Podosphaerapannosa is the most widespread disease in global cut-rose production, as well as a major disease in garden and pot roses. In this study, the powdery mildew resistance of different wild rose varieties was evaluated. Rose varieties with high resistance and high sensitivity were used for cytological observation and transcriptome and expression profile analyses to study changes at the morphological and molecular levels during the interaction between Rosa multiflora and P. pannosa. There were significant differences in powdery mildew resistance among three R. multiflora plants; R. multiflora ‘13’ had high resistance, while R. multiflora ‘4’ and ‘1’ had high susceptibility. Cytological observations showed that in susceptible plants, 96 and 144 h after inoculation, hyphae were observed in infected leaves; hyphae infected the leaf tissue through the stoma of the lower epidermis, while papillae were formed on the upper epidermis of susceptible leaf tissue. Gene ontology enrichment analysis showed that the differentially expressed genes that were significantly enriched in biological process functions were related to the secondary metabolic process, the most significantly enriched cellular component function was cell wall, and the most significantly enriched molecular function was chitin binding. Changes in the transcript levels of important defense-related genes were analyzed. The results showed that chitinase may have played an important role in the interactions between resistant R. multiflora and P. pannosa. Jasmonic acid and ethylene (JA/ET) signaling pathways might be triggered in the interaction between susceptible R. multiflora and P. pannosa. In the resistant R. multiflora, the salicylic acid (SA) signaling pathway was induced earlier. Between susceptible plants and resistant plants, key phenylpropanoid pathway genes were induced and upregulated after P. pannosa inoculation, demonstrating that the phenylpropanoid pathway and secondary metabolites may play important and active roles in R. multiflora defense against powdery mildew infection.Powdery mildew disease caused by  Rosa sp.) have become increasingly important to the floriculture industry as ornamental plants [Podosphaera pannosa, is one of the most serious and widespread rose diseases [Roses -regulated defense response, and the ethylene (ET)-regulated defense response . Plants Rosa multiflora transcriptome in the process of infection with powdery mildew, and the genes with powdery mildew resistance were identified. The temporal and spatial expression levels of these genes were analyzed using real-time polymerase chain reaction (PCR). These results provide a framework for the study of the mechanisms underlying the resistance to powdery mildew in roses.However, relatively few studies have compared the morphological or transcription-level responses to rose powdery mildew between mildew-resistant and mildew-susceptible rose varieties. In this study, the responses to powdery mildew were compared between two different wild rose varieties. This study focused on characterizing changes at the morphological and molecular levels during the interaction between the wild rose varieties and powdery mildew. In order to examine the RNA levels of plant defense-related genes after powdery mildew infection, high-throughput sequencing was used to screen the Rosamultiflora plants, denoted ‘1’, ‘4’, and ‘13’, were collected from the Shennongjia Nature Reserve in central China’s Hubei province. They were planted in the rose germplasm resource nursery of Huazhong Agricultural University, Wuhan, China, for preservation. The leaves infected with powdery mildew of Rosa chinensis and R. multiflora were collected and placed into centrifugal tubes filled with fixed liquid :absolute ethanol (V) = 1:3). After 22 h of fixation, the fixation solution was poured out. The centrifugal nozzle was sealed with gauze and rinsed under running water for 4 h to fully remove the residual fixed liquid. Then, a pipette gun was used to remove the residual liquid from the centrifugal tube, and the preservation liquid (lactic acid (V):water (V):glycerol = 1:1:1) was injected into the centrifugal tube, then stored at 4 °C. Then, a temporary slide was set up for observation and stained with staining solution (0.2 g aniline blue was added to 100 mL distilled water and diluted with twice the volume of absolute ethanol before use) during observation.Three 4, 5 μL DNTPs, 1 μL cDNA, 1.5 μL each of upstream and downstream primers, 1 μL KOD plus high-fidelity enzyme, and sterile ddH2O to a total volume of 20 μL. The PCR reaction procedure was as follows: 94 °C for 5 min, 94 °C for 30 s, 56.0 °C for 30 s, and 68 °C for 1 min, repeated for 35 cycles, with a final step of 68 °C for 5 min. The PCR products were detected by agarose gel electrophoresis, and the target bands were recovered using the TaKaRa Mini BESTAgarose Gel DNA Extraction Kit  and connected with pMD18-T vector. The products were transformed into Escherichia coli, the blue and white spots were screened, and the white single colonies were selected for identification. According to the PCR results, the identified positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China, for sequencing. The sequences were compared on the NCBI website .The DNA of powdery mildew was extracted using the CTAB method. The primers used in this experiment were ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), which were used as universal primers for the ITS region of fungal ribosomes . The PCRR. multiflora collected from the Shennongjia Nature Reserve in central China’s Hubei province were employed in this research. From September 2018 to June 2019, the disease progression of three R. multiflora plants, denoted ‘1’, ‘4’, and ‘13’, was observed and recorded every 15 d in the field according to the grading standard shown in R. multiflora to powdery mildew was also determined based on microscopic observations. The grade standards used in the microscopic observation method are shown in P. pannosa that infected roses were employed as an inoculum. P. pannosa spores in a solution of 0.1% Tween 20 (the spore concentration was adjusted to 10−4–10−5·mL−1) were applied to whole plants using a sprayer. Five leaves were collected from each R. multiflora variety at 24 h and 96 h post-inoculation (hpi), then placed into centrifuge tubes containing stationary liquid. Temporary slides were set up for observation according to the above methods. The observations included the mycelial growth rate (24 hpi) and the mycelial coverage area and conidiophore formation grade (96 hpi). The experiment was repeated three times. The disease index = Σ(number of diseased leaves × disease grade)/ × most severe disease (grade), and resistance index = 1 − disease index. The average value of these two indexes was used as the final basis for evaluating the disease resistance of R. multiflora. The degree of disease resistance could be divided into five categories: immune (I), high resistance (HR), moderate resistance (MR), moderate sensitivity (MS), and high sensitivity (HS), with resistance indexes of 1.00, 0.80–1.00, 0.40–0.80, 0.20–0.40, and less than 0.20, respectively.The resistance of R. multiflora to powdery mildew, the cutting seedlings of R. multiflora ‘4’ and ‘13’ were inoculated with powdery mildew. Hard branches with good growth and without diseases and insect pests were selected to obtain cuttings, and one rooted cutting was grown in each pot. Young plants with three to five leaves (leaf age 20–35 d) that exhibited good growth without diseases and pests were used in the following experiments. No fungicide was used during the growth stage of the tested leaves. P. pannosa spores were employed as inoculum. The powdery mildew inoculation method is shown above 2.2. Each treatment included seven rooted cuttings of each line, with three replicates. Ten days after inoculation, according to the percentage of the total leaf area occupied by pathogens, grades 0–6 were used to describe the development of the disease. The evaluation criteria are shown in In order to further evaluate the resistance of R. multiflora ‘4’ was highly susceptible, while R. multiflora ‘13’ was highly resistant to powdery mildew. The stem segments with nodes of these two plants were washed under tap water for 30 min, surface-sterilized in 70% ethanol for 30 sec and 0.1% HgCl2 for 15 min, rinsed three times in sterile distilled water, dried, and then used as explants. The optimum medium for the in vitro growth and multiplication of R. multiflora from nodal segments was MS + 6-BA1.0 mg/L + NAA 0.01 mg/L + 30 g/L sucrose + 7.5 g/L agar. The explants were placed in an incubator at 23 °C (±2 °C), relative humidity 60%, with 1500–2000 lux light intensity until sampled.According to the research results shown in P. pannosa were employed as an inoculum. The powdery mildew inoculation method is shown above in (R. multiflora resistance to powdery mildew). Sterile water was applied in the non-inoculated treatments. For microscopic studies, the leaves of tissue-culture seedlings were sampled at 0, 12, 24, 48, 96, and 144 hpi. The leaves were gently washed and cut into small pieces (0.2 cm × 0.2 cm). The leaf pieces were fixed in FAA  standard fixative. The slices were obtained with a Leica RM2265 slicer , spread on the slides with the addition of distilled water, and then stained with 1% toluidine blue stain. The stain was rinsed off for about 3 min before baking the slices, and then semi-thin slices were made for the cytological observation of the host–powdery mildew interaction. The methods used followed those described by Li [Pathogen spores from Resistant and susceptible plants were separately inoculated with a spore suspension of powdery mildew when the seedlings had grown 6–10 leaves. Sterile water was used in the control treatment. Leaf samples were taken from the disease-susceptible and disease-resistant plants at 24 h and 96 h. Total RNA was extracted using an OminiPlant RNA kit  according to the manufacturer’s instructions, with an additional sonication step after the addition of RLT buffer (Qiagen). Strand-specific RNA libraries were constructed and sequenced on a HiSeq 2500 system (Illumina), according to the manufacturer’s instructions. The samples were analyzed using high-throughput transcription group and express spectrum sequencing by Shanghai Genergy Biotechnology Company . Bioinformatics analyses were conducted by Guangzhou Kidio Biotechnology Co., Ltd. .