance alkaloids production in mango (cultivar Ubá) [MF23 infection can increase alkaloids accumulation in D. nobile [DcBGLUs. MF23 induced BGLU1, 7, 19, and 20 expressions  , Ceratocvar Ubá) , and MF2. nobile . Accordions Fig. a with inAtBGLU45 and AtBGLU46 in A. thaliana are responsible for hydrolysis of lignin precursors in response to various stresses [DcBGLU16 was mainly expressed in leaves of D. catenatum  expressed in roots, it failed to respond to MF23 infection as revealed by the RNA-seq analysis  encoded enzymes from carnation (Dianthus caryophyllus), delphinium (Delphinium grandiflorum), and A. taliana can transglycosylate monogalactosyl diacylglyceride to di-, tri-, and tetra-galactosyl diacylglycerides [DcBGLU3 and DcBGLU16 as BGLU-like SFR2 genes in cluster I (Table SBGLU into Artemisia annua vacuole increased trichome numbers and artemisinin production [Plant BGLUs are groups of GH1 enzymes that remove the nonreducing terminal β-D-glucosyl residue from glucoconjugates. BGLU enzymes contribute to various biological functions in plants, including cell wall remodeling, defense and stress response, scent release, phytohormone activation, and secondary metabolism . BGLUs cthaliana , 58. Thiatenatum , suggestycerides , 60. Theoduction . These wDcBGLUs in different tissues and under stress conditions. Although some family members of AtBGLUs and DcBGLUs shared high sequence similarities, the tissue and stress responsiveness were diversified. Therefore, the present study paves the way for further dissection of the distinct role of DcBGLUs in secondary metabolism and other functions.The present study has investigated the classification and expression profile of 22 GH1 Additional file 1.Additional file 2: Fig. S1. Transient expression of DcBGLUs in leaves of D. catenatum.Fig. S2. Amino acid sequence alignment of 22 DcBGLU enzymes in D. catenatum.Fig. S3. Analysis of the numbers and types of cis-acting elements in DcBGLU genes. Table S1. Accession numbers for proteins used in the phylogenetic tree. Table S2. Primers used for qRT-PCR validation. Table S3. Primers used for gene cloning. Table S4. Abbreviations.


Ixodes ricinus nymphs which were or were not infected with horizontally transmitted human pathogens.Microbial communities can affect disease risk by interfering with the transmission or maintenance of pathogens in blood-feeding arthropods. Here, we investigated whether bacterial communities vary between Borrelia burgdorferi sensu lato, Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis by quantitative polymerase chain reaction (qPCR), and their microbiomes were determined by 16S rRNA amplicon sequencing. Tick bacterial communities clustered poorly by pathogen infection status but better by geography. As a second approach, we analysed variation in tick microorganism community structure (in terms of species co-infection) across space using hierarchical modelling of species communities. For that, we analysed almost 14,000 nymphs, which were tested for the presence of horizontally transmitted pathogens B. burgdorferi s.l., A. phagocytophilum, and N. mikurensis, and the vertically transmitted tick symbionts Rickettsia helvetica, Rickettsiella spp., Spiroplasma ixodetis, and CandidatusMidichloria mitochondrii.Ticks from eight forest sites were tested for the presence of Rickettsiella spp., all microorganisms had either significant negative (R. helvetica and A. phagocytophilum) or positive  associations with M. mitochondrii. Two tick symbionts, R. helvetica and S. ixodetis, were negatively associated with each other. As expected, both B. burgdorferi s.l. and N. mikurensis had a significant positive association with each other and a negative association with A. phagocytophilum. Although these few specific associations do not appear to have a large effect on the entire microbiome composition, they can still be relevant for tick-borne pathogen dynamics.With the exception of M. mitochondrii alters the propensity of ticks to acquire or maintain horizontally acquired pathogens. The underlying mechanisms for some of these remarkable interactions are discussed herein and merit further investigation.Based on our results, we propose that The online version contains supplementary material available at 10.1186/s13071-022-05519-w. Enterobacter bacterium isolated from wild Anopheles gambiae mosquitoes in Zambia confers resistance to infection with the malaria parasite in 99% of mosquitoes within the population by interfering with its development prior to the invasion of the midgut epithelium [An. gambiae complex is thought to play a similar role [There is increasing evidence that members of the arthropod microbiome can decrease vector-borne disease risk –3. Microithelium . A microlar role .Like mosquitoes, ticks can transmit a plethora of pathogens causing disease in humans and domestic animals , 6. ThusRickettsia species in Amblyomma maculatum and Dermacentor andersoni ticks [Rickettsia bellii and Anaplasma marginale in D. andersoni ticks [Candidatus Midichloria mitochondrii (hereafter M. mitochondrii) has been shown to be a successful colonizing partner of pathogenic Rickettsia parkeri in A. maculatum ticks [In ticks, bacteria are the most commonly studied organisms in the microbiome-disease risk context. To date, studies have shown the mutually exclusive occurrence of pathogenic and non-pathogenic ni ticks –12, as wni ticks . Regardium ticks .Ixodes ricinus, the best-studied symbionts include obligate intracellular bacteria belonging to the Rickettsia, Midichloria, Rickettsiella, and Spiroplasma genera, which are predominantly transmitted vertically from a female tick to her eggs [Borrelia, Anaplasma, and Neoehrlichia. Although Borrelia burgdorferi sensu lato, responsible for Lyme disease, has been occasionally detected in larvae [Borrelia miyamotoi, a relapsing fever spirochete, in which vertical transmission is more efficient than horizontal [In her eggs . Neverthn larvae , 17, then larvae –20. In orizontal .Ixodes ticks, observed associations between Rickettsia and Borrelia have ranged from negative to positive [The colonization of ticks by pathogens may be inhibited by vertically transmitted symbionts, which are already present in larvae. Evidence from other arthropod–pathogen systems suggests the involvement of several potential mechanisms, including direct killing, competition, and enhancement of host immune responses , 23. Befpositive –26.Anaplasma and Borrelia may alter the tick gut microbiome to increase their colonization success by adjusting a tick antibacterial protein expression [At the same time, horizontally transmitted pathogens may engage in strategies to promote their replication in the presence of other microbes. Some studies have shown that pression , 28. HowB. burgdorferi s. l., possess a different bacterial microbiome than uninfected ticks or ticks infected with the pathogenic protozoan Babesia, bacterial B. miyamotoi, Anaplasma phagocytophilum, or N. mikurensis. To test this, we performed 16S rRNA amplicon sequencing on pools of I. ricinus nymphs infected with one of these pathogens and compared them with each other.Here, we tested two hypotheses. The first hypothesis is that ticks infected with pathogenic bacteria such as I. ricinus is a three-host generalist tick, feeding once per life stage  on various vertebrates, all of which may be amplification hosts of human pathogens [Importantly, athogens . BecauseB. burgdorferi s.l., A. phagocytophilum, and N. mikurensis as well as the predominantly vertically transmitted symbionts M. mitochondrii, Rickettsia helvetica, Rickettsiella spp. and Spiroplasma ixodetis [The second hypothesis is that vertically transmitted members of the tick microbiome