−5. KEGG pathway enrichment analysis of DTGs was performed using KOBAS. GO term enrichment was analyzed using the GOseq R package based on Wallenius non-central hyper-geometric distribution.To annotate the identified genes, the sequences were analyzed with various nucleotide and protein databases, including the NR (NCBI non-redundant protein), NT (NCBI non-redundant transcript), Swiss-Prot, KEGG (Kyoto Encyclopedia of Genes and Genomes), KOG (eukaryotic Ortholog Groups), and GO (Gene Ontology) libraries. The BLASTX algorithm was used with a significance threshold of E-value ≤ 10non-race-specific disease resistance (NDR1), enhanced disease susceptibility (EDS1), senescence-associated gene 101 (SAG101), ethylene-responsive-element (ERF10), kinase, pathogenesis related protein 1 (PR1), PR5, callose synthase (CS), chitinase, catalase (CAT), peroxidase (POD), chorismate mutase (CM), phenylalanine ammonialyase (PAL), phospholipase D (PLD), lipoxygenase (LOX), allene oxide cyclase (AOC), adenosythomocysteinase (Ade), ACC (1-aminocyclopropane-1-carboxylic) synthase (ACS), ACC oxidase (ACO), 4-coumarate coenzyme A ligase (4CL), chalcone synthase (CHS), and cinnamyl alcohol dehydrogenase (CAD)) were screened for gene expression analysis. The expression levels of candidate genes . Total RNA was isolated from leaves with the EASY spin rapid RNA extraction kit , following the manufacturer’s instructions. cDNA was produced by reverse transcription using PrimeScript RT reagent kits with gDNA Eraser . GADPH primers .As shown in R. multiflora ‘1’, ‘4’, and ‘13’ is shown in R. multiflora ‘1’, ‘4’, and ‘13’ were recorded, and the disease grade index and resistance index were calculated. The results showed that there were significant differences in powdery mildew resistance among the three plants; R. multiflora ‘13’ had high resistance, and R. multiflora ‘4’ and ‘1’ had high susceptibility  volcanic distribution map  functions, 54 cellular component (CC) functions, and 112 molecular function (MF) functions were significantly enriched in the differentially expressed genes in resistant plants invaded by P. pannosa for 96 h, 513 BP functions, 66 CC functions, and 161 MF functions were significantly enriched in the differentially expressed genes. As shown in P. pannosa for 96 h. As shown in When the resistant plants were invaded by P. pannosa for 24 h, and 254 identical BP functions of differentially expressed genes were found in susceptible and resistant plants when invaded by powdery mildew for 96 h . In contrast, chitinase was downregulated 1.5-fold in the susceptible plants at 12 hpi; the chitinase expression level then steadily increased from 24 to 96 hpi, decreasing again at 144 hpi. In resistant plants, chitinase was significantly upregulated 0–48 hpi (2.86-fold); the chitinase transcript levels in resistant plants rose more quickly, and to higher maxima, than levels in susceptible plants  were affected by powdery mildew inoculation  and slower upregulation in the resistant plants (96 hpi). LOX was upregulated in both the resistant and the susceptible plants, although the expression levels of LOX were greater in the resistant plants than in the susceptible plants at all time points.The expression of nfection . In the Ade was rapidly upregulated in the susceptible plants (at 24 hpi), and more slowly in the resistant plants (at 96 hpi)  . ACS wasP. pannosa infection. Second-generation high-throughput sequencing was used to fully screen the plant transcriptomes during the R. multiflora–powdery mildew interaction in order to identify the genes associated with powdery mildew resistance. The temporal and spatial expression levels of some genes important for pathogen defense were analyzed with real-time PCR. Obvious morphological differences were identified between the susceptible and resistant plants, as well as time-dependent differences in resistance-related gene expression patterns.This study characterized the morphological and molecular responses of two wild rose varieties (powdery mildew-susceptible and powdery mildew-resistant) to R. multiflora at 96 hpi in the susceptible plants and at 144 hpi in the resistant plants. More callose was deposited in the resistant plants compared to the susceptible plants. In addition, in this study, the differentially expressed genes that were significantly enriched in BP functions were related to the secondary metabolic process, the most significantly enriched CC function was cell wall, and the most significantly enriched MF function was chitin binding in the resistant plants compared to the susceptible plants. This was consistent with research results on the defense response of Camellia oleifera against the anthracnose fungal disease [In plants, the cell wall is a structural barrier of passive defense that also acts as an active and dynamic defense system. The active defense responses of plant cell walls include callose deposition, phenol accumulation, and lignin synthesis . Therefo disease . AltogetPAL expression peaked at 12 hpi in resistant plants, and PAL was expressed earlier and more strongly in resistant plants compared to susceptible plants.The phenylpropanoid metabolic pathway has a close relationship with plant disease resistance . The uprCAD upregulation increased the resistance of Arabidopsis thaliana to bacterial spot disease. In the present study, the key genes in the phenylpropanoid synthesis pathway were upregulated in both susceptible and resistant plants, which indicated that different phenylpropanoid pathways were activated after induction. As POD is required for the polymerization reaction at the final step of lignin synthesis, this enzyme is closely correlated with plant disease resistance [chorismate mutase, PAL, 4-CL, CAD, and POD) were significantly upregulated after the inoculation of leaves with P. pannosa. The expression levels of these genes were higher in the resistant plants than in the susceptible plants. Furthermore, these genes were more quickly and strongly upregulated in resistant plants compared to susceptible plants. This suggests that plant cell wall-mediated disease resistance plays a crucial role in the defense against powdery mildew in roses, and that the upregulation of key genes in the lignin synthesis pathway is one of the most important factors conferring disease resistance to disease-resistant roses. In addition, secondary metabolites in the phenylpropanoid pathway play an important role in the resistance of R. multiflora to powdery mildew.After pathogenic bacterial infection, the repair of the cell wall is performed through lignification of the plant cell walls, so as to improve the mechanical strength of the cell walls and the plant’s disease resistance . Bhuiyansistance . Here, tPR1 and PR5) [PR4 and PR5 [PR1 is upregulated in response to biotrophic pathogens [P. pannosa, the expression levels of NPR1, EDS1, SAG101, PR1, PR5, CAT, chorismate mutase, and PAL increased rapidly, resulting in the rapid synthesis of SA and increasing the acquired resistance of the plant at the early stages of pathogen infection. This was consistent with a previous study on downy mildew in pearl millet [p < 0.05) and significantly more strongly (p < 0.05) in resistant plants.A previous study showed that early morphological resistance was accompanied by a pronounced transcriptional response, including the upregulation of the defense-related genes associated with the SA-, JA-, and ET-mediated defense signal transduction pathways , which aand PR5) . The accand PR5) . Endogenand PR5) . SA playand PR5) ,51, incl and PR5 ,53. In pathogens . After il millet . Genes aR. multiflora–powdery mildew interaction. The SA pathway was activated earlier and more effectively in the resistant plants, while many genes related to the JA and ET defense pathways were upregulated in susceptible plants (PLD, LOX, and AOC are key enzymes in the JA synthesis pathway ,56. Adene plants .P. pannosa fungal penetration, global gene expression, defense-associated genes, and signaling pathway genes between resistant and susceptible varieties of R. multiflora. Similar resistance pathways were activated by both resistant and susceptible R. multiflora varieties. However, time-course experiments indicated that individual genes were expressed earlier in resistant plants and later in susceptible plants. Furthermore, callose played an important role in the defense reactions of the resistant plants. Both the timing of callose accumulation and the total amount of callose deposited appeared to be critical for optimal disease resistance. The findings of this study increase the understanding of the complex mechanisms underlying the response of R. multiflora to powdery mildew and provide a framework for future in-depth characterizations of the pathways and key constituents imparting pathogen resistance.In summary, this study compared


Ehrlichia and Anaplasma in coati’s blood samples in Midwestern Brazil. Twenty-five samples (25/165—15.1%) were positive in the screening PCR based on the dsb gene of Ehrlichia spp. and were characterized using 16S rRNA, sodB, groEL, and gltA genes and the 23S-5S intergenic space region (ITS). Phylogenetic analyses based on all six molecular markers positioned the sequences into a new clade, with a common origin of Ehrlichia ruminantium. Haplotype analyses of 16S RNA sequences revealed the presence of two distinct Ehrlichia genotypes. Six samples  were positive in the screening nPCR for the 16S rRNA gene of Anaplasma spp. and were submitted to an additional PCR targeting the ITS for molecular characterization. Phylogenetic analyses based on both 16S rRNA gene and ITS positioned the Anaplasma sp. detected in the present study in a large clade with other Anaplasma sp. previously detected in ticks and wild animals and in a clade with ‘Candidatus Anaplasma brasiliensis’, respectively. Based on distinct molecular markers, the present work described a putative novel Anaplasmataceae agent, namely ‘Candidatus Ehrlichia dumleri’, and Anaplasma sp. closely related to the previously described ‘Candidatus Anaplasma brasiliensis’.The Anaplasmataceae family encompasses obligate intracellular α-proteobacteria of human and veterinary medicine importance. This study performed multi-locus sequencing to characterize  Nasua nasua from Brazil, can easily adapt to peri-urban areas   24] to oer 2022)  was useder 2022) . The beson (AIC) ,28. The on (AIC)  via the software .Additionally, an analysis of nucleotide polymorphisms of the sequences obtained in the present study was performed. The number of haplotypes, haplotype diversity (Hd), and nucleotide diversity (Pi) were determined using the program DnaSP 5, version 5.10.01 . Also, tIn total, 165 blood samples were collected (63 in PEP and 102 in VBA). In PEP, blood samples were obtained from 47 different individuals . In VBA, samples were obtained from 57 different individuals  . All recgapdh endogenous gene. Out of 165 coatis’ blood samples, 25 (15.1%) were positive in the screening PCR for the