either facilitate or impede the acquisition and maintenance of pathogens. To test this, we used data obtained in previous studies , 31, wheixodetis . SubsequI. ricinus from eight forest sites in the Netherlands , which were designed to detect Babesia divergens, B. venatorum , B. capreoli, and B. odocoilei [B. burgdorferi s.l. were subjected to conventional PCR followed by sequencing for genotype identification [For the microbiome analyses, we collected a total of 7874 questing eri s.l. , B. miyaiyamotoi , N. mikukurensis , A. phagtophilum , and Babdocoilei . A detaification . After tIn all samples, absolute bacterial density was quantified, and proportions were multiplied by a load to convert relative into absolute abundance. The quantification of total bacterial DNA load was determined by 16S rRNA qPCR as previously described –41. The www.clcbio.com). Overlapping pairs of raw reads were merged into single longer reads and trimmed with a quality score limit of 0.05 and two ambiguous nucleotides. At this stage, primer sequences were trimmed. Subsequently, reads were fixed-length trimmed (~ 400 bp). To identify operational taxonomic units (OTUs), reads were clustered using the SILVA 16S version 128 reference database with 97% identity as the clustering criterion. Chimeras were removed with a built-in tool in the CLC Genomics Workbench.Illumina MiSeq sequencing libraries targeting the V3-V4 region of the 16S rRNA amplicon were generated and sequenced by BaseClear . In short, barcoded amplicons from the V3-V4 region of 16S rRNA genes were generated using a two-step PCR. Between 10 and 25 ng of genomic (g)DNA was used as a template for the first PCR with a total volume of 50 µl using the 341F (5′-CCTACGGGNGGCWGCAG-3′) and the 785R (5′-GACTACHVGGGTATCTAATCC-3′) primers appended with Illumina adaptor sequences. Control PCR reactions were performed alongside each separate amplification without the addition of a template. PCR products were purified, and the size of the PCR products was checked on a fragment analyser  and quantified by fluorometric analysis. Purified PCR products were used for the second PCR in combination with sample-specific barcoded primers . Subsequently, PCR products were purified, checked on the fragment analyser  and quantified, followed by multiplexing, clustering, and sequencing on an Illumina MiSeq with the paired-end (2×) 300-base-pair (bp) protocol and indexing. The sequencing run was analysed with the Illumina CASAVA pipeline (v1.8.3) with demultiplexing based on sample-specific barcodes. The raw sequencing data produced were processed by removing the sequence reads of inadequate quality (only “passing filter” reads were selected) and discarding reads containing adaptor sequences or PhiX control using an in-house filtering protocol. A quality assessment on the remaining reads was performed using the FastQC version 0.10.0 quality control tool. Sequenced reads were imported to the QIAGEN CLC Genomics Workbench 10.0.1 supplemented with CLC Microbial Genomics Module 3.6.1 (vegan (version 2.5-6) for ordination [All analyses were carried out in R 3.6.3 . We useddination . A princdination ).adonis function from R package vegan to assess whether pathogen presence significantly affected community variation. We tested for multivariate dispersion through the betadisper function.Additionally, we carried out permutational multivariate analysis of variance (PERMANOVA)  with theI. ricinus were collected from 19 forest sites in the Netherlands in 2013 and 2014 . AnThe R package Hmsc  was usedgelman.diag function. We performed variance portioning with the computeVariancePartitioning function to investigate which random effect was most responsible for the observed variation in the prevalence of individual microorganisms. Lastly, to compute the microorganism association matrices associated with each random level, we used the computeAssociations function. The corrplot function from the corrplot package was used to plot only those associations for which the posterior probability of being negative or positive was at least 0.95 [We then examined MCMC convergence by assessing the effective size of the posterior sample and Gelman and Rubin's  convergeast 0.95 .Borrelia, Midichloria, Neoehrlichia, Methylobacterium, Mycobacterium, Pseudomonas, Rickettsia, Rickettsiella, Sphingomonas, and Wolbachia . However, significant overdispersion (P < 0.001) indicates that the P-values may be overinflated. Although the results suggest that microbiome composition was significantly different across samples with different pathogens, the R2 demonstrates that most variation could not be attributed to a pathogen status.The PCoA did not convincingly show that community composition was structured by pathogen-specific pools, or separated along the pools with or without pathogens Fig. A. InsteaS. ixodetis, R. helvetica, Rickettsiella spp., M. mitochondrii, A. phagocytophilum, N. mikurensis, and B. burgdorferi s.l. All microorganisms were present in all locations; however, infection rates varied strongly among the bacterial species and locations. The prevalence of the tick microorganisms per location and region is provided in Table S6, as well as in Krawczyk et al. [Data on the prevalence of tick microorganisms in questing nymphs generated in previous studies were analysed for co-infections , 31, 46.k et al.  and TakuP < 0.001 in all cases), and infections with at least five, six, or seven microorganisms occurred as often as expected  tested questing nymphs were free of any of the seven studied microorganisms, and 12,225 (87.6%) nymphs were infected with one or more. The exact binomial test showed that infections with at least one, two, three, or four tick microorganisms were significantly less frequent than expected (M. mitochondrii (80.0%), S. ixodetis (80.1%), N. mikurensis (70.6%), and B. burgdorferi s.l. (84.7%). Location explained variation in the prevalence of Rickettsiella spp. (99.6%), R. helvetica (84.3%), and A. phagocytophilum (61.2%).To investigate co-infections of tick microorganisms, we constructed an HMSC model in which we included the tick’s identity as a sampling unit, which was nested as a random effect within the location where the tick was collected. The MCMC convergence of the model was satisfactory: the potential scale reduction factors for the β-parameters were 1.056 on average (max = 1.211). Variance partitioning showed that the tick’s identity explained a substantial proportion of the observed variation in the prevalence of Rickettsiella spp. had either significant negative (R. helvetica and A. phagocytophilum) or positive  associations with M. mitochondrii  transovarial transmission of a second Rickettsia species. However, to our knowledge, this so-called rickettsial interference has never been related to any other tick symbiont genus. If this interference between R. helvetica and S. ixodetis exists, to persist in a tick population, S. ixodetis would have to occasionally utilize horizontal transmission, which has been suggested but not undisputedly proven [It is possible that ticks cannot simultaneously maintain two symbiont species by vertical transmission, which may affect their distribution in tick populations. The phenomenon of interference between two vertically transmitted symbionts in hard ticks (Ixodidae) has been described in previous studies , 65, 66.y proven , 68.Acyrthosiphon pisum, both Serratia symbiotica and Hamiltonella defensa provide resistance to parasitoids. Although a double infection provided increased resistance in the laboratory, it was rarely observed under natural conditions [The mutual exclusion among symbionts may also indicate that the roles which they play in ticks overlap