dsb gene of Ehrlichia spp. [4/63 samples—6.3% from PEP area (from 4 different coatis) and 21/102 samples—20.6% from VBA (obtained from 16 different coatis). No recaptured animal showed positivity in more than one collection time at PEP, while coatis from VBA showed positivity in more than one recapture (All DNA samples obtained from coati blood (n = 165) were positive in the PCR targeting the ecapture .Transiently positive PCR results were detected in three animals from VBA: VBA03 was positive in four out of six samplings; VBA21 was positive in 2/6 samplings, and VBA44 in 3/3 samplings. No animal from PEP showed PCR positive results for more than one sampling . Animal dsb-based PCR screening for Ehrlichia spp., three were properly sequenced, nine were positive in the PCR targeting the near-complete 16S rRNA gene, one for groEL gene, three for sodB gene, two for 23S–5S ITS, and six for gltA gene. Results from Blast analyses, including sequence size, query coverage, E-value, and % of identity, are shown in Out of 25 positive samples in the dsb gene  grouped the three sequenced samples in a new clade separated (100% posterior probability) from other Ehrlichia genotypes previously detected in wild animals from Brazil and Amblyomma ticks from Brazil and Argentina  and TIM2+I evolutionary model and based on the rgentina . The genrgentina .Ehrlichia genotypes detected in coatis from PEP and ‘Candidatus Ehrlichia occidentalis’ and Ehrlichia sp. Eira barbara ranged from 1.2 to 1.7%  and HKY+I+G evolutionary model and based on the 16S rRNA gene  grouped the sequences detected in the present study in a new large clade (92% posterior probability) . The gen to 1.7% .groEL gene    on the y model) , sodB gey model) , and 23Sy model)  grouped  markers .gltA gene  showed similar topology to 16S rRNA-based phylogenetic inference, positioning sequences from the present study in a new large clade (92% posterior probability) divided into two sub-clades  and TN+G4 evolutionary model and based on the b-clades . The genb-clades .Ehrlichia sp. sub-clades detected in the present study (PEP x VBA), median joining network (MJN) and haplotype analyses with fourteen 16S rRNA sequences that clustered together or in a clade near those sequences detected in coatis  were represented by the same haplotype (haplotype #4). On the other hand, sequences detected in PEP clustered in two different genotypes (haplotype #1 and #2), also apart from haplotype 3, which was represented by ‘Ca. Ehrlichia occidentalis’ (KY425523). Sequences positioned in a clade near sequences detected in coatis  in the BI were represented by different genotypes   . The gen and #7) .Anaplasma spp. [1/63 samples—1.6% from PEP area and 5/102 samples—4.9% from VBA (obtained from five different coatis). No recaptured animal showed positivity in more than one sampling time. Co-positivity for Ehrlichia spp. and Anaplasma spp. was not found in the sampled animals. Four samples were properly sequenced, and Blast analyses showed 100% of query coverage, 0.0 E-value, and 100% of identity with Anaplasma spp. sequences previously detected in ticks and wild animals from Brazil . Only one sample amplified for the 23S–5S ITS and Blast results showed 100% of query coverage, 0.0 E-value, and 100% of identity with ‘Candidatus Anaplasma brasiliensis’. All six samples failed to amplify the groEL gene fragment.Out of 165 coati blood samples, six (3.6%) were positive in the screening nPCR for the 16S rRNA gene of Anaplasma sp. detected in ticks and wild animals from Brazil   and TPM2+I+G evolutionary model and based on the 16S rRNA gene  grouped the sequences detected in the present study in a large clade (100% posterior probability) with other sequences of Y499183) . Phylogeliensis’ .Ehrlichia spp. and Anaplasma spp. in different orders of wild animals [Previous studies conducted in Brazil indicated the occurrence of putative new genotypes of  animals ,40,41,42Ehrlichia DNA in 25 (15.1%) blood samples from coatis using a dsb-based PCR screening. Such findings represent a higher percentage of positivity when compared with two previous studies performed in Brazil. In the Brazilian Pantanal, a previous study reported the occurrence of antibodies to E. canis in one coati  (titer of 640) as well as the molecular detection of a Ehrlichia 16S rDNA genotype phylogenetically related to multiple Ehrlichia spp. previously detected in ticks and wild rodents from Brazil [Ehrlichia/Anaplasma 16S rDNA was detected in 1/18 coati blood sampled in Iguazu National Park, southern Brazil, albeit without sequencing [Amblyomma sp. larvae, Amblyomma dubitatum, and Amblyomma sculptum nymphs, and A. sculptum and Amblyomma ovale adults [Ehrlichia sp. is not known yet, the studied biome and, consequently, tick diversity, together with the higher number of sampled animals, might have influenced the higher percentage of positive animals in the Cerrado biome. It is also worth mentioning that the applied technique might have influenced the percentage of positive animals. For instance, nested PCR is highly sensitive, and in the case of E. canis detection, it is capable of detecting 0.2 pg of purified DNA, while a conventional PCR can only detect values above 20 pg [groEL gene and validated for the main Ehrlichia and Anaplasma species of medical and veterinary importance [Ehrlichia and Anaplasma genotypes described in mammals from the Superorder Xenarthra in Brazil [Herein, we were able to detect m Brazil . Moreovequencing . In the quencing  and 18 bquencing ). This hquencing  took plaquencing  was carr coati 1/1, 3.2%  and 21/102 samples—20.6% from VBA (obtained from 16 different coatis). Although sampling areas are in Campo Grande city, Cerrado biome, they present very different characteristics. PEP is a preserved forest area, being an important refuge for wildlife, where many species of mammals can be found, such as ring-tailed coatis, anteaters, capybaras, opossums, bats, and also many species of birds and reptiles ,49,50,51Ehrlichia sp. in coati blood samples. Three animals sampled at VBA were PCR-positive at different times of sampling. All three animals were positive in March 2019 and two animals were in April 2019. Due to the small number of recaptured/positive animals, and since the captures were performed by chance on different days, it is not possible to infer any type of correlation according to the sampling period and PCR positivity. Besides that, the results found herein are influenced by the limit of the detection of the PCR protocols used. Nevertheless, these data indicate that N. nasua may play a role in the maintenance of this new Anaplasmataceae agent in the wild environment.Interestingly, the present study shows, for the first time, transient positivity for Ehrlichia sp. detected from coatis in the present study in a new clade, originated as a sister clade to E. ruminantium. It is noteworthy that all phylogenetic inferences based on different molecular markers showed the same topology, with the establishment of a new distinct clade. Interestingly, two branches were observed on the phylogenetic inferences based on 16S rRNA and gltA genes and 23S-5S intergenic region, according to the sampling spot (PEP and VBA). These findings may indicate the occurrence of distinct genotypes of a unique putative novel Anaplasmataceae agent, which is proposed here as ‘Candidatus Ehrlichia dumleri’, in honor of Dr. John Stephen Dumler and his great contribution to the knowledge of this group of proteobacteria. Indeed, while the genetic divergence between these two genotypes ranged from 0.010 to 0.013, values ranging from 1.2 to 1.3% were found when comparing the newly described genotypes and ‘Candidatus Ehrlichia occidentalis’. Moreover, the haplotype analyses based on the 16S rRNA sequences corroborate the presence of two distinct genotypes, also according to the sampling spot.Phylogenetic analyses positioned Candidatus Ehrlichia’ have been proposed and detected in biological samples from wild animals by using different molecular target genes. For instance, ‘Candidatus Ehrlichia regneryi’ was detected in spleen samples of dromedary camels (Camelus dromedarius) in Saudi Arabia using two genetic markers (16SrRNA and groEL) [Candidatus Ehrlichia pampeana’ was detected in Haemaphysalis juxtakochi and in gray Brocket Deer (Mazama gouazoubira) from Uruguay using three loci  [Candidatus Ehrlichia hydrochoerus’ was detected in blood samples from capybaras (Hydrochoerus hydrochaeris) from Brazil, using four loci  [dsb, sodB, groEL, gltA genes and the 23S-5S intergenic space region (ITS). Phylogenetic analyses based on all six molecular markers showed the same topology, supporting the description of the new ‘Candidatus Ehrlichia dumleri’ presented herein.Recently, several ‘d groEL) . ‘Candidd groEL) . ‘Candidnd sodB) . In the Anaplasma spp. (6/165—3.6%) by using a screening nPCR based on the 16S rRNA gene. Sousa et al. [Anaplasma spp. in 22% (7/31) coatis sampled in the Brazilian Pantanal, using an nPCR also based on the 16S rRNA gene. Phylogenetic inferences grouped the sequences detected in coatis from Pantanal in two different clades: one clade related to A. phagocytophilum and another one clustering with Anaplasma sp. previously detected in different wild animal species, including rodents, marsupials, and carnivores [Anaplasma sp. closely related to ‘Candidatus Anaplasma brasiliensis’, which was previously detected in anteaters in the state of São Paulo [Anaplasma sp. detected in other wild animals from Brazil. Recently, an unusual case of human anaplasmosis caused by ‘Candidatus Anaplasma sparouinense’, which was genetically related to ‘Candidatus Anaplasma amazonensis’ was recently described in sloths from the Brazilian Amazon [Anaplasma to humans.The present work describes a low percentage of positive animals for a et al.  detectedrnivores . The preão Paulo , and Anan Amazon , was repEhrlichia spp. was found in the sampled coatis when compared to that one found for Anaplasma spp. Phylogenetic analyses based on six molecular markers  positioned the novel Ehrlichia sp. detected in the sampled coatis into a new clade and supported the description of ‘Candidatus Ehrlichia dumleri’, a putative novel Anaplasmataceae species that appeared as a sister clade to E. ruminantium. Phylogenetic analyses based on both 16S rRNA gene and 23S–5S intergenic region grouped the Anaplasma sp. detected in coatis with the previously described ‘Candidatus Anaplasma brasiliensis’.Anaplasmataceae agents were detected in blood samples from free-ranging coatis sampled in Midwestern Brazil. A higher positivity for


Observational studies have demonstrated associations between gout and hypertension, but whether they are causal remains unclear. Our work aims to assess the causal relationship between gout and hypertension.We obtained genetic information from the Taiwan Biobank, including 88,347 participants and 686,439 single-nucleotide polymorphisms (SNPs). A novel model of Mendelian randomisation (MR) with coarsened exposures was used to examine the causality between the liability of gout on hypertension and vice versa, using 4 SNPs associated with gout and 10 SNPs associated with hypertension after removal of SNPs associated with measured confounders. The binary exposure (gout/hypertension) can be considered a coarsened approximation of a latent continuous trait. The inverse-variance weighted (IVW) and polygenic risk score (PRS) methods were used to estimate effect size. The MR analysis with coarsened exposures was performed with and without adjustments for covariates.p = 0.0077) and the PRS method . The result of causality was the same before and after involving measured confounders. However, there was no causal effect of the liability of hypertension on gout.Of the 88,347 participants, 3253 (3.68%) had gout and 11,948 (13.52%) had hypertension . After adjusting to measured confounders, MR analysis with coarsened exposures showed a significant positive causal effect of the liability of gout on hypertension in both the IVW method (relative risk [RR], 1.10; 95% confidence interval [CI], 1.03–1.19; In this study, we showed that the liability of gout has a causal effect on hypertension, but the liability of hypertension does not have a causal effect on gout. Adequate management of gout may reduce the risk of developing hypertension.The online version contains supplementary material available at 10.1186/s13075-022-02933-4. Gout is a common inflammatory arthritis caused by the deposition of monosodium urate around the joints due to persistent elevation of serum urate (uric acid) levels above the saturation point for crystal formation. Gout has an incidence of 2.74 per 1000 person-years and is linked to increased risks of mortality and multiple comorbidities . MultiplRandomised controlled trials are the “gold standard” for inferring causal effects in medical research because random group assignments can minimise confounding bias; however, this may be expensive and not easy to implement. Mendelian randomisation (MR) mimics the random group assignment process by using randomly distributed single-nucleotide polymorphisms (SNPs) in genetic data as instrumental variables (IV) for group assignment –13. BecaNotably, the MR study design is used primarily for continuous exposures such as urate level and BMI, and its application to dichotomous variables may lead to erroneous measurement of effect size . TherefoThe study was based on genetic and clinical data from the Taiwan Biobank, which was established in 2012. The participants are adult Taiwan residents greater than 20 years of age. The Taiwan Biobank prospectively enrolls individuals from communities across Taiwan, with 142,882 participants as of 28 February 2021. All participants attended 1 of 39 enrollment centers; at these centers, each participant completed a series of genetic, physical, sociodemographic, and medical assessments. A standardised questionnaire was completed by each participant to record past medical history, medication, family history, and home environmental factors. Additionally, the Taiwan Biobank includes a physical examination and laboratory tests, including blood and urine tests performed at enrollment and follow-up visits, which provide sufficient variables for covariate examination. Another benefit of the Taiwan Biobank is its relatively homogenous population group compared to other large biobanks .Genotyping was based on the C2-58 Axiom Genome-Wide TWB 2.0 Array containing 686,439 SNPs, specifically designed for people of Taiwanese descent . The data were anonymized, and no tracking was possible. Information regarding each participant’s personal and clinical history was collected by questionnaires at enrollment, then every 2 years thereafter. We obtained data concerning 88,347 participants. We also obtained various data associated with gout and hypertension, including each participant’s age, sex, BMI, and serum creatinine levels. In Taiwan, the diagnosis of gout is based on clinical observation of rapidly developing monoarticular arthritis, and the Taiwan Biobank then used questionnaires to ask whether the participants have a history of gouty arthritis.http://www.cog-genomics.org/plink/1.9/) [p-value for the Hardy–Weinberg equilibrium test of < 1.0 × 10−6 for controls, and a minor allele frequency of < 0.01. The Hardy–Weinberg equilibrium test offers additional support to confirm whether SNPs were randomly distributed, in a manner analogous to randomised controlled trials. For the GWAS procedure, we selected significant SNPs associated with gout or hypertension at a threshold p-value of < 5.0 × 10−8 in logistic regression analyses in three scenarios: unadjusted, adjusted to age and sex, and adjusted to age, sex, and BMI. For evaluation of linkage disequilibrium coefficients and haplotype association statistics, we used Haploview version 4.2 software  and LDLinkR [http://www.phenoscanner.medschl.cam.ac.uk/) [A standard quality control (QC) procedure and genome-wide association studies (GWASs) were conducted by PLINK 1.9 (nk/1.9/) . For the LDLinkR , and the.ac.uk/) , 26 to eMR is an analytical method that manages the causal structure that involves unmeasured confounders by using genetic variants as IVs to investigate causal effects from the exposure to the outcome. There are three core assumptions in conventional MR for genetic variants to be valid IVs: relevance, the genetic variants are predictive of the exposure; exogeneity, the genetic variants are independent of any unmeasured confounders of the exposure-outcome association under the condition on all measured confounders; and exclusion restriction, the genetic variants are conditionally independent of the outcome, considering the exposure and all confounders.Conventional MR analysis requires that the effects of exposure on the outcome and the genetic variants’ associations with the risk factors/outcome be linear without effect modification. The MR methods supporting binary or dichotomous exposure have not been clarified, despite IV analysis for continuous exposure/outcome and potential strategies to overcome the linearity assumption . The appFurthermore, of the five SNPs selected as IVs for gout, one rs671) may have pleiotropic effects on HTN, but in contrast, there were no such SNPs among the ten SNPs selected for hypertension for this study. SNP information are found in the “SNP selection” section and Supplementary Table  may haveMendelianRandomization for analysis [p-value obtained in LRT does not guarantee the strength of the genetic share and valid IVs; it increases the probability that the genetic share is composed of valid IVs. Moreover, to avoid biased results, we also considered another approach, using the PRS method to combine multiple genetic variants into a single score is a common approach for increasing IV strength [To estimate the causal effect of the latent variable of exposure on the outcome, we considered two standard methods, the IVW method and the PRS method  before and 892.41 (p < 2.2 × 10−16) after adjusting to measured confounders, supporting the credibility of the genetic share composed of the selected SNPs. The Cochran’s Q test for gout-associated SNPs before adjusting to measured confounders was 3.42 (p = 0.3314), suggesting that the effects of gout-associated SNPs were homogenous, and the fixed-effect IVW method is more suitable than the random-effects method. At the estimated p = 0.033) for the fixed-effect IVW method and 1.08  for the PRS method. After adjustments for measured confounders, Cochran’s Q test continued to support homogenous effects of the selected SNPs ; the results remained significant for the fixed-effect IVW method  and the PRS method . Collectively, the results support the causality of the liability of gout on hypertension.The results of MR analysis with coarsened exposures for gout on hypertension are presented in Fig. 1p < 2.2 × 10−16) and 514.63 (p < 2.2 × 10−16) before and after adjustment to measured confounders, supporting the credibility of the genetic share being composed of the selected valid IVs. The estimated values of p = 0.1194), suggesting homogenous effects of SNPs; hence, the fixed-effect method was applied. After adjustments for measured confounders, Cochran’s Q test value remained nonsignificant . At the specified The results of the MR analysis with coarsened exposures for hypertension on gout are shown in Fig. p < 0.05, which resulted in only one SNP rs671 associated with gout have been excluded in conducting an MR analysis with coarsened exposures to evaluate the causal effects of the liability of gout on hypertension, including measured confounders , and the Cochran’s Q test was 5.22 , supporting the application of the fixed-effect method. The results remained significant after adjustment for measured confounders for the fixed-effect IVW method  and the PRS method . The results are shown in Fig. To evaluate modified assumption 5 of modified MR with coarsened exposure (the genetic variants should be independent with measured confounder) and multicollinearity, we performed the chi-square test of independence and Spearman correlation tests between selected SNPs and measured confounders. Most of the selected SNPs were independent of all measured confounders, and only a few SNPs were associated with creatinine  and [MendelianRandomization). R version(4.0.3), LDLinkR (https://github.com/CBIIT/LDlinkR) [http://www.cog-genomics.org/plink/1.9/) [http://www.phenoscanner.medschl.cam.ac.uk/) [The code for modified MR with coarsened exposure in this study is mainly based on  (https:/ion) and  (R packaLDlinkR) , PLINK 1nk/1.9/) , and Phe.ac.uk/) , 26. TheAdditional file 1: Supplementary methods: Assumptions and mathematical proof for the modified MR with coarsened exposure.Supplementary Table 1.  Detailed information on SNPs associated with gout and hypertension contains SNPs associated with gout, heterogeneous SNPs associated with gout, and SNPs associated with HTN. Supplementary Table 2.  P-values for the association between SNPs and measured confounders. Supplementary Table 3. Comparing no remove and remove SNPs associated with exposure correlated with measured confounders. Supplementary Figure 1: Flow chart for GWAS procedure of SNP selection under three different settings. Supplementary Figure 2. The results of MR analysis without coarsened exposures after removing SNPs associated without measured confounders. Supplementary Code: Modified MR with coarsened exposures.