and that a double infection comes with fitness costs without providing sufficient increases in fitness. For instance, in nditions .M. mitochondrii and S. ixodetis associations imply that these pairs of symbionts serve complementary functions and/or exhibit different tissue tropism in ticks. Different host tissues may constitute distinct microhabitats and be nutritionally favourable, immunotolerant, and easy to colonize for some but not for other microorganisms [The positive association between rganisms .M. mitochondrii, was positively associated with the pathogens B. burgdorferi s.l. and N. mikurensis, and negatively associated with the pathogen A. phagocytophilum  are ampherlands , 75, 76.Anaplasma phagocytophilum may be acquired by I. ricinus from various vertebrate species, but it is most commonly acquired from roe deer [A. phagocytophilum with B. burgdorferi s.l. and N. mikurensis pathogens implies that nymphs that fed on deer had no chance to acquire rodent-borne pathogens [roe deer . Therefoathogens .Although here a few specific associations did not have a large effect on the composition of the entire microbiome, they can still be relevant for tick-borne pathogen dynamics. We observed that tick symbionts are heterogeneously distributed across the Dutch tick population and revealed a novel pattern of associations. Although the underlying mechanisms are unknown, they are important for understanding the role of vertically transmitted symbionts in the control of tick-borne diseases.M. mitochondrii contribute more to the transmission cycle and the acarological risk of Lyme borreliosis and neoehrlichiosis than ticks lacking M. mitochondrii. Future studies on microorganism interference should investigate the dynamics of symbionts through the ontogeny of pre-infected ticks in comparison with non-infected ticks, applying bacterial quantification methods and identifying the microbiomes of ticks during different life events such as moulting, questing, and feeding.For instance, our findings imply that ticks carrying Additional file 1: Table S1. Details on studied forest sites. Table S2. The qPCR protocol for detection of tick symbionts and pathogens. Table S3. Bacterial loads of samples for the 16S rRNA sequencing; sample scheme. Table S4. Details on a positive control, primers, protocol, and in silico analysis used in the 16S rRNA quantification analyses. Table S5. OTU table. Table S6. Prevalence of veritcally and horizontally transmitted symbionts and pathogens per location. Table S7. Expected and observed co-infections in questing nymphs and results of the exact binomial test. Table S8. Tick microorganism species-to-species associations (co-infections) in ticks.Additional file 2: Figure S1. Abundance of the most abundant taxa, separated by quartiles of absolute bacterial density (16S rRNA content in ng/µL). Figure S2. Principal coordinate analysis of Bray–Curtis dissimilarities for tick microbiomes compared to blanks.


Antrodia cinnamomea is a precious and popular edible and medicinal mushroom. It has attracted increasing attention due to its various and excellent bioactivities, such as hepatoprotection, hypoglycemic, antioxidant, antitumor, anticancer, anti-inflammatory, immunomodulation, and gut microbiota regulation properties. To elucidate its bioactivities and develop novel functional foods or medicines, numerous studies have focused on the isolation and identification of the bioactive compounds of A. cinnamomea. In this review, the recent advances in bioactivity, isolation, purification, and identification methods of active compounds from A. cinnamomea were summarized. The present work is beneficial to the further isolation and discovery of new active compounds from A. cinnamomea. Antrodia cinnamomea (syn. Antrodia camphorate) is a precious edible and medicinal mushroom; it belongs to phylum Basidiomycetes, family Polyporaceae, and genus Antrodia   [70]A. cinnamomea is extracted with 95% ethanol and then condensed at decreased pressure. The residues are suspended in water before being partitioned with n-hexane and ethyl acetate. Antrodin C is isolated from the ethyl acetate fraction by subsequent chromatography on silica gel and Sephadex LH-20 columns [2, 5 µm) is used to separate crude antrodin C. The mobile phase comprises distilled water H2O (0.5% acetic acid) and acetonitrile  (10 mL/min) to obtain antrodin C with a purity exceeding 95% [2O (0.5% acetic acid) and acetonitrile at a flow rate of 7.2 mL/min, and their content is only 0.004% and 0.001%, respectively [A. cinnamomea in recent publications are summarized in Later studies have confirmed five maleic and succinic acid derivatives, which were named antrodins A–E . Researc columns . The semding 95% . The mixectively . The preA. cinnamomea possess potent anti-inflammatory properties. 4,7-Dimethoxy-5-methyl-1,3-benzodioxole (DMB) is a representative benzenoid compound and was first discovered in the crude methanol extract of A. cinnamomea fruiting bodies and cultured mycelia. Previous research has revealed that DMB exerts a significant anti-inflammatory properties by inhibiting superoxide production in human neutrophils. Furthermore, DMB therapy reduces dendritic cell-mediated Th2 allergic responses [A. cinnamomea mycelia, has an excellent anti-inflammatory effect, indicating its possible use in the development of novel anti-inflammatory drugs [0  is another important benzenoid compound of A. cinnamomea, and it possesses antitumor and anti-inflammatory activities [Several benzenoid compounds from esponses . Antrolory drugs . Coenzymtivities ,81,82.A. cinnamomea. Briefly, freeze-dried and powdered mycelia are extracted with methanol and then filtered. The filtrate is concentrated by evaporation under decreased pressure to produce the methanol extract. The extract is suspended in distilled water, and the aqueous solution is partitioned with n-hexane, ethyl acetate, and n-butanol in sequence. The ethyl acetate layer is evaporated to dryness, and the residue is chromatographed on a silica gel with n-hexane:ethyl acetate (7:1) to elute a crude fraction containing DMB. For the acquisition of pure DMB, this fraction is purified using a Sephadex LH-20 column with methanol as the eluent [A. cinnamomea in recent publications are summarized in At present, a limited number of studies have been conducted on the isolation and purification of benzenoid compounds. Shie et al.  reportede eluent . The DMBe eluent . In addie eluent , antroloe eluent . The preA. cinnamomea. This polysaccharide is identified as an antrodan with a complex union of α- and β-glucans, which have (1 → 4)-linked α-Glcp and (1 → 3)-linked β-Glcp linkages to the carbohydrate chains though the asparagine connection with protein sites. Antrodan at low dosages (40 mg/kg) may be a potential hepatoprotective agent. However, increased doses of antrodan may cause certain unpleasant responses [According to reports, minimal variations in dextran glycosidic linkages, high-order structure, molecular weight, solubility, protein and lipid side chains, and/or high-order aggregates can result in significant differences in bioactivity ,85. Chiuesponses . Antrodaesponses ,89. Furtesponses .A. cinnamomea [2) and eluted with DDW at pH 11.0 (adjusted using 1 M NaOH) to separate polysaccharides at a flow rate of 0.5 mL/min. The target was collected with a fraction collector. High-performance SEC yielded the product at roughly 10% with an average molecular weight of 442 kD. The protein content of antrodan was 71.0%, which was evidently larger than the carbohydrate quantity (14.1%); thus, this antrodan was classified as a glycoprotein [A. cinnamomea are listed in Glycoprotein is a binding protein and a complex