Cuscuta japonica Choisy (Japanese dodder) is a parasitic weed that damages many plants and affects agricultural production. The haustorium of C. japonica plays a key role during parasitism in host plants; in contrast, some non-host plants effectively inhibit its formation. However, the metabolic differences between normal dodder in host plants and dodder inhibition in non-host plants are largely unknown. Here, we utilized an integrative analysis of transcriptomes and metabolomes to compare the differential regulatory mechanisms between C. japonica interacting with the host plant Ficus microcarpa and the non-host plant Mangifera indica.C. japonica in host and non-host plants.After parasitization for 24 h and 72 h, the differentially abundant metabolites between these two treatments were enriched in pathways associated with α-linolenic acid metabolism, linoleic acid metabolism, phenylpropanoid biosynthesis, and pyrimidine metabolism. At the transcriptome level, the flavor biosynthesis pathway was significantly enriched at 24 h, whereas the plant–pathogen interaction, arginine and proline metabolism, and MARK signaling-plant pathways were significantly enriched at 72 h, based on the differentially expressed genes between these two treatments. Subsequent temporal analyses identified multiple genes and metabolites that showed different trends in dodder interactions between the host and non-host plants. In particular, the phenylpropanoid biosynthesis pathway showed significant differential regulation between C. japonica parasitism.These results provide insights into the metabolic mechanisms of dodder–host interactions, which will facilitate future plant protection from The online version contains supplementary material available at 10.1186/s12870-022-03773-9. Cuscuta japonica Choisy, commonly known as the Japanese dodder, is an annual or perennial holoparasitic plant that mainly endangers woody plants, such as tropical and subtropical fruit trees, economic trees, and ornamental trees [Cuscuta japonica is an all-parasitic seed plant that absorbs water, nutrients, and other macromolecules from the host plant through its haustorium, which is a special absorption organ [al trees . The plaon organ . The hauon organ , 60. Wheon organ . Some chon organ ; despiteon organ . Thus, sCuscuta has a broad host spectrum and several possible plant “victims.” However, few plants have defense mechanisms that can successfully prevent Cuscuta infestation [C. japonica have mainly focused on the stress response and resistance of resistant hosts. Werner et al. [Mangifera indica successfully mounted a post-attachment resistance response to dodder. In summary, in direct contact with haustoria, epidermal tomato cells elongate and die following a hypersensitive response, whereas a secondarily modified tissue is formed in the hypodermis to protect against haustoria penetration [estation . At presr et al.  found thr et al. . Hegenaur et al.  found thr et al. . Masangar et al.  found thetration .TvPirin, which responds to haustoria-inducing factors, was identified in Triphysaria versicolor through transcriptome research. Interference with TvPirin using RNAi significantly reduced haustorium formation [Because of the vigorous metabolism of haustorium tissue cells, the carbon and nitrogen compounds and organic acids entering the haustorium from the host plant can be processed into more easily absorbed substances for the use of parasitic plants , and theormation . Moreoveormation  found thormation .C. japonica parasitizations on host and non-host plants to investigate the regulatory mechanism of the interaction between C. japonica and host plants. After parasitization, the differentially abundant metabolites (DAMs) and differentially expressed genes (DEGs) of C. japonica were enriched in pathways associated with fatty acid metabolism, phenylpropanoid biosynthesis, and pyrimidine metabolism. In particular, the phenylpropanoid biosynthesis pathway showed significant differential regulation between C. japonica in host and non-host plants. These results provide insights into the metabolic mechanisms of dodder–host interactions.We hypothesized that non-host plants might be resistant to dodder by interfering with the normal metabolism of dodder during haustoria formation. However, most studies have explored the causes of haustorial inhibition based on macro-tissue structure or physiological changes, without clear targets and mechanisms. With the development of omics technologies, the metabolome and transcriptome have become important research tools for investigating plant–plant interactions , 32. In C. japonica lacks leaves and roots, it can grow several stems to twist around the host and develop haustoria for host attachment and intrusion, which can extract inorganic compounds, nutrients, and water from the host. Cuscuta japonica exhibited distinct morphological features after attachment to F. microcarpa ‘Golden Leaves’ (host) and M. indica (non-host) plants. We observed that although the seedlings of dodder warped around M. indica, the haustorial roots did not fully penetrate the bark of M. indica  to obtain the metabolome profile of C. japonica  than at 0 and 24 haa.To evaluate the metabolome differences between groups and variation status among the three replicates, we performed principal component analysis (PCA) of all samples. The PCA plot showed that initial seedling samples clearly distinguished between samples after 24 h and 72 h in the host (HTD_24h and HTD_72h) and non-host (MTD_24h and MTD_72h) plants, indicating large differences in metabolite concentrations in C. japonica attached to the two host types, we compared the abundance of metabolites at 24 h and 72 h. The abundance of phenolic acids in the host plants was lower than that in the non-host plants at 24 h. At 72 h, we found that nucleotides and derivatives, amino acids and derivatives, and phenolic acids were the top three differential classes based on their abundances  pathway enrichment analysis. α-linolenic acid metabolism, linoleic acid metabolism, and phenylpropanoid biosynthesis pathways were significantly enriched, containing multiple upregulated DAMs in MTD at 24 h compared with HTD at 24 h , arginine and proline metabolism (ko00330), and MARK signaling pathway plant (ko04016), were identified by KEGG analysis of HTD versus MTD at 72 h . However, few DEGs and no DAMs were found between the HTD and MTD in this pathway.To assess gene expression patterns during haustoria development, 12 919 genes in the HTD and 12 900 genes in the MTD were separately clustered into eight profiles, in which profiles 0, 1, and 7 were common among the two types of samples significantly enriched by genes Fig. S. The majP = 0.0001, chi-square test). Our non-targeted metabolome analysis indicated the highest abundance of p-coumaryl alcohol and coniferyl alcohol in HTD at 72 haa, whereas the accumulation of ferulic acid, sinapaldehyde and 1-O-sinapoyl-D-glucose gradually increased and peaked at 72 haa. Moreover, we observed that the gene expression patterns of DEGs in HTD and MTD were similar to those of the metabolites. The levels of cinnamoyl-CoA reductase, phenylalanine ammonia-lyase, caffeoyl-CoA O-methyltransferase, and peroxidase in HTD were significantly high in the inoculated stems at 24 and 72 ha. The peak expression value of cinnamyl alcohol dehydrogenase appeared in the MTD at 72 haa. These results suggest that DAMs and DEGs present various regulatory trends based on hosts inoculated with C. japonica.Phenylpropanoids play an important role in plant responses to biotic and abiotic stimuli . They meC. japonica are identical to those of autotrophic plant roots, indicating that the evolution of C. japonica may be related to changes in the expression of genes involved in autotrophic plant root development [C. japonica were wrapped around young mango plants, although papillary protrusions (superior haustoria) could be formed at the contact points, endogenous haustoria, where nutrients or water could be transferred, failed to form. In our study, we also observed that the dodder stem became black and eventually died after haustorial connection with non-hosts, in contrast to the green color of dodder stems on binding with host plants, suggesting that nutrition compounds or water could not be transferred from M. indica (non-host) to dodder  and downstream key gene acyl-CoA oxidase (ACX) decreased, reducing the consumption of linolenic acid in fatty acid metabolism [LOX is a key gene in α-linolenic acid metabolism and regulates the oxidation of α-linolenic acid to form fatty acid hydroperoxide, which is the first step in the conversion of linolenic acid to other substances [α-Linolenic acid metabolism and linoleic acid metabolism were significantly enriched pathways with upregulated DAMs in MTD compared with HTD at 24 haa Fig. B, suggeshaa Fig. B. Moreovtabolism . LOX is bstances , 55. ACXbstances . In our Cuscuta reflexa induced tomato cells at the site of infection to secrete soluble phenylpropanol, eliciting a defense program in tomato and showing increased accumulation and activity of peroxidases associated with phenylpropanoids and other components of the cell wall, such as proteins, pectins, or cellulose fibers. The chemical composition of the wound tissue was found to contain substances belonging to the phenylpropanoids, as previously described [Phenylpropanoids play an important role in plant responses to biotic and abiotic stimuli . They meescribed , 38. In escribed . This suPinus taeda MYB4 resulted in increased expression of C3H, COMT, CCR, CCoAOMT, and CAD; the expression of 4CL and C4H genes was reportedly unaffected, although there was a decline in the expression of PAL genes [C. japonica and its hosts may also be a result of stress responses in host plants.In addition, many prediction methods, including cis-regulatory elements of monolignol biosynthesis pathway