polysaccharide. Thus, it has certain properties of proteins and polysaccharides. Most glycoproteins are soluble in water, dilute salt, and dilute alkali solution. Therefore, the extraction method of proteins or polysaccharides can be used based on its properties. A glycoprotein named antrodan was isolated from cultured mycelia of nnamomea . The resoprotein . The maiA. cinnamomea is critical. This knowledge is essential for understanding the biological activities of these metabolites and discovering new A. cinnamomea bioactive compounds. Typically, the characterization and quantification of natural products can use various detection techniques, including nuclear magnetic resonance (NMR), infrared spectra, and mass spectrometry (MS) detection. NMR has steadily developed and has become an essential technology for chemical identification and structural characterization. This technique enables the acquisition of data from extremely mass-limited samples and the investigation of very complex materials [1H-NMR and 13C-NMR analyses were used to identify compounds from solid-state cultured mycelia of A. cinnamomea [A. cinnamomea due to its superiority to other standard chromatographic techniques in detecting the amounts of specific herbal mixture elements [Aside from isolation and purification, the identification of particular bioactive compounds of aterials . 1H-NMR nnamomea . Finallynnamomea . NMR canelements .A. cinnamomea was established [A. cinnamomea, and different cultivation techniques exhibit significant variations. Qiao et al. [A. cinnamomea samples derived from cutting wood culture, solid-state fermentation, submerged fermentation, and dish culture. It was determined by ultra-high performance liquid chromatography/ultraviolet (UPLC/UV) or supercritical fluid chromatography coupled with mass spectrometry  within 16 min. The result showed that the 18 kinds of triterpenoids accounted for 118.2 ± 28.2, 89.4 ± 30.8, and 116.5 ± 1.1 mg·g−1 in wood-cultured fruiting bodies, wood-cultured mycelia, and dish-cultured, respectively. However, no triterpenoids were detected in the solid support cultivation or submerged fermentation samples in this research [A. cinnamomea benefits from the precise and rapid measurement of signature compounds in various samples using this method. Ultra-performance liquid chromatography quadrupole time-of-flight MS (UPLC/Q-TOF/MS) has been used to characterize and quantify mixture compounds. For the first time, the UPLC/Q-TOF/MS method was used to identify the extracts from A. cinnamomea and led to the discovery of 139 chemical compounds, including 102 terpenoids, 8 benzenoids, 2 purine nucleosides, and 27 other classes [A. cinnamomea products.Fingerprint analysis is used for the thorough assessment of the quality of herbal medicines and their associated products. Recently, the HPLC fingerprinting approach supplemented by ultraviolet (UV) and photodiode array (PDA) has been widely utilized for herbal-quality testing. However, UV and PDA detectors present several problems in the isolation of target analytes from interfering impurities. Thus, these techniques involve time-consuming sample preparation and long HPLC run times. To resolve these issues, scientists use the more sensitive liquid chromatography–tandem MS (HPLC-MS/MS), which can offer better overall information, such as molecular weight, retention time, and analyte collision fragments. Accordingly, a validated HPLC-MS/MS method for the quick and precise measurement of compounds from ablished . The metablished . Triterpo et al.  reportedresearch . The dev classes . The devA. cinnamomea as a burgeoning medicinal fungus has multiple significant biological activity and pharmacological functions. Researchers have isolated and purified various bioactive compounds from its fruiting bodies and cultured mycelia. Despite recent advances, critical concerns regarding the fundamental research and use of A. cinnamomea remain unresolved.Pharmacologically active natural compounds have achieved extraordinary appeal in recent decades, and certain bioactive molecules have historically made significant contributions to medication development. A. cinnamomea by conventional solvent extraction has a number of drawbacks, particularly solvent wastage and low extraction efficiency. The extensive use of organic solvents is bound to cause pollution and harm people’s health. Thus, the identification of novel solvents to replace traditional organic solvents is necessary. Alternative solvents aim to provide an extraction method that is environment friendly and of high quality [A. cinnamomea. The current patterns in isolation, purification, and identification of characteristic metabolites are the bottlenecks in the development of high-quality A. cinnamomea products. Optimizing such isolation and purification methods is necessary to improve the product quality.First, obtaining active substances from  quality . ResearcA. cinnamomea active constituents inhibits their development in the field of medicine. Apart from improving the efficiency of isolation and purification, the yield of bioactive compounds should be increased. Moreover, waste is inevitable in the extraction of active substances. China produces about hundreds of metric tons of A. cinnamomea extraction waste every year [A. cinnamomea resources and reduces environmental pollution, thereby providing an accurate research idea to further study the use of extraction residue and improve its utilization value and paving the way for green sustainable development in the future.Second, the low content of ery year . Interesery year . The reuA. cinnamomea remain unclear. One of the main reasons for this dilemma is that researchers usually use extracts or mixtures rather than pure compounds to test the effect, which causes difficulty in pinpointing compounds with specific roles. The combined effects of natural bioactive substances include synergistic, antagonistic, and independent effects. Antagonistic effect means the inactive compounds contained in these mixes may cause harmful side effects. Thus, the isolation and identification of pure bioactive compounds from A. cinnamomea are beneficial to revealing the mechanism underlying the effects of its compounds. Moreover, defining the related mechanisms can improve the use of functional compounds from A. cinnamomea in drug development and clinical applications.Finally, the mechanisms underlying the effects of certain active compounds from


Argas ticks are primary parasites of birds with veterinary importance. Nevertheless, these ticks have received little attention regarding molecular identification studies. DNA barcoding is a powerful technique for identifying tick species besides traditional morphological identification. The present work is a first effort to divulge DNA sequences of Argas (Persicargas) arboreus from Egypt and worldwide. We used cytochrome c oxidase subunit I (COI) from A. arboreus infesting herons, and from the fowl tick Argas (Persicargas) persicus. Our results pointed out another success for the Folmer primers that are widely used in DNA barcoding, permitting the discrimination of morphologically similar A. arboreus and A. persicus. Babesia, Theileria, Rickettsia, and Borrelia to humans and animals, ranking second after mosquitoes in disease transmission  arboreus is a common parasite of the cattle egret (Bubulcus ibis) and other water birds in Egypt and all over the African continent  persicus is an ectoparasite of poultry birds, which causes severe irritation to poultry, harming their production . At the same time, 150 tick specimens were collected from domestic chicken houses in the Abo-Rawash, Giza governorate . The collected ticks were transferred to the laboratory, morphologically identified according to Kaiser et al. , according to the manufacturer’s protocol.The COI gene was amplified using Folmer universal primers LCO1490 (GGTCAACAAATCATAAAGATATTGG) and HCO2198 (TAAACTTCAGGGTGACCAAAA AATCA) . Ticks (n = 150) collected from domestic chicken houses were A. persicus . Collected ticks were morphologically differentiated by the lateral integumental cells  collected from trees supporting the rookeries of the cattle egret 00 collecA. arboreus, and 14 for A. persicus, with the expected PCR product of 710 bp. However, due to technical issues, we successfully amplified and trimmed the sequences to 603 and 573 bp from A. arboreus and A. persicus, respectively.For each species identified, 15 specimens were submitted to DNA extraction and PCR amplification. Mitochondrial COI region of nearly all involved samples were successfully PCR amplified—13 samples were successfully amplified for A. arboreus sequenced gene was retrieved. When searched against BLASTn, the highest similarity scores were 90.5% , 85.1% , and 84.8% .Resulting sequences were searched against the BLAST database , 98.4% , and 97.8% . Sequences presently revealed have been deposited in the GenBank database with accession numbers OM177656 [A. arboreus COX1] gene and OM177661 [A. persicus COX1] gene.On the other hand, for A. persicus, OM177661, against A. arboreus OM177656 revealed 76.28% sequence identity. Phylogenetic analysis was established based on COI sequences of as many Argas species as available on the GenBank. A neighbor-joining tree of the COI gene from Argas species worldwide was constructed using MEGA11 ; Kenya (KJ133581.2); Romania (FN394341); USA (MK287892.1); Iran (KX879770.1), Pakistan (MW077849); Kazakhstan (MN900726) and China (LC209195)’; A. arboreus COI (OM177656) ‘Egypt, current study’; Argas sp. Spain  transgariepinus South Africa (KX431961); A. africolumbae South Africa (NC_019642); A. lagenoplastis Australia (KC769587); A. (Carios) vespertilionis Vietnam (KX431960); A. miniatus Brazil (KC769590); A. reflexus Spain (MW288388); A. walkerae South Africa (KJ133584); A. hermanni Iran (ON090294). In the constructed tree, Dermanyssus sp. (FN650615) was used as an out-group.Multiple sequence alignment of the COI gene nucleotide sequence for A. persicus groups, including A. persicus from collected specimens, were monophyletic and shared the same clade with haplotypes of Argas sp. from Spain, conferring their relatedness. Another result is the uniqueness of A. arboreus genotype in the present study. Indeed, such genotype shares the same molecular basis with A. miniatus as reported by Muñoz-Leal et al.   robertsi from Australia and A. (P.) miniatus from Brazil. Similarly, A. (P.) robertsi is considered to be the Oriental Australian counterpart of the Ethiopian A. (P.) arboreus  arboreus and the Asian-Australian A. (P.) robertsi originated from a common ancestor and geographic isolation caused genetic incompatibility. The present analysis helped reveal the significant genetic divergence between the two species in this study. Our phylogenetic analysis assured the uniqueness of the molecular marker COI in A. arboreus and its validity to be used in the differentiation process between the other species. In a try to assign the five sequences identified by Palomar et al. (A. persicus, one genotype ‘MW288385’ was clustered with both A. africolumbae and A. hermanni, two genotypes ‘MW288380 and MW288384’ were clustered with A. lagenoplastis, all of these mentioned clustered in the tree in this study, supporting the sequence identities shown in Palomar et al. (According to Khalil et al. , the Afrr et al.  to the Gr et al. , as wellr et al. A. arboreus and A. persicus. A large-scale multi-marker study with a large sample size from different localities of soft ticks covering various hosts and populations across Egypt in different seasons is needed in the future. Such a study will provide a better and broader understanding of the population structure and dynamics, host range, physical environment, and habitat of these important medical and veterinary species. Additionally, their economic importance and effects on public health throughout the country will undoubtedly be better understood.This study provides the first molecular descriptive barcoding of the two soft tick species that are major bird ectoparasites, especially on herons and poultry from the Giza governorate in Egypt,


The role of MMP14 in the TME was investigated in a retrospective analysis of tumor samples from 86 individuals with stage III colorectal cancer by single cell–based spatial profiling of MMP14 expression as performed by 12-color multiplex immunohistochemistry (mIHC). Analysis of gene expression data revealed that high MMP14 expression was associated with tumor progression and implicated both cancer-associated fibroblasts (CAFs) and tumor-associated macrophages in such progression. Spatial profiling by mIHC revealed that a higher percentage of MMP14+ cells among intratumoral CAFs (MMP14+ CAF/CAF ratio) was associated with poorer relapse-free survival. Multivariable analysis including key clinical factors identified the MMP14+ CAF/CAF ratio as an independent poor prognostic factor. Moreover, the patient subset with both a high MMP14+ CAF/CAF ratio and a low tumor-infiltrating lymphocyte density showed the worst prognosis. Our results suggest that MMP14+ CAFs play an important role in progression of stage III colorectal cancer and may therefore be a promising therapeutic target.Matrix metalloproteinase 14 (MMP14) expression is implicated in progression of colorectal cancer, but its role in the tumor microenvironment (TME) has been unclear. The relevance of MMP14 to colorectal cancer progression was explored by analysis of transcriptomic data for colorectal adenocarcinoma patients ( Colorectal cancer is the second most common cause of cancer-related deaths worldwide . Stage I+ and CD8+ T cells has been found to classify stage III colorectal cancer into recurrent and nonrecurrent subgroups (Increasing evidence has suggested that the tumor microenvironment (TME), including immune cells and cancer-associated fibroblasts (CAFs), plays an important role in cancer fate including therapeutic response and clinical outcome –14. An Iubgroups –19. In aubgroups . CAFs coubgroups . These cubgroups , and theubgroups –29. ThesMMP14 expression level tend to have a mesenchymal phenotype such as CMS4  is a transmembrane proteolytic enzyme  that pla as CMS4 , 32. In l cancer . Further+ CAFs may act in collaboration with TAMs to promote the progression of colorectal cancer. Our results suggest a novel risk stratification of stage III colorectal cancer based on MMP14 expression in CAFs.We have now conducted a retrospective biomarker analysis of surgically resected tumor specimens from patients with stage III colorectal cancer attending Kindai University Hospital as well as an analysis of TCGA transcriptomic data in order to clarify this role of MMP14. We performed 12-color multiplex immunohistochemistry (mIHC) to evaluate the relation of MMP14 protein expression in multiple cell subsets including intratumoral immune cells to colorectal cancer progression. Our digital pathology platform thus allowed a single cell–based quantitative spatial profiling of MMP14 expression in various cell lineages in the stage III colorectal tumors. We found that CAFs are an important source of MMP14 and that MMP14We consecutively reviewed the medical records of patients with stage III colorectal cancer who underwent definitive surgery followed by adjuvant chemotherapy at Kindai University Hospital between January 2013 and December 2017. Patients who received