genes, have helped reveal the mystery of the transcriptional regulation of phenylpropanoid pathways . TranscrAL genes . In the C. japonica when parasitizing host and non-host plants. Complex changes were observed in transcriptome and metabolome regulation suggesting that metabolic pathways, such as α-linolenic acid, linoleic acid metabolism, and phenylpropanoid biosynthesis, might play important roles during haustoria development, and non-host plants might be resistant to dodder through interfering with these metabolic pathways during the formation of haustoria. Related pathway genes such as LOX, ACX, CCR, PAL, and CCoAOMT presented various regulatory trends and might be potential targets for inhibiting haustorium formation. The results provide insights for a better understanding of the metabolic mechanism of dodder–host interactions, and will therefore, contribute to inhibitory activity by identifying and analyzing the structures, functions, and metabolic pathways of differentially expressed metabolites.This study describes the transcriptomic and metabolomic differences of Ficus microcarpa ‘Golden Leaves’ (HTD) and Mangifera indica (MTD) plants were cultivated on Jiffy peat pellets under 8 h light and 16 h dark at 22 °C for one month and then individually placed at the bottom of deep pots with a diameter of 16 cm and 14 cm in height. The temperature was maintained at 25 °C (day) and 18 °C (night) under natural light . Dodder seeds were purchased from a traditional Chinese medicinal herb store in Kunming, China. The seeds were dipped in 95% sulfuric acid for 30 min. The treated seeds were then washed 10 times with water and placed on a wet filter paper in the dark at 25 °C, following a previously published method [Cuscuta japonica seedlings were attached to the stems of the host (HTD) and non-host (MTD) plants to form growing stocks. For attachment experiments, the plants with similar age, and the stems of host or non-host plants with similar size and close development stage were selected. All stem attachment were carried out at the same time. Normally, the attached position of C. japonica would begin to form haustoria and invade the stems of host plant within 24 h post attachment.All plants were grown in a greenhouse. The plant was identified by Prof. Ma and deposited in the germplasm resources nursery of the Guangxi Academy of Agricultural Sciences (deposition number: tsz-03). Permission to use this line was obtained. All methods were performed in accordance with relevant guidelines and regulations. d method . Four-daC. japonica during stock growth, proximal stem segments were harvested at 24 h and 72 haa. C. japonica usually grows ~ 3 cm per day if it successfully parasite and get nutrients from host plant, and it would grow ~ 1 cm per day without successful parasitism. Therefore, a 2 cm long proximal stem segment of C. japonica was cut 1 cm away from the attachment region to host or non-host plants. Three replicates were used for each stage, each of which was pooled from at least five stem segments. We used HTD and MTD strands for the dodder segments captured from the attachment of host-dodders and non-host-dodders, respectively. All specimens were harvested using sterile razor blades, and then frozen in liquid nitrogen and stored at − 80 °C for subsequent metabolomic and transcriptomic analyses.To investigate dynamic changes in C. japonica proximal stem samples including three biological replicates, were used for metabolite preparation. The stem samples were freeze-dried under vacuum and crushed at 30 Hz for 1.5 min using a Scientz-100F lyophilizer  and an MM 400 grinder  to produce the sample powder. After dehydration and crushing of the samples, approximately 100 mg of each sample powder was weighed and dissolved in 1.2 mL of 70% methanol. The mixtures were vortexed six times, each time for 30 s for 30 min, and stored at 4 °C overnight. The mixtures were then centrifuged at 12 000×g for 10 min. We implemented a microporous membrane (pore size:0.02) to filter the supernatant of each sample collected from the extracts in sample bottles for UPLC–MC analysis.Fifteen https://www.shimadzu.com.cn/) and tandem mass spectrometry  were performed. The liquid phase included an Agilent SB-C18 chromatographic column (2.1 × 100 mm), and the mobile phase, solvent A (0.1% acetic acid in water) and solvent B (0.1% acetic acid in water). The elution gradient program was 0 min with 95% A and 5% B; B linearly increased to 95% in 9 min and maintained at 95% for 1 min, then B dropped 5% from minute 10 to 11.10, and maintained at that ratio from minute 11.1 to 14. An injection volume of 4 μL was loaded onto the column at a flow rate of 0.35 mL/min at 40 °C. The MS parameters were as follows: temperature of the positive electrospray ionization, 550 °C; voltage, 5500 V/− 4500 V; ion source gas I, 50 psi; gas II, 60 psi; curtain gas, 25 psi; collision-activated dissociation, high. Triple quadrupole system (QQQ) scans were acquired using multiple reaction monitoring (MRM) experiments. We further evaluated the declustering potential and collision energy for each MRM ion pair. Finally, the set of MRM transitions was recorded in each period during the elution of the metabolite.To analyze the extracts, UPLC . Quantitative analyses of metabolites were performed using MRM. The isotope signal and the repeat signals of K+, Na+, and NH4+ were removed during the quantitative analysis. We computed the mass spectrum peak area integral of all samples and performed an integral correction for each metabolite across all samples. To assess the reliability and reproducibility of the data, we performed quality control by monitoring the changes in the mixtures of the extracted samples.Secondary spectrum information of the metabolites in the P-values of the hypergeometric test. The PCA was performed using the statistical function prcomp on the R platform.Metabolomic analysis was performed using the MetaboAnalystR (1.0.1) package in R , 34. To C. japonica stems from three biological replicates at each stage using the Qiagen RNeasy Plant Kit  according to the manufacturer’s protocol. DNA contamination, quality, concentration, and integrity of the total RNA were confirmed using agarose gel electrophoresis, a NanoPhotometer, qubit 2.0 fluorometer, and Agilent 2100 BioAnalyzer.Total RNA was extracted from C. japonica stem RNA-seq libraries were prepared using the Illumina TruSeq RNA Sample Prep Kit following the manufacturer’s instructions, and the quality of the library was detected using Qubit2.0 and Q-PCR. The cDNA library products that passed quality tests were sequenced on an Illumina HiSeq-2500 platform.The C. japonica retained after the filtering pipeline described above were used for de novo transcriptome assembly using Trinity (2.6.6) with default parameters, performed in a Linux cluster with 48 computing nodes and 1.5 TB memory to construct the unigenes.To obtain high-quality clean reads, read sets obtained from dodder stems were subjected to adapter removal and quality analysis using CASAVA . Read sets with N content exceeding 10% of the number of read bases were considered low-quality sequences and filtered out. The Trinity software package was used for the efficient and robust de novo assembly of clean reads. All read pairs from The unigenes were functionally annotated and classified using different databases, including nr protein, Swiss-Prot, KEGG, Trembl, Gene Ontology (GO), and Clusters of Orthologous Groups of proteins (COG), using BLAST software. First, we selected the nr, Swiss-Prot, KEGG, and COG databases to confirm sequence directions. Then, the alignment of unigenes and protein databases was implemented using BLASTx. Finally, the protein sequences of unigenes with the highest similarity were retrieved for functional annotation and classification.C. japonica were aligned to assemble the transcripts using Botwie2, and the quantity of gene expression was calculated by RSEM (RNA-seq by Expectation Maximization). The expression value of each unigene was normalized to fragments per kilobase of transcript per million fragments mapped reads (FPKM). To identify differentially expressed genes, we used the DEseq2 package (1.22.2) in R to analyze unstandardized read count data between two samples based on a false discovery rate (FDR) < 0.05, and absolute log2 FC ≥ 1.Clean reads from C. japonica during the formation of a haustorial connection. The DEGs of C. japonica were clustered according to their P-values. Clustered profiles with P ≤ 0.05 were considered differentially expressed. Genes within the selected clusters were enriched in GO terms and KEGG pathways for functional annotation using the hypergeometric distribution test. Functional items of each selected cluster with Q-values ≤0.05 were retained.Short time-series expression miner (STEM) software can process short time-series data for clustering and statistical biological explanations using exclusive approaches and integrating them with GO and KEGG databases . We usedAdditional file 1: Figure S1. Comparison of metabolite abundance in Cuscuta japonica attached to the host and non-host plants at 24 h (A) and 72 h (B). Figure S2. Number of differentially clustered metabolites in the HTD (A) and MTD (B) groups. The metabolite abundance trends of the differentially clustered profiles in the HTD (C) and MTD (D) groups are shown. Figure S3. Number of differentially clustered genes in the HTD (A) and MTD (B) groups. The gene expression trends of the differentially clustered profiles in the HTD (C) and MTD (D) groups are shown. Table S1. Metabolome profiles in Cuscuta japonica in the HTD and MTD groups. Table S2. Metabolite differences in Cuscuta japonica in MTD vs. _HTD at 24 haa. Table S3. Metabolite differences of C. japonica in MTD vs. _HTD at 72 haa. Table S4. Statistic of RNA-seq data. Table S5. DEGs of MTD vs HTD at 24 haa. Table S6. DEGs of MTD vs HTD at 72 haa.


The hepatitis A virus (HAV) is a highly hepatotropic virus transmitted mainly via the fecal–oral route. The purpose of this study is to describe a prolonged HAV outbreak in HIV-infected men who have sex with men (MSM) and pre-exposure prophylaxis (PrEP) users in Croatia in 2022. Croatia has a centralized system of HIV care and the PrEP service is only available at the University Hospital for Infectious Diseases (UHID), Zagreb. We reviewed all MSM living with HIV and MSM PrEP users at UHID and identified those diagnosed with HAV between January and October 2022. During this period, a total of 1036 MSM living with HIV and 361 PrEP users were followed, and 45 (4.4%) and 32 (8.9%) were diagnosed with HAV, respectively. Most cases were diagnosed in mid-February. A total of 70.1% (726/1036) MSM living with HIV and 82.3% (297/361) PrEP users were susceptible to HAV. Sequencing information was available for 34 persons; in all cases the HAV subtype IA was found. Our findings indicate that both MSM living with HIV and HIV-uninfected PrEP users are vulnerable to HAV infection and might be a potential source for a more widespread HAV epidemic.  Hepatovirus within the family Picornaviridae, transmitted via the faecal–oral route through direct contact and through contaminated food or water. In 2016 and before, European Union and European Economic Area (EU/EEA) countries have had a low incidence of HAV infection with most cases reported in children with slightly higher notification rates among males than females [The hepatitis A virus (HAV) is a small, non-enveloped noncytopathic and highly hepatotropic virus from the genus A large and prolonged epidemic in men who have sex with men (MSM) was noticed in many EU/EEA countries starting in 2016 and lasting until 2018 ,4. In 20The aim of this study is to describe an ongoing hepatitis A outbreak among HIV-infected MSM and MSM who use pre-exposure prophylaxis (PrEP) in Croatia that started in 2022. We were interested in describing the outbreak in the context of immunity to HAV and also report the main molecular findings of the HAV in the current epidemic. A brief overview of the HAV outbreaks among MSM in Europe from 2016 to 2021 is also given.Croatia has a centralized system of HIV care, and all persons living with HIV (PLWH) are treated at the University Hospital for Infectious Diseases (UHID) in Zagreb ,6,7. The® HAV IgM, DiaSorin, Saluggia, Italy). We also reviewed the HAV antibody status and assessed whether evidence of past syphilis was present by a positive Treponema pallidum hemagglutination test . If antibodies to HAV  were present before 2022, the person was considered to be immune to hepatitis A. We reviewed all PLWH and PrEP users who used the service at UHID and identified those who were diagnosed with hepatitis A between January and October 2022. Included in the analysis were MSM ≥ 18 years old. The diagnosis of HAV was established upon the detection of serum HAV-specific IgM antibodies . Detection of HAV RNA was performed by using Altostar HAV RT-PCR kit  on LightCycler 480 instrument . TM III One-Step RT-PCR System with Platinum® Taq High Fidelity”  and primersVP1f1 5′GTTTTGCTCCTCTTTATCATGCTATGU3′ VP1r1 at 50 °C for 30 min, 94 °C for 2 min, 45×  and 68 °C for 5 min. Nested PCR was carried out with the “FastStart™ High Fidelity PCR System” , primers VP1Nf2 5′TATCATGCTATGGATGTTAC3′ and VP1Nr2 5′TTCATTATTTCATGCTCCTC3′ at 94 °C for 2 min, 45×  and 72 °C for 7 min. Sequencing reaction was performed by using BigDyeTM Terminator v3.1 Cycle Sequencing Kit  with primers used for nested PCR. The reaction was carried out at 25× . The sequences were analysed in the “Vector NTI” program, and the genotype and subtype of VP1-2A HAV genome region was determined by using the Hepatitis A Virus Genotyping Tool Version 1.0 algorithm . CustalW within MEGA X was used for the alignment of the 45 obtained sequences, along with 12 sequences from the NCBI gene bank where 878 nucleotides was the final length of the sequences. The phylogenetic tree was constructed in the MEGA X program using the maximum likelihood method, Tamura-Nei evolutionary model with 1000 bootstrap replicates. Sequences of the VP1-2A region from the 2022 epidemics are available in the GenBank under accession numbers OP807977-OP808012. Phylogenetic analysis also included 35 sequences from the Croatian HAV epidemics in the years 2017 and 2018 available in the GenBank under accession number MK396845-MK396889.For HAV RNA sequencing, RT-PCR was performed by using “SuperScriptWe describe our findings with the median and first and third quartiles for continuous variables and by frequencies for categorical variables. Continuous variables were compared using the Mann–Whitney test; categorical variables were compared using the Chi-square test. The trend in the susceptibility to hepatitis A by age categories was assessed using the Cochrane–Armitage test. We used GraphPad Prism version 9.4 for Windows  to make the graphs and SAS software 9.4  to perform the analyses.We searched PubMed for research articles published up to October 31, 2022, with no language restrictions, using the following sets of search terms: “HAV”, “MSM” and “Europe”. In addition, European Centre for Disease Prevention and Control epidemiological reports and surveillance data were included. Studies conducted in Europe with abstracts published in English were included. Studies not conducted in Europe, and studies that did not include any relevant data on epidemic characteristics and included only serology, molecular, vaccination, behavior or epidemiological data were excluded from the review. Finally, 28 studies were included and described in the discussion section.p = 0.087). The median duration of HIV infection was 5.5 years, and more than 90% were using antiretroviral treatment (ART). Of 45 PLWH, 41 had a VL < 50 copies/mL. Of four participants with plasma HIV-RNA > 50 copies/mL, two had a concomitant HIV and hepatitis A diagnosis, one had interrupted antiretroviral therapy, and one had only 1-month of ART. Among persons with hepatitis A, more than ¾ of PLWH and less than half of PrEP users had syphilis in the past , RIVM-HAV-090 strain cluster from the 2018 epidemic  and VRD_521_2016 strain cluster from the 2017/2018 epidemic  .We report an increase in the number of persons diagnosed with hepatitis A among MSM living with HIV and MSM PrEP users in Croatia from January to October 2022. Most cases were diagnosed in mid-February . During p < 0.001) [Hepatitis A outbreaks were described among PLWH and PrEP users during the 2017/2018 European epidemic ,11,12,13< 0.001) . p = 0.48) [p < 0.001). This could be partially explained by the older age of PLWH and possibly by some behavioral factors and willingness to receive the vaccine. However, the HAV vaccine in Croatia is recommended only for travelers and as post-exposure prophylaxis, but due to limited amounts and frequent shortages, it was not possible to provide the vaccine for all MSM at risk at UHID in the current epidemic.In southwest Hungary during the period 2010–2020, the HAV seropositivity was low (less than 18%) up to the age of 40 years and increased to 75–80% at age group 66–70 years . SimilarPrevious studies showed that patients diagnosed with HAV are often coinfected with another sexually transmitted disease (STD) ,20. Almohttps://www.ecdc.europa.eu/en/hepatitis-a/surveillance/atlas, access date 5 November 2022). After 96 HAV cases were reported in Croatia in 2018, there were only 9, 5 and 5 cases reported in 2019, 2020 and 2021, respectively (https://www.ecdc.europa.eu/en/hepatitis-a/surveillance/atlas). However, there is a possibility of under-reporting, and these numbers might underestimate actual case numbers.After an increase of confirmed HAV cases in 2017 in EU/EEA countries, including Croatia, which was associated with younger MSM , the numOur report on the HIV outbreak has some limitations. We included only patients diagnosed with HAV followed at the HIV Outpatient Department at UHID. This could result in underestimation of case numbers as some persons might seek help elsewhere. In addition, we did not have any data on recent travel or other risk behaviors.p < 0.001), came from an HAV endemic country (p < 0.001) and were HBV seropositive (p = 0.001) [p < 0.001 [The European outbreak of hepatitis A during 2017/2018 was reported from a number of countries. The increase in HAV cases was observed mostly among younger MSM  4,8,9,1,32,33,34= 0.001) . During = 0.001) . In Vien < 0.001 . Between < 0.001 . Table 3The response to an HAV outbreak among MSM should include expanded vaccination accessibility as well as involvement and good communication with the community, including using social media. For example, in Normandy, France, effective vaccination campaigns included HAV vaccination sexually transmitted infection clinics, at community-based sites and on mobile trucks. Vaccination and sexual health promotion through social media and gay dating apps have also been implemented . BecauseIn October and December 2016, the Netherlands, followed by the United Kingdom, reported two clusters of hepatitis A virus sub-genotype IA infections. Later, the investigation of these cases uncovered an HAV outbreak disproportionally affecting MSM . During In 2017, a total of 19,947 HAV cases (mostly genotype IA) were reported by 24 EU/EEA countries which represented more than a fourfold increase compared with the average of 4671 cases reported in 2012–2015. The confirmed number of cases provides an underestimation of the true extent of the outbreak due to the lack of sequencing information. This outbreak was also associated with MSM . There wIn the period July 2016 to February 2017, 48 HAV cases were confirmed in the Netherlands; 17 were MSM, and 10/13 cases with available typing information were infected with RIVM-HAV16-090 (EuroPride strain) .Similar to the epidemiological situation in the abovementioned European countries, HAV strains RIVM-HAV16-090 and VRD_521_2016 were detected among MSM from Croatia in the period 2017-2018. HAV genotyping of IA sequences from the current Croatian epidemics among MSM revealed a cluster of IA genotype sequences that was closely related but not identical to the RIVM-HAV16-090 and VRD_521_2016 local strains from 2017–2018, suggesting a possibility of a distinct source of infection that needs to be a subject of additional epidemiological monitoring and characterization in the near future. We described a prolonged hepatitis A outbreak among HIV-infected MSM and PrEP users in Zagreb, Croatia, that started in January 2022. Our findings indicate that both MSM living with HIV and HIV-uninfected PrEP users in Croatia are vulnerable to HAV infection and might be a potential source for a more widespread HAV epidemic. To avoid future hepatitis A outbreaks, HAV vaccination for all MSM should be implemented. There is an opportunity to implement vaccination “on-site” at the HIV Outpatient Department and PrEP service at UHID, Zagreb, Croatia.