adjuvant chemotherapy for <3 months and those without sufficient tumor tissue available for our study were excluded. Colorectal cancer was staged according to the eighth edition of the Union for International Cancer Control (UICC) TNM Classification of Malignant Tumors . From thmIHC staining was performed as described previously , 36. In De Novo Software) as described previously  and center of the tumor (CT), were randomly selected from the whole digital slides with the use of Aperio ImageScope v.12 software (Leica Biosystems). The IF was defined as the area within 300 µm external to the boundary separating the tumor cell area from the surrounding connective tissue . The CT eviously , 36. In + and CD8+ T cells in both the CT or ISA and the IF regions were calculated, and tumors were then categorized into two groups based on the median values of the densities of each T cell subset in each region. Values lower than or equal to the median and those higher than the median were scored as 0 or 1, respectively. The sum of all scores  was calculated as the final Immunoscore. A final score based on CT and IF regions was determined as the conventional Immunoscore. In addition, an IS-Immunoscore based on scores in IF and ISA regions was provided by our Tissue Segmentation platform. Patients with final scores of ≥2 or <2 were classified as having a high or low Immunoscore, respectively.The Immunoscore was determined as described previously . In brien = 592) of the TCGA database were downloaded via the cBioportal website  in August 2021. The downloaded value (x) was converted to x + 1 for subsequent differential gene expression analysis. Genes expressed at a low level  were filtered out. Gene set enrichment analysis (GSEA) (http://www.broadinstitute.org/gsea/index.jsp) was performed with hallmark gene sets v7.4 and GSEA software version 4.0.3 (Broad Institute) (https://cibersortx.stanford.edu). Correlation coefficients for gene expression levels were estimated with the cBioportal website platform.Normalized gene expression data (RNA Seq v2) for the colorectal adenocarcinoma cohort (stitute) . The extstitute)   or GraphPad Prism version 9.1.1 (GraphPad Software).Differences between survival curves constructed by the Kaplan-Meier method were assessed with the log-rank test. The hazard ratio (HR) and its 95% confidence interval (CI) were calculated according to the Cox proportional hazards model. Correlations were examined with the Spearman rank correlation test. The Benjamini-Hochberg method was applied to calculate the false discovery rate (FDR) for multiple testing unless specified otherwise. The Wilcoxon rank-sum test was adopted to compare continuous variables, with the Bonferroni correction being applied to adjust  studies –45. All This retrospective study was approved by the Institutional Review Board (R02-070) and the Ethics Committee of Kindai University Faculty of Medicine. The Institutional Review Board waived the need to obtain written consent.MMP14 expression in tumors was associated with a poor survival outcome in this cohort  as well as that of many collagen genes  value. This list included M2-TAM–related genes such as MARCO . MMP14 expression was also associated with that of other M2-TAM genes such as CD163  and CD206 .To explore the clinical relevance of MMP14 in colorectal cancer, we first analyzed publicly available gene expression data for tumor tissue of the colorectal adenocarcinoma data set in TCGA. A high level of en genes , suggestted gene , was mosas MARCO  and SPP1CO  was in the top five gene sets that were significantly up-regulated in tumors expressing MMP14 at a high level  were also evaluated in the same sections, given that previous studies based on the Immunoscore have implicated these cells in colorectal cancer progression –19. In a+ CAF/CAF ratio) was associated with a poorer RFS , whereas a similar trend was not apparent for MMP14 expression in tumor cells, TAMs, or TILs  was more clearly prognostic  , whereas a high IS-Immunoscore was an independent prognostic factor for a better RFS  (+ CAF/CAF ratio). Among these four groups , group C showed the worst prognosis whereas the other three groups each showed a similarly better prognosis  Table 2.+ CAF/CAF ratio according to adjuvant chemotherapy regimen in order to investigate whether oxaliplatin is able to overcome the poor clinical outcome associated with MMP14+ CAFs. There was no significant difference in RFS between patients treated with 5-FU alone and those treated with both oxaliplatin and 5-FU , however  and the Ethics Committee of Kindai University Faculty of Medicine. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Conception and design, YM, KH, HK, and KN. Development of methodology, YM, KH, KY, and TT. Acquisition of data (such as management of patients and provision of facilities), YM, KH, HK, and JK. Analysis and interpretation of data , YM, KH, HK, AI, YC, and KN. Writing, review, and/or revision of the manuscript, YM, KH, and HK. Administrative, technical, or material support (such as reporting or organization of data and database construction), YM, KH, HK, KY, and TT. Study supervision, KH, HK, AI, JK, and KN. Funding, JK, and KN. All authors contributed to the article and approved the submitted version.We thank Kyoko Itoh of the Department of Pathology and Applied Neurobiology, Kyoto Prefectural University of Medicine, as well as Hiroshi Ogi of SCREEN Holdings Co. Ltd. for technical support.HK has received consulting fees from Bristol-Myers Squibb Co. Ltd., Eli Lilly Japan K.K., MSD K.K., Ono Pharmaceutical Co. Ltd., Daiichi-Sankyo Co. Ltd., and Taiho Pharmaceutical Co. Ltd.YC honoraria from Bristol-Myers Squibb Co. Ltd., Bayer Yakuhin Ltd., Eli Lilly Japan K.K., MSD K.K., Ono Pharmaceutical Co. Ltd., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co. Ltd., Merck Biopharma Co. Ltd., Takeda Pharmaceutical Co. Ltd., Yakult Pharmaceutical Industry, Teijin Pharma Ltd., and Taiho Pharmaceutical Co. Ltd.YC lecture fees from Glaxo Smith Kline K.K. and Otsuka Pharmaceutical Co. Ltd.YC and research funding from Chugai Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co. Ltd., Kobayashi Pharmaceutical Co. Ltd., and Eisai Co. Ltd. TT has received speaker fees from MSD K.K., Ono Pharmaceutical Co. Ltd., and Bristol-Myers Squibb Co. Ltd. YC has received honoraria from Chugai Pharmaceutical Co. Ltd. KH has received lecture fees from AS ONE Corp., AstraZeneca K.K., Bristol-Myers Squibb Co. Ltd., MSD K.K., and Ono Pharmaceutical Co. Ltd. as well as research funding from AstraZeneca K.K. and MSD K.K. KN has received honoraria from Astellas Pharma Inc., Takeda Pharmaceutical Co. Ltd., Nanzando Co. Ltd., AstraZeneca K.K., Chugai Pharmaceutical Co. Ltd., Roche Diagnostics K.K., MSD K.K., Eli Lilly Japan K.K., Nippon Kayaku Co. Ltd., Daiichi Sankyo Co. Ltd., Novartis Pharma K.K., Kyowa Kirin Co. Ltd., Taiho Pharmaceutical Co. Ltd., Pfizer Japan Inc., AbbVie Inc., Bristol-Myers Squibb Co. Ltd., CareNet Inc., Amgen Inc., Medical Review Co. Ltd., Yodosha Co. Ltd., 3H Clinical Trial Inc., Thermo Fisher Scientific K.K., Hisamitsu Pharmaceutical Co. Inc., Nichi-Iko Pharmaceutical Co. Ltd., Kyorin Pharmaceutical Co. Ltd., Medicus Shuppan Publishers Co. Ltd., Nippon Boehringer Ingelheim Co. Ltd., Nikkei Business Publications Inc., Yomiuri Telecasting Corp., and Medical Mobile Communications Co. Ltd.YC research funding from MSD K.K., AstraZeneca K.K., Pfizer Japan Inc., ICON Japan K.K., Astellas Pharma Inc., Bayer Yakuhin Ltd., Takeda Pharmaceutical Co. Ltd., Novartis Pharma K.K., Otsuka Pharmaceutical Co. Ltd., Eli Lilly Japan K.K., EPS International Co. Ltd., Bristol Myers Squibb Co. Ltd., CMIC Shift Zero K.K., PRA Health Sciences, Taiho Pharmaceutical Co. Ltd., Eisai Co. Ltd., Merck Biopharma Co. Ltd., Parexel International Corp., Mochida Pharmaceutical Co. Ltd., Covance Japan Inc., Ono Pharmaceutical Co. Ltd., Kissei Pharmaceutical Co. Ltd., Medical Research Support, Sysmex Corp., GlaxoSmithKline K.K., Sanofi K.K., A2 Healthcare Corp., Kyowa Hakko Kirin Co. Ltd., Syneos Health, AbbVie Inc., EPS Corp., Pfizer R&D Japan G.K., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co. Ltd., PPD-SNBL K.K., Nippon Boehringer Ingelheim Co. Ltd., IQVIA Services Japan K.K./Quintiles Inc., Japan Clinical Research Operations, and SymBio Pharmaceuticals Ltd.YC and consulting fees from Astellas Pharma Inc., Takeda Pharmaceutical Co. Ltd., Eli Lilly Japan K.K., Pfizer Japan Inc., Kyorin Pharmaceutical Co. Ltd., and Ono Pharmaceutical Co. Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Article title: Zinc Pyrithione is a Potent Inhibitor of PLPro and Cathepsin L Enzymes with Ex Vivo Inhibition of SARS-CoV-2 Entry and ReplicationAuthors: Jerneja Kladnik, Ana Dolinar, Jakob Kljun, David Perea, Judith Grau-Expósito, Meritxell Genescà, Marko Novinec, Maria J. Buzon, Iztok TurelJournal:Journal of Enzyme Inhibition and Medicinal ChemistryBibliometrics: Volume 37, Number 1, pages 2158–2168DOI:10.1080/14756366.2022.21084171) When this paper has been published online, the Graphical Abstract is missing. Below is the Graphical Abstract of the manuscript.2) The correct and updated version of the Acknowledgement is as follows1d. Schematic representation of the zinc pyrithione antiviral activity (Table of Content Graphic) was created by the artist Simon Kajtna.The authors acknowledge EATRIS Academic Collaboration and their COVID-19 Fast Response Service for the coordination of new collaboration establishment between research group of Prof. Dr. I.Turel and Dr. Marıa J. Buzon. We would also like to thank Masa Masic for the help with the synthesis and Tja_sa Rijavec for the crystallization of complex


Guidelines recommend measuring blood pressure (BP) in both arms, adopting the higher arm readings for diagnosis and management. Data to support this recommendation are lacking. We evaluated associations of higher and lower arm systolic BPs with diagnostic and treatment thresholds, and prognosis in hypertension, using data from the Inter-arm Blood Pressure Difference—Individual Participant Data Collaboration.One-stage multivariable Cox regression models, stratified by study, were used to examine associations of higher or lower reading arm BPs with cardiovascular mortality, all-cause mortality, and cardiovascular events, in individual participant data meta-analyses pooled from 23 cohorts. Cardiovascular events were modelled for Framingham and atherosclerotic cardiovascular disease risk scores. Model fit was compared throughout using Akaike information criteria. Proportions reclassified across guideline recommended intervention thresholds were also compared.P<0.001). Higher arm BP models fitted better for all-cause mortality, cardiovascular mortality, and cardiovascular events . Higher arm BP models better predicted cardiovascular events with Framingham and atherosclerotic cardiovascular disease risk scores (both P<0.001) and reclassified 4.6% and 3.5% of participants respectively to higher risk categories compared with lower arm BPs).We analyzed 53 172 participants: mean age 60 years; 48% female. Higher arm BP, compared with lower arm, reclassified 12% of participants at either 130 or 140 mm Hg systolic BP thresholds (both Using BP from higher instead of lower reading arms reclassified 12% of people over thresholds used to diagnose hypertension. All prediction models performed better when using the higher arm BP. Both arms should be measured for accurate diagnosis and management of hypertension.https://www.clinicaltrials.gov; Unique identifier: CRD42015031227.URL:  Guidelines advise adoption of the higher reading arm blood pressure for diagnosis and management of hypertension, based on expert opinion. We studied data on over 53 000 participants from 23 studies around the world to examine the implications of choosing the higher or lower arm blood pressure.The higher arm blood pressure better predicts all-cause mortality, cardiovascular mortality, and cardiovascular events, compared with the lower arm reading.Up to 12% of people classified with hypertension fall below recommended thresholds for diagnosis and treatment of hypertension if the lower, rather than the higher reading arm, is used.Routine adoption of the higher arm blood pressure measurement allows better cardiovascular risk prediction and classification, more accurate diagnosis and treatment of hypertensionWhen considering diagnosis or treatment of hypertension, blood pressure should be measured in both arms and all decisions based on the higher arm measurement. Failure to do this risks under-diagnosis and under-treatment for many millions of people globally.3 Only 20% to 50% of patients in primary care reportedly have both arms measured and general practitioners do not always adopt the higher reading arm for management decisions.5 Inter-arm differences may arise due to stenosis, making adoption of the higher arm reading logical; however, in most instances, there is no clearly demonstrable stenosis.6 Studies that compare the prognostic values associated with higher and lower reading arm blood pressures are lacking.Differences in blood pressure readings between arms have been recognised for over a century. This raises the question of which arm blood pressure—the higher or the lower arm reading—should be routinely adopted for the diagnosis and management of hypertension? International guidelines advise checking blood pressure in both arms, when assessing people for hypertension, and adoption of the higher arm reading where differences exist; this is, however, based on expert consensus opinion alone.7 Individual-level data on systolic blood pressure in each arm allowed us to compare associations of the higher and lower arm systolic blood pressures with cardiovascular events, mortality, and hypertension diagnoses. Since treatment decisions for primary prevention of cardiovascular events are based on cardiovascular risk score thresholds, the impact of using the higher compared to the lower arm reading on reclassification across intervention thresholds was also examined.This study examined data from the Inter-arm Blood Pressure Difference—Individual Participant Data (INTERPRESS-IPD) Collaboration.8 In brief, systematic searches for prospective studies that recorded systolic blood pressure in both arms at recruitment were undertaken in Medline, Old Medline, Medline in process, Embase, and CINAHL databases until January 2017; unpublished data were also sought. Inclusion criteria were participants aged ≥18 years, recruited from community, primary care, or general clinic settings. Required primary outcomes were one or more of all-cause mortality, cardiovascular mortality, or fatal and nonfatal cardiovascular events.Data supporting the findings of this study are available from the corresponding author upon reasonable request. The INTERPRESS-IPD Collaboration was established to examine the independent associations of systolic inter-arm difference (IAD) with cardiovascular events, all-cause mortality, and cardiovascular mortality using individual participant data (IPD) meta-analyses and has been described in detail previously.Covidence . Disagreements were resolved by discussion between authors. Lead authors of potentially eligible studies were invited to participate, with a data sharing agreement and pro-forma request for data required. Nonresponders received 2 further invitations.Studies were independently selected by 2 