Stress hyperglycemia is common in critical and severe diseases. However, few studies have examined the association between stress hyperglycemia and the functional outcomes of patients with anterior circulation stroke, after mechanical thrombectomy (MT), in different diabetes status. This study therefore aimed to determine the relationship between stress hyperglycemia and the risk of adverse neurological functional outcomes in anterior circulation stroke patients with and without diabetes after MT.Data of 408 patients with acute anterior circulation stroke treated with MT through the green-channel treatment system for emergency stroke at the First Affiliated Hospital of Jinan University between January 2016 and December 2020 were reviewed retrospectively. The stress hyperglycemia ratio (SHR) was calculated as fasting plasma glucose (mmol/L) divided by glycosylated hemoglobin (%). The patients were stratified into four groups by quartiles of SHR (Q1-Q4). The primary outcome was an excellent (nondisabled) functional outcome at 3 months after admission . The relationship between stress hyperglycemia and neurological outcome after stroke was assessed using multivariate logistic regression.p = 0.003), a good outcome at 3 months , and major neurological improvement . Severe stress hyperglycemia increased risks of 3-months all-cause mortality  and ICH .After adjusting for potential confounders, compared with patients in Q1, those in Q4 were less likely to have an excellent outcome at 3 months (odds ratio [OR], 0.32, 95% confidence interval [CI], 0.14–0.66, Stress hyperglycemia was associated with a reduced rate of excellent neurological outcomes, and increased mortality and ICH risks in patients with anterior circulation stroke after MT regardless of diabetes status. Altered glucose metabolism and hyperglycemia at admission are common in patients with acute ischemic stroke (AIS), regardless of the status of diabetes. Several previous studies showed that stress hyperglycemia predicted a poor prognosis of stroke . They deEndovascular therapy has been shown to be beneficial for patients with AIS . HoweverFurthermore, the premorbid diabetic status may be a potential influence for stress hyperglycemia on the outcomes for AIS patients or AIS patients treated with intravenous thrombolysis . HoweverThis retrospective study enrolled consecutive patients with AIS treated with MT at the stroke center of the First Affiliated Hospital of Jinan University between January 2016 and December 2020. The inclusion criteria for the patients screened for enrollment were (1) AIS diagnosis and admission within 6 h after symptom onset or more than 6 h after illness onset, but with MT performed as an emergency measure after evaluation by professional neurology experts; (2) proximal large-vessel occlusion at the internal carotid artery, middle cerebral artery M1 or M2, or anterior cerebral artery A1 or A2; and (3) treatment with MT and successful reperfusion. Patients were excluded if they conformed to any of the following criteria: (1) posterior circulation stroke; (2) lacking integral laboratory data, such as FPG on the second day, or HbA1c or computed tomography (CT)/magnetic resonance imaging (MRI) scans, or (3) did not receive follow-up.This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Jinan University. Given the retrospective design of this study, there was no requirement to obtained informed consent. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki.We collected the following clinical data: age, sex, systolic blood pressure at admission, vascular risk factors, stroke severity, laboratory findings and information on therapy. Vascular risk factors included previous transient ischemic attack (TIA) or stroke, cardiovascular disease, atrial fibrillation, hypertension, diabetes, and previous or current smoking status. Stroke severity was assessed at admission and 24 h thereafter by physicians using the NIHSS. Functional outcome was assessed by means of the modified Rankin Scale (mRS) at admission, ground on pre-stroke disability, and 3 months after stroke. The mRS score after discharge was collected through telephone interview with patients or their immediate caregivers. Levels of FPG, HbA1c, hemoglobin, creatinine, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride, homocysteine, and high-sensitivity C-reactive protein, and the international normalized ratio at admission were extracted from the hospital system.Participants were diagnosed with diabetes according to a prior history of diabetes or an HbA1c level of ≥6.5% . Stroke FPG and HbA1c were tested within 24 h of hospitalization, and venous blood samples were drawn during the morning (05:00–09:00) after an overnight fast lasting at least 8 h. FPG levels were tested using the hexokinase method when HbA1c was measured using ion-exchange chromatography. According to the instructions of the manufacturer, the reference intervals were glucose level = 3.89–6.11 mmol/l and HbA1c = 4.0–6.1%. Stress hyperglycemia was estimated using the stress hyperglycemia ratio (SHR), which was calculated as FPG (mmol/L) divided by HbA1c (%). The patients enrolled were stratified into four groups according to SHR quartiles (Q1–Q4) for further comparisons. The extent of stress hyperglycemia was quantified by the relative stress hyperglycemia considering the background glucose levels.The primary outcome in the current analyses was an excellent (nondisabled) functional outcome at 3 months after admission, defined as a mRS score of 0–1 . SecondaH test as appropriate. Patients in the lowest SHR quartile  were compared with those in the other three SHR quartile (Q2–Q4) groups. Baseline variables that were considered clinically relevant or had a univariate relationship with the prognosis were included in a multivariate logistic regression model. Given the number of available events, the variables included were all carefully selected to ensure the refinement of the final model.Percentage, mean (± standard deviation), or median (interquartile range) values were reported depending on the characteristics of the variables. Categorical variables were analyzed using the chi-square or Fisher’s exact tests. Continuous variables were analyzed using one-way ANOVA or the Kruskal–Wallis p value of the interaction between the subgroup and the four cohorts was calculated from the Log-likelihood ratio test, and the p value for interaction of <0.05 was deemed statistically significant for effect modification.To test whether diabetes (yes/no) modified relationships between stress hyperglycemia status and neurological function outcomes, the risks of mortality or ICH, subgroup analyses of participants with or without diabetes were performed separately. The p < 0.05 was considered significant.Receiver operating characteristic (ROC) curves were used to examine the predictive value of FBG, HbA1c or SHR with no excellent functional outcome based on the area under the ROC curve (AUC) of patients with anterior circulation stroke after MT. Statistical analyses were conducted using R software, MedCalc (Version 20.121) and SPSS (version 27.0). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated, and a two-tailed probability value of During the study period, 515 patients who underwent MT met the inclusion criteria. Of these, 43 with posterior circulation stroke, 41 without FPG, HbA1c or CT/MRI data, and 19 who were not followed up were excluded. The study cohort therefore comprised 408 patients. These data are summarized in the study flow diagram in p < 0.001 for trend), good outcomes at 3 months , major neurological improvement , all-cause mortality at 3 months , and ICH  differed significantly among the four quartiles.p = 0.004 for trend; p = 0.015), and major neurological improvement . Furthermore, SHR increased risks of 3-month all-cause mortality  and ICH . Similarly, rates of sICH were numerically higher among patients in Q4 than that in Q1 (33.3% versus 4.76%), though differences did not reach statistical significance in multivariate analysis(p = 0.060).After adjusting for potential confounders, significant associations emerged between an excellent outcome and sex, age, hypertension, previous TIA/stroke, smoking, pre-stroke mRS, NIHSS score at admission, and SHR . Patientp-value of different groups are diverse among the outcomes. However, the p-values of the interaction test results are all >0.05, indicating whether the patient had diabetes or not has no significant impact on the relationship between SHR and neurological function outcomes, all-cause mortality and hemorrhage outcomes. Due to the small subgroup sample size, the result for the diabetes status on the association of SHR and sICH are lacking.In the subgroup analysis , the OR p < 0.0001; SHR versus HbA1c, p = 0.0020). However, the association of the predictive value between FBG and SHR was not significant  and FBG  for less excellent neurological outcomes was stronger than HbA1c /HbA1c (%), is an indicator of relative hyperglycemia, which avoids deviations in prognosis assessments of absolute hyperglycemia resulting from unknown background glucose levels. FPG, which reflects the blood glucose status at admission, avoids the effects of food intake on the immediate blood glucose levels at admission. HbA1c reflects blood glucose levels during the previous 2–3 months, and we adjusted for factors that may affect HbA1c measurements  to improve the validity of the results. Another recent study also found that the SHR calculated as FPG (mmol/L)/HbA1c (%) had a better predictive power for outcomes than other SHR definitions, such as being calculated as FPG (mmol/L)/ ([1.59 × HbA1c] −2.59) and admission blood glucose (mmol/L)//HbA1c (%), was associated with a reduced rate of excellent neurological outcomes and increased mortality and ICH risks in patients with acute anterior circulation stroke after MT regardless of diabetes status. Different glucose control targets can be adopted for blood glucose after admission according to different background glucose.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRB of the First Affiliated Hospital of Jinan University, the First Affiliated Hospital of Jinan University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.LH and HZ conceptualized the study and supervised the study. JZ, D-wD, YZ, BY, FL, XC, JL, MG, NH, HQ, KL, and AX contributed to the acquisition of data. JZ, D-wD, YZ, and FL performed the statistical analysis and interpreted data. JZ, D-wD, and YZ prepared the manuscript. BY, FL, XC, JL, MG, NH, HQ, KL, AX, LH, and HZ revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Natural Science Foundation of China (81901200), Science and Technology Program in Guangzhou (202002030425 and 202002020003), the Clinical Frontier Technology Program of the First Affiliated Hospital of Jinan University, China (JNU1AF-CFTP-2022-a01215 and JNU1AF-CFTP-2022-a01217) and Key Field Program of Colleges and Universities in Guangdong Province (2021ZDZX2025).The 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.