authors using Anonymised datasets were cleaned individually and then combined to form a single dataset for the INTERPRESS-IPD Collaboration. Analyses were undertaken using .8 We compared models based on higher or lower arm blood pressures alone, then adjusted for age and sex. Finally, we compared a fully adjusted model previously derived and validated in the INTERPRESS-IPD dataset.7 This model adjusted for age, sex, current smoking status, ethnicity, total cholesterol and preexisting diagnoses of diabetes and/or hypertension. In sensitivity analyses, we added terms for preexisting cardiovascular disease and/or renal disease to the fully adjusted model to explore differences associated with higher preexisting risks of mortality and cardiovascular events.For these analyses, we used the first pair of left and right arm systolic blood pressure readings. Reclassification across clinically important blood pressure thresholds, according to choice of arm, was examined. Follow-up was truncated at 10 years to minimize censorship while permitting comparison of 10-year outcomes for risk scores. Baseline descriptive data were calculated for all participants with available data; no imputation of missing data was performed. Multilevel mixed-effects linear regression models, with a random effect for study, were used to compare values based on higher and lower arms. Due to low between-study heterogeneity within the dataset, which caused failure of previous 1-stage random effects flexible parametric models to converge, we used fixed effect 1-stage Cox proportional hazards modelling, stratified by study.10 The European Systematic Coronary Risk Evaluation (SCORE) score for cardiovascular mortality was also examined. However, it has recently been superceded by SCORE2; therefore, these results are summarised, but presented in full in the Supplemental Material.12 We calculated each score from the higher and lower arm blood pressures. We restricted analyses to participants within age ranges and ethnicities applicable to each risk score .11 We assessed classification across the American (10%), Canadian (15%), and European  guideline recommended 10-year cardiovascular risk thresholds.3For participants free of preexisting cardiovascular disease, we compared the atherosclerotic cardiovascular disease (ASCVD) scores and Framingham risk scores for 10-year risks of cardiovascular events.7 we also repeated the primary outcomes and risk score categorisation restricted to participants with a systolic IAD ≥ 10 mm Hg.More than 1 pair of blood pressure readings were available for a minority of participants. We conducted a sensitivity analysis of the primary outcomes, and risk score categorization, using the mean of all available pairs of recruitment blood pressures to account for repeated measures. To specifically address implications for clinically important inter-arm systolic blood pressure differences,13 This measures the information lost when approximating reality, thus qualitatively model fit is considered better with a lower AIC estimate indicating improved risk prediction. As a rule of thumb a difference in AIC (ΔAIC) between models <2 indicates that they are comparable, ΔAIC between 4 and 7 indicates considerably less support, and ΔAIC >10 essentially no support for the likelihood that the higher scoring model is better.14 To facilitate interpretation, raw differences in AIC between arm models were assigned probabilities using Akaike weights.15 Likelihood ratios were also compared, and concordance of models assessed with Harrell C statistic.16Regression model fit using higher or the lower reading arm blood pressures was compared using the Akaike information criterion (AIC).17 The protocol is registered at PROSPERO (record no: CRD42015031227).Data-sharing agreements were signed with each collaborating study lead author. All data were originally collected under cohort specific ethical approvals so further approval was not required for this IPD meta-analysis. This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses of IPD (PRISMA-IPD) statement.Table S1). Mean age was 60 (SD 12.5) years and 48% were female. The mean difference in systolic blood pressure between higher and lower reading arms was 6.6 mm Hg .The current analyses were conducted using 53 172 participants with blood pressure recorded in both arms from 23 cohorts in the INTERPRESS-IPD Collaboration  deaths; 1 cohort only collected a combined fatal and nonfatal outcome. For all-cause mortality, all models returned lower AIC values when based on the higher arm systolic blood pressure compared with the lower reading arm. The weighted difference in AICs was significant in all analyses, except for the sensitivity analysis with preexisting renal disease. Harrell’s P<0.001). However, model fit according to higher or lower arm did not differ .Mean European SCORE values for 18 017 eligible participants free of preexisting cardiovascular disease were significantly higher when calculated using the higher arm blood pressure in comparison to the lower arm reading . Reclassification, from below to above guideline-recommended risk thresholds within this subgroup, was observed for 9.3% of participants with Framingham scores and 4.5% with ASCVD scores  participants with a systolic IAD≥10 mm Hg, based on a single pair of readings. Higher compared with lower arm blood pressure measurement resulted in reclassification of 3644 (24.2%) of these participants’ systolic blood pressures from below to above 130 mm Hg, and 3784 (25.2%) from below to above 140 mm Hg , Gary Collins and Tom Fahey; Data management Group: Tim Eames and Sofia Sanabria; Administrators: Ellie Kingsland and Chloe Thomas.C.E. Clark proposed the current analyses; C.E. Clark, K. Boddy, F.C. Warren, V. Aboyans, L. Cloutier, R.J. McManus, A.C. Shore, R.S. Taylor, and J.L. Campbell were co-applicants on the INTERPRESS-IPD grant; SMcD and SM are researchers on the project. KB led public and patient involvement throughout the study. S.T.J. McDonagh, S.F. Moore, J.L. Campbell, A.C. Shore, and C.E. Clark undertook literature searches, C.E. Clark negotiated data access with collaborating authors. F.C. Warren led data cleaning, initial analyses, and supervised current analyses. C.E. Clark undertook analyses presented here. A.C. Shore, L. Cloutier, R.J. McManus, R.S. Taylor, and V. Aboyans advised on study conduct, analysis, and interpretation of findings. All authors contributed to development of the article and read and reviewed the final article. C.E. Clark has full access to data and acts as guarantor for this study.The INTERPRESS-IPD Collaboration was funded by the National institute for Health Research (NIHR) Research for Patient Benefit Programme (PB-PG-0215-36009). C.E. Clark is funded by NIHR School for Primary Care Research (SPCR) Grant (Ref: 512). S.T.J. McDonagh is funded by NIHR SPCR Fellowship. R.J. McManus is supported by NIHR Oxford CLAHRC. A.C. Shore is supported by NIHR Exeter Clinical Research Facility. J.-G. Wang supported by grants from National Natural Science Foundation of China (91639203 and 82070435), Beijing and Shanghai Commission of Science and Technology (19DZ2340200), Shanghai, China. Views expressed are those of the authors and not necessarily those of the NIHR, the NHS, the Department of Health and Social Care or the funding bodies acknowledged.C.E. Clark received honoraria from Bayer and ReCor medical for unrelated work. R.L. McClelland received monitors for research from Omron and works with them on a telemonitoring project. J.-G. Wang received lecture and consulting fees from Novartis, Omron, Servier and Takeda. No company had, or will have, any involvement in design, conduct